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Sample records for replication complex reveals

  1. Evolution of complexity in RNA-like replicator systems

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    Hogeweg Paulien

    2008-03-01

    Full Text Available Abstract Background The evolution of complexity is among the most important questions in biology. The evolution of complexity is often observed as the increase of genetic information or that of the organizational complexity of a system. It is well recognized that the formation of biological organization – be it of molecules or ecosystems – is ultimately instructed by the genetic information, whereas it is also true that the genetic information is functional only in the context of the organization. Therefore, to obtain a more complete picture of the evolution of complexity, we must study the evolution of both information and organization. Results Here we investigate the evolution of complexity in a simulated RNA-like replicator system. The simplicity of the system allows us to explicitly model the genotype-phenotype-interaction mapping of individual replicators, whereby we avoid preconceiving the functionality of genotypes (information or the ecological organization of replicators in the model. In particular, the model assumes that interactions among replicators – to replicate or to be replicated – depend on their secondary structures and base-pair matching. The results showed that a population of replicators, originally consisting of one genotype, evolves to form a complex ecosystem of up to four species. During this diversification, the species evolve through acquiring unique genotypes with distinct ecological functionality. The analysis of this diversification reveals that parasitic replicators, which have been thought to destabilize the replicator's diversity, actually promote the evolution of diversity through generating a novel "niche" for catalytic replicators. This also makes the current replicator system extremely stable upon the evolution of parasites. The results also show that the stability of the system crucially depends on the spatial pattern formation of replicators. Finally, the evolutionary dynamics is shown to

  2. Centromeric DNA replication reconstitution reveals DNA loops and ATR checkpoint suppression.

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    Aze, Antoine; Sannino, Vincenzo; Soffientini, Paolo; Bachi, Angela; Costanzo, Vincenzo

    2016-06-01

    Half of the human genome is made up of repetitive DNA. However, mechanisms underlying replication of chromosome regions containing repetitive DNA are poorly understood. We reconstituted replication of defined human chromosome segments using bacterial artificial chromosomes in Xenopus laevis egg extract. Using this approach we characterized the chromatin assembly and replication dynamics of centromeric alpha-satellite DNA. Proteomic analysis of centromeric chromatin revealed replication-dependent enrichment of a network of DNA repair factors including the MSH2-6 complex, which was required for efficient centromeric DNA replication. However, contrary to expectations, the ATR-dependent checkpoint monitoring DNA replication fork arrest could not be activated on highly repetitive DNA due to the inability of the single-stranded DNA binding protein RPA to accumulate on chromatin. Electron microscopy of centromeric DNA and supercoil mapping revealed the presence of topoisomerase I-dependent DNA loops embedded in a protein matrix enriched for SMC2-4 proteins. This arrangement suppressed ATR signalling by preventing RPA hyper-loading, facilitating replication of centromeric DNA. These findings have important implications for our understanding of repetitive DNA metabolism and centromere organization under normal and stressful conditions.

  3. In Situ Tagged nsp15 Reveals Interactions with Coronavirus Replication/Transcription Complex-Associated Proteins

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    Jeremiah Athmer

    2017-01-01

    Full Text Available Coronavirus (CoV replication and transcription are carried out in close proximity to restructured endoplasmic reticulum (ER membranes in replication/transcription complexes (RTC. Many of the CoV nonstructural proteins (nsps are required for RTC function; however, not all of their functions are known. nsp15 contains an endoribonuclease domain that is conserved in the CoV family. While the enzymatic activity and crystal structure of nsp15 are well defined, its role in replication remains elusive. nsp15 localizes to sites of RNA replication, but whether it acts independently or requires additional interactions for its function remains unknown. To begin to address these questions, we created an in situ tagged form of nsp15 using the prototypic CoV, mouse hepatitis virus (MHV. In MHV, nsp15 contains the genomic RNA packaging signal (P/S, a 95-bp RNA stem-loop structure that is not required for viral replication or nsp15 function. Utilizing this knowledge, we constructed an internal hemagglutinin (HA tag that replaced the P/S. We found that nsp15-HA was localized to discrete perinuclear puncta and strongly colocalized with nsp8 and nsp12, both well-defined members of the RTC, but not the membrane (M protein, involved in virus assembly. Finally, we found that nsp15 interacted with RTC-associated proteins nsp8 and nsp12 during infection, and this interaction was RNA independent. From this, we conclude that nsp15 localizes and interacts with CoV proteins in the RTC, suggesting it plays a direct or indirect role in virus replication. Furthermore, the use of in situ epitope tags could be used to determine novel nsp-nsp interactions in coronaviruses.

  4. Complex Dynamic Development of Poliovirus Membranous Replication Complexes

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    Nair, Vinod; Hansen, Bryan T.; Hoyt, Forrest H.; Fischer, Elizabeth R.; Ehrenfeld, Ellie

    2012-01-01

    Replication of all positive-strand RNA viruses is intimately associated with membranes. Here we utilize electron tomography and other methods to investigate the remodeling of membranes in poliovirus-infected cells. We found that the viral replication structures previously described as “vesicles” are in fact convoluted, branching chambers with complex and dynamic morphology. They are likely to originate from cis-Golgi membranes and are represented during the early stages of infection by single-walled connecting and branching tubular compartments. These early viral organelles gradually transform into double-membrane structures by extension of membranous walls and/or collapsing of the luminal cavity of the single-membrane structures. As the double-membrane regions develop, they enclose cytoplasmic material. At this stage, a continuous membranous structure may have double- and single-walled membrane morphology at adjacent cross-sections. In the late stages of the replication cycle, the structures are represented mostly by double-membrane vesicles. Viral replication proteins, double-stranded RNA species, and actively replicating RNA are associated with both double- and single-membrane structures. However, the exponential phase of viral RNA synthesis occurs when single-membrane formations are predominant in the cell. It has been shown previously that replication complexes of some other positive-strand RNA viruses form on membrane invaginations, which result from negative membrane curvature. Our data show that the remodeling of cellular membranes in poliovirus-infected cells produces structures with positive curvature of membranes. Thus, it is likely that there is a fundamental divergence in the requirements for the supporting cellular membrane-shaping machinery among different groups of positive-strand RNA viruses. PMID:22072780

  5. Assembly of Slx4 signaling complexes behind DNA replication forks.

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    Balint, Attila; Kim, TaeHyung; Gallo, David; Cussiol, Jose Renato; Bastos de Oliveira, Francisco M; Yimit, Askar; Ou, Jiongwen; Nakato, Ryuichiro; Gurevich, Alexey; Shirahige, Katsuhiko; Smolka, Marcus B; Zhang, Zhaolei; Brown, Grant W

    2015-08-13

    Obstructions to replication fork progression, referred to collectively as DNA replication stress, challenge genome stability. In Saccharomyces cerevisiae, cells lacking RTT107 or SLX4 show genome instability and sensitivity to DNA replication stress and are defective in the completion of DNA replication during recovery from replication stress. We demonstrate that Slx4 is recruited to chromatin behind stressed replication forks, in a region that is spatially distinct from that occupied by the replication machinery. Slx4 complex formation is nucleated by Mec1 phosphorylation of histone H2A, which is recognized by the constitutive Slx4 binding partner Rtt107. Slx4 is essential for recruiting the Mec1 activator Dpb11 behind stressed replication forks, and Slx4 complexes are important for full activity of Mec1. We propose that Slx4 complexes promote robust checkpoint signaling by Mec1 by stably recruiting Dpb11 within a discrete domain behind the replication fork, during DNA replication stress. © 2015 The Authors.

  6. A NuRD Complex from Xenopus laevis Eggs Is Essential for DNA Replication during Early Embryogenesis

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    Christo P. Christov

    2018-02-01

    Full Text Available DNA replication in the embryo of Xenopus laevis changes dramatically at the mid-blastula transition (MBT, with Y RNA-independent random initiation switching to Y RNA-dependent initiation at specific origins. Here, we identify xNuRD, an MTA2-containing assemblage of the nucleosome remodeling and histone deacetylation complex NuRD, as an essential factor in pre-MBT Xenopus embryos that overcomes a functional requirement for Y RNAs during DNA replication. Human NuRD complexes have a different subunit composition than xNuRD and do not support Y RNA-independent initiation of DNA replication. Blocking or immunodepletion of xNuRD inhibits DNA replication initiation in isolated nuclei in vitro and causes inhibition of DNA synthesis, developmental delay, and embryonic lethality in early embryos. xNuRD activity declines after the MBT, coinciding with dissociation of the complex and emergence of Y RNA-dependent initiation. Our data thus reveal an essential role for a NuRD complex as a DNA replication factor during early Xenopus development.

  7. Chromosomal Replication Complexity: A Novel DNA Metrics and Genome Instability Factor.

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    Andrei Kuzminov

    2016-10-01

    Full Text Available As the ratio of the copy number of the most replicated to the unreplicated regions in the same chromosome, the definition of chromosomal replication complexity (CRC appears to leave little room for variation, being either two during S-phase or one otherwise. However, bacteria dividing faster than they replicate their chromosome spike CRC to four and even eight. A recent experimental inquiry about the limits of CRC in Escherichia coli revealed two major reasons to avoid elevating it further: (i increased chromosomal fragmentation and (ii complications with subsequent double-strand break repair. Remarkably, examples of stable elevated CRC in eukaryotic chromosomes are well known under various terms like "differential replication," "underreplication," "DNA puffs," "onion-skin replication," or "re-replication" and highlight the phenomenon of static replication fork (sRF. To accurately describe the resulting "amplification by overinitiation," I propose a new term: "replification" (subchromosomal overreplication. In both prokaryotes and eukaryotes, replification, via sRF processing, causes double-strand DNA breaks and, with their repair elevating chromosomal rearrangements, represents a novel genome instability factor. I suggest how static replication bubbles could be stabilized and speculate that some tandem duplications represent such persistent static bubbles. Moreover, I propose how static replication bubbles could be transformed into tandem duplications, double minutes, or inverted triplications. Possible experimental tests of these models are discussed.

  8. Organizational changes of the daughter basal complex during the parasite replication of Toxoplasma gondii.

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    Ke Hu

    2008-01-01

    Full Text Available The apicomplexans are a large group of parasitic protozoa, many of which are important human and animal pathogens, including Plasmodium falciparum and Toxoplasma gondii. These parasites cause disease only when they replicate, and their replication is critically dependent on the proper assembly of the parasite cytoskeletons during cell division. In addition to their importance in pathogenesis, the apicomplexan parasite cytoskeletons are spectacular structures. Therefore, understanding the cytoskeletal biogenesis of these parasites is important not only for parasitology but also of general interest to broader cell biology. Previously, we found that the basal end of T. gondii contains a novel cytoskeletal assembly, the basal complex, a cytoskeletal compartment constructed in concert with the daughter cortical cytoskeleton during cell division. This study focuses on key events during the biogenesis of the basal complex using high resolution light microscopy, and reveals that daughter basal complexes are established around the duplicated centrioles independently of the structural integrity of the daughter cortical cytoskeleton, and that they are dynamic "caps" at the growing ends of the daughters. Compartmentation and polarization of the basal complex is first revealed at a late stage of cell division upon the recruitment of an EF-hand containing calcium binding protein, TgCentrin2. This correlates with the constriction of the basal complex, a process that can be artificially induced by increasing cellular calcium concentration. The basal complex is therefore likely to be a new kind of centrin-based contractile apparatus.

  9. Regulation of adeno-associated virus DNA replication by the cellular TAF-I/set complex.

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    Pegoraro, Gianluca; Marcello, Alessandro; Myers, Michael P; Giacca, Mauro

    2006-07-01

    The Rep proteins of the adeno-associated virus (AAV) are required for viral replication in the presence of adenovirus helper functions and as yet poorly characterized cellular factors. In an attempt to identify such factors, we purified Flag-Rep68-interacting proteins from human cell lysates. Several polypeptides were identified by mass spectrometry, among which was ANP32B, a member of the acidic nuclear protein 32 family which takes part in the formation of the template-activating factor I/Set oncoprotein (TAF-I/Set) complex. The N terminus of Rep was found to specifically bind the acidic domain of ANP32B; through this interaction, Rep was also able to recruit other members of the TAF-I/Set complex, including the ANP32A protein and the histone chaperone TAF-I/Set. Further experiments revealed that silencing of ANP32A and ANP32B inhibited AAV replication, while overexpression of all of the components of the TAF-I/Set complex increased de novo AAV DNA synthesis in permissive cells. Besides being the first indication that the TAF-I/Set complex participates in wild-type AAV replication, these findings have important implications for the generation of recombinant AAV vectors since overexpression of the TAF-I/Set components was found to markedly increase viral vector production.

  10. Evolution of DNA replication protein complexes in eukaryotes and Archaea.

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    Nicholas Chia

    Full Text Available BACKGROUND: The replication of DNA in Archaea and eukaryotes requires several ancillary complexes, including proliferating cell nuclear antigen (PCNA, replication factor C (RFC, and the minichromosome maintenance (MCM complex. Bacterial DNA replication utilizes comparable proteins, but these are distantly related phylogenetically to their archaeal and eukaryotic counterparts at best. METHODOLOGY/PRINCIPAL FINDINGS: While the structures of each of the complexes do not differ significantly between the archaeal and eukaryotic versions thereof, the evolutionary dynamic in the two cases does. The number of subunits in each complex is constant across all taxa. However, they vary subtly with regard to composition. In some taxa the subunits are all identical in sequence, while in others some are homologous rather than identical. In the case of eukaryotes, there is no phylogenetic variation in the makeup of each complex-all appear to derive from a common eukaryotic ancestor. This is not the case in Archaea, where the relationship between the subunits within each complex varies taxon-to-taxon. We have performed a detailed phylogenetic analysis of these relationships in order to better understand the gene duplications and divergences that gave rise to the homologous subunits in Archaea. CONCLUSION/SIGNIFICANCE: This domain level difference in evolution suggests that different forces have driven the evolution of DNA replication proteins in each of these two domains. In addition, the phylogenies of all three gene families support the distinctiveness of the proposed archaeal phylum Thaumarchaeota.

  11. Replication-mediated disassociation of replication protein A-XPA complex upon DNA damage: implications for RPA handing off.

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    Jiang, Gaofeng; Zou, Yue; Wu, Xiaoming

    2012-08-01

    RPA (replication protein A), the eukaryotic ssDNA (single-stranded DNA)-binding protein, participates in most cellular processes in response to genotoxic insults, such as NER (nucleotide excision repair), DNA, DSB (double-strand break) repair and activation of cell cycle checkpoint signalling. RPA interacts with XPA (xeroderma pigmentosum A) and functions in early stage of NER. We have shown that in cells the RPA-XPA complex disassociated upon exposure of cells to high dose of UV irradiation. The dissociation required replication stress and was partially attributed to tRPA hyperphosphorylation. Treatment of cells with CPT (camptothecin) and HU (hydroxyurea), which cause DSB DNA damage and replication fork collapse respectively and also leads to the disruption of RPA-XPA complex. Purified RPA and XPA were unable to form complex in vitro in the presence of ssDNA. We propose that the competition-based RPA switch among different DNA metabolic pathways regulates the dissociation of RPA with XPA in cells after DNA damage. The biological significances of RPA-XPA complex disruption in relation with checkpoint activation, DSB repair and RPA hyperphosphorylation are discussed.

  12. Replication-mediated disassociation of replication protein A–XPA complex upon DNA damage: implications for RPA handing off

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    Jiang, Gaofeng; Zou, Yue; Wu, Xiaoming

    2013-01-01

    RPA (replication protein A), the eukaryotic ssDNA (single-stranded DNA)-binding protein, participates in most cellular processes in response to genotoxic insults, such as NER (nucleotide excision repair), DNA, DSB (double-strand break) repair and activation of cell cycle checkpoint signalling. RPA interacts with XPA (xeroderma pigmentosum A) and functions in early stage of NER. We have shown that in cells the RPA–XPA complex disassociated upon exposure of cells to high dose of UV irradiation. The dissociation required replication stress and was partially attributed to tRPA hyperphosphorylation. Treatment of cells with CPT (camptothecin) and HU (hydroxyurea), which cause DSB DNA damage and replication fork collapse respectively and also leads to the disruption of RPA–XPA complex. Purified RPA and XPA were unable to form complex in vitro in the presence of ssDNA. We propose that the competition-based RPA switch among different DNA metabolic pathways regulates the dissociation of RPA with XPA in cells after DNA damage. The biological significances of RPA–XPA complex disruption in relation with checkpoint activation, DSB repair and RPA hyperphosphorylation are discussed. PMID:22578086

  13. Prereplicative complexes assembled in vitro support origin-dependent and independent DNA replication

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    On, Kin Fan; Beuron, Fabienne; Frith, David; Snijders, Ambrosius P; Morris, Edward P; Diffley, John F X

    2014-01-01

    Eukaryotic DNA replication initiates from multiple replication origins. To ensure each origin fires just once per cell cycle, initiation is divided into two biochemically discrete steps: the Mcm2-7 helicase is first loaded into prereplicative complexes (pre-RCs) as an inactive double hexamer by the origin recognition complex (ORC), Cdt1 and Cdc6; the helicase is then activated by a set of “firing factors.” Here, we show that plasmids containing pre-RCs assembled with purified proteins support complete and semi-conservative replication in extracts from budding yeast cells overexpressing firing factors. Replication requires cyclin-dependent kinase (CDK) and Dbf4-dependent kinase (DDK). DDK phosphorylation of Mcm2-7 does not by itself promote separation of the double hexamer, but is required for the recruitment of firing factors and replisome components in the extract. Plasmid replication does not require a functional replication origin; however, in the presence of competitor DNA and limiting ORC concentrations, replication becomes origin-dependent in this system. These experiments indicate that Mcm2-7 double hexamers can be precursors of replication and provide insight into the nature of eukaryotic DNA replication origins. PMID:24566989

  14. Elg1 forms an alternative RFC complex important for DNA replication and genome integrity

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    Bellaoui, Mohammed; Chang, Michael; Ou, Jiongwen; Xu, Hong; Boone, Charles; Brown, Grant W

    2003-01-01

    Genome-wide synthetic genetic interaction screens with mutants in the mus81 and mms4 replication fork-processing genes identified a novel replication factor C (RFC) homolog, Elg1, which forms an alternative RFC complex with Rfc2-5. This complex is distinct from the DNA replication RFC, the DNA

  15. Optical tweezers reveal how proteins alter replication

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    Chaurasiya, Kathy

    acids. We use single molecule DNA stretching to show that the nucleocapsid protein (NC) of the yeast retrotransposon Ty3, which is likely to be an ancestor of HIV NC, has optimal nucleic acid chaperone activity with only a single zinc finger. We also show that the chaperone activity of the ORF1 protein is responsible for successful replication of the mouse LINE-1 retrotransposon. LINE-1 is also 17% of the human genome, where it generates insertion mutations and alters gene expression. Retrotransposons such as LINE-1 and Ty3 are likely to be ancestors of retroviruses such as HIV. Human APOBEC3G (A3G) inhibits HIV-1 replication via cytidine deamination of the viral ssDNA genome, as well as via a distinct deamination-independent mechanism. Efficient deamination requires rapid on-off binding kinetics, but a slow dissociation rate is required for the proposed deaminase-independent mechanism. We resolve this apparent contradiction with a new quantitative single molecule method, which shows that A3G initially binds ssDNA with fast on-off rates and subsequently converts to a slow binding mode. This suggests that oligomerization transforms A3G from a fast enzyme to a slow binding protein, which is the biophysical mechanism that allows A3G to inhibit HIV replication. A complete understanding of the mechanism of A3G-mediated antiviral activity is required to design drugs that disrupt the viral response to A3G, enhance A3G packaging inside the viral core, and other potential strategies for long-term treatment of HIV infection. We use single molecule biophysics to explore the function of proteins involved in bacterial DNA replication, endogenous retrotransposition of retroelements in eukaryotic hosts such yeast and mice, and HIV replication in human cells. Our quantitative results provide insight into protein function in a range of complex biological systems and have wide-ranging implications for human health.

  16. H4K20me0 marks post-replicative chromatin and recruits the TONSL₋MMS22L DNA repair complex

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    Saredi, Giulia; Huang, Hongda; Hammond, Colin M.; Alabert, Constance; Bekker-Jensen, Simon; Forne, Ignasi; Reverón-Gómez, Nazaret; Foster, Benjamin M.; Mlejnkova, Lucie; Bartke, Till; Cejka, Petr; Mailand, Niels; Imhof, Axel; Patel, Dinshaw J.; Groth, Anja [UCopenhagen; (MSKCC); (ICL); (LMU); (Zurich)

    2016-06-22

    Here, we report that after DNA replication, chromosomal processes including DNA repair and transcription take place in the context of sister chromatids. While cell cycle regulation can guide these processes globally, mechanisms to distinguish pre- and post-replicative states locally remain unknown. In this paper we reveal that new histones incorporated during DNA replication provide a signature of post-replicative chromatin, read by the human TONSL–MMS22L1, 2, 3, 4 homologous recombination complex. We identify the TONSL ankyrin repeat domain (ARD) as a reader of histone H4 tails unmethylated at K20 (H4K20me0), which are specific to new histones incorporated during DNA replication and mark post-replicative chromatin until the G2/M phase of the cell cycle. Accordingly, TONSL–MMS22L binds new histones H3–H4 both before and after incorporation into nucleosomes, remaining on replicated chromatin until late G2/M. H4K20me0 recognition is required for TONSL–MMS22L binding to chromatin and accumulation at challenged replication forks and DNA lesions. Consequently, TONSL ARD mutants are toxic, compromising genome stability, cell viability and resistance to replication stress. Finally, together, these data reveal a histone-reader-based mechanism for recognizing the post-replicative state, offering a new angle to understand DNA repair with the potential for targeted cancer therapy.

  17. Phosphorylated STAT5 directly facilitates parvovirus B19 DNA replication in human erythroid progenitors through interaction with the MCM complex.

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    Ganaie, Safder S; Zou, Wei; Xu, Peng; Deng, Xuefeng; Kleiboeker, Steve; Qiu, Jianming

    2017-05-01

    Productive infection of human parvovirus B19 (B19V) exhibits high tropism for burst forming unit erythroid (BFU-E) and colony forming unit erythroid (CFU-E) progenitor cells in human bone marrow and fetal liver. This exclusive restriction of the virus replication to human erythroid progenitor cells is partly due to the intracellular factors that are essential for viral DNA replication, including erythropoietin signaling. Efficient B19V replication also requires hypoxic conditions, which upregulate the signal transducer and activator of transcription 5 (STAT5) pathway, and phosphorylated STAT5 is essential for virus replication. In this study, our results revealed direct involvement of STAT5 in B19V DNA replication. Consensus STAT5-binding elements were identified adjacent to the NS1-binding element within the minimal origins of viral DNA replication in the B19V genome. Phosphorylated STAT5 specifically interacted with viral DNA replication origins both in vivo and in vitro, and was actively recruited within the viral DNA replication centers. Notably, STAT5 interacted with minichromosome maintenance (MCM) complex, suggesting that STAT5 directly facilitates viral DNA replication by recruiting the helicase complex of the cellular DNA replication machinery to viral DNA replication centers. The FDA-approved drug pimozide dephosphorylates STAT5, and it inhibited B19V replication in ex vivo expanded human erythroid progenitors. Our results demonstrated that pimozide could be a promising antiviral drug for treatment of B19V-related diseases.

  18. Partial Purification of a Megadalton DNA Replication Complex by Free Flow Electrophoresis.

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    Caroline M Li

    Full Text Available We describe a gentle and rapid method to purify the intact multiprotein DNA replication complex using free flow electrophoresis (FFE. In particular, we applied FFE to purify the human cell DNA synthesome, which is a multiprotein complex that is fully competent to carry-out all phases of the DNA replication process in vitro using a plasmid containing the simian virus 40 (SV40 origin of DNA replication and the viral large tumor antigen (T-antigen protein. The isolated native DNA synthesome can be of use in studying the mechanism by which mammalian DNA replication is carried-out and how anti-cancer drugs disrupt the DNA replication or repair process. Partially purified extracts from HeLa cells were fractionated in a native, liquid based separation by FFE. Dot blot analysis showed co-elution of many proteins identified as part of the DNA synthesome, including proliferating cell nuclear antigen (PCNA, DNA topoisomerase I (topo I, DNA polymerase δ (Pol δ, DNA polymerase ɛ (Pol ɛ, replication protein A (RPA and replication factor C (RFC. Previously identified DNA synthesome proteins co-eluted with T-antigen dependent and SV40 origin-specific DNA polymerase activity at the same FFE fractions. Native gels show a multiprotein PCNA containing complex migrating with an apparent relative mobility in the megadalton range. When PCNA containing bands were excised from the native gel, mass spectrometric sequencing analysis identified 23 known DNA synthesome associated proteins or protein subunits.

  19. Partial Purification of a Megadalton DNA Replication Complex by Free Flow Electrophoresis.

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    Li, Caroline M; Miao, Yunan; Lingeman, Robert G; Hickey, Robert J; Malkas, Linda H

    2016-01-01

    We describe a gentle and rapid method to purify the intact multiprotein DNA replication complex using free flow electrophoresis (FFE). In particular, we applied FFE to purify the human cell DNA synthesome, which is a multiprotein complex that is fully competent to carry-out all phases of the DNA replication process in vitro using a plasmid containing the simian virus 40 (SV40) origin of DNA replication and the viral large tumor antigen (T-antigen) protein. The isolated native DNA synthesome can be of use in studying the mechanism by which mammalian DNA replication is carried-out and how anti-cancer drugs disrupt the DNA replication or repair process. Partially purified extracts from HeLa cells were fractionated in a native, liquid based separation by FFE. Dot blot analysis showed co-elution of many proteins identified as part of the DNA synthesome, including proliferating cell nuclear antigen (PCNA), DNA topoisomerase I (topo I), DNA polymerase δ (Pol δ), DNA polymerase ɛ (Pol ɛ), replication protein A (RPA) and replication factor C (RFC). Previously identified DNA synthesome proteins co-eluted with T-antigen dependent and SV40 origin-specific DNA polymerase activity at the same FFE fractions. Native gels show a multiprotein PCNA containing complex migrating with an apparent relative mobility in the megadalton range. When PCNA containing bands were excised from the native gel, mass spectrometric sequencing analysis identified 23 known DNA synthesome associated proteins or protein subunits.

  20. Mechanisms and regulation of DNA replication initiation in eukaryotes.

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    Parker, Matthew W; Botchan, Michael R; Berger, James M

    2017-04-01

    Cellular DNA replication is initiated through the action of multiprotein complexes that recognize replication start sites in the chromosome (termed origins) and facilitate duplex DNA melting within these regions. In a typical cell cycle, initiation occurs only once per origin and each round of replication is tightly coupled to cell division. To avoid aberrant origin firing and re-replication, eukaryotes tightly regulate two events in the initiation process: loading of the replicative helicase, MCM2-7, onto chromatin by the origin recognition complex (ORC), and subsequent activation of the helicase by its incorporation into a complex known as the CMG. Recent work has begun to reveal the details of an orchestrated and sequential exchange of initiation factors on DNA that give rise to a replication-competent complex, the replisome. Here, we review the molecular mechanisms that underpin eukaryotic DNA replication initiation - from selecting replication start sites to replicative helicase loading and activation - and describe how these events are often distinctly regulated across different eukaryotic model organisms.

  1. GINS complex protein Sld5 recruits SIK1 to activate MCM helicase during DNA replication.

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    Joshi, Kiranmai; Shah, Varun Jayeshkumar; Maddika, Subbareddy

    2016-12-01

    In eukaryotes, proper loading and activation of MCM helicase at chromosomal origins plays a central role in DNA replication. Activation of MCM helicase requires its association with CDC45-GINS complex, but the mechanism of how this complex activates MCM helicase is poorly understood. Here we identified SIK1 (salt-inducible kinase 1), an AMPK related protein kinase, as a molecular link that connects GINS complex with MCM helicase activity. We demonstrated that Sld5 a component of GINS complex interacts with SIK1 and recruits it to the sites of DNA replication at the onset of S phase. Depletion of SIK1 leads to defective DNA replication. Further, we showed that SIK1 phosphorylates MCM2 at five conserved residues at its N-terminus, which is essential for the activation of MCM helicase. Collectively, our results suggest SIK1 as a novel integral component of CMG replicative helicase during eukaryotic DNA replication. Copyright © 2016 Elsevier Inc. All rights reserved.

  2. Plum Pox Virus 6K1 Protein Is Required for Viral Replication and Targets the Viral Replication Complex at the Early Stage of Infection.

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    Cui, Hongguang; Wang, Aiming

    2016-05-15

    proteins, with the majority of them having been at least partially functionally characterized. However, the functional role of a small protein named 6K1 remains obscure. In this study, we showed that deletion of 6K1 or a short motif/region of 6K1 in the full-length cDNA clones of plum pox virus abolishes viral replication and that mutation of the N- or C-terminal cleavage sites of 6K1 to prevent its release from the polyprotein greatly attenuates or completely inhibits viral replication, suggesting its important role in potyviral infection. We report that 6K1 forms punctate structures and targets the replication vesicles in PPV-infected plant leaf cells at the early infection stage. Our data reveal that 6K1 is an important viral protein of the potyviral replication complex. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  3. A Polycomb complex remains bound through DNA replication in the absence of other eukaryotic proteins

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    Lengsfeld, Bettina M.; Berry, Kayla N.; Ghosh, Sharmistha; Takahashi, Masateru; Francis, Nicole J.

    2012-01-01

    Propagation of chromatin states through DNA replication is central to epigenetic regulation and can involve recruitment of chromatin proteins to replicating chromatin through interactions with replication fork components. Here we show using a fully reconstituted T7 bacteriophage system that eukaryotic proteins are not required to tether the Polycomb complex PRC1 to templates during DNA replication. Instead, DNA binding by PRC1 can withstand passage of a simple replication fork.

  4. A Polycomb complex remains bound through DNA replication in the absence of other eukaryotic proteins

    KAUST Repository

    Lengsfeld, Bettina M.

    2012-09-17

    Propagation of chromatin states through DNA replication is central to epigenetic regulation and can involve recruitment of chromatin proteins to replicating chromatin through interactions with replication fork components. Here we show using a fully reconstituted T7 bacteriophage system that eukaryotic proteins are not required to tether the Polycomb complex PRC1 to templates during DNA replication. Instead, DNA binding by PRC1 can withstand passage of a simple replication fork.

  5. An Adenovirus DNA Replication Factor, but Not Incoming Genome Complexes, Targets PML Nuclear Bodies.

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    Komatsu, Tetsuro; Nagata, Kyosuke; Wodrich, Harald

    2016-02-01

    Promyelocytic leukemia protein nuclear bodies (PML-NBs) are subnuclear domains implicated in cellular antiviral responses. Despite the antiviral activity, several nuclear replicating DNA viruses use the domains as deposition sites for the incoming viral genomes and/or as sites for viral DNA replication, suggesting that PML-NBs are functionally relevant during early viral infection to establish productive replication. Although PML-NBs and their components have also been implicated in the adenoviral life cycle, it remains unclear whether incoming adenoviral genome complexes target PML-NBs. Here we show using immunofluorescence and live-cell imaging analyses that incoming adenovirus genome complexes neither localize at nor recruit components of PML-NBs during early phases of infection. We further show that the viral DNA binding protein (DBP), an early expressed viral gene and essential DNA replication factor, independently targets PML-NBs. We show that DBP oligomerization is required to selectively recruit the PML-NB components Sp100 and USP7. Depletion experiments suggest that the absence of one PML-NB component might not affect the recruitment of other components toward DBP oligomers. Thus, our findings suggest a model in which an adenoviral DNA replication factor, but not incoming viral genome complexes, targets and modulates PML-NBs to support a conducive state for viral DNA replication and argue against a generalized concept that PML-NBs target incoming viral genomes. The immediate fate upon nuclear delivery of genomes of incoming DNA viruses is largely unclear. Early reports suggested that incoming genomes of herpesviruses are targeted and repressed by PML-NBs immediately upon nuclear import. Genome localization and/or viral DNA replication has also been observed at PML-NBs for other DNA viruses. Thus, it was suggested that PML-NBs may immediately sense and target nuclear viral genomes and hence serve as sites for deposition of incoming viral genomes and

  6. Absence of Non-histone Protein Complexes at Natural Chromosomal Pause Sites Results in Reduced Replication Pausing in Aging Yeast Cells

    Directory of Open Access Journals (Sweden)

    Marleny Cabral

    2016-11-01

    Full Text Available There is substantial evidence that genomic instability increases during aging. Replication pausing (and stalling at difficult-to-replicate chromosomal sites may induce genomic instability. Interestingly, in aging yeast cells, we observed reduced replication pausing at various natural replication pause sites (RPSs in ribosomal DNA (rDNA and non-rDNA locations (e.g., silent replication origins and tRNA genes. The reduced pausing occurs independent of the DNA helicase Rrm3p, which facilitates replication past these non-histone protein-complex-bound RPSs, and is independent of the deacetylase Sir2p. Conditions of caloric restriction (CR, which extend life span, also cause reduced replication pausing at the 5S rDNA and at tRNA genes. In aged and CR cells, the RPSs are less occupied by their specific non-histone protein complexes (e.g., the preinitiation complex TFIIIC, likely because members of these complexes have primarily cytosolic localization. These conditions may lead to reduced replication pausing and may lower replication stress at these sites during aging.

  7. Specificity and function of Archaeal DNA replication initiator proteins

    DEFF Research Database (Denmark)

    Samson, Rachel Y.; Xu, Yanqun; Gadelha, Catarina

    2013-01-01

    Chromosomes with multiple DNA replication origins are a hallmark of Eukaryotes and some Archaea. All eukaryal nuclear replication origins are defined by the origin recognition complex (ORC) that recruits the replicative helicase MCM(2-7) via Cdc6 and Cdt1. We find that the three origins...... to investigate the role of ATP binding and hydrolysis in initiator function in vivo and in vitro. We find that the ATP-bound form of Orc1-1 is proficient for replication and implicates hydrolysis of ATP in downregulation of origin activity. Finally, we reveal that ATP and DNA binding by Orc1-1 remodels...

  8. H3K9me3 demethylase Kdm4d facilitates the formation of pre-initiative complex and regulates DNA replication.

    Science.gov (United States)

    Wu, Rentian; Wang, Zhiquan; Zhang, Honglian; Gan, Haiyun; Zhang, Zhiguo

    2017-01-09

    DNA replication is tightly regulated to occur once and only once per cell cycle. How chromatin, the physiological substrate of DNA replication machinery, regulates DNA replication remains largely unknown. Here we show that histone H3 lysine 9 demethylase Kdm4d regulates DNA replication in eukaryotic cells. Depletion of Kdm4d results in defects in DNA replication, which can be rescued by the expression of H3K9M, a histone H3 mutant transgene that reverses the effect of Kdm4d on H3K9 methylation. Kdm4d interacts with replication proteins, and its recruitment to DNA replication origins depends on the two pre-replicative complex components (origin recognition complex [ORC] and minichromosome maintenance [MCM] complex). Depletion of Kdm4d impairs the recruitment of Cdc45, proliferating cell nuclear antigen (PCNA), and polymerase δ, but not ORC and MCM proteins. These results demonstrate a novel mechanism by which Kdm4d regulates DNA replication by reducing the H3K9me3 level to facilitate formation of pre-initiative complex. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

  9. Electron microscopic analysis of rotavirus assembly-replication intermediates

    International Nuclear Information System (INIS)

    Boudreaux, Crystal E.; Kelly, Deborah F.; McDonald, Sarah M.

    2015-01-01

    Rotaviruses (RVs) replicate their segmented, double-stranded RNA genomes in tandem with early virion assembly. In this study, we sought to gain insight into the ultrastructure of RV assembly-replication intermediates (RIs) using transmission electron microscopy (EM). Specifically, we examined a replicase-competent, subcellular fraction that contains all known RV RIs. Three never-before-seen complexes were visualized in this fraction. Using in vitro reconstitution, we showed that ~15-nm doughnut-shaped proteins in strings were nonstructural protein 2 (NSP2) bound to viral RNA transcripts. Moreover, using immunoaffinity-capture EM, we revealed that ~20-nm pebble-shaped complexes contain the viral RNA polymerase (VP1) and RNA capping enzyme (VP3). Finally, using a gel purification method, we demonstrated that ~30–70-nm electron-dense, particle-shaped complexes represent replicase-competent core RIs, containing VP1, VP3, and NSP2 as well as capsid proteins VP2 and VP6. The results of this study raise new questions about the interactions among viral proteins and RNA during the concerted assembly–replicase process. - Highlights: • Rotaviruses replicate their genomes in tandem with early virion assembly. • Little is known about rotavirus assembly-replication intermediates. • Assembly-replication intermediates were imaged using electron microscopy

  10. Electron microscopic analysis of rotavirus assembly-replication intermediates

    Energy Technology Data Exchange (ETDEWEB)

    Boudreaux, Crystal E.; Kelly, Deborah F. [Virginia Tech Carilion School of Medicine and Research Institute, Roanoke, VA (United States); McDonald, Sarah M., E-mail: mcdonaldsa@vtc.vt.edu [Virginia Tech Carilion School of Medicine and Research Institute, Roanoke, VA (United States); Department of Biomedical Sciences and Pathobiology, Virginia—Maryland Regional College of Veterinary Medicine, Blacksburg, VA (United States)

    2015-03-15

    Rotaviruses (RVs) replicate their segmented, double-stranded RNA genomes in tandem with early virion assembly. In this study, we sought to gain insight into the ultrastructure of RV assembly-replication intermediates (RIs) using transmission electron microscopy (EM). Specifically, we examined a replicase-competent, subcellular fraction that contains all known RV RIs. Three never-before-seen complexes were visualized in this fraction. Using in vitro reconstitution, we showed that ~15-nm doughnut-shaped proteins in strings were nonstructural protein 2 (NSP2) bound to viral RNA transcripts. Moreover, using immunoaffinity-capture EM, we revealed that ~20-nm pebble-shaped complexes contain the viral RNA polymerase (VP1) and RNA capping enzyme (VP3). Finally, using a gel purification method, we demonstrated that ~30–70-nm electron-dense, particle-shaped complexes represent replicase-competent core RIs, containing VP1, VP3, and NSP2 as well as capsid proteins VP2 and VP6. The results of this study raise new questions about the interactions among viral proteins and RNA during the concerted assembly–replicase process. - Highlights: • Rotaviruses replicate their genomes in tandem with early virion assembly. • Little is known about rotavirus assembly-replication intermediates. • Assembly-replication intermediates were imaged using electron microscopy.

  11. Rpa4, a homolog of the 34-kilodalton subunit of the replication protein A complex.

    OpenAIRE

    Keshav, K F; Chen, C; Dutta, A

    1995-01-01

    Replication protein A (RPA) is a complex of three polypeptides of 70, 34, and 13 kDa isolated from diverse eukaryotes. The complex is a single-stranded DNA-binding protein essential for simian virus 40-based DNA replication in vitro and for viability in the yeast Saccharomyces cerevisiae. We have identified a new 30-kDa human protein which interacts with the 70- and 13-kDa subunits of RPA, with a yeast two-hybrid/interaction trap method. This protein, Rpa4, has 47% identity with Rpa2, the 34-...

  12. Chromatin Immunoprecipitation of Replication Factors Moving with the Replication Fork

    OpenAIRE

    Rapp, Jordan B.; Ansbach, Alison B.; Noguchi, Chiaki; Noguchi, Eishi

    2009-01-01

    Replication of chromosomes involves a variety of replication proteins including DNA polymerases, DNA helicases, and other accessory factors. Many of these proteins are known to localize at replication forks and travel with them as components of the replisome complex. Other proteins do not move with replication forks but still play an essential role in DNA replication. Therefore, in order to understand the mechanisms of DNA replication and its controls, it is important to examine localization ...

  13. Checkpoint responses to replication stalling: inducing tolerance and preventing mutagenesis

    Energy Technology Data Exchange (ETDEWEB)

    Kai, Mihoko; Wang, Teresa S.-F

    2003-11-27

    Replication mutants often exhibit a mutator phenotype characterized by point mutations, single base frameshifts, and the deletion or duplication of sequences flanked by homologous repeats. Mutation in genes encoding checkpoint proteins can significantly affect the mutator phenotype. Here, we use fission yeast (Schizosaccharomyces pombe) as a model system to discuss the checkpoint responses to replication perturbations induced by replication mutants. Checkpoint activation induced by a DNA polymerase mutant, aside from delay of mitotic entry, up-regulates the translesion polymerase DinB (Pol{kappa}). Checkpoint Rad9-Rad1-Hus1 (9-1-1) complex, which is loaded onto chromatin by the Rad17-Rfc2-5 checkpoint complex in response to replication perturbation, recruits DinB onto chromatin to generate the point mutations and single nucleotide frameshifts in the replication mutator. This chain of events reveals a novel checkpoint-induced tolerance mechanism that allows cells to cope with replication perturbation, presumably to make possible restarting stalled replication forks. Fission yeast Cds1 kinase plays an essential role in maintaining DNA replication fork stability in the face of DNA damage and replication fork stalling. Cds1 kinase is known to regulate three proteins that are implicated in maintaining replication fork stability: Mus81-Eme1, a hetero-dimeric structure-specific endonuclease complex; Rqh1, a RecQ-family helicase involved in suppressing inappropriate recombination during replication; and Rad60, a protein required for recombinational repair during replication. These Cds1-regulated proteins are thought to cooperatively prevent mutagenesis and maintain replication fork stability in cells under replication stress. These checkpoint-regulated processes allow cells to survive replication perturbation by preventing stalled replication forks from degenerating into deleterious DNA structures resulting in genomic instability and cancer development.

  14. Checkpoint responses to replication stalling: inducing tolerance and preventing mutagenesis

    International Nuclear Information System (INIS)

    Kai, Mihoko; Wang, Teresa S.-F.

    2003-01-01

    Replication mutants often exhibit a mutator phenotype characterized by point mutations, single base frameshifts, and the deletion or duplication of sequences flanked by homologous repeats. Mutation in genes encoding checkpoint proteins can significantly affect the mutator phenotype. Here, we use fission yeast (Schizosaccharomyces pombe) as a model system to discuss the checkpoint responses to replication perturbations induced by replication mutants. Checkpoint activation induced by a DNA polymerase mutant, aside from delay of mitotic entry, up-regulates the translesion polymerase DinB (Polκ). Checkpoint Rad9-Rad1-Hus1 (9-1-1) complex, which is loaded onto chromatin by the Rad17-Rfc2-5 checkpoint complex in response to replication perturbation, recruits DinB onto chromatin to generate the point mutations and single nucleotide frameshifts in the replication mutator. This chain of events reveals a novel checkpoint-induced tolerance mechanism that allows cells to cope with replication perturbation, presumably to make possible restarting stalled replication forks. Fission yeast Cds1 kinase plays an essential role in maintaining DNA replication fork stability in the face of DNA damage and replication fork stalling. Cds1 kinase is known to regulate three proteins that are implicated in maintaining replication fork stability: Mus81-Eme1, a hetero-dimeric structure-specific endonuclease complex; Rqh1, a RecQ-family helicase involved in suppressing inappropriate recombination during replication; and Rad60, a protein required for recombinational repair during replication. These Cds1-regulated proteins are thought to cooperatively prevent mutagenesis and maintain replication fork stability in cells under replication stress. These checkpoint-regulated processes allow cells to survive replication perturbation by preventing stalled replication forks from degenerating into deleterious DNA structures resulting in genomic instability and cancer development

  15. Phosphorylation of NS5A Serine-235 is essential to hepatitis C virus RNA replication and normal replication compartment formation

    Energy Technology Data Exchange (ETDEWEB)

    Eyre, Nicholas S., E-mail: nicholas.eyre@adelaide.edu.au [School of Biological Sciences and Research Centre for Infectious Diseases, University of Adelaide, Adelaide (Australia); Centre for Cancer Biology, SA Pathology, Adelaide (Australia); Hampton-Smith, Rachel J.; Aloia, Amanda L. [School of Biological Sciences and Research Centre for Infectious Diseases, University of Adelaide, Adelaide (Australia); Centre for Cancer Biology, SA Pathology, Adelaide (Australia); Eddes, James S. [Adelaide Proteomics Centre, School of Biological Sciences, University of Adelaide, Adelaide (Australia); Simpson, Kaylene J. [Victorian Centre for Functional Genomics, Peter MacCallum Cancer Centre, East Melbourne (Australia); The Sir Peter MacCallum Department of Oncology, University of Melbourne, Parkville (Australia); Hoffmann, Peter [Adelaide Proteomics Centre, School of Biological Sciences, University of Adelaide, Adelaide (Australia); Institute for Photonics and Advanced Sensing (IPAS), University of Adelaide, Adelaide (Australia); Beard, Michael R. [School of Biological Sciences and Research Centre for Infectious Diseases, University of Adelaide, Adelaide (Australia); Centre for Cancer Biology, SA Pathology, Adelaide (Australia)

    2016-04-15

    Hepatitis C virus (HCV) NS5A protein is essential for HCV RNA replication and virus assembly. Here we report the identification of NS5A phosphorylation sites Ser-222, Ser-235 and Thr-348 during an infectious HCV replication cycle and demonstrate that Ser-235 phosphorylation is essential for HCV RNA replication. Confocal microscopy revealed that both phosphoablatant (S235A) and phosphomimetic (S235D) mutants redistribute NS5A to large juxta-nuclear foci that display altered colocalization with known replication complex components. Using electron microscopy (EM) we found that S235D alters virus-induced membrane rearrangements while EM using ‘APEX2’-tagged viruses demonstrated S235D-mediated enrichment of NS5A in irregular membranous foci. Finally, using a customized siRNA screen of candidate NS5A kinases and subsequent analysis using a phospho-specific antibody, we show that phosphatidylinositol-4 kinase III alpha (PI4KIIIα) is important for Ser-235 phosphorylation. We conclude that Ser-235 phosphorylation of NS5A is essential for HCV RNA replication and normal replication complex formation and is regulated by PI4KIIIα. - Highlights: • NS5A residues Ser-222, Ser-235 and Thr-348 are phosphorylated during HCV infection. • Phosphorylation of Ser-235 is essential to HCV RNA replication. • Mutation of Ser-235 alters replication compartment localization and morphology. • Phosphatidylinositol-4 kinase III alpha is important for Ser-235 phosphorylation.

  16. A crystal structure of the Dengue virus NS5 protein reveals a novel inter-domain interface essential for protein flexibility and virus replication.

    Directory of Open Access Journals (Sweden)

    Yongqian Zhao

    2015-03-01

    Full Text Available Flavivirus RNA replication occurs within a replication complex (RC that assembles on ER membranes and comprises both non-structural (NS viral proteins and host cofactors. As the largest protein component within the flavivirus RC, NS5 plays key enzymatic roles through its N-terminal methyltransferase (MTase and C-terminal RNA-dependent-RNA polymerase (RdRp domains, and constitutes a major target for antivirals. We determined a crystal structure of the full-length NS5 protein from Dengue virus serotype 3 (DENV3 at a resolution of 2.3 Å in the presence of bound SAH and GTP. Although the overall molecular shape of NS5 from DENV3 resembles that of NS5 from Japanese Encephalitis Virus (JEV, the relative orientation between the MTase and RdRp domains differs between the two structures, providing direct evidence for the existence of a set of discrete stable molecular conformations that may be required for its function. While the inter-domain region is mostly disordered in NS5 from JEV, the NS5 structure from DENV3 reveals a well-ordered linker region comprising a short 310 helix that may act as a swivel. Solution Hydrogen/Deuterium Exchange Mass Spectrometry (HDX-MS analysis reveals an increased mobility of the thumb subdomain of RdRp in the context of the full length NS5 protein which correlates well with the analysis of the crystallographic temperature factors. Site-directed mutagenesis targeting the mostly polar interface between the MTase and RdRp domains identified several evolutionarily conserved residues that are important for viral replication, suggesting that inter-domain cross-talk in NS5 regulates virus replication. Collectively, a picture for the molecular origin of NS5 flexibility is emerging with profound implications for flavivirus replication and for the development of therapeutics targeting NS5.

  17. Overcoming natural replication barriers: differential helicase requirements.

    Science.gov (United States)

    Anand, Ranjith P; Shah, Kartik A; Niu, Hengyao; Sung, Patrick; Mirkin, Sergei M; Freudenreich, Catherine H

    2012-02-01

    DNA sequences that form secondary structures or bind protein complexes are known barriers to replication and potential inducers of genome instability. In order to determine which helicases facilitate DNA replication across these barriers, we analyzed fork progression through them in wild-type and mutant yeast cells, using 2-dimensional gel-electrophoretic analysis of the replication intermediates. We show that the Srs2 protein facilitates replication of hairpin-forming CGG/CCG repeats and prevents chromosome fragility at the repeat, whereas it does not affect replication of G-quadruplex forming sequences or a protein-bound repeat. Srs2 helicase activity is required for hairpin unwinding and fork progression. Also, the PCNA binding domain of Srs2 is required for its in vivo role of replication through hairpins. In contrast, the absence of Sgs1 or Pif1 helicases did not inhibit replication through structural barriers, though Pif1 did facilitate replication of a telomeric protein barrier. Interestingly, replication through a protein barrier but not a DNA structure barrier was modulated by nucleotide pool levels, illuminating a different mechanism by which cells can regulate fork progression through protein-mediated stall sites. Our analyses reveal fundamental differences in the replication of DNA structural versus protein barriers, with Srs2 helicase activity exclusively required for fork progression through hairpin structures.

  18. Control of Genome Integrity by RFC Complexes; Conductors of PCNA Loading onto and Unloading from Chromatin during DNA Replication

    Directory of Open Access Journals (Sweden)

    Yasushi Shiomi

    2017-01-01

    Full Text Available During cell division, genome integrity is maintained by faithful DNA replication during S phase, followed by accurate segregation in mitosis. Many DNA metabolic events linked with DNA replication are also regulated throughout the cell cycle. In eukaryotes, the DNA sliding clamp, proliferating cell nuclear antigen (PCNA, acts on chromatin as a processivity factor for DNA polymerases. Since its discovery, many other PCNA binding partners have been identified that function during DNA replication, repair, recombination, chromatin remodeling, cohesion, and proteolysis in cell-cycle progression. PCNA not only recruits the proteins involved in such events, but it also actively controls their function as chromatin assembles. Therefore, control of PCNA-loading onto chromatin is fundamental for various replication-coupled reactions. PCNA is loaded onto chromatin by PCNA-loading replication factor C (RFC complexes. Both RFC1-RFC and Ctf18-RFC fundamentally function as PCNA loaders. On the other hand, after DNA synthesis, PCNA must be removed from chromatin by Elg1-RFC. Functional defects in RFC complexes lead to chromosomal abnormalities. In this review, we summarize the structural and functional relationships among RFC complexes, and describe how the regulation of PCNA loading/unloading by RFC complexes contributes to maintaining genome integrity.

  19. The progression of replication forks at natural replication barriers in live bacteria

    NARCIS (Netherlands)

    Moolman, M.C.; Tiruvadi Krishnan, S; Kerssemakers, J.W.J.; de Leeuw, R.; Lorent, V.J.F.; Sherratt, David J.; Dekker, N.H.

    2016-01-01

    Protein-DNA complexes are one of the principal barriers the replisome encounters during replication. One such barrier is the Tus-ter complex, which is a direction dependent barrier for replication fork progression. The details concerning the dynamics of the replisome when encountering these

  20. An amphipathic alpha-helix controls multiple roles of brome mosaic virus protein 1a in RNA replication complex assembly and function.

    Directory of Open Access Journals (Sweden)

    Ling Liu

    2009-03-01

    Full Text Available Brome mosaic virus (BMV protein 1a has multiple key roles in viral RNA replication. 1a localizes to perinuclear endoplasmic reticulum (ER membranes as a peripheral membrane protein, induces ER membrane invaginations in which RNA replication complexes form, and recruits and stabilizes BMV 2a polymerase (2a(Pol and RNA replication templates at these sites to establish active replication complexes. During replication, 1a provides RNA capping, NTPase and possibly RNA helicase functions. Here we identify in BMV 1a an amphipathic alpha-helix, helix A, and use NMR analysis to define its structure and propensity to insert in hydrophobic membrane-mimicking micelles. We show that helix A is essential for efficient 1a-ER membrane association and normal perinuclear ER localization, and that deletion or mutation of helix A abolishes RNA replication. Strikingly, mutations in helix A give rise to two dramatically opposite 1a function phenotypes, implying that helix A acts as a molecular switch regulating the intricate balance between separable 1a functions. One class of helix A deletions and amino acid substitutions markedly inhibits 1a-membrane association and abolishes ER membrane invagination, viral RNA template recruitment, and replication, but doubles the 1a-mediated increase in 2a(Pol accumulation. The second class of helix A mutations not only maintains efficient 1a-membrane association but also amplifies the number of 1a-induced membrane invaginations 5- to 8-fold and enhances viral RNA template recruitment, while failing to stimulate 2a(Pol accumulation. The results provide new insights into the pathways of RNA replication complex assembly and show that helix A is critical for assembly and function of the viral RNA replication complex, including its central role in targeting replication components and controlling modes of 1a action.

  1. Coevolution between Nuclear-Encoded DNA Replication, Recombination, and Repair Genes and Plastid Genome Complexity.

    Science.gov (United States)

    Zhang, Jin; Ruhlman, Tracey A; Sabir, Jamal S M; Blazier, John Chris; Weng, Mao-Lun; Park, Seongjun; Jansen, Robert K

    2016-02-17

    Disruption of DNA replication, recombination, and repair (DNA-RRR) systems has been hypothesized to cause highly elevated nucleotide substitution rates and genome rearrangements in the plastids of angiosperms, but this theory remains untested. To investigate nuclear-plastid genome (plastome) coevolution in Geraniaceae, four different measures of plastome complexity (rearrangements, repeats, nucleotide insertions/deletions, and substitution rates) were evaluated along with substitution rates of 12 nuclear-encoded, plastid-targeted DNA-RRR genes from 27 Geraniales species. Significant correlations were detected for nonsynonymous (dN) but not synonymous (dS) substitution rates for three DNA-RRR genes (uvrB/C, why1, and gyrA) supporting a role for these genes in accelerated plastid genome evolution in Geraniaceae. Furthermore, correlation between dN of uvrB/C and plastome complexity suggests the presence of nucleotide excision repair system in plastids. Significant correlations were also detected between plastome complexity and 13 of the 90 nuclear-encoded organelle-targeted genes investigated. Comparisons revealed significant acceleration of dN in plastid-targeted genes of Geraniales relative to Brassicales suggesting this correlation may be an artifact of elevated rates in this gene set in Geraniaceae. Correlation between dN of plastid-targeted DNA-RRR genes and plastome complexity supports the hypothesis that the aberrant patterns in angiosperm plastome evolution could be caused by dysfunction in DNA-RRR systems. © The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

  2. Knockdown of RMI1 impairs DNA repair under DNA replication stress.

    Science.gov (United States)

    Xu, Chang; Fang, Lianying; Kong, Yangyang; Xiao, Changyan; Yang, Mengmeng; Du, Li-Qing; Liu, Qiang

    2017-12-09

    RMI1 (RecQ-mediated genome instability protein 1) forms a conserved BTR complex with BLM, Topo IIIα, and RMI2, and its absence causes genome instability. It has been revealed that RMI1 localizes to nuclear foci with BLM and Topo IIIα in response to replication stress, and that RMI1 functions downstream of BLM in promoting replication elongation. However, the precise functions of RMI1 during replication stress are not completely understood. Here we report that RMI1 knockdown cells are hypersensitive to hydroxyurea (HU). Using comet assay, we show that RMI1 knockdown cells exhibit accumulation of broken DNAs after being released from HU treatment. Moreover, we demonstrate that RMI1 facilitates the recovery from activated checkpoint and resuming the cell cycle after replicative stress. Surprisingly, loss of RMI1 results in a failure of RAD51 loading onto DNA damage sites. These findings reveal the importance of RMI1 in response to replication stress, which could explain the molecular basis for its function in maintaining genome integrity. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  3. Suppression of gross chromosomal rearrangements by a new alternative replication factor C complex

    International Nuclear Information System (INIS)

    Banerjee, Soma; Sikdar, Nilabja; Myung, Kyungjae

    2007-01-01

    Defects in DNA replication fidelity lead to genomic instability. Gross chromosomal rearrangement (GCR), a type of genomic instability, is highly enhanced by various initial mutations affecting DNA replication. Frequent observations of GCRs in many cancers strongly argue the importance of maintaining high fidelity of DNA replication to suppress carcinogenesis. Recent genome wide screens in Saccharomyces cerevisiae identified a new GCR suppressor gene, ELG1, enhanced level of genome instability gene 1. Its physical interaction with proliferating cell nuclear antigen (PCNA) and complex formation with Rfc2-5p proteins suggest that Elg1 functions to load/unload PCNA onto DNA during a certain DNA metabolism. High level of DNA damage accumulation and enhanced phenotypes with mutations in genes involved in cell cycle checkpoints, homologous recombination (HR), or chromatin assembly in the elg1 strain suggest that Elg1p-Rfc2-5p functions in a fundamental DNA metabolism to suppress genomic instability

  4. High-Resolution Profiling of Drosophila Replication Start Sites Reveals a DNA Shape and Chromatin Signature of Metazoan Origins

    Directory of Open Access Journals (Sweden)

    Federico Comoglio

    2015-05-01

    Full Text Available At every cell cycle, faithful inheritance of metazoan genomes requires the concerted activation of thousands of DNA replication origins. However, the genetic and chromatin features defining metazoan replication start sites remain largely unknown. Here, we delineate the origin repertoire of the Drosophila genome at high resolution. We address the role of origin-proximal G-quadruplexes and suggest that they transiently stall replication forks in vivo. We dissect the chromatin configuration of replication origins and identify a rich spatial organization of chromatin features at initiation sites. DNA shape and chromatin configurations, not strict sequence motifs, mark and predict origins in higher eukaryotes. We further examine the link between transcription and origin firing and reveal that modulation of origin activity across cell types is intimately linked to cell-type-specific transcriptional programs. Our study unravels conserved origin features and provides unique insights into the relationship among DNA topology, chromatin, transcription, and replication initiation across metazoa.

  5. Visualization of the structures of the hepatitis C virus replication complex

    International Nuclear Information System (INIS)

    Chan, Shih-Ching; Lo, Shih-Yen; Liou, Je-Wen; Lin, Min-Ching; Syu, Ciao-Ling; Lai, Meng-Jiun; Chen, Yi- Cheng; Li, Hui-Chun

    2011-01-01

    Research highlights: → Lipid rafts are known to play an important role in virus entry and virus assembly of many viruses. → However, HCV is the first example of the association of lipid raft with viral RNA replication. → Our results in this manuscript demonstrate that purified HCV RCs with associated lipid raft membrane appeared as distinct particles of around 0.7 um under EM and AFM. → Knockdown of proteins associated with lipid raft suppressed the HCV replication and reduced the number of these particles. → To our knowledge, structures of HCV RCs were demonstrated at its first time in this manuscript. -- Abstract: Hepatitis C viral RNA synthesis has been demonstrated to occur on a lipid raft membrane structure. Lipid raft membrane fraction purified by membrane flotation analysis was observed using transmission electron microscopy and atomic force microscopy. Particles around 0.7 um in size were found in lipid raft membrane fraction purified from hepatitis C virus (HCV) replicon but not their parental HuH7 cells. HCV NS5A protein was associated with these specialized particles. After several cycles of freezing-thawing, these particles would fuse into larger sizes up to 10 um. Knockdown of seven proteins associated with lipid raft (VAPA, COPG, RAB18, COMT, CDC42, DPP4, and KDELR2) of HCV replicon cells reduced the observed number of these particles and suppressed the HCV replication. Results in this study indicated that HCV replication complexes with associated lipid raft membrane form distinct particle structures of around 0.7 um as observed from transmission electron microscopy and atomic force microscopy.

  6. Infidelity of SARS-CoV Nsp14-exonuclease mutant virus replication is revealed by complete genome sequencing.

    Directory of Open Access Journals (Sweden)

    Lance D Eckerle

    2010-05-01

    Full Text Available Most RNA viruses lack the mechanisms to recognize and correct mutations that arise during genome replication, resulting in quasispecies diversity that is required for pathogenesis and adaptation. However, it is not known how viruses encoding large viral RNA genomes such as the Coronaviridae (26 to 32 kb balance the requirements for genome stability and quasispecies diversity. Further, the limits of replication infidelity during replication of large RNA genomes and how decreased fidelity impacts virus fitness over time are not known. Our previous work demonstrated that genetic inactivation of the coronavirus exoribonuclease (ExoN in nonstructural protein 14 (nsp14 of murine hepatitis virus results in a 15-fold decrease in replication fidelity. However, it is not known whether nsp14-ExoN is required for replication fidelity of all coronaviruses, nor the impact of decreased fidelity on genome diversity and fitness during replication and passage. We report here the engineering and recovery of nsp14-ExoN mutant viruses of severe acute respiratory syndrome coronavirus (SARS-CoV that have stable growth defects and demonstrate a 21-fold increase in mutation frequency during replication in culture. Analysis of complete genome sequences from SARS-ExoN mutant viral clones revealed unique mutation sets in every genome examined from the same round of replication and a total of 100 unique mutations across the genome. Using novel bioinformatic tools and deep sequencing across the full-length genome following 10 population passages in vitro, we demonstrate retention of ExoN mutations and continued increased diversity and mutational load compared to wild-type SARS-CoV. The results define a novel genetic and bioinformatics model for introduction and identification of multi-allelic mutations in replication competent viruses that will be powerful tools for testing the effects of decreased fidelity and increased quasispecies diversity on viral replication

  7. DNA-PK/Ku complex binds to latency-associated nuclear antigen and negatively regulates Kaposi's sarcoma-associated herpesvirus latent replication

    International Nuclear Information System (INIS)

    Cha, Seho; Lim, Chunghun; Lee, Jae Young; Song, Yoon-Jae; Park, Junsoo; Choe, Joonho; Seo, Taegun

    2010-01-01

    During latent infection, latency-associated nuclear antigen (LANA) of Kaposi's sarcoma-associated herpesvirus (KSHV) plays important roles in episomal persistence and replication. Several host factors are associated with KSHV latent replication. Here, we show that the catalytic subunit of DNA protein kinase (DNA-PKcs), Ku70, and Ku86 bind the N-terminal region of LANA. LANA was phosphorylated by DNA-PK and overexpression of Ku70, but not Ku86, impaired transient replication. The efficiency of transient replication was significantly increased in the HCT116 (Ku86 +/-) cell line, compared to the HCT116 (Ku86 +/+) cell line, suggesting that the DNA-PK/Ku complex negatively regulates KSHV latent replication.

  8. Phosphoproteomic Analysis of KSHV-Infected Cells Reveals Roles of ORF45-Activated RSK during Lytic Replication.

    Directory of Open Access Journals (Sweden)

    Denis Avey

    2015-07-01

    Full Text Available Kaposi's Sarcoma-Associated Herpesvirus (KSHV is an oncogenic virus which has adapted unique mechanisms to modulate the cellular microenvironment of its human host. The pathogenesis of KSHV is intimately linked to its manipulation of cellular signaling pathways, including the extracellular signal-regulated kinase (ERK mitogen-activated protein kinase (MAPK pathway. We have previously shown that KSHV ORF45 contributes to the sustained activation of both ERK and p90 ribosomal S6 kinase (RSK, a major functional mediator of ERK/MAPK signaling during KSHV lytic replication. ORF45-activated RSK is required for optimal KSHV lytic gene expression and progeny virion production, though the underlying mechanisms downstream of this activation are still unclear. We hypothesized that the activation of RSK by ORF45 causes differential phosphorylation of cellular and viral substrates, affecting biological processes essential for efficient KSHV lytic replication. Accordingly, we observed widespread and significant differences in protein phosphorylation upon induction of lytic replication. Mass-spectrometry-based phosphoproteomic screening identified putative substrates of ORF45-activated RSK in KSHV-infected cells. Bioinformatic analyses revealed that nuclear proteins, including several transcriptional regulators, were overrepresented among these candidates. We validated the ORF45/RSK-dependent phosphorylation of several putative substrates by employing KSHV BAC mutagenesis, kinase inhibitor treatments, and/or CRISPR-mediated knockout of RSK in KSHV-infected cells. Furthermore, we assessed the consequences of knocking out these substrates on ORF45/RSK-dependent regulation of gene expression and KSHV progeny virion production. Finally, we show data to support that ORF45 regulates the translational efficiency of a subset of viral/cellular genes with complex secondary structure in their 5' UTR. Altogether, these data shed light on the mechanisms by which KSHV ORF45

  9. Crystallization and preliminary X-ray characterization of the eukaryotic replication terminator Reb1-Ter DNA complex.

    Science.gov (United States)

    Jaiswal, Rahul; Singh, Samarendra K; Bastia, Deepak; Escalante, Carlos R

    2015-04-01

    The Reb1 protein from Schizosaccharomyces pombe is a member of a family of proteins that control programmed replication termination and/or transcription termination in eukaryotic cells. These events occur at naturally occurring replication fork barriers (RFBs), where Reb1 binds to termination (Ter) DNA sites and coordinates the polar arrest of replication forks and transcription approaching in opposite directions. The Reb1 DNA-binding and replication-termination domain was expressed in Escherichia coli, purified and crystallized in complex with a 26-mer DNA Ter site. Batch crystallization under oil was required to produce crystals of good quality for data collection. Crystals grew in space group P2₁, with unit-cell parameters a = 68.9, b = 162.9, c = 71.1 Å, β = 94.7°. The crystals diffracted to a resolution of 3.0 Å. The crystals were mosaic and required two or three cycles of annealing. This study is the first to yield structural information about this important family of proteins and will provide insights into the mechanism of replication and transcription termination.

  10. Structure of p15PAF-PCNA complex and implications for clamp sliding during DNA replication and repair

    DEFF Research Database (Denmark)

    De Biasio, Alfredo; de Opakua, Alain Ibáñez; Mortuza, Gulnahar B

    2015-01-01

    The intrinsically disordered protein p15(PAF) regulates DNA replication and repair by binding to the proliferating cell nuclear antigen (PCNA) sliding clamp. We present the structure of the human p15(PAF)-PCNA complex. Crystallography and NMR show the central PCNA-interacting protein motif (PIP...... the DNA and facilitates the switch from replicative to translesion synthesis polymerase binding....... free and PCNA-bound p15(PAF) binds DNA mainly through its histone-like N-terminal tail, while PCNA does not, and a model of the ternary complex with DNA inside the PCNA ring is consistent with electron micrographs. We propose that p15(PAF) acts as a flexible drag that regulates PCNA sliding along...

  11. H4K20me0 marks post-replicative chromatin and recruits the TONSL–MMS22L DNA repair complex

    DEFF Research Database (Denmark)

    Saredi, Giulia; Huang, Hongda; Hammond, Colin M

    2016-01-01

    After DNA replication, chromosomal processes including DNA repair and transcription take place in the context of sister chromatids. While cell cycle regulation can guide these processes globally, mechanisms to distinguish pre- and post-replicative states locally remain unknown. Here we reveal...

  12. Replication-Coupled PCNA Unloading by the Elg1 Complex Occurs Genome-wide and Requires Okazaki Fragment Ligation

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    Takashi Kubota

    2015-08-01

    Full Text Available The sliding clamp PCNA is a crucial component of the DNA replication machinery. Timely PCNA loading and unloading are central for genome integrity and must be strictly coordinated with other DNA processing steps during replication. Here, we show that the S. cerevisiae Elg1 replication factor C-like complex (Elg1-RLC unloads PCNA genome-wide following Okazaki fragment ligation. In the absence of Elg1, PCNA is retained on chromosomes in the wake of replication forks, rather than at specific sites. Degradation of the Okazaki fragment ligase Cdc9 leads to PCNA accumulation on chromatin, similar to the accumulation caused by lack of Elg1. We demonstrate that Okazaki fragment ligation is the critical prerequisite for PCNA unloading, since Chlorella virus DNA ligase can substitute for Cdc9 in yeast and simultaneously promotes PCNA unloading. Our results suggest that Elg1-RLC acts as a general PCNA unloader and is dependent upon DNA ligation during chromosome replication.

  13. Rif1 controls DNA replication by directing Protein Phosphatase 1 to reverse Cdc7-mediated phosphorylation of the MCM complex.

    Science.gov (United States)

    Hiraga, Shin-Ichiro; Alvino, Gina M; Chang, Fujung; Lian, Hui-Yong; Sridhar, Akila; Kubota, Takashi; Brewer, Bonita J; Weinreich, Michael; Raghuraman, M K; Donaldson, Anne D

    2014-02-15

    Initiation of eukaryotic DNA replication requires phosphorylation of the MCM complex by Dbf4-dependent kinase (DDK), composed of Cdc7 kinase and its activator, Dbf4. We report here that budding yeast Rif1 (Rap1-interacting factor 1) controls DNA replication genome-wide and describe how Rif1 opposes DDK function by directing Protein Phosphatase 1 (PP1)-mediated dephosphorylation of the MCM complex. Deleting RIF1 partially compensates for the limited DDK activity in a cdc7-1 mutant strain by allowing increased, premature phosphorylation of Mcm4. PP1 interaction motifs within the Rif1 N-terminal domain are critical for its repressive effect on replication. We confirm that Rif1 interacts with PP1 and that PP1 prevents premature Mcm4 phosphorylation. Remarkably, our results suggest that replication repression by Rif1 is itself also DDK-regulated through phosphorylation near the PP1-interacting motifs. Based on our findings, we propose that Rif1 is a novel PP1 substrate targeting subunit that counteracts DDK-mediated phosphorylation during replication. Fission yeast and mammalian Rif1 proteins have also been implicated in regulating DNA replication. Since PP1 interaction sites are evolutionarily conserved within the Rif1 sequence, it is likely that replication control by Rif1 through PP1 is a conserved mechanism.

  14. An isolated Hda-clamp complex is functional in the regulatory inactivation of DnaA and DNA replication.

    Science.gov (United States)

    Kawakami, Hironori; Su'etsugu, Masayuki; Katayama, Tsutomu

    2006-10-01

    In Escherichia coli, a complex consisting of Hda and the DNA-loaded clamp-subunit of the DNA polymerase III holoenzyme promotes hydrolysis of DnaA-ATP. The resultant ADP-DnaA is inactive for initiation of chromosomal DNA replication, thereby repressing excessive initiations. As the cellular content of the clamp is 10-100 times higher than that of Hda, most Hda molecules might be complexed with the clamp in vivo. Although Hda predominantly forms irregular aggregates when overexpressed, in the present study we found that co-overexpression of the clamp with Hda enhances Hda solubility dramatically and we efficiently isolated the Hda-clamp complex. A single molecule of the complex appears to consist of two Hda molecules and a single clamp. The complex is competent in DnaA-ATP hydrolysis and DNA replication in the presence of DNA and the clamp deficient subassembly of the DNA polymerase III holoenzyme (pol III*). These findings indicate that the clamp contained in the complex is loaded onto DNA through an interaction with the pol III* and that the Hda activity is preserved in these processes. The complex consisting of Hda and the DNA-unloaded clamp may play a specific role in a process proceeding to the DnaA-ATP hydrolysis in vivo.

  15. Selective recruitment of nuclear factors to productively replicating herpes simplex virus genomes.

    Science.gov (United States)

    Dembowski, Jill A; DeLuca, Neal A

    2015-05-01

    Much of the HSV-1 life cycle is carried out in the cell nucleus, including the expression, replication, repair, and packaging of viral genomes. Viral proteins, as well as cellular factors, play essential roles in these processes. Isolation of proteins on nascent DNA (iPOND) was developed to label and purify cellular replication forks. We adapted aspects of this method to label viral genomes to both image, and purify replicating HSV-1 genomes for the identification of associated proteins. Many viral and cellular factors were enriched on viral genomes, including factors that mediate DNA replication, repair, chromatin remodeling, transcription, and RNA processing. As infection proceeded, packaging and structural components were enriched to a greater extent. Among the more abundant proteins that copurified with genomes were the viral transcription factor ICP4 and the replication protein ICP8. Furthermore, all seven viral replication proteins were enriched on viral genomes, along with cellular PCNA and topoisomerases, while other cellular replication proteins were not detected. The chromatin-remodeling complexes present on viral genomes included the INO80, SWI/SNF, NURD, and FACT complexes, which may prevent chromatinization of the genome. Consistent with this conclusion, histones were not readily recovered with purified viral genomes, and imaging studies revealed an underrepresentation of histones on viral genomes. RNA polymerase II, the mediator complex, TFIID, TFIIH, and several other transcriptional activators and repressors were also affinity purified with viral DNA. The presence of INO80, NURD, SWI/SNF, mediator, TFIID, and TFIIH components is consistent with previous studies in which these complexes copurified with ICP4. Therefore, ICP4 is likely involved in the recruitment of these key cellular chromatin remodeling and transcription factors to viral genomes. Taken together, iPOND is a valuable method for the study of viral genome dynamics during infection and

  16. The progression of replication forks at natural replication barriers in live bacteria.

    Science.gov (United States)

    Moolman, M Charl; Tiruvadi Krishnan, Sriram; Kerssemakers, Jacob W J; de Leeuw, Roy; Lorent, Vincent; Sherratt, David J; Dekker, Nynke H

    2016-07-27

    Protein-DNA complexes are one of the principal barriers the replisome encounters during replication. One such barrier is the Tus-ter complex, which is a direction dependent barrier for replication fork progression. The details concerning the dynamics of the replisome when encountering these Tus-ter barriers in the cell are poorly understood. By performing quantitative fluorescence microscopy with microfuidics, we investigate the effect on the replisome when encountering these barriers in live Escherichia coli cells. We make use of an E. coli variant that includes only an ectopic origin of replication that is positioned such that one of the two replisomes encounters a Tus-ter barrier before the other replisome. This enables us to single out the effect of encountering a Tus-ter roadblock on an individual replisome. We demonstrate that the replisome remains stably bound after encountering a Tus-ter complex from the non-permissive direction. Furthermore, the replisome is only transiently blocked, and continues replication beyond the barrier. Additionally, we demonstrate that these barriers affect sister chromosome segregation by visualizing specific chromosomal loci in the presence and absence of the Tus protein. These observations demonstrate the resilience of the replication fork to natural barriers and the sensitivity of chromosome alignment to fork progression. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

  17. Rare genomic structural variants in complex disease: lessons from the replication of associations with obesity.

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    Robin G Walters

    Full Text Available The limited ability of common variants to account for the genetic contribution to complex disease has prompted searches for rare variants of large effect, to partly explain the 'missing heritability'. Analyses of genome-wide genotyping data have identified genomic structural variants (GSVs as a source of such rare causal variants. Recent studies have reported multiple GSV loci associated with risk of obesity. We attempted to replicate these associations by similar analysis of two familial-obesity case-control cohorts and a population cohort, and detected GSVs at 11 out of 18 loci, at frequencies similar to those previously reported. Based on their reported frequencies and effect sizes (OR≥25, we had sufficient statistical power to detect the large majority (80% of genuine associations at these loci. However, only one obesity association was replicated. Deletion of a 220 kb region on chromosome 16p11.2 has a carrier population frequency of 2×10(-4 (95% confidence interval [9.6×10(-5-3.1×10(-4]; accounts overall for 0.5% [0.19%-0.82%] of severe childhood obesity cases (P = 3.8×10(-10; odds ratio = 25.0 [9.9-60.6]; and results in a mean body mass index (BMI increase of 5.8 kg.m(-2 [1.8-10.3] in adults from the general population. We also attempted replication using BMI as a quantitative trait in our population cohort; associations with BMI at or near nominal significance were detected at two further loci near KIF2B and within FOXP2, but these did not survive correction for multiple testing. These findings emphasise several issues of importance when conducting rare GSV association, including the need for careful cohort selection and replication strategy, accurate GSV identification, and appropriate correction for multiple testing and/or control of false discovery rate. Moreover, they highlight the potential difficulty in replicating rare CNV associations across different populations. Nevertheless, we show that such studies are potentially

  18. Characterization of the adenoassociated virus Rep protein complex formed on the viral origin of DNA replication

    International Nuclear Information System (INIS)

    Li Zengi; Brister, J. Rodney; Im, Dong-Soo; Muzyczka, Nicholas

    2003-01-01

    Interaction between the adenoassociated virus (AAV) replication proteins, Rep68 and 78, and the viral terminal repeats (TRs) is mediated by a DNA sequence termed the Rep-binding element (RBE). This element is necessary for Rep-mediated unwinding of duplex DNA substrates, directs Rep catalyzed cleavage of the AAV origin of DNA replication, and is required for viral transcription and proviral integration. Six discrete Rep complexes with the AAV TR substrates have been observed in vitro, and cross-linking studies suggest these complexes contain one to six molecules of Rep. However, the functional relationship between Rep oligomerization and biochemical activity is unclear. Here we have characterized Rep complexes that form on the AAV TR. Both Rep68 and Rep78 appear to form the same six complexes with the AAV TR, and ATP seems to stimulate formation of specific, higher order complexes. When the sizes of these Rep complexes were estimated on native polyacrylamide gels, the four slower migrating complexes were larger than predicted by an amount equivalent to one or two TRs. To resolve this discrepancy, the molar ratio of protein and DNA was calculated for the three largest complexes. Data from these experiments indicated that the larger complexes included multiple TRs in addition to multiple Rep molecules and that the Rep-to-TR ratio was approximately 2. The two largest complexes were also associated with increased Rep-mediated, origin cleavage activity. Finally, we characterized a second, Rep-mediated cleavage event that occurs adjacent to the normal nicking site, but on the opposite strand. This second site nicking event effectively results in double-stranded DNA cleavage at the normal nicking site

  19. Mcm10 regulates DNA replication elongation by stimulating the CMG replicative helicase.

    Science.gov (United States)

    Lõoke, Marko; Maloney, Michael F; Bell, Stephen P

    2017-02-01

    Activation of the Mcm2-7 replicative DNA helicase is the committed step in eukaryotic DNA replication initiation. Although Mcm2-7 activation requires binding of the helicase-activating proteins Cdc45 and GINS (forming the CMG complex), an additional protein, Mcm10, drives initial origin DNA unwinding by an unknown mechanism. We show that Mcm10 binds a conserved motif located between the oligonucleotide/oligosaccharide fold (OB-fold) and A subdomain of Mcm2. Although buried in the interface between these domains in Mcm2-7 structures, mutations predicted to separate the domains and expose this motif restore growth to conditional-lethal MCM10 mutant cells. We found that, in addition to stimulating initial DNA unwinding, Mcm10 stabilizes Cdc45 and GINS association with Mcm2-7 and stimulates replication elongation in vivo and in vitro. Furthermore, we identified a lethal allele of MCM10 that stimulates initial DNA unwinding but is defective in replication elongation and CMG binding. Our findings expand the roles of Mcm10 during DNA replication and suggest a new model for Mcm10 function as an activator of the CMG complex throughout DNA replication. © 2017 Lõoke et al.; Published by Cold Spring Harbor Laboratory Press.

  20. Class I ADP-ribosylation factors are involved in enterovirus 71 replication.

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    Jianmin Wang

    Full Text Available Enterovirus 71 is one of the major causative agents of hand, foot, and mouth disease in infants and children. Replication of enterovirus 71 depends on host cellular factors. The viral replication complex is formed in novel, cytoplasmic, vesicular compartments. It has not been elucidated which cellular pathways are hijacked by the virus to create these vesicles. Here, we investigated whether proteins associated with the cellular secretory pathway were involved in enterovirus 71 replication. We used a loss-of-function assay, based on small interfering RNA. We showed that enterovirus 71 RNA replication was dependent on the activity of Class I ADP-ribosylation factors. Simultaneous depletion of ADP-ribosylation factors 1 and 3, but not three others, inhibited viral replication in cells. We also demonstrated with various techniques that the brefeldin-A-sensitive guanidine nucleotide exchange factor, GBF1, was critically important for enterovirus 71 replication. Our results suggested that enterovirus 71 replication depended on GBF1-mediated activation of Class I ADP-ribosylation factors. These results revealed a connection between enterovirus 71 replication and the cellular secretory pathway; this pathway may represent a novel target for antiviral therapies.

  1. Class I ADP-ribosylation factors are involved in enterovirus 71 replication.

    Science.gov (United States)

    Wang, Jianmin; Du, Jiang; Jin, Qi

    2014-01-01

    Enterovirus 71 is one of the major causative agents of hand, foot, and mouth disease in infants and children. Replication of enterovirus 71 depends on host cellular factors. The viral replication complex is formed in novel, cytoplasmic, vesicular compartments. It has not been elucidated which cellular pathways are hijacked by the virus to create these vesicles. Here, we investigated whether proteins associated with the cellular secretory pathway were involved in enterovirus 71 replication. We used a loss-of-function assay, based on small interfering RNA. We showed that enterovirus 71 RNA replication was dependent on the activity of Class I ADP-ribosylation factors. Simultaneous depletion of ADP-ribosylation factors 1 and 3, but not three others, inhibited viral replication in cells. We also demonstrated with various techniques that the brefeldin-A-sensitive guanidine nucleotide exchange factor, GBF1, was critically important for enterovirus 71 replication. Our results suggested that enterovirus 71 replication depended on GBF1-mediated activation of Class I ADP-ribosylation factors. These results revealed a connection between enterovirus 71 replication and the cellular secretory pathway; this pathway may represent a novel target for antiviral therapies.

  2. Global profiling of DNA replication timing and efficiency reveals that efficient replication/firing occurs late during S-phase in S. pombe.

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    Majid Eshaghi

    Full Text Available BACKGROUND: During S. pombe S-phase, initiation of DNA replication occurs at multiple sites (origins that are enriched with AT-rich sequences, at various times. Current studies of genome-wide DNA replication profiles have focused on the DNA replication timing and origin location. However, the replication and/or firing efficiency of the individual origins on the genomic scale remain unclear. METHODOLOGY/PRINCIPAL FINDINGS: Using the genome-wide ORF-specific DNA microarray analysis, we show that in S. pombe, individual origins fire with varying efficiencies and at different times during S-phase. The increase in DNA copy number plotted as a function of time is approximated to the near-sigmoidal model, when considering the replication start and end timings at individual loci in cells released from HU-arrest. Replication efficiencies differ from origin to origin, depending on the origin's firing efficiency. We have found that DNA replication is inefficient early in S-phase, due to inefficient firing at origins. Efficient replication occurs later, attributed to efficient but late-firing origins. Furthermore, profiles of replication timing in cds1Delta cells are abnormal, due to the failure in resuming replication at the collapsed forks. The majority of the inefficient origins, but not the efficient ones, are found to fire in cds1Delta cells after HU removal, owing to the firing at the remaining unused (inefficient origins during HU treatment. CONCLUSIONS/SIGNIFICANCE: Taken together, our results indicate that efficient DNA replication/firing occurs late in S-phase progression in cells after HU removal, due to efficient late-firing origins. Additionally, checkpoint kinase Cds1p is required for maintaining the efficient replication/firing late in S-phase. We further propose that efficient late-firing origins are essential for ensuring completion of DNA duplication by the end of S-phase.

  3. Viral replication. Structural basis for RNA replication by the hepatitis C virus polymerase.

    Science.gov (United States)

    Appleby, Todd C; Perry, Jason K; Murakami, Eisuke; Barauskas, Ona; Feng, Joy; Cho, Aesop; Fox, David; Wetmore, Diana R; McGrath, Mary E; Ray, Adrian S; Sofia, Michael J; Swaminathan, S; Edwards, Thomas E

    2015-02-13

    Nucleotide analog inhibitors have shown clinical success in the treatment of hepatitis C virus (HCV) infection, despite an incomplete mechanistic understanding of NS5B, the viral RNA-dependent RNA polymerase. Here we study the details of HCV RNA replication by determining crystal structures of stalled polymerase ternary complexes with enzymes, RNA templates, RNA primers, incoming nucleotides, and catalytic metal ions during both primed initiation and elongation of RNA synthesis. Our analysis revealed that highly conserved active-site residues in NS5B position the primer for in-line attack on the incoming nucleotide. A β loop and a C-terminal membrane-anchoring linker occlude the active-site cavity in the apo state, retract in the primed initiation assembly to enforce replication of the HCV genome from the 3' terminus, and vacate the active-site cavity during elongation. We investigated the incorporation of nucleotide analog inhibitors, including the clinically active metabolite formed by sofosbuvir, to elucidate key molecular interactions in the active site. Copyright © 2015, American Association for the Advancement of Science.

  4. DNA-PK/Ku complex binds to latency-associated nuclear antigen and negatively regulates Kaposi's sarcoma-associated herpesvirus latent replication

    Energy Technology Data Exchange (ETDEWEB)

    Cha, Seho [Department of Life Science, Dongguk Univ-Seoul, Seoul 100-715 (Korea, Republic of); Lim, Chunghun [Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon 305-701 (Korea, Republic of); Lee, Jae Young [Department of Life Science, Dongguk Univ-Seoul, Seoul 100-715 (Korea, Republic of); Song, Yoon-Jae [Department of Life Science, Kyungwon University, Seongnam-Si, Kyeonggi-Do 461-701 (Korea, Republic of); Park, Junsoo [Division of Biological Science and Technology, Yonsei University, Wonju 220-100 (Korea, Republic of); Choe, Joonho [Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon 305-701 (Korea, Republic of); Seo, Taegun, E-mail: tseo@dongguk.edu [Department of Life Science, Dongguk Univ-Seoul, Seoul 100-715 (Korea, Republic of)

    2010-04-16

    During latent infection, latency-associated nuclear antigen (LANA) of Kaposi's sarcoma-associated herpesvirus (KSHV) plays important roles in episomal persistence and replication. Several host factors are associated with KSHV latent replication. Here, we show that the catalytic subunit of DNA protein kinase (DNA-PKcs), Ku70, and Ku86 bind the N-terminal region of LANA. LANA was phosphorylated by DNA-PK and overexpression of Ku70, but not Ku86, impaired transient replication. The efficiency of transient replication was significantly increased in the HCT116 (Ku86 +/-) cell line, compared to the HCT116 (Ku86 +/+) cell line, suggesting that the DNA-PK/Ku complex negatively regulates KSHV latent replication.

  5. Solution structure of the N-terminal domain of a replication restart primosome factor, PriC, in Escherichia coli

    Science.gov (United States)

    Aramaki, Takahiko; Abe, Yoshito; Katayama, Tsutomu; Ueda, Tadashi

    2013-01-01

    In eubacterial organisms, the oriC-independent primosome plays an essential role in replication restart after the dissociation of the replication DNA-protein complex by DNA damage. PriC is a key protein component in the replication restart primosome. Our recent study suggested that PriC is divided into two domains: an N-terminal and a C-terminal domain. In the present study, we determined the solution structure of the N-terminal domain, whose structure and function have remained unknown until now. The revealed structure was composed of three helices and one extended loop. We also observed chemical shift changes in the heteronuclear NMR spectrum and oligomerization in the presence of ssDNA. These abilities may contribute to the PriC-ssDNA complex, which is important for the replication restart primosome. PMID:23868391

  6. Cytology of DNA Replication Reveals Dynamic Plasticity of Large-Scale Chromatin Fibers.

    Science.gov (United States)

    Deng, Xiang; Zhironkina, Oxana A; Cherepanynets, Varvara D; Strelkova, Olga S; Kireev, Igor I; Belmont, Andrew S

    2016-09-26

    In higher eukaryotic interphase nuclei, the 100- to >1,000-fold linear compaction of chromatin is difficult to reconcile with its function as a template for transcription, replication, and repair. It is challenging to imagine how DNA and RNA polymerases with their associated molecular machinery would move along the DNA template without transient decondensation of observed large-scale chromatin "chromonema" fibers [1]. Transcription or "replication factory" models [2], in which polymerases remain fixed while DNA is reeled through, are similarly difficult to conceptualize without transient decondensation of these chromonema fibers. Here, we show how a dynamic plasticity of chromatin folding within large-scale chromatin fibers allows DNA replication to take place without significant changes in the global large-scale chromatin compaction or shape of these large-scale chromatin fibers. Time-lapse imaging of lac-operator-tagged chromosome regions shows no major change in the overall compaction of these chromosome regions during their DNA replication. Improved pulse-chase labeling of endogenous interphase chromosomes yields a model in which the global compaction and shape of large-Mbp chromatin domains remains largely invariant during DNA replication, with DNA within these domains undergoing significant movements and redistribution as they move into and then out of adjacent replication foci. In contrast to hierarchical folding models, this dynamic plasticity of large-scale chromatin organization explains how localized changes in DNA topology allow DNA replication to take place without an accompanying global unfolding of large-scale chromatin fibers while suggesting a possible mechanism for maintaining epigenetic programming of large-scale chromatin domains throughout DNA replication. Copyright © 2016 Elsevier Ltd. All rights reserved.

  7. Specificity and Function of Archaeal DNA Replication Initiator Proteins

    Directory of Open Access Journals (Sweden)

    Rachel Y. Samson

    2013-02-01

    Full Text Available Chromosomes with multiple DNA replication origins are a hallmark of Eukaryotes and some Archaea. All eukaryal nuclear replication origins are defined by the origin recognition complex (ORC that recruits the replicative helicase MCM(2-7 via Cdc6 and Cdt1. We find that the three origins in the single chromosome of the archaeon Sulfolobus islandicus are specified by distinct initiation factors. While two origins are dependent on archaeal homologs of eukaryal Orc1 and Cdc6, the third origin is instead reliant on an archaeal Cdt1 homolog. We exploit the nonessential nature of the orc1-1 gene to investigate the role of ATP binding and hydrolysis in initiator function in vivo and in vitro. We find that the ATP-bound form of Orc1-1 is proficient for replication and implicates hydrolysis of ATP in downregulation of origin activity. Finally, we reveal that ATP and DNA binding by Orc1-1 remodels the protein’s structure rather than that of the DNA template.

  8. Selective recruitment of nuclear factors to productively replicating herpes simplex virus genomes.

    Directory of Open Access Journals (Sweden)

    Jill A Dembowski

    2015-05-01

    Full Text Available Much of the HSV-1 life cycle is carried out in the cell nucleus, including the expression, replication, repair, and packaging of viral genomes. Viral proteins, as well as cellular factors, play essential roles in these processes. Isolation of proteins on nascent DNA (iPOND was developed to label and purify cellular replication forks. We adapted aspects of this method to label viral genomes to both image, and purify replicating HSV-1 genomes for the identification of associated proteins. Many viral and cellular factors were enriched on viral genomes, including factors that mediate DNA replication, repair, chromatin remodeling, transcription, and RNA processing. As infection proceeded, packaging and structural components were enriched to a greater extent. Among the more abundant proteins that copurified with genomes were the viral transcription factor ICP4 and the replication protein ICP8. Furthermore, all seven viral replication proteins were enriched on viral genomes, along with cellular PCNA and topoisomerases, while other cellular replication proteins were not detected. The chromatin-remodeling complexes present on viral genomes included the INO80, SWI/SNF, NURD, and FACT complexes, which may prevent chromatinization of the genome. Consistent with this conclusion, histones were not readily recovered with purified viral genomes, and imaging studies revealed an underrepresentation of histones on viral genomes. RNA polymerase II, the mediator complex, TFIID, TFIIH, and several other transcriptional activators and repressors were also affinity purified with viral DNA. The presence of INO80, NURD, SWI/SNF, mediator, TFIID, and TFIIH components is consistent with previous studies in which these complexes copurified with ICP4. Therefore, ICP4 is likely involved in the recruitment of these key cellular chromatin remodeling and transcription factors to viral genomes. Taken together, iPOND is a valuable method for the study of viral genome dynamics

  9. RFWD3-Dependent Ubiquitination of RPA Regulates Repair at Stalled Replication Forks.

    Science.gov (United States)

    Elia, Andrew E H; Wang, David C; Willis, Nicholas A; Boardman, Alexander P; Hajdu, Ildiko; Adeyemi, Richard O; Lowry, Elizabeth; Gygi, Steven P; Scully, Ralph; Elledge, Stephen J

    2015-10-15

    We have used quantitative proteomics to profile ubiquitination in the DNA damage response (DDR). We demonstrate that RPA, which functions as a protein scaffold in the replication stress response, is multiply ubiquitinated upon replication fork stalling. Ubiquitination of RPA occurs on chromatin, involves sites outside its DNA binding channel, does not cause proteasomal degradation, and increases under conditions of fork collapse, suggesting a role in repair at stalled forks. We demonstrate that the E3 ligase RFWD3 mediates RPA ubiquitination. RFWD3 is necessary for replication fork restart, normal repair kinetics during replication stress, and homologous recombination (HR) at stalled replication forks. Mutational analysis suggests that multisite ubiquitination of the entire RPA complex is responsible for repair at stalled forks. Multisite protein group sumoylation is known to promote HR in yeast. Our findings reveal a similar requirement for multisite protein group ubiquitination during HR at stalled forks in mammalian cells. Copyright © 2015 Elsevier Inc. All rights reserved.

  10. The fission yeast minichromosome maintenance (MCM)-binding protein (MCM-BP), Mcb1, regulates MCM function during prereplicative complex formation in DNA replication.

    Science.gov (United States)

    Santosa, Venny; Martha, Sabrina; Hirose, Noriaki; Tanaka, Katsunori

    2013-03-08

    The minichromosome maintenance (MCM) complex is a replicative helicase, which is essential for chromosome DNA replication. In recent years, the identification of a novel MCM-binding protein (MCM-BP) in most eukaryotes has led to numerous studies investigating its function and its relationship to the MCM complex. However, the mechanisms by which MCM-BP functions and associates with MCM complexes are not well understood; in addition, the functional role of MCM-BP remains controversial and may vary between model organisms. The present study aims to elucidate the nature and biological function of the MCM-BP ortholog, Mcb1, in fission yeast. The Mcb1 protein continuously interacts with MCM proteins during the cell cycle in vivo and can interact with any individual MCM subunit in vitro. To understand the detailed characteristics of mcb1(+), two temperature-sensitive mcb1 gene mutants (mcb1(ts)) were isolated. Extensive genetic analysis showed that the mcb1(ts) mutants were suppressed by a mcm5(+) multicopy plasmid and displayed synthetic defects with many S-phase-related gene mutants. Moreover, cyclin-dependent kinase modulation by Cig2 repression or Rum1 overproduction suppressed the mcb1(ts) mutants, suggesting the involvement of Mcb1 in pre-RC formation during DNA replication. These data are consistent with the observation that Mcm7 loading onto replication origins is reduced and S-phase progression is delayed in mcb1(ts) mutants. Furthermore, the mcb1(ts) mutation led to the redistribution of MCM subunits to the cytoplasm, and this redistribution was dependent on an active nuclear export system. These results strongly suggest that Mcb1 promotes efficient pre-RC formation during DNA replication by regulating the MCM complex.

  11. The Fission Yeast Minichromosome Maintenance (MCM)-binding Protein (MCM-BP), Mcb1, Regulates MCM Function during Prereplicative Complex Formation in DNA Replication*

    Science.gov (United States)

    Santosa, Venny; Martha, Sabrina; Hirose, Noriaki; Tanaka, Katsunori

    2013-01-01

    The minichromosome maintenance (MCM) complex is a replicative helicase, which is essential for chromosome DNA replication. In recent years, the identification of a novel MCM-binding protein (MCM-BP) in most eukaryotes has led to numerous studies investigating its function and its relationship to the MCM complex. However, the mechanisms by which MCM-BP functions and associates with MCM complexes are not well understood; in addition, the functional role of MCM-BP remains controversial and may vary between model organisms. The present study aims to elucidate the nature and biological function of the MCM-BP ortholog, Mcb1, in fission yeast. The Mcb1 protein continuously interacts with MCM proteins during the cell cycle in vivo and can interact with any individual MCM subunit in vitro. To understand the detailed characteristics of mcb1+, two temperature-sensitive mcb1 gene mutants (mcb1ts) were isolated. Extensive genetic analysis showed that the mcb1ts mutants were suppressed by a mcm5+ multicopy plasmid and displayed synthetic defects with many S-phase-related gene mutants. Moreover, cyclin-dependent kinase modulation by Cig2 repression or Rum1 overproduction suppressed the mcb1ts mutants, suggesting the involvement of Mcb1 in pre-RC formation during DNA replication. These data are consistent with the observation that Mcm7 loading onto replication origins is reduced and S-phase progression is delayed in mcb1ts mutants. Furthermore, the mcb1ts mutation led to the redistribution of MCM subunits to the cytoplasm, and this redistribution was dependent on an active nuclear export system. These results strongly suggest that Mcb1 promotes efficient pre-RC formation during DNA replication by regulating the MCM complex. PMID:23322785

  12. H4 replication-dependent diacetylation and Hat1 promote S-phase chromatin assembly in vivo

    Science.gov (United States)

    Ejlassi-Lassallette, Aïda; Mocquard, Eloïse; Arnaud, Marie-Claire; Thiriet, Christophe

    2011-01-01

    While specific posttranslational modification patterns within the H3 and H4 tail domains are associated with the S-phase, their actual functions in replication-dependent chromatin assembly have not yet been defined. Here we used incorporation of trace amounts of recombinant proteins into naturally synchronous macroplasmodia of Physarum polycephalum to examine the function of H3 and H4 tail domains in replication-coupled chromatin assembly. We found that the H3/H4 complex lacking the H4 tail domain was not efficiently recovered in nuclei, whereas depletion of the H3 tail domain did not impede nuclear import but chromatin assembly failed. Furthermore, our results revealed that the proper pattern of acetylation on the H4 tail domain is required for nuclear import and chromatin assembly. This is most likely due to binding of Hat1, as coimmunoprecipitation experiments showed Hat1 associated with predeposition histones in the cytoplasm and with replicating chromatin. These results suggest that the type B histone acetyltransferase assists in shuttling the H3/H4 complex from cytoplasm to the replication forks. PMID:21118997

  13. DNA Replication in Engineered Escherichia coli Genomes with Extra Replication Origins.

    Science.gov (United States)

    Milbredt, Sarah; Farmani, Neda; Sobetzko, Patrick; Waldminghaus, Torsten

    2016-10-21

    The standard outline of bacterial genomes is a single circular chromosome with a single replication origin. From the bioengineering perspective, it appears attractive to extend this basic setup. Bacteria with split chromosomes or multiple replication origins have been successfully constructed in the last few years. The characteristics of these engineered strains will largely depend on the respective DNA replication patterns. However, the DNA replication has not been investigated systematically in engineered bacteria with multiple origins or split replicons. Here we fill this gap by studying a set of strains consisting of (i) E. coli strains with an extra copy of the native replication origin (oriC), (ii) E. coli strains with an extra copy of the replication origin from the secondary chromosome of Vibrio cholerae (oriII), and (iii) a strain in which the E. coli chromosome is split into two linear replicons. A combination of flow cytometry, microarray-based comparative genomic hybridization (CGH), and modeling revealed silencing of extra oriC copies and differential timing of ectopic oriII copies compared to the native oriC. The results were used to derive construction rules for future multiorigin and multireplicon projects.

  14. Two mechanisms coordinate replication termination by the Escherichia coli Tus–Ter complex

    KAUST Repository

    Pandey, Manjula

    2015-07-13

    The Escherichia coli replication terminator protein (Tus) binds to Ter sequences to block replication forks approaching from one direction. Here, we used single molecule and transient state kinetics to study responses of the heterologous phage T7 replisome to the Tus–Ter complex. The T7 replisome was arrested at the non-permissive end of Tus–Ter in a manner that is explained by a composite mousetrap and dynamic clamp model. An unpaired C(6) that forms a lock by binding into the cytosine binding pocket of Tus was most effective in arresting the replisome and mutation of C(6) removed the barrier. Isolated helicase was also blocked at the non-permissive end, but unexpectedly the isolated polymerase was not, unless C(6) was unpaired. Instead, the polymerase was blocked at the permissive end. This indicates that the Tus–Ter mechanism is sensitive to the translocation polarity of the DNA motor. The polymerase tracking along the template strand traps the C(6) to prevent lock formation; the helicase tracking along the other strand traps the complementary G(6) to aid lock formation. Our results are consistent with the model where strand separation by the helicase unpairs the GC(6) base pair and triggers lock formation immediately before the polymerase can sequester the C(6) base.

  15. Quantitative Proteomics Reveals Dynamic Interactions of the Minichromosome Maintenance Complex (MCM) in the Cellular Response to Etoposide Induced DNA Damage.

    Science.gov (United States)

    Drissi, Romain; Dubois, Marie-Line; Douziech, Mélanie; Boisvert, François-Michel

    2015-07-01

    The minichromosome maintenance complex (MCM) proteins are required for processive DNA replication and are a target of S-phase checkpoints. The eukaryotic MCM complex consists of six proteins (MCM2-7) that form a heterohexameric ring with DNA helicase activity, which is loaded on chromatin to form the pre-replication complex. Upon entry in S phase, the helicase is activated and opens the DNA duplex to recruit DNA polymerases at the replication fork. The MCM complex thus plays a crucial role during DNA replication, but recent work suggests that MCM proteins could also be involved in DNA repair. Here, we employed a combination of stable isotope labeling with amino acids in cell culture (SILAC)-based quantitative proteomics with immunoprecipitation of green fluorescent protein-tagged fusion proteins to identify proteins interacting with the MCM complex, and quantify changes in interactions in response to DNA damage. Interestingly, the MCM complex showed very dynamic changes in interaction with proteins such as Importin7, the histone chaperone ASF1, and the Chromodomain helicase DNA binding protein 3 (CHD3) following DNA damage. These changes in interactions were accompanied by an increase in phosphorylation and ubiquitination on specific sites on the MCM proteins and an increase in the co-localization of the MCM complex with γ-H2AX, confirming the recruitment of these proteins to sites of DNA damage. In summary, our data indicate that the MCM proteins is involved in chromatin remodeling in response to DNA damage. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  16. Genome-wide mapping reveals single-origin chromosome replication in Leishmania, a eukaryotic microbe.

    Science.gov (United States)

    Marques, Catarina A; Dickens, Nicholas J; Paape, Daniel; Campbell, Samantha J; McCulloch, Richard

    2015-10-19

    DNA replication initiates on defined genome sites, termed origins. Origin usage appears to follow common rules in the eukaryotic organisms examined to date: all chromosomes are replicated from multiple origins, which display variations in firing efficiency and are selected from a larger pool of potential origins. To ask if these features of DNA replication are true of all eukaryotes, we describe genome-wide origin mapping in the parasite Leishmania. Origin mapping in Leishmania suggests a striking divergence in origin usage relative to characterized eukaryotes, since each chromosome appears to be replicated from a single origin. By comparing two species of Leishmania, we find evidence that such origin singularity is maintained in the face of chromosome fusion or fission events during evolution. Mapping Leishmania origins suggests that all origins fire with equal efficiency, and that the genomic sites occupied by origins differ from related non-origins sites. Finally, we provide evidence that origin location in Leishmania displays striking conservation with Trypanosoma brucei, despite the latter parasite replicating its chromosomes from multiple, variable strength origins. The demonstration of chromosome replication for a single origin in Leishmania, a microbial eukaryote, has implications for the evolution of origin multiplicity and associated controls, and may explain the pervasive aneuploidy that characterizes Leishmania chromosome architecture.

  17. Mechanisms of bacterial DNA replication restart

    Science.gov (United States)

    Windgassen, Tricia A; Wessel, Sarah R; Bhattacharyya, Basudeb

    2018-01-01

    Abstract Multi-protein DNA replication complexes called replisomes perform the essential process of copying cellular genetic information prior to cell division. Under ideal conditions, replisomes dissociate only after the entire genome has been duplicated. However, DNA replication rarely occurs without interruptions that can dislodge replisomes from DNA. Such events produce incompletely replicated chromosomes that, if left unrepaired, prevent the segregation of full genomes to daughter cells. To mitigate this threat, cells have evolved ‘DNA replication restart’ pathways that have been best defined in bacteria. Replication restart requires recognition and remodeling of abandoned replication forks by DNA replication restart proteins followed by reloading of the replicative DNA helicase, which subsequently directs assembly of the remaining replisome subunits. This review summarizes our current understanding of the mechanisms underlying replication restart and the proteins that drive the process in Escherichia coli (PriA, PriB, PriC and DnaT). PMID:29202195

  18. The proteasomal Rpn11 metalloprotease suppresses tombusvirus RNA recombination and promotes viral replication via facilitating assembly of the viral replicase complex.

    Science.gov (United States)

    Prasanth, K Reddisiva; Barajas, Daniel; Nagy, Peter D

    2015-03-01

    RNA viruses co-opt a large number of cellular proteins that affect virus replication and, in some cases, viral genetic recombination. RNA recombination helps viruses in an evolutionary arms race with the host's antiviral responses and adaptation of viruses to new hosts. Tombusviruses and a yeast model host are used to identify cellular factors affecting RNA virus replication and RNA recombination. In this study, we have examined the role of the conserved Rpn11p metalloprotease subunit of the proteasome, which couples deubiquitination and degradation of proteasome substrates, in tombusvirus replication and recombination in Saccharomyces cerevisiae and plants. Depletion or mutations of Rpn11p lead to the rapid formation of viral RNA recombinants in combination with reduced levels of viral RNA replication in yeast or in vitro based on cell extracts. Rpn11p interacts with the viral replication proteins and is recruited to the viral replicase complex (VRC). Analysis of the multifunctional Rpn11p has revealed that the primary role of Rpn11p is to act as a "matchmaker" that brings the viral p92(pol) replication protein and the DDX3-like Ded1p/RH20 DEAD box helicases into VRCs. Overexpression of Ded1p can complement the defect observed in rpn11 mutant yeast by reducing TBSV recombination. This suggests that Rpn11p can suppress tombusvirus recombination via facilitating the recruitment of the cellular Ded1p helicase, which is a strong suppressor of viral recombination, into VRCs. Overall, this work demonstrates that the co-opted Rpn11p, which is involved in the assembly of the functional proteasome, also functions in the proper assembly of the tombusvirus VRCs. RNA viruses evolve rapidly due to genetic changes based on mutations and RNA recombination. Viral genetic recombination helps viruses in an evolutionary arms race with the host's antiviral responses and facilitates adaptation of viruses to new hosts. Cellular factors affect viral RNA recombination, although the role

  19. A loss of function analysis of host factors influencing Vaccinia virus replication by RNA interference.

    Directory of Open Access Journals (Sweden)

    Philippa M Beard

    Full Text Available Vaccinia virus (VACV is a large, cytoplasmic, double-stranded DNA virus that requires complex interactions with host proteins in order to replicate. To explore these interactions a functional high throughput small interfering RNA (siRNA screen targeting 6719 druggable cellular genes was undertaken to identify host factors (HF influencing the replication and spread of an eGFP-tagged VACV. The experimental design incorporated a low multiplicity of infection, thereby enhancing detection of cellular proteins involved in cell-to-cell spread of VACV. The screen revealed 153 pro- and 149 anti-viral HFs that strongly influenced VACV replication. These HFs were investigated further by comparisons with transcriptional profiling data sets and HFs identified in RNAi screens of other viruses. In addition, functional and pathway analysis of the entire screen was carried out to highlight cellular mechanisms involved in VACV replication. This revealed, as anticipated, that many pro-viral HFs are involved in translation of mRNA and, unexpectedly, suggested that a range of proteins involved in cellular transcriptional processes and several DNA repair pathways possess anti-viral activity. Multiple components of the AMPK complex were found to act as pro-viral HFs, while several septins, a group of highly conserved GTP binding proteins with a role in sequestering intracellular bacteria, were identified as strong anti-viral VACV HFs. This screen has identified novel and previously unexplored roles for cellular factors in poxvirus replication. This advancement in our understanding of the VACV life cycle provides a reliable knowledge base for the improvement of poxvirus-based vaccine vectors and development of anti-viral theraputics.

  20. Self-enhancement of hepatitis C virus replication by promotion of specific sphingolipid biosynthesis.

    Directory of Open Access Journals (Sweden)

    Yuichi Hirata

    Full Text Available Lipids are key components in the viral life cycle that affect host-pathogen interactions. In this study, we investigated the effect of HCV infection on sphingolipid metabolism, especially on endogenous SM levels, and the relationship between HCV replication and endogenous SM molecular species. We demonstrated that HCV induces the expression of the genes (SGMS1 and 2 encoding human SM synthases 1 and 2. We observed associated increases of both total and individual sphingolipid molecular species, as assessed in human hepatocytes and in the detergent-resistant membrane (DRM fraction in which HCV replicates. SGMS1 expression had a correlation with HCV replication. Inhibition of sphingolipid biosynthesis with a hepatotropic serine palmitoyltransferase (SPT inhibitor, NA808, suppressed HCV-RNA production while also interfering with sphingolipid metabolism. Further, we identified the SM molecular species that comprise the DRM fraction and demonstrated that these endogenous SM species interacted with HCV nonstructural 5B polymerase to enhance viral replication. Our results reveal that HCV alters sphingolipid metabolism to promote viral replication, providing new insights into the formation of the HCV replication complex and the involvement of host lipids in the HCV life cycle.

  1. Self-enhancement of hepatitis C virus replication by promotion of specific sphingolipid biosynthesis.

    Science.gov (United States)

    Hirata, Yuichi; Ikeda, Kazutaka; Sudoh, Masayuki; Tokunaga, Yuko; Suzuki, Akemi; Weng, Leiyun; Ohta, Masatoshi; Tobita, Yoshimi; Okano, Ken; Ozeki, Kazuhisa; Kawasaki, Kenichi; Tsukuda, Takuo; Katsume, Asao; Aoki, Yuko; Umehara, Takuya; Sekiguchi, Satoshi; Toyoda, Tetsuya; Shimotohno, Kunitada; Soga, Tomoyoshi; Nishijima, Masahiro; Taguchi, Ryo; Kohara, Michinori

    2012-01-01

    Lipids are key components in the viral life cycle that affect host-pathogen interactions. In this study, we investigated the effect of HCV infection on sphingolipid metabolism, especially on endogenous SM levels, and the relationship between HCV replication and endogenous SM molecular species. We demonstrated that HCV induces the expression of the genes (SGMS1 and 2) encoding human SM synthases 1 and 2. We observed associated increases of both total and individual sphingolipid molecular species, as assessed in human hepatocytes and in the detergent-resistant membrane (DRM) fraction in which HCV replicates. SGMS1 expression had a correlation with HCV replication. Inhibition of sphingolipid biosynthesis with a hepatotropic serine palmitoyltransferase (SPT) inhibitor, NA808, suppressed HCV-RNA production while also interfering with sphingolipid metabolism. Further, we identified the SM molecular species that comprise the DRM fraction and demonstrated that these endogenous SM species interacted with HCV nonstructural 5B polymerase to enhance viral replication. Our results reveal that HCV alters sphingolipid metabolism to promote viral replication, providing new insights into the formation of the HCV replication complex and the involvement of host lipids in the HCV life cycle.

  2. A positive-strand RNA virus uses alternative protein-protein interactions within a viral protease/cofactor complex to switch between RNA replication and virion morphogenesis.

    Science.gov (United States)

    Dubrau, Danilo; Tortorici, M Alejandra; Rey, Félix A; Tautz, Norbert

    2017-02-01

    The viruses of the family Flaviviridae possess a positive-strand RNA genome and express a single polyprotein which is processed into functional proteins. Initially, the nonstructural (NS) proteins, which are not part of the virions, form complexes capable of genome replication. Later on, the NS proteins also play a critical role in virion formation. The molecular basis to understand how the same proteins form different complexes required in both processes is so far unknown. For pestiviruses, uncleaved NS2-3 is essential for virion morphogenesis while NS3 is required for RNA replication but is not functional in viral assembly. Recently, we identified two gain of function mutations, located in the C-terminal region of NS2 and in the serine protease domain of NS3 (NS3 residue 132), which allow NS2 and NS3 to substitute for uncleaved NS2-3 in particle assembly. We report here the crystal structure of pestivirus NS3-4A showing that the NS3 residue 132 maps to a surface patch interacting with the C-terminal region of NS4A (NS4A-kink region) suggesting a critical role of this contact in virion morphogenesis. We show that destabilization of this interaction, either by alanine exchanges at this NS3/4A-kink interface, led to a gain of function of the NS3/4A complex in particle formation. In contrast, RNA replication and thus replicase assembly requires a stable association between NS3 and the NS4A-kink region. Thus, we propose that two variants of NS3/4A complexes exist in pestivirus infected cells each representing a basic building block required for either RNA replication or virion morphogenesis. This could be further corroborated by trans-complementation studies with a replication-defective NS3/4A double mutant that was still functional in viral assembly. Our observations illustrate the presence of alternative overlapping surfaces providing different contacts between the same proteins, allowing the switch from RNA replication to virion formation.

  3. A positive-strand RNA virus uses alternative protein-protein interactions within a viral protease/cofactor complex to switch between RNA replication and virion morphogenesis

    Science.gov (United States)

    Rey, Félix A.

    2017-01-01

    The viruses of the family Flaviviridae possess a positive-strand RNA genome and express a single polyprotein which is processed into functional proteins. Initially, the nonstructural (NS) proteins, which are not part of the virions, form complexes capable of genome replication. Later on, the NS proteins also play a critical role in virion formation. The molecular basis to understand how the same proteins form different complexes required in both processes is so far unknown. For pestiviruses, uncleaved NS2-3 is essential for virion morphogenesis while NS3 is required for RNA replication but is not functional in viral assembly. Recently, we identified two gain of function mutations, located in the C-terminal region of NS2 and in the serine protease domain of NS3 (NS3 residue 132), which allow NS2 and NS3 to substitute for uncleaved NS2-3 in particle assembly. We report here the crystal structure of pestivirus NS3-4A showing that the NS3 residue 132 maps to a surface patch interacting with the C-terminal region of NS4A (NS4A-kink region) suggesting a critical role of this contact in virion morphogenesis. We show that destabilization of this interaction, either by alanine exchanges at this NS3/4A-kink interface, led to a gain of function of the NS3/4A complex in particle formation. In contrast, RNA replication and thus replicase assembly requires a stable association between NS3 and the NS4A-kink region. Thus, we propose that two variants of NS3/4A complexes exist in pestivirus infected cells each representing a basic building block required for either RNA replication or virion morphogenesis. This could be further corroborated by trans-complementation studies with a replication-defective NS3/4A double mutant that was still functional in viral assembly. Our observations illustrate the presence of alternative overlapping surfaces providing different contacts between the same proteins, allowing the switch from RNA replication to virion formation. PMID:28151973

  4. A positive-strand RNA virus uses alternative protein-protein interactions within a viral protease/cofactor complex to switch between RNA replication and virion morphogenesis.

    Directory of Open Access Journals (Sweden)

    Danilo Dubrau

    2017-02-01

    Full Text Available The viruses of the family Flaviviridae possess a positive-strand RNA genome and express a single polyprotein which is processed into functional proteins. Initially, the nonstructural (NS proteins, which are not part of the virions, form complexes capable of genome replication. Later on, the NS proteins also play a critical role in virion formation. The molecular basis to understand how the same proteins form different complexes required in both processes is so far unknown. For pestiviruses, uncleaved NS2-3 is essential for virion morphogenesis while NS3 is required for RNA replication but is not functional in viral assembly. Recently, we identified two gain of function mutations, located in the C-terminal region of NS2 and in the serine protease domain of NS3 (NS3 residue 132, which allow NS2 and NS3 to substitute for uncleaved NS2-3 in particle assembly. We report here the crystal structure of pestivirus NS3-4A showing that the NS3 residue 132 maps to a surface patch interacting with the C-terminal region of NS4A (NS4A-kink region suggesting a critical role of this contact in virion morphogenesis. We show that destabilization of this interaction, either by alanine exchanges at this NS3/4A-kink interface, led to a gain of function of the NS3/4A complex in particle formation. In contrast, RNA replication and thus replicase assembly requires a stable association between NS3 and the NS4A-kink region. Thus, we propose that two variants of NS3/4A complexes exist in pestivirus infected cells each representing a basic building block required for either RNA replication or virion morphogenesis. This could be further corroborated by trans-complementation studies with a replication-defective NS3/4A double mutant that was still functional in viral assembly. Our observations illustrate the presence of alternative overlapping surfaces providing different contacts between the same proteins, allowing the switch from RNA replication to virion formation.

  5. Two mechanisms coordinate replication termination by the Escherichia coli Tus-Ter complex.

    Science.gov (United States)

    Pandey, Manjula; Elshenawy, Mohamed M; Jergic, Slobodan; Takahashi, Masateru; Dixon, Nicholas E; Hamdan, Samir M; Patel, Smita S

    2015-07-13

    The Escherichia coli replication terminator protein (Tus) binds to Ter sequences to block replication forks approaching from one direction. Here, we used single molecule and transient state kinetics to study responses of the heterologous phage T7 replisome to the Tus-Ter complex. The T7 replisome was arrested at the non-permissive end of Tus-Ter in a manner that is explained by a composite mousetrap and dynamic clamp model. An unpaired C(6) that forms a lock by binding into the cytosine binding pocket of Tus was most effective in arresting the replisome and mutation of C(6) removed the barrier. Isolated helicase was also blocked at the non-permissive end, but unexpectedly the isolated polymerase was not, unless C(6) was unpaired. Instead, the polymerase was blocked at the permissive end. This indicates that the Tus-Ter mechanism is sensitive to the translocation polarity of the DNA motor. The polymerase tracking along the template strand traps the C(6) to prevent lock formation; the helicase tracking along the other strand traps the complementary G(6) to aid lock formation. Our results are consistent with the model where strand separation by the helicase unpairs the GC(6) base pair and triggers lock formation immediately before the polymerase can sequester the C(6) base. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  6. Membrane fractions active in poliovirus RNA replication contain VPg precursor polypeptides

    International Nuclear Information System (INIS)

    Takegami, T.; Semler, B.L.; Anderson, C.W.; Wimmer, E.

    1983-01-01

    The poliovirus specific polypeptide P3-9 is of special interest for studies of viral RNA replication because it contains a hydrophobic region and, separated by only seven amino acids from that region, the amino acid sequence of the genome-linked protein VPg. Membraneous complexes of poliovirus-infected HeLa cells that contain poliovirus RNA replicating proteins have been analyzed for the presence of P3-9 by immunoprecipitation. Incubation of a membrane fraction rich in P3-9 with proteinase leaves the C-terminal 69 amino acids of P3-9 intact, an observation suggesting that this portion is protected by its association with the cellular membrane. These studies have also revealed two hitherto undescribed viral polypeptides consisting of amino acid sequences of the P2 andf P3 regions of the polyprotein. Sequence analysis by stepwise Edman degradation show that these proteins are 3b/9 (M/sub r/77,000) and X/9 (M/sub r/50,000). 3b/9 and X/9 are membrane bound and are turned over rapidly and may be direct precursors to proteins P2-X and P3-9 of the RNA replication complex. P2-X, a polypeptide void of hydrophobic amino acid sequences but also found associated with membranes, is rapidly degraded when the membraneous complex is treated with trypsin. It is speculated that P2-X is associated with membranes by its affinity to the N-terminus of P3-9

  7. Constitutive role of the Fanconi anemia D2 gene in the replication stress response.

    Science.gov (United States)

    Tian, Yanyan; Shen, Xi; Wang, Rui; Klages-Mundt, Naeh L; Lynn, Erica J; Martin, Sara K; Ye, Yin; Gao, Min; Chen, Junjie; Schlacher, Katharina; Li, Lei

    2017-12-08

    In response to DNA cross-linking damage, the Fanconi anemia (FA) core complex activates the FA pathway by monoubiquitinating Fanconi anemia complementation group D2 (FANCD2) for the initiation of the nucleolytic processing of the DNA cross-links and stabilization of stalled replication forks. Given that all the classic FA proteins coordinately monoubiquitinate FANCD2, it is unclear why losses of individual classic FA genes yield varying cellular sensitivities to cross-linking damage. To address this question, we generated cellular knock-out models of FA core complex components and FANCD2 and found that FANCD2-null mutants display higher levels of spontaneous chromosomal damage and hypersensitivity to replication-blocking lesions than Fanconi anemia complementation group L (FANCL)-null mutants, suggesting that FANCD2 provides a basal level of DNA protection countering endogenous lesions in the absence of monoubiquitination. FANCD2's ubiquitination-independent function is likely involved in optimized recruitment of nucleolytic activities for the processing and protection of stressed replication forks. Our results reveal that FANCD2 has a ubiquitination-independent role in countering endogenous levels of replication stress, a function that is critical for the maintenance of genomic stability. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  8. Adaptation to High Ethanol Reveals Complex Evolutionary Pathways.

    Directory of Open Access Journals (Sweden)

    Karin Voordeckers

    2015-11-01

    Full Text Available Tolerance to high levels of ethanol is an ecologically and industrially relevant phenotype of microbes, but the molecular mechanisms underlying this complex trait remain largely unknown. Here, we use long-term experimental evolution of isogenic yeast populations of different initial ploidy to study adaptation to increasing levels of ethanol. Whole-genome sequencing of more than 30 evolved populations and over 100 adapted clones isolated throughout this two-year evolution experiment revealed how a complex interplay of de novo single nucleotide mutations, copy number variation, ploidy changes, mutator phenotypes, and clonal interference led to a significant increase in ethanol tolerance. Although the specific mutations differ between different evolved lineages, application of a novel computational pipeline, PheNetic, revealed that many mutations target functional modules involved in stress response, cell cycle regulation, DNA repair and respiration. Measuring the fitness effects of selected mutations introduced in non-evolved ethanol-sensitive cells revealed several adaptive mutations that had previously not been implicated in ethanol tolerance, including mutations in PRT1, VPS70 and MEX67. Interestingly, variation in VPS70 was recently identified as a QTL for ethanol tolerance in an industrial bio-ethanol strain. Taken together, our results show how, in contrast to adaptation to some other stresses, adaptation to a continuous complex and severe stress involves interplay of different evolutionary mechanisms. In addition, our study reveals functional modules involved in ethanol resistance and identifies several mutations that could help to improve the ethanol tolerance of industrial yeasts.

  9. The Cdc45/RecJ-like protein forms a complex with GINS and MCM, and is important for DNA replication in Thermococcus kodakarensis.

    Science.gov (United States)

    Nagata, Mariko; Ishino, Sonoko; Yamagami, Takeshi; Ogino, Hiromi; Simons, Jan-Robert; Kanai, Tamotsu; Atomi, Haruyuki; Ishino, Yoshizumi

    2017-10-13

    The archaeal minichromosome maintenance (MCM) has DNA helicase activity, which is stimulated by GINS in several archaea. In the eukaryotic replicative helicase complex, Cdc45 forms a complex with MCM and GINS, named as CMG (Cdc45-MCM-GINS). Cdc45 shares sequence similarity with bacterial RecJ. A Cdc45/RecJ-like protein from Thermococcus kodakarensis shows a bacterial RecJ-like exonuclease activity, which is stimulated by GINS in vitro. Therefore, this archaeal Cdc45/RecJ is designated as GAN, from GINS-associated nuclease. In this study, we identified the CMG-like complex in T. kodakarensis cells. The GAN·GINS complex stimulated the MCM helicase, but MCM did not affect the nuclease activity of GAN in vitro. The gene disruption analysis showed that GAN was non-essential for its viability but the Δgan mutant did not grow at 93°C. Furthermore, the Δgan mutant showed a clear retardation in growth as compared with the parent cells under optimal conditions at 85°C. These deficiencies were recovered by introducing the gan gene encoding the nuclease deficient GAN protein back to the genome. These results suggest that the replicative helicase complex without GAN may become unstable and ineffective in replication fork progression. The nuclease activity of GAN is not related to the growth defects of the Δgan mutant cells. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  10. Architecture and ssDNA interaction of the Timeless-Tipin-RPA complex.

    Science.gov (United States)

    Witosch, Justine; Wolf, Eva; Mizuno, Naoko

    2014-11-10

    The Timeless-Tipin (Tim-Tipin) complex, also referred to as the fork protection complex, is involved in coordination of DNA replication. Tim-Tipin is suggested to be recruited to replication forks via Replication Protein A (RPA) but details of the interaction are unknown. Here, using cryo-EM and biochemical methods, we characterized complex formation of Tim-Tipin, RPA and single-stranded DNA (ssDNA). Tim-Tipin and RPA form a 258 kDa complex with a 1:1:1 stoichiometry. The cryo-EM 3D reconstruction revealed a globular architecture of the Tim-Tipin-RPA complex with a ring-like and a U-shaped domain covered by a RPA lid. Interestingly, RPA in the complex adopts a horse shoe-like shape resembling its conformation in the presence of long ssDNA (>30 nucleotides). Furthermore, the recruitment of the Tim-Tipin-RPA complex to ssDNA is modulated by the RPA conformation and requires RPA to be in the more compact 30 nt ssDNA binding mode. The dynamic formation and disruption of the Tim-Tipin-RPA-ssDNA complex implicates the RPA-based recruitment of Tim-Tipin to the replication fork. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

  11. Surface Microstructure Replication in Injection Moulding

    DEFF Research Database (Denmark)

    Hansen, Hans Nørgaard; Arlø, Uffe Rolf

    2005-01-01

    topography is transcribed onto the plastic part through complex mechanisms. This replication however, is not perfect, and the replication quality depends on the plastic material properties, the topography itself, and the process conditions. This paper describes and discusses an investigation of injection...... moulding of surface microstructures. Emphasis is put on the ability to replicate surface microstructures under normal injection moulding conditions, notably with low cost materials at low mould temperatures. The replication of surface microstructures in injection moulding has been explored...... for Polypropylene at low mould temperatures. The process conditions were varied over the recommended process window for the material. The geometry of the obtained structures was analyzed. Evidence suggests that step height replication quality depends linearly on structure width in a certain range. Further...

  12. Epigenetically-inherited centromere and neocentromere DNA replicates earliest in S-phase.

    Directory of Open Access Journals (Sweden)

    Amnon Koren

    2010-08-01

    Full Text Available Eukaryotic centromeres are maintained at specific chromosomal sites over many generations. In the budding yeast Saccharomyces cerevisiae, centromeres are genetic elements defined by a DNA sequence that is both necessary and sufficient for function; whereas, in most other eukaryotes, centromeres are maintained by poorly characterized epigenetic mechanisms in which DNA has a less definitive role. Here we use the pathogenic yeast Candida albicans as a model organism to study the DNA replication properties of centromeric DNA. By determining the genome-wide replication timing program of the C. albicans genome, we discovered that each centromere is associated with a replication origin that is the first to fire on its respective chromosome. Importantly, epigenetic formation of new ectopic centromeres (neocentromeres was accompanied by shifts in replication timing, such that a neocentromere became the first to replicate and became associated with origin recognition complex (ORC components. Furthermore, changing the level of the centromere-specific histone H3 isoform led to a concomitant change in levels of ORC association with centromere regions, further supporting the idea that centromere proteins determine origin activity. Finally, analysis of centromere-associated DNA revealed a replication-dependent sequence pattern characteristic of constitutively active replication origins. This strand-biased pattern is conserved, together with centromere position, among related strains and species, in a manner independent of primary DNA sequence. Thus, inheritance of centromere position is correlated with a constitutively active origin of replication that fires at a distinct early time. We suggest a model in which the distinct timing of DNA replication serves as an epigenetic mechanism for the inheritance of centromere position.

  13. An Alternative Form of Replication Protein A Prevents Viral Replication in Vitro*

    OpenAIRE

    Mason, Aaron C.; Haring, Stuart J.; Pryor, John M.; Staloch, Cathy A.; Gan, Tze Fei; Wold, Marc S.

    2009-01-01

    Replication protein A (RPA), the eukaryotic single-stranded DNA-binding complex, is essential for multiple processes in cellular DNA metabolism. The “canonical” RPA is composed of three subunits (RPA1, RPA2, and RPA3); however, there is a human homolog to the RPA2 subunit, called RPA4, that can substitute for RPA2 in complex formation. We demonstrate that the resulting “alternative” RPA (aRPA) complex has solution and DNA binding properties indistinguishable from the c...

  14. SARS-coronavirus replication is supported by a reticulovesicular network of modified endoplasmic reticulum.

    Directory of Open Access Journals (Sweden)

    Kèvin Knoops

    2008-09-01

    Full Text Available Positive-strand RNA viruses, a large group including human pathogens such as SARS-coronavirus (SARS-CoV, replicate in the cytoplasm of infected host cells. Their replication complexes are commonly associated with modified host cell membranes. Membrane structures supporting viral RNA synthesis range from distinct spherular membrane invaginations to more elaborate webs of packed membranes and vesicles. Generally, their ultrastructure, morphogenesis, and exact role in viral replication remain to be defined. Poorly characterized double-membrane vesicles (DMVs were previously implicated in SARS-CoV RNA synthesis. We have now applied electron tomography of cryofixed infected cells for the three-dimensional imaging of coronavirus-induced membrane alterations at high resolution. Our analysis defines a unique reticulovesicular network of modified endoplasmic reticulum that integrates convoluted membranes, numerous interconnected DMVs (diameter 200-300 nm, and "vesicle packets" apparently arising from DMV merger. The convoluted membranes were most abundantly immunolabeled for viral replicase subunits. However, double-stranded RNA, presumably revealing the site of viral RNA synthesis, mainly localized to the DMV interior. Since we could not discern a connection between DMV interior and cytosol, our analysis raises several questions about the mechanism of DMV formation and the actual site of SARS-CoV RNA synthesis. Our data document the extensive virus-induced reorganization of host cell membranes into a network that is used to organize viral replication and possibly hide replicating RNA from antiviral defense mechanisms. Together with biochemical studies of the viral enzyme complex, our ultrastructural description of this "replication network" will aid to further dissect the early stages of the coronavirus life cycle and its virus-host interactions.

  15. Human Parvovirus B19 Utilizes Cellular DNA Replication Machinery for Viral DNA Replication.

    Science.gov (United States)

    Zou, Wei; Wang, Zekun; Xiong, Min; Chen, Aaron Yun; Xu, Peng; Ganaie, Safder S; Badawi, Yomna; Kleiboeker, Steve; Nishimune, Hiroshi; Ye, Shui Qing; Qiu, Jianming

    2018-03-01

    Human parvovirus B19 (B19V) infection of human erythroid progenitor cells (EPCs) induces a DNA damage response and cell cycle arrest at late S phase, which facilitates viral DNA replication. However, it is not clear exactly which cellular factors are employed by this single-stranded DNA virus. Here, we used microarrays to systematically analyze the dynamic transcriptome of EPCs infected with B19V. We found that DNA metabolism, DNA replication, DNA repair, DNA damage response, cell cycle, and cell cycle arrest pathways were significantly regulated after B19V infection. Confocal microscopy analyses revealed that most cellular DNA replication proteins were recruited to the centers of viral DNA replication, but not the DNA repair DNA polymerases. Our results suggest that DNA replication polymerase δ and polymerase α are responsible for B19V DNA replication by knocking down its expression in EPCs. We further showed that although RPA32 is essential for B19V DNA replication and the phosphorylated forms of RPA32 colocalized with the replicating viral genomes, RPA32 phosphorylation was not necessary for B19V DNA replication. Thus, this report provides evidence that B19V uses the cellular DNA replication machinery for viral DNA replication. IMPORTANCE Human parvovirus B19 (B19V) infection can cause transient aplastic crisis, persistent viremia, and pure red cell aplasia. In fetuses, B19V infection can result in nonimmune hydrops fetalis and fetal death. These clinical manifestations of B19V infection are a direct outcome of the death of human erythroid progenitors that host B19V replication. B19V infection induces a DNA damage response that is important for cell cycle arrest at late S phase. Here, we analyzed dynamic changes in cellular gene expression and found that DNA metabolic processes are tightly regulated during B19V infection. Although genes involved in cellular DNA replication were downregulated overall, the cellular DNA replication machinery was tightly

  16. Structural properties of replication origins in yeast DNA sequences

    International Nuclear Information System (INIS)

    Cao Xiaoqin; Zeng Jia; Yan Hong

    2008-01-01

    Sequence-dependent DNA flexibility is an important structural property originating from the DNA 3D structure. In this paper, we investigate the DNA flexibility of the budding yeast (S. Cerevisiae) replication origins on a genome-wide scale using flexibility parameters from two different models, the trinucleotide and the tetranucleotide models. Based on analyzing average flexibility profiles of 270 replication origins, we find that yeast replication origins are significantly rigid compared with their surrounding genomic regions. To further understand the highly distinctive property of replication origins, we compare the flexibility patterns between yeast replication origins and promoters, and find that they both contain significantly rigid DNAs. Our results suggest that DNA flexibility is an important factor that helps proteins recognize and bind the target sites in order to initiate DNA replication. Inspired by the role of the rigid region in promoters, we speculate that the rigid replication origins may facilitate binding of proteins, including the origin recognition complex (ORC), Cdc6, Cdt1 and the MCM2-7 complex

  17. Regulated eukaryotic DNA replication origin firing with purified proteins.

    Science.gov (United States)

    Yeeles, Joseph T P; Deegan, Tom D; Janska, Agnieszka; Early, Anne; Diffley, John F X

    2015-03-26

    Eukaryotic cells initiate DNA replication from multiple origins, which must be tightly regulated to promote precise genome duplication in every cell cycle. To accomplish this, initiation is partitioned into two temporally discrete steps: a double hexameric minichromosome maintenance (MCM) complex is first loaded at replication origins during G1 phase, and then converted to the active CMG (Cdc45-MCM-GINS) helicase during S phase. Here we describe the reconstitution of budding yeast DNA replication initiation with 16 purified replication factors, made from 42 polypeptides. Origin-dependent initiation recapitulates regulation seen in vivo. Cyclin-dependent kinase (CDK) inhibits MCM loading by phosphorylating the origin recognition complex (ORC) and promotes CMG formation by phosphorylating Sld2 and Sld3. Dbf4-dependent kinase (DDK) promotes replication by phosphorylating MCM, and can act either before or after CDK. These experiments define the minimum complement of proteins, protein kinase substrates and co-factors required for regulated eukaryotic DNA replication.

  18. Acute Smc5/6 depletion reveals its primary role in rDNA replication by restraining recombination at fork pausing sites.

    Directory of Open Access Journals (Sweden)

    Xiao P Peng

    2018-01-01

    Full Text Available Smc5/6, a member of the conserved SMC family of complexes, is essential for growth in most organisms. Its exact functions in a mitotic cell cycle are controversial, as chronic Smc5/6 loss-of-function alleles produce varying phenotypes. To circumvent this issue, we acutely depleted Smc5/6 in budding yeast and determined the first cell cycle consequences of Smc5/6 removal. We found a striking primary defect in replication of the ribosomal DNA (rDNA array. Each rDNA repeat contains a programmed replication fork barrier (RFB established by the Fob1 protein. Fob1 removal improves rDNA replication in Smc5/6 depleted cells, implicating Smc5/6 in the management of programmed fork pausing. A similar improvement is achieved by removing the DNA helicase Mph1 whose recombinogenic activity can be inhibited by Smc5/6 under DNA damage conditions. DNA 2D gel analyses further show that Smc5/6 loss increases recombination structures at RFB regions; moreover, mph1∆ and fob1∆ similarly reduce this accumulation. These findings point to an important mitotic role for Smc5/6 in restraining recombination events when protein barriers in rDNA stall replication forks. As rDNA maintenance influences multiple essential cellular processes, Smc5/6 likely links rDNA stability to overall mitotic growth.

  19. Surface microstructure replication in injection molding

    DEFF Research Database (Denmark)

    Theilade, Uffe Arlø; Hansen, Hans Nørgaard

    2006-01-01

    topography is transcribed onto the plastic part through complex mechanisms. This replication, however, is not perfect, and the replication quality depends on the plastic material properties, the topography itself, and the process conditions. This paper describes and discusses an investigation of injection...... molding of surface microstructures. The fundamental problem of surface microstructure replication has been studied. The research is based on specific microstructures as found in lab-on-a-chip products and on rough surfaces generated from EDM (electro discharge machining) mold cavities. Emphasis is put...... on the ability to replicate surface microstructures under normal injection-molding conditions, i.e., with commodity materials within typical process windows. It was found that within typical process windows the replication quality depends significantly on several process parameters, and especially the mold...

  20. Structural disorder in proteins of the rhabdoviridae replication complex.

    Science.gov (United States)

    Leyrat, Cédric; Gérard, Francine C A; de Almeida Ribeiro, Euripedes; Ivanov, Ivan; Ruigrok, Rob W H; Jamin, Marc

    2010-08-01

    Rhabdoviridae are single stranded negative sense RNA viruses. The viral RNA condensed by the nucleoprotein (N), the phosphoprotein (P) and the large subunit (L) of the RNA-dependent RNA polymerase are the viral components of the transcription/replication machineries. Both P and N contain intrinsically disordered regions (IDRs) that play different roles in the virus life cycle. Here, we describe the modular organization of P based on recent structural, biophysical and bioinformatics data. We show how flexible loops in N participate in the attachment of P to the N-RNA template by an induced-fit mechanism. Finally, we discuss the roles of IDRs in the mechanism of replication/transcription, and propose a new model for the interaction of the L subunit with its N-RNA template.

  1. Rolling replication of UV-irradiated duplex DNA in the phi X174 replicative-form----single-strand replication system in vitro

    International Nuclear Information System (INIS)

    Shavitt, O.; Livneh, Z.

    1989-01-01

    Cloning of the phi X174 viral origin of replication into phage M13mp8 produced an M13-phi X174 chimera, the DNA of which directed efficient replicative-form----single-strand rolling replication in vitro. This replication assay was performed with purified phi X174-encoded gene A protein, Escherichia coli rep helicase, single-stranded DNA-binding protein, and DNA polymerase III holoenzyme. The nicking of replicative-form I (RFI) DNA by gene A protein was essentially unaffected by the presence of UV lesions in the DNA. However, unwinding of UV-irradiated DNA by the rep helicase was inhibited twofold as compared with unwinding of the unirradiated substrate. UV irradiation of the substrate DNA caused a strong inhibition in its ability to direct DNA synthesis. However, even DNA preparations that contained as many as 10 photodimers per molecule still supported the synthesis of progeny full-length single-stranded DNA. The appearance of full-length radiolabeled products implied at least two full rounds of replication, since the first round released the unlabeled plus viral strand of the duplex DNA. Pretreatment of the UV-irradiated DNA substrate with purified pyrimidine dimer endonuclease from Micrococcus luteus, which converted photodimer-containing supercoiled RFI DNA into relaxed, nicked RFII DNA and thus prevented its replication, reduced DNA synthesis by 70%. Analysis of radiolabeled replication products by agarose gel electrophoresis followed by autoradiography revealed that this decrease was due to a reduction in the synthesis of progeny full-length single-stranded DNA. This implies that 70 to 80% of the full-length DNA products produced in this system were synthesized on molecules that carried photodimers

  2. Two mechanisms coordinate replication termination by the Escherichia coli Tus–Ter complex

    KAUST Repository

    Pandey, Manjula; Elshenawy, Mohamed; Jergic, Slobodan; Takahashi, Masateru; Dixon, Nicholas E.; Hamdan, Samir; Patel, Smita S.

    2015-01-01

    The Escherichia coli replication terminator protein (Tus) binds to Ter sequences to block replication forks approaching from one direction. Here, we used single molecule and transient state kinetics to study responses of the heterologous phage T7

  3. Recruitment of a SAP18-HDAC1 complex into HIV-1 virions and its requirement for viral replication.

    Directory of Open Access Journals (Sweden)

    Masha Sorin

    2009-06-01

    Full Text Available HIV-1 integrase (IN is a virally encoded protein required for integration of viral cDNA into host chromosomes. INI1/hSNF5 is a component of the SWI/SNF complex that interacts with HIV-1 IN, is selectively incorporated into HIV-1 (but not other retroviral virions, and modulates multiple steps, including particle production and infectivity. To gain further insight into the role of INI1 in HIV-1 replication, we screened for INI1-interacting proteins using the yeast two-hybrid system. We found that SAP18 (Sin3a associated protein 18 kD, a component of the Sin3a-HDAC1 complex, directly binds to INI1 in yeast, in vitro and in vivo. Interestingly, we found that IN also binds to SAP18 in vitro and in vivo. SAP18 and components of a Sin3A-HDAC1 complex were specifically incorporated into HIV-1 (but not SIV and HTLV-1 virions in an HIV-1 IN-dependent manner. Using a fluorescence-based assay, we found that HIV-1 (but not SIV virion preparations harbour significant deacetylase activity, indicating the specific recruitment of catalytically active HDAC into the virions. To determine the requirement of virion-associated HDAC1 to HIV-1 replication, an inactive, transdominant negative mutant of HDAC1 (HDAC1(H141A was utilized. Incorporation of HDAC1(H141A decreased the virion-associated histone deacetylase activity. Furthermore, incorporation of HDAC1(H141A decreased the infectivity of HIV-1 (but not SIV virions. The block in infectivity due to virion-associated HDAC1(H141A occurred specifically at the early reverse transcription stage, while entry of the virions was unaffected. RNA-interference mediated knock-down of HDAC1 in producer cells resulted in decreased virion-associated HDAC1 activity and a reduction in infectivity of these virions. These studies indicate that HIV-1 IN and INI1/hSNF5 bind SAP18 and selectively recruit components of Sin3a-HDAC1 complex into HIV-1 virions. Furthermore, HIV-1 virion-associated HDAC1 is required for efficient early post

  4. Initiation of DNA replication: functional and evolutionary aspects

    Science.gov (United States)

    Bryant, John A.; Aves, Stephen J.

    2011-01-01

    Background The initiation of DNA replication is a very important and highly regulated step in the cell division cycle. It is of interest to compare different groups of eukaryotic organisms (a) to identify the essential molecular events that occur in all eukaryotes, (b) to start to identify higher-level regulatory mechanisms that are specific to particular groups and (c) to gain insights into the evolution of initiation mechanisms. Scope This review features a wide-ranging literature survey covering replication origins, origin recognition and usage, modification of origin usage (especially in response to plant hormones), assembly of the pre-replication complex, loading of the replisome, genomics, and the likely origin of these mechanisms and proteins in Archaea. Conclusions In all eukaryotes, chromatin is organized for DNA replication as multiple replicons. In each replicon, replication is initiated at an origin. With the exception of those in budding yeast, replication origins, including the only one to be isolated so far from a plant, do not appear to embody a specific sequence; rather, they are AT-rich, with short tracts of locally bent DNA. The proteins involved in initiation are remarkably similar across the range of eukaryotes. Nevertheless, their activity may be modified by plant-specific mechanisms, including regulation by plant hormones. The molecular features of initiation are seen in a much simpler form in the Archaea. In particular, where eukaryotes possess a number of closely related proteins that form ‘hetero-complexes’ (such as the origin recognition complex and the MCM complex), archaeans typically possess one type of protein (e.g. one MCM) that forms a homo-complex. This suggests that several eukaryotic initiation proteins have evolved from archaeal ancestors by gene duplication and divergence. PMID:21508040

  5. Functions of alternative Replication Protein A (aRPA) in initiation and elongation

    OpenAIRE

    Mason, Aaron C.; Roy, Rupa; Simmons, Daniel T.; Wold, Marc S.

    2010-01-01

    Replication protein A (RPA) is a single-stranded DNA-binding complex that is essential for DNA replication, repair and recombination in eukaryotic cells. In addition to this canonical complex, we have recently characterized an alternative Replication Protein A complex (aRPA) that is unique to primates. aRPA is composed of three subunits: RPA1 and RPA3, also present in canonical RPA, and a primate-specific subunit RPA4, homologous to canonical RPA2. aRPA has biochemical properties similar to t...

  6. A Temporal Proteomic Map of Epstein-Barr Virus Lytic Replication in B Cells

    Directory of Open Access Journals (Sweden)

    Ina Ersing

    2017-05-01

    Full Text Available Epstein-Barr virus (EBV replication contributes to multiple human diseases, including infectious mononucleosis, nasopharyngeal carcinoma, B cell lymphomas, and oral hairy leukoplakia. We performed systematic quantitative analyses of temporal changes in host and EBV proteins during lytic replication to gain insights into virus-host interactions, using conditional Burkitt lymphoma models of type I and II EBV infection. We quantified profiles of >8,000 cellular and 69 EBV proteins, including >500 plasma membrane proteins, providing temporal views of the lytic B cell proteome and EBV virome. Our approach revealed EBV-induced remodeling of cell cycle, innate and adaptive immune pathways, including upregulation of the complement cascade and proteasomal degradation of the B cell receptor complex, conserved between EBV types I and II. Cross-comparison with proteomic analyses of human cytomegalovirus infection and of a Kaposi-sarcoma-associated herpesvirus immunoevasin identified host factors targeted by multiple herpesviruses. Our results provide an important resource for studies of EBV replication.

  7. Reduced evolutionary rates in HIV-1 reveal extensive latency periods among replicating lineages.

    Science.gov (United States)

    Immonen, Taina T; Leitner, Thomas

    2014-10-16

    HIV-1 can persist for the duration of a patient's life due in part to its ability to hide from the immune system, and from antiretroviral drugs, in long-lived latent reservoirs. Latent forms of HIV-1 may also be disproportionally involved in transmission. Thus, it is important to detect and quantify latency in the HIV-1 life cycle. We developed a novel molecular clock-based phylogenetic tool to investigate the prevalence of HIV-1 lineages that have experienced latency. The method removes alternative sources that may affect evolutionary rates, such as hypermutation, recombination, and selection, to reveal the contribution of generation-time effects caused by latency. Our method was able to recover latent lineages with high specificity and sensitivity, and low false discovery rates, even on relatively short branches on simulated phylogenies. Applying the tool to HIV-1 sequences from 26 patients, we show that the majority of phylogenetic lineages have been affected by generation-time effects in every patient type, whether untreated, elite controller, or under effective or failing treatment. Furthermore, we discovered extensive effects of latency in sequence data (gag, pol, and env) from reservoirs as well as in the replicating plasma population. To better understand our phylogenetic findings, we developed a dynamic model of virus-host interactions to investigate the proportion of lineages in the actively replicating population that have ever been latent. Assuming neutral evolution, our dynamic modeling showed that under most parameter conditions, it is possible for a few activated latent viruses to propagate so that in time, most HIV-1 lineages will have been latent at some time in their past. These results suggest that cycling in and out of latency plays a major role in the evolution of HIV-1. Thus, no aspect of HIV-1 evolution can be fully understood without considering latency - including treatment, drug resistance, immune evasion, transmission, and pathogenesis.

  8. Proteomics Reveals Global Regulation of Protein SUMOylation by ATM and ATR Kinases during Replication Stress

    Directory of Open Access Journals (Sweden)

    Stephanie Munk

    2017-10-01

    Full Text Available The mechanisms that protect eukaryotic DNA during the cumbersome task of replication depend on the precise coordination of several post-translational modification (PTM-based signaling networks. Phosphorylation is a well-known regulator of the replication stress response, and recently an essential role for SUMOs (small ubiquitin-like modifiers has also been established. Here, we investigate the global interplay between phosphorylation and SUMOylation in response to replication stress. Using SUMO and phosphoproteomic technologies, we identify thousands of regulated modification sites. We find co-regulation of central DNA damage and replication stress responders, of which the ATR-activating factor TOPBP1 is the most highly regulated. Using pharmacological inhibition of the DNA damage response kinases ATR and ATM, we find that these factors regulate global protein SUMOylation in the protein networks that protect DNA upon replication stress and fork breakage, pointing to integration between phosphorylation and SUMOylation in the cellular systems that protect DNA integrity.

  9. Initiation of chromosomal replication in predatory bacterium Bdellovibrio bacteriovorus

    Directory of Open Access Journals (Sweden)

    Lukasz Makowski

    2016-11-01

    Full Text Available Bdellovibrio bacteriovorus is a small Gram-negative predatory bacterium that attacks other Gram-negative bacteria, including many animal, human, and plant pathogens. This bacterium exhibits a peculiar biphasic life cycle during which two different types of cells are produced: non-replicating highly motile cells (the free-living phase and replicating cells (the intracellular-growth phase. The process of chromosomal replication in B. bacteriovorus must therefore be temporally and spatially regulated to ensure that it is coordinated with cell differentiation and cell cycle progression. Recently, B. bacteriovorus has received considerable research interest due to its intriguing life cycle and great potential as a prospective antimicrobial agent. Although we know that chromosomal replication in bacteria is mainly regulated at the initiation step, no data exists about this process in B. bacteriovorus. We report the first characterization of key elements of initiation of chromosomal replication – DnaA protein and oriC region from the predatory bacterium, B. bacteriovorus. In vitro studies using different approaches demonstrate that the B. bacteriovorus oriC (BdoriC is specifically bound and unwound by the DnaA protein. Sequence comparison of the DnaA-binding sites enabled us to propose a consensus sequence for the B. bacteriovorus DnaA box (5’-NN(A/TTCCACA-3’. Surprisingly, in vitro analysis revealed that BdoriC is also bound and unwound by the host DnaA proteins (relatively distantly related from B. bacteriovorus. We compared the architecture of the DnaA–oriC complexes (orisomes in homologous (oriC and DnaA from B. bacteriovorus and heterologous (BdoriC and DnaA from prey, E. coli or P. aeruginosa systems. This work provides important new entry points toward improving our understanding of the initiation of chromosomal replication in this predatory bacterium.

  10. Replication and Inhibitors of Enteroviruses and Parechoviruses

    Directory of Open Access Journals (Sweden)

    Lonneke van der Linden

    2015-08-01

    Full Text Available The Enterovirus (EV and Parechovirus genera of the picornavirus family include many important human pathogens, including poliovirus, rhinovirus, EV-A71, EV-D68, and human parechoviruses (HPeV. They cause a wide variety of diseases, ranging from a simple common cold to life-threatening diseases such as encephalitis and myocarditis. At the moment, no antiviral therapy is available against these viruses and it is not feasible to develop vaccines against all EVs and HPeVs due to the great number of serotypes. Therefore, a lot of effort is being invested in the development of antiviral drugs. Both viral proteins and host proteins essential for virus replication can be used as targets for virus inhibitors. As such, a good understanding of the complex process of virus replication is pivotal in the design of antiviral strategies goes hand in hand with a good understanding of the complex process of virus replication. In this review, we will give an overview of the current state of knowledge of EV and HPeV replication and how this can be inhibited by small-molecule inhibitors.

  11. Identification of Proteins Bound to Dengue Viral RNA In Vivo Reveals New Host Proteins Important for Virus Replication

    Directory of Open Access Journals (Sweden)

    Stacia L. Phillips

    2016-01-01

    Full Text Available Dengue virus is the most prevalent cause of arthropod-borne infection worldwide. Due to the limited coding capacity of the viral genome and the complexity of the viral life cycle, host cell proteins play essential roles throughout the course of viral infection. Host RNA-binding proteins mediate various aspects of virus replication through their physical interactions with viral RNA. Here we describe a technique designed to identify such interactions in the context of infected cells using UV cross-linking followed by antisense-mediated affinity purification and mass spectrometry. Using this approach, we identified interactions, several of them novel, between host proteins and dengue viral RNA in infected Huh7 cells. Most of these interactions were subsequently validated using RNA immunoprecipitation. Using small interfering RNA (siRNA-mediated gene silencing, we showed that more than half of these host proteins are likely involved in regulating virus replication, demonstrating the utility of this method in identifying biologically relevant interactions that may not be identified using traditional in vitro approaches.

  12. Replicative manufacturing of complex lighting optics by non-isothermal glass molding

    Science.gov (United States)

    Kreilkamp, Holger; Vu, Anh Tuan; Dambon, Olaf; Klocke, Fritz

    2016-09-01

    The advantages of LED lighting, especially its energy efficiency and the long service life have led to a wide distribution of LED technology in the world. However, in order to make fully use of the great potential that LED lighting offers, complex optics are required to distribute the emitted light from the LED efficiently. Nowadays, many applications use polymer optics which can be manufactured at low costs. However, due to ever increasing luminous power, polymer optics reach their technological limits. Due to its outstanding properties, especially its temperature resistance, resistance against UV radiation and its long term stability, glass is the alternative material of choice for the use in LED optics. This research is introducing a new replicative glass manufacturing approach, namely non-isothermal glass molding (NGM) which is able to manufacture complex lighting optics in high volumes at competitive prices. The integration of FEM simulation at the early stage of the process development is presented and helps to guarantee a fast development cycle. A coupled thermo-mechanical model is used to define the geometry of the glass preform as well as to define the mold surface geometry. Furthermore, simulation is used to predict main process outcomes, especially in terms of resulting form accuracy of the molded optics. Experiments conducted on a commercially available molding machine are presented to validate the developed simulation model. Finally, the influence of distinct parameters on important process outcomes like form accuracy, surface roughness, birefringence, etc. is discussed.

  13. PRC1 Prevents Replication Stress during Chondrogenic Transit Amplification

    Directory of Open Access Journals (Sweden)

    Frank Spaapen

    2017-12-01

    Full Text Available Transit amplification (TA, a state of combined, rapid proliferative expansion and differentiation of stem cell-descendants, remains poorly defined at the molecular level. The Polycomb Repressive Complex 1 (PRC1 protein BMI1 has been localized to TA compartments, yet its exact role in TA is unclear. PRC1 proteins control gene expression, cell proliferation and DNA-damage repair. Coordination of such DNA-templated activities during TA is predicted to be crucial to support DNA replication and differentiation-associated transcriptional programming. We here examined whether chondrogenesis provides a relevant biological context for synchronized coordination of these chromatin-based tasks by BMI1. Taking advantage of a prominently featuring TA-phase during chondrogenesis in vitro and in vivo, we here report that TA is completely dependent on intact PRC1 function. BMI1-depleted chondrogenic progenitors rapidly accumulate double strand DNA breaks during DNA replication, present massive non-H3K27me3-directed transcriptional deregulation and fail to undergo chondrogenic TA. Genome-wide accumulation of Topoisomerase 2α and Geminin suggests a model in which PRC1 synchronizes replication and transcription during rapid chondrogenic progenitor expansion. Our combined data reveals for the first time a vital cell-autonomous role for PRC1 during chondrogenesis. We provide evidence that chondrocyte hyper-replication and hypertrophy represent a unique example of programmed senescence in vivo. These findings provide new perspectives on PRC1 function in development and disease.

  14. Proteomics Reveals Global Regulation of Protein SUMOylation by ATM and ATR Kinases during Replication Stress

    DEFF Research Database (Denmark)

    Munk, Stephanie; Sigurðsson, Jón Otti; Xiao, Zhenyu

    2017-01-01

    The mechanisms that protect eukaryotic DNA during the cumbersome task of replication depend on the precise coordination of several post-translational modification (PTM)-based signaling networks. Phosphorylation is a well-known regulator of the replication stress response, and recently an essentia....... They analyze changes in the SUMO and phosphoproteome after MMC and hydroxyurea treatments and find that the DNA damage response kinases ATR and ATM globally regulate SUMOylation upon replication stress and fork breakage....

  15. Effector-Triggered Self-Replication in Coupled Subsystems.

    Science.gov (United States)

    Komáromy, Dávid; Tezcan, Meniz; Schaeffer, Gaël; Marić, Ivana; Otto, Sijbren

    2017-11-13

    In living systems processes like genome duplication and cell division are carefully synchronized through subsystem coupling. If we are to create life de novo, similar control over essential processes such as self-replication need to be developed. Here we report that coupling two dynamic combinatorial subsystems, featuring two separate building blocks, enables effector-mediated control over self-replication. The subsystem based on the first building block shows only self-replication, whereas that based on the second one is solely responsive toward a specific external effector molecule. Mixing the subsystems arrests replication until the effector molecule is added, resulting in the formation of a host-effector complex and the liberation of the building block that subsequently engages in self-replication. The onset, rate and extent of self-replication is controlled by the amount of effector present. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  16. The Role of the Transcriptional Response to DNA Replication Stress.

    Science.gov (United States)

    Herlihy, Anna E; de Bruin, Robertus A M

    2017-03-02

    During DNA replication many factors can result in DNA replication stress. The DNA replication stress checkpoint prevents the accumulation of replication stress-induced DNA damage and the potential ensuing genome instability. A critical role for post-translational modifications, such as phosphorylation, in the replication stress checkpoint response has been well established. However, recent work has revealed an important role for transcription in the cellular response to DNA replication stress. In this review, we will provide an overview of current knowledge of the cellular response to DNA replication stress with a specific focus on the DNA replication stress checkpoint transcriptional response and its role in the prevention of replication stress-induced DNA damage.

  17. The Role of the Transcriptional Response to DNA Replication Stress

    Science.gov (United States)

    Herlihy, Anna E.; de Bruin, Robertus A.M.

    2017-01-01

    During DNA replication many factors can result in DNA replication stress. The DNA replication stress checkpoint prevents the accumulation of replication stress-induced DNA damage and the potential ensuing genome instability. A critical role for post-translational modifications, such as phosphorylation, in the replication stress checkpoint response has been well established. However, recent work has revealed an important role for transcription in the cellular response to DNA replication stress. In this review, we will provide an overview of current knowledge of the cellular response to DNA replication stress with a specific focus on the DNA replication stress checkpoint transcriptional response and its role in the prevention of replication stress-induced DNA damage. PMID:28257104

  18. [Single-molecule detection and characterization of DNA replication based on DNA origami].

    Science.gov (United States)

    Wang, Qi; Fan, Youjie; Li, Bin

    2014-08-01

    To investigate single-molecule detection and characterization of DNA replication. Single-stranded DNA (ssDNA) as the template of DNA replication was attached to DNA origami by a hybridization reaction based on the complementary base-pairing principle. DNA replication catalyzed by E.coli DNA polymerase I Klenow Fragment (KF) was detected using atomic force microscopy (AFM). The height variations between the ssDNA and the double-stranded DNA (dsDNA), the distribution of KF during DNA replication and biotin-streptavidin (BA) complexes on the DNA strand after replication were detected. Agarose gel electrophoresis was employed to analyze the changes in the DNA after replication. The designed ssDNA could be anchored on the target positions of over 50% of the DNA origami. The KF was capable of binding to the ssDNA fixed on DNA origami and performing its catalytic activities, and was finally dissociated from the DNA after replication. The height of DNA strand increased by about 0.7 nm after replication. The addition of streptavidin also resulted in an DNA height increase to about 4.9 nm due to the formation of BA complexes on the biotinylated dsDNA. The resulting dsDNA and BA complex were subsequently confirmed by agarose gel electrophoresis. The combination of AFM and DNA origami allows detection and characterization of DNA replication at the single molecule level, and this approach provides better insights into the mechanism of DNA polymerase and the factors affecting DNA replication.

  19. Reactivation and Lytic Replication of Kaposi’s Sarcoma-Associated Herpesvirus: An Update

    Science.gov (United States)

    Aneja, Kawalpreet K.; Yuan, Yan

    2017-01-01

    The life cycle of Kaposi’s sarcoma-associated herpesvirus (KSHV) consists of two phases, latent and lytic. The virus establishes latency as a strategy for avoiding host immune surveillance and fusing symbiotically with the host for lifetime persistent infection. However, latency can be disrupted and KSHV is reactivated for entry into the lytic replication. Viral lytic replication is crucial for efficient dissemination from its long-term reservoir to the sites of disease and for the spread of the virus to new hosts. The balance of these two phases in the KSHV life cycle is important for both the virus and the host and control of the switch between these two phases is extremely complex. Various environmental factors such as oxidative stress, hypoxia, and certain chemicals have been shown to switch KSHV from latency to lytic reactivation. Immunosuppression, unbalanced inflammatory cytokines, and other viral co-infections also lead to the reactivation of KSHV. This review article summarizes the current understanding of the initiation and regulation of KSHV reactivation and the mechanisms underlying the process of viral lytic replication. In particular, the central role of an immediate-early gene product RTA in KSHV reactivation has been extensively investigated. These studies revealed multiple layers of regulation in activation of RTA as well as the multifunctional roles of RTA in the lytic replication cascade. Epigenetic regulation is known as a critical layer of control for the switch of KSHV between latency and lytic replication. The viral non-coding RNA, PAN, was demonstrated to play a central role in the epigenetic regulation by serving as a guide RNA that brought chromatin remodeling enzymes to the promoters of RTA and other lytic genes. In addition, a novel dimension of regulation by microPeptides emerged and has been shown to regulate RTA expression at the protein level. Overall, extensive investigation of KSHV reactivation and lytic replication has revealed

  20. Intramolecular telomeric G-quadruplexes dramatically inhibit DNA synthesis by replicative and translesion polymerases, revealing their potential to lead to genetic change.

    Directory of Open Access Journals (Sweden)

    Deanna N Edwards

    Full Text Available Recent research indicates that hundreds of thousands of G-rich sequences within the human genome have the potential to form secondary structures known as G-quadruplexes. Telomeric regions, consisting of long arrays of TTAGGG/AATCCC repeats, are among the most likely areas in which these structures might form. Since G-quadruplexes assemble from certain G-rich single-stranded sequences, they might arise when duplex DNA is unwound such as during replication. Coincidentally, these bulky structures when present in the DNA template might also hinder the action of DNA polymerases. In this study, single-stranded telomeric templates with the potential to form G-quadruplexes were examined for their effects on a variety of replicative and translesion DNA polymerases from humans and lower organisms. Our results demonstrate that single-stranded templates containing four telomeric GGG runs fold into intramolecular G-quadruplex structures. These intramolecular G quadruplexes are somewhat dynamic in nature and stabilized by increasing KCl concentrations and decreasing temperatures. Furthermore, the presence of these intramolecular G-quadruplexes in the template dramatically inhibits DNA synthesis by various DNA polymerases, including the human polymerase δ employed during lagging strand replication of G-rich telomeric strands and several human translesion DNA polymerases potentially recruited to sites of replication blockage. Notably, misincorporation of nucleotides is observed when certain translesion polymerases are employed on substrates containing intramolecular G-quadruplexes, as is extension of the resulting mismatched base pairs upon dynamic unfolding of this secondary structure. These findings reveal the potential for blockage of DNA replication and genetic changes related to sequences capable of forming intramolecular G-quadruplexes.

  1. Identification of the determinants of efficient Pestivirus replication

    OpenAIRE

    Risager, Peter Christian; Belsham, Graham; Rasmussen, Thomas Bruun

    2013-01-01

    The key for the survival of a virus is to copy its own genome into progeny genomes that allows continued reproduction. The mechanism behind this "copy function" or "replication" is a wellorganized process that involves the formation of a replication complex in the cell and interactions between the viral proteins. The replication process in single-stranded RNA viruses of positive polarity requires a particular enzyme, an RNA dependent RNA polymerase, that has no direct counterpart elsewhere in...

  2. Dynamics of Escherichia coli Chromosome Segregation during Multifork Replication

    DEFF Research Database (Denmark)

    Nielsen, Henrik Jørck; Youngren, Brenda; Hansen, Flemming G.

    2007-01-01

    Slowly growing Escherichia coli cells have a simple cell cycle, with replication and progressive segregation of the chromosome completed before cell division. In rapidly growing cells, initiation of replication occurs before the previous replication rounds are complete. At cell division, the chro......Slowly growing Escherichia coli cells have a simple cell cycle, with replication and progressive segregation of the chromosome completed before cell division. In rapidly growing cells, initiation of replication occurs before the previous replication rounds are complete. At cell division......, the chromosomes contain multiple replication forks and must be segregated while this complex pattern of replication is still ongoing. Here, we show that replication and segregation continue in step, starting at the origin and progressing to the replication terminus. Thus, early-replicated markers on the multiple......-branched chromosomes continue to separate soon after replication to form separate protonucleoids, even though they are not segregated into different daughter cells until later generations. The segregation pattern follows the pattern of chromosome replication and does not follow the cell division cycle. No extensive...

  3. Centromere replication timing determines different forms of genomic instability in Saccharomyces cerevisiae checkpoint mutants during replication stress.

    Science.gov (United States)

    Feng, Wenyi; Bachant, Jeff; Collingwood, David; Raghuraman, M K; Brewer, Bonita J

    2009-12-01

    Yeast replication checkpoint mutants lose viability following transient exposure to hydroxyurea, a replication-impeding drug. In an effort to understand the basis for this lethality, we discovered that different events are responsible for inviability in checkpoint-deficient cells harboring mutations in the mec1 and rad53 genes. By monitoring genomewide replication dynamics of cells exposed to hydroxyurea, we show that cells with a checkpoint deficient allele of RAD53, rad53K227A, fail to duplicate centromeres. Following removal of the drug, however, rad53K227A cells recover substantial DNA replication, including replication through centromeres. Despite this recovery, the rad53K227A mutant fails to achieve biorientation of sister centromeres during recovery from hydroxyurea, leading to secondary activation of the spindle assembly checkpoint (SAC), aneuploidy, and lethal chromosome segregation errors. We demonstrate that cell lethality from this segregation defect could be partially remedied by reinforcing bipolar attachment. In contrast, cells with the mec1-1 sml1-1 mutations suffer from severely impaired replication resumption upon removal of hydroxyurea. mec1-1 sml1-1 cells can, however, duplicate at least some of their centromeres and achieve bipolar attachment, leading to abortive segregation and fragmentation of incompletely replicated chromosomes. Our results highlight the importance of replicating yeast centromeres early and reveal different mechanisms of cell death due to differences in replication fork progression.

  4. BPIFB6 Regulates Secretory Pathway Trafficking and Enterovirus Replication.

    Science.gov (United States)

    Morosky, Stefanie; Lennemann, Nicholas J; Coyne, Carolyn B

    2016-05-15

    Bactericidal/permeability-increasing protein (BPI) fold-containing family B, member 3 (BPIFB3) is an endoplasmic reticulum (ER)-localized host factor that negatively regulates coxsackievirus B (CVB) replication through its control of the autophagic pathway. Here, we show that another member of the BPIFB family, BPIFB6, functions as a positive regulator of CVB, and other enterovirus, replication by controlling secretory pathway trafficking and Golgi complex morphology. We show that similar to BPIFB3, BPIFB6 localizes exclusively to the ER, where it associates with other members of the BPIFB family. However, in contrast to our findings that RNA interference (RNAi)-mediated silencing of BPIFB3 greatly enhances CVB replication, we show that silencing of BPIFB6 expression dramatically suppresses enterovirus replication in a pan-viral manner. Mechanistically, we show that loss of BPIFB6 expression induces pronounced alterations in retrograde and anterograde trafficking, which correlate with dramatic fragmentation of the Golgi complex. Taken together, these data implicate BPIFB6 as a key regulator of secretory pathway trafficking and viral replication and suggest that members of the BPIFB family participate in diverse host cell functions to regulate virus infections. Enterovirus infections are associated with a number of severe pathologies, such as aseptic meningitis, dilated cardiomyopathy, type I diabetes, paralysis, and even death. These viruses, which include coxsackievirus B (CVB), poliovirus (PV), and enterovirus 71 (EV71), co-opt the host cell secretory pathway, which controls the transport of proteins from the endoplasmic reticulum to the Golgi complex, to facilitate their replication. Here we report on the identification of a novel regulator of the secretory pathway, bactericidal/permeability-increasing protein (BPI) fold-containing family B, member 6 (BPIFB6), whose expression is required for enterovirus replication. We show that loss of BPIFB6 expression

  5. BPIFB6 Regulates Secretory Pathway Trafficking and Enterovirus Replication

    Science.gov (United States)

    Morosky, Stefanie; Lennemann, Nicholas J.

    2016-01-01

    ABSTRACT Bactericidal/permeability-increasing protein (BPI) fold-containing family B, member 3 (BPIFB3) is an endoplasmic reticulum (ER)-localized host factor that negatively regulates coxsackievirus B (CVB) replication through its control of the autophagic pathway. Here, we show that another member of the BPIFB family, BPIFB6, functions as a positive regulator of CVB, and other enterovirus, replication by controlling secretory pathway trafficking and Golgi complex morphology. We show that similar to BPIFB3, BPIFB6 localizes exclusively to the ER, where it associates with other members of the BPIFB family. However, in contrast to our findings that RNA interference (RNAi)-mediated silencing of BPIFB3 greatly enhances CVB replication, we show that silencing of BPIFB6 expression dramatically suppresses enterovirus replication in a pan-viral manner. Mechanistically, we show that loss of BPIFB6 expression induces pronounced alterations in retrograde and anterograde trafficking, which correlate with dramatic fragmentation of the Golgi complex. Taken together, these data implicate BPIFB6 as a key regulator of secretory pathway trafficking and viral replication and suggest that members of the BPIFB family participate in diverse host cell functions to regulate virus infections. IMPORTANCE Enterovirus infections are associated with a number of severe pathologies, such as aseptic meningitis, dilated cardiomyopathy, type I diabetes, paralysis, and even death. These viruses, which include coxsackievirus B (CVB), poliovirus (PV), and enterovirus 71 (EV71), co-opt the host cell secretory pathway, which controls the transport of proteins from the endoplasmic reticulum to the Golgi complex, to facilitate their replication. Here we report on the identification of a novel regulator of the secretory pathway, bactericidal/permeability-increasing protein (BPI) fold-containing family B, member 6 (BPIFB6), whose expression is required for enterovirus replication. We show that loss of

  6. The Inner Membrane Complex Sub-compartment Proteins Critical for Replication of the Apicomplexan Parasite Toxoplasma gondii Adopt a Pleckstrin Homology Fold*

    Science.gov (United States)

    Tonkin, Michelle L.; Beck, Josh R.; Bradley, Peter J.; Boulanger, Martin J.

    2014-01-01

    Toxoplasma gondii, an apicomplexan parasite prevalent in developed nations, infects up to one-third of the human population. The success of this parasite depends on several unique structures including an inner membrane complex (IMC) that lines the interior of the plasma membrane and contains proteins important for gliding motility and replication. Of these proteins, the IMC sub-compartment proteins (ISPs) have recently been shown to play a role in asexual T. gondii daughter cell formation, yet the mechanism is unknown. Complicating mechanistic characterization of the ISPs is a lack of sequence identity with proteins of known structure or function. In support of elucidating the function of ISPs, we first determined the crystal structures of representative members TgISP1 and TgISP3 to a resolution of 2.10 and 2.32 Å, respectively. Structural analysis revealed that both ISPs adopt a pleckstrin homology fold often associated with phospholipid binding or protein-protein interactions. Substitution of basic for hydrophobic residues in the region that overlays with phospholipid binding in related pleckstrin homology domains, however, suggests that ISPs do not retain phospholipid binding activity. Consistent with this observation, biochemical assays revealed no phospholipid binding activity. Interestingly, mapping of conserved surface residues combined with crystal packing analysis indicates that TgISPs have functionally repurposed the phospholipid-binding site likely to coordinate protein partners. Recruitment of larger protein complexes may also be aided through avidity-enhanced interactions resulting from multimerization of the ISPs. Overall, we propose a model where TgISPs recruit protein partners to the IMC to ensure correct progression of daughter cell formation. PMID:24675080

  7. In Vivo fitness associated with high virulence in a vertebrate virus is a complex trait regulated by host entry, replication, and shedding

    Science.gov (United States)

    Wargo, Andrew R.; Kurath, Gael

    2011-01-01

    The relationship between pathogen fitness and virulence is typically examined by quantifying only one or two pathogen fitness traits. More specifically, it is regularly assumed that within-host replication, as a precursor to transmission, is the driving force behind virulence. In reality, many traits contribute to pathogen fitness, and each trait could drive the evolution of virulence in different ways. Here, we independently quantified four viral infection cycle traits, namely, host entry, within-host replication, within-host coinfection fitness, and shedding, in vivo, in the vertebrate virus Infectious hematopoietic necrosis virus (IHNV). We examined how each of these stages of the viral infection cycle contributes to the fitness of IHNV genotypes that differ in virulence in rainbow trout. This enabled us to determine how infection cycle fitness traits are independently associated with virulence. We found that viral fitness was independently regulated by each of the traits examined, with the largest impact on fitness being provided by within-host replication. Furthermore, the more virulent of the two genotypes of IHNV we used had advantages in all of the traits quantified. Our results are thus congruent with the assumption that virulence and within-host replication are correlated but suggest that infection cycle fitness is complex and that replication is not the only trait associated with virulence.

  8. Nonequilibrium Entropic Bounds for Darwinian Replicators

    Directory of Open Access Journals (Sweden)

    Jordi Piñero

    2018-01-01

    Full Text Available Life evolved on our planet by means of a combination of Darwinian selection and innovations leading to higher levels of complexity. The emergence and selection of replicating entities is a central problem in prebiotic evolution. Theoretical models have shown how populations of different types of replicating entities exclude or coexist with other classes of replicators. Models are typically kinetic, based on standard replicator equations. On the other hand, the presence of thermodynamical constraints for these systems remain an open question. This is largely due to the lack of a general theory of statistical methods for systems far from equilibrium. Nonetheless, a first approach to this problem has been put forward in a series of novel developements falling under the rubric of the extended second law of thermodynamics. The work presented here is twofold: firstly, we review this theoretical framework and provide a brief description of the three fundamental replicator types in prebiotic evolution: parabolic, malthusian and hyperbolic. Secondly, we employ these previously mentioned techinques to explore how replicators are constrained by thermodynamics. Finally, we comment and discuss where further research should be focused on.

  9. From structure to mechanism—understanding initiation of DNA replication

    Science.gov (United States)

    Riera, Alberto; Barbon, Marta; Noguchi, Yasunori; Reuter, L. Maximilian; Schneider, Sarah; Speck, Christian

    2017-01-01

    DNA replication results in the doubling of the genome prior to cell division. This process requires the assembly of 50 or more protein factors into a replication fork. Here, we review recent structural and biochemical insights that start to explain how specific proteins recognize DNA replication origins, load the replicative helicase on DNA, unwind DNA, synthesize new DNA strands, and reassemble chromatin. We focus on the minichromosome maintenance (MCM2–7) proteins, which form the core of the eukaryotic replication fork, as this complex undergoes major structural rearrangements in order to engage with DNA, regulate its DNA-unwinding activity, and maintain genome stability. PMID:28717046

  10. DNA Replication and Cell Cycle Progression Regulatedby Long Range Interaction between Protein Complexes bound to DNA.

    Science.gov (United States)

    Matsson, L

    2001-12-01

    A nonstationary interaction that controlsDNA replication and the cell cycle isderived from many-body physics in achemically open T cell. The model predictsa long range force F'(ξ) =- (κ/2) ξ(1 - ξ)(2 - ξ)between thepre-replication complexes (pre-RCs) boundby the origins in DNA, ξ = ϕ/N being the relativedisplacement of pre-RCs, ϕ the number of pre-RCs, N the number of replicons to be replicated,and κ the compressibilitymodulus in the lattice of pre-RCs whichbehaves dynamically like an elasticallybraced string. Initiation of DNAreplication is induced at the thresholdϕ = N by a switch ofsign of F''(ξ), fromattraction (-) and assembly in the G(1) phase (0force at ϕ = 2N, from repulsion inS phase back to attraction in G(2), when all primed replicons havebeen duplicated once. F'(0) = 0corresponds to a resting cell in theabsence of driving force at ϕ= 0. The model thus ensures that the DNAcontent in G(2) cells is exactlytwice that of G(1) cells. The switch of interaction at the R-point, at which N pre-RCs have been assembled, starts the release of Rb protein thus also explaining the shift in the Rb phosphorylation from mitogen-dependent cyclinD to mitogen-independent cyclin E.Shape,slope and scale of the response curvesderived agree well with experimental datafrom dividing T cells and polymerising MTs,the variable length of which is due to anonlinear dependence of the growthamplitude on the initial concentrations oftubulin dimers and guanosine-tri-phosphate(GTP). The model also explains the dynamic instabilityin growing MTs.

  11. Large-scale replication study reveals a limit on probabilistic prediction in language comprehension.

    Science.gov (United States)

    Nieuwland, Mante S; Politzer-Ahles, Stephen; Heyselaar, Evelien; Segaert, Katrien; Darley, Emily; Kazanina, Nina; Von Grebmer Zu Wolfsthurn, Sarah; Bartolozzi, Federica; Kogan, Vita; Ito, Aine; Mézière, Diane; Barr, Dale J; Rousselet, Guillaume A; Ferguson, Heather J; Busch-Moreno, Simon; Fu, Xiao; Tuomainen, Jyrki; Kulakova, Eugenia; Husband, E Matthew; Donaldson, David I; Kohút, Zdenko; Rueschemeyer, Shirley-Ann; Huettig, Falk

    2018-04-03

    Do people routinely pre-activate the meaning and even the phonological form of upcoming words? The most acclaimed evidence for phonological prediction comes from a 2005 Nature Neuroscience publication by DeLong, Urbach and Kutas, who observed a graded modulation of electrical brain potentials (N400) to nouns and preceding articles by the probability that people use a word to continue the sentence fragment ('cloze'). In our direct replication study spanning 9 laboratories ( N =334), pre-registered replication-analyses and exploratory Bayes factor analyses successfully replicated the noun-results but, crucially, not the article-results. Pre-registered single-trial analyses also yielded a statistically significant effect for the nouns but not the articles. Exploratory Bayesian single-trial analyses showed that the article-effect may be non-zero but is likely far smaller than originally reported and too small to observe without very large sample sizes. Our results do not support the view that readers routinely pre-activate the phonological form of predictable words. © 2018, Nieuwland et al.

  12. Identification and characterization of the host protein DNAJC14 as a broadly active flavivirus replication modulator.

    Directory of Open Access Journals (Sweden)

    Zhigang Yi

    2011-01-01

    Full Text Available Viruses in the Flavivirus genus of the Flaviviridae family are arthropod-transmitted and contribute to staggering numbers of human infections and significant deaths annually across the globe. To identify cellular factors with antiviral activity against flaviviruses, we screened a cDNA library using an iterative approach. We identified a mammalian Hsp40 chaperone protein (DNAJC14 that when overexpressed was able to mediate protection from yellow fever virus (YFV-induced cell death. Further studies revealed that DNAJC14 inhibits YFV at the step of viral RNA replication. Since replication of bovine viral diarrhea virus (BVDV, a member of the related Pestivirus genus, is also known to be modulated by DNAJC14, we tested the effect of this host factor on diverse Flaviviridae family members. Flaviviruses, including the pathogenic Asibi strain of YFV, Kunjin, and tick-borne Langat virus, as well as a Hepacivirus, hepatitis C virus (HCV, all were inhibited by overexpression of DNAJC14. Mutagenesis showed that both the J-domain and the C-terminal domain, which mediates self-interaction, are required for anti-YFV activity. We found that DNAJC14 does not block YFV nor HCV NS2-3 cleavage, and using non-inhibitory mutants demonstrate that DNAJC14 is recruited to YFV replication complexes. Immunofluorescence analysis demonstrated that endogenous DNAJC14 rearranges during infection and is found in replication complexes identified by dsRNA staining. Interestingly, silencing of endogenous DNAJC14 results in impaired YFV replication suggesting a requirement for DNAJC14 in YFV replication complex assembly. Finally, the antiviral activity of overexpressed DNAJC14 occurs in a time- and dose-dependent manner. DNAJC14 overexpression may disrupt the proper stoichiometry resulting in inhibition, which can be overcome upon restoration of the optimal ratios due to the accumulation of viral nonstructural proteins. Our findings, together with previously published work

  13. Sterol Binding by the Tombusviral Replication Proteins Is Essential for Replication in Yeast and Plants.

    Science.gov (United States)

    Xu, Kai; Nagy, Peter D

    2017-04-01

    Membranous structures derived from various organelles are important for replication of plus-stranded RNA viruses. Although the important roles of co-opted host proteins in RNA virus replication have been appreciated for a decade, the equally important functions of cellular lipids in virus replication have been gaining full attention only recently. Previous work with Tomato bushy stunt tombusvirus (TBSV) in model host yeast has revealed essential roles for phosphatidylethanolamine and sterols in viral replication. To further our understanding of the role of sterols in tombusvirus replication, in this work we showed that the TBSV p33 and p92 replication proteins could bind to sterols in vitro The sterol binding by p33 is supported by cholesterol recognition/interaction amino acid consensus (CRAC) and CARC-like sequences within the two transmembrane domains of p33. Mutagenesis of the critical Y amino acids within the CRAC and CARC sequences blocked TBSV replication in yeast and plant cells. We also showed the enrichment of sterols in the detergent-resistant membrane (DRM) fractions obtained from yeast and plant cells replicating TBSV. The DRMs could support viral RNA synthesis on both the endogenous and exogenous templates. A lipidomic approach showed the lack of enhancement of sterol levels in yeast and plant cells replicating TBSV. The data support the notion that the TBSV replication proteins are associated with sterol-rich detergent-resistant membranes in yeast and plant cells. Together, the results obtained in this study and the previously published results support the local enrichment of sterols around the viral replication proteins that is critical for TBSV replication. IMPORTANCE One intriguing aspect of viral infections is their dependence on efficient subcellular assembly platforms serving replication, virion assembly, or virus egress via budding out of infected cells. These assembly platforms might involve sterol-rich membrane microdomains, which are

  14. The Design of Finite State Machine for Asynchronous Replication Protocol

    Science.gov (United States)

    Wang, Yanlong; Li, Zhanhuai; Lin, Wei; Hei, Minglei; Hao, Jianhua

    Data replication is a key way to design a disaster tolerance system and to achieve reliability and availability. It is difficult for a replication protocol to deal with the diverse and complex environment. This means that data is less well replicated than it ought to be. To reduce data loss and to optimize replication protocols, we (1) present a finite state machine, (2) run it to manage an asynchronous replication protocol and (3) report a simple evaluation of the asynchronous replication protocol based on our state machine. It's proved that our state machine is applicable to guarantee the asynchronous replication protocol running in the proper state to the largest extent in the event of various possible events. It also can helpful to build up replication-based disaster tolerance systems to ensure the business continuity.

  15. Non‐Canonical Replication Initiation: You’re Fired!

    Directory of Open Access Journals (Sweden)

    Bazilė Ravoitytė

    2017-01-01

    Full Text Available The division of prokaryotic and eukaryotic cells produces two cells that inherit a perfect copy of the genetic material originally derived from the mother cell. The initiation of canonical DNA replication must be coordinated to the cell cycle to ensure the accuracy of genome duplication. Controlled replication initiation depends on a complex interplay of cis‐acting DNA sequences, the so‐called origins of replication (ori, with trans‐acting factors involved in the onset of DNA synthesis. The interplay of cis‐acting elements and trans‐acting factors ensures that cells initiate replication at sequence‐specific sites only once, and in a timely order, to avoid chromosomal endoreplication. However, chromosome breakage and excessive RNA:DNA hybrid formation can cause breakinduced (BIR or transcription‐initiated replication (TIR, respectively. These non‐canonical replication events are expected to affect eukaryotic genome function and maintenance, and could be important for genome evolution and disease development. In this review, we describe the difference between canonical and non‐canonical DNA replication, and focus on mechanistic differences and common features between BIR and TIR. Finally, we discuss open issues on the factors and molecular mechanisms involved in TIR.

  16. The transcription elongation factor Bur1-Bur2 interacts with replication protein A and maintains genome stability during replication stress

    DEFF Research Database (Denmark)

    Clausing, Emanuel; Mayer, Andreas; Chanarat, Sittinan

    2010-01-01

    Multiple DNA-associated processes such as DNA repair, replication, and recombination are crucial for the maintenance of genome integrity. Here, we show a novel interaction between the transcription elongation factor Bur1-Bur2 and replication protein A (RPA), the eukaryotic single-stranded DNA......-binding protein with functions in DNA repair, recombination, and replication. Bur1 interacted via its C-terminal domain with RPA, and bur1-¿C mutants showed a deregulated DNA damage response accompanied by increased sensitivity to DNA damage and replication stress as well as increased levels of persisting Rad52...... foci. Interestingly, the DNA damage sensitivity of an rfa1 mutant was suppressed by bur1 mutation, further underscoring a functional link between these two protein complexes. The transcription elongation factor Bur1-Bur2 interacts with RPA and maintains genome integrity during DNA replication stress....

  17. Regulation of replication fork progression through histone supply and demand

    DEFF Research Database (Denmark)

    Groth, Anja; Corpet, Armelle; Cook, Adam J L

    2007-01-01

    DNA replication in eukaryotes requires nucleosome disruption ahead of the replication fork and reassembly behind. An unresolved issue concerns how histone dynamics are coordinated with fork progression to maintain chromosomal stability. Here, we characterize a complex in which the human histone c...... progression and histone supply and demand.......1 chaperone function, histone supply, and replicative unwinding of DNA in chromatin. We propose that Asf1, as a histone acceptor and donor, handles parental and new histones at the replication fork via an Asf1-(H3-H4)-MCM2-7 intermediate and thus provides a means to fine-tune replication fork...

  18. A Molecular Toolbox to Engineer Site-Specific DNA Replication Perturbation.

    Science.gov (United States)

    Larsen, Nicolai B; Hickson, Ian D; Mankouri, Hocine W

    2018-01-01

    Site-specific arrest of DNA replication is a useful tool for analyzing cellular responses to DNA replication perturbation. The E. coli Tus-Ter replication barrier can be reconstituted in eukaryotic cells as a system to engineer an unscheduled collision between a replication fork and an "alien" impediment to DNA replication. To further develop this system as a versatile tool, we describe a set of reagents and a detailed protocol that can be used to engineer Tus-Ter barriers into any locus in the budding yeast genome. Because the Tus-Ter complex is a bipartite system with intrinsic DNA replication-blocking activity, the reagents and protocols developed and validated in yeast could also be optimized to engineer site-specific replication fork barriers into other eukaryotic cell types.

  19. A Molecular Toolbox to Engineer Site-Specific DNA Replication Perturbation

    DEFF Research Database (Denmark)

    Larsen, Nicolai B; Hickson, Ian D; Mankouri, Hocine W

    2018-01-01

    " impediment to DNA replication. To further develop this system as a versatile tool, we describe a set of reagents and a detailed protocol that can be used to engineer Tus-Ter barriers into any locus in the budding yeast genome. Because the Tus-Ter complex is a bipartite system with intrinsic DNA replication......Site-specific arrest of DNA replication is a useful tool for analyzing cellular responses to DNA replication perturbation. The E. coli Tus-Ter replication barrier can be reconstituted in eukaryotic cells as a system to engineer an unscheduled collision between a replication fork and an "alien......-blocking activity, the reagents and protocols developed and validated in yeast could also be optimized to engineer site-specific replication fork barriers into other eukaryotic cell types....

  20. From structure to mechanism-understanding initiation of DNA replication.

    Science.gov (United States)

    Riera, Alberto; Barbon, Marta; Noguchi, Yasunori; Reuter, L Maximilian; Schneider, Sarah; Speck, Christian

    2017-06-01

    DNA replication results in the doubling of the genome prior to cell division. This process requires the assembly of 50 or more protein factors into a replication fork. Here, we review recent structural and biochemical insights that start to explain how specific proteins recognize DNA replication origins, load the replicative helicase on DNA, unwind DNA, synthesize new DNA strands, and reassemble chromatin. We focus on the minichromosome maintenance (MCM2-7) proteins, which form the core of the eukaryotic replication fork, as this complex undergoes major structural rearrangements in order to engage with DNA, regulate its DNA-unwinding activity, and maintain genome stability. © 2017 Riera et al.; Published by Cold Spring Harbor Laboratory Press.

  1. Mechanisms of mutagenesis: DNA replication in the presence of DNA damage.

    Science.gov (United States)

    Liu, Binyan; Xue, Qizhen; Tang, Yong; Cao, Jia; Guengerich, F Peter; Zhang, Huidong

    2016-01-01

    Environmental mutagens cause DNA damage that disturbs replication and produces mutations, leading to cancer and other diseases. We discuss mechanisms of mutagenesis resulting from DNA damage, from the level of DNA replication by a single polymerase to the complex DNA replisome of some typical model organisms (including bacteriophage T7, T4, Sulfolobus solfataricus, Escherichia coli, yeast and human). For a single DNA polymerase, DNA damage can affect replication in three major ways: reducing replication fidelity, causing frameshift mutations, and blocking replication. For the DNA replisome, protein interactions and the functions of accessory proteins can yield rather different results even with a single DNA polymerase. The mechanism of mutation during replication performed by the DNA replisome is a long-standing question. Using new methods and techniques, the replisomes of certain organisms and human cell extracts can now be investigated with regard to the bypass of DNA damage. In this review, we consider the molecular mechanism of mutagenesis resulting from DNA damage in replication at the levels of single DNA polymerases and complex DNA replisomes, including translesion DNA synthesis. Copyright © 2016 Elsevier B.V. All rights reserved.

  2. Replication of urban innovations - prioritization of strategies for the replication of Dhaka's community-based decentralized composting model.

    Science.gov (United States)

    Yedla, Sudhakar

    2012-01-01

    Dhaka's community-based decentralized composting (DCDC) is a successful demonstration of solid waste management by adopting low-cost technology, local resources community participation and partnerships among the various actors involved. This paper attempts to understand the model, necessary conditions, strategies and their priorities to replicate DCDC in the other developing cities of Asia. Thirteen strategies required for its replication are identified and assessed based on various criteria, namely transferability, longevity, economic viability, adaptation and also overall replication. Priority setting by multi-criteria analysis by applying analytic hierarchy process revealed that immediate transferability without long-term and economic viability consideration is not advisable as this would result in unsustainable replication of DCDC. Based on the analysis, measures to ensure the product quality control; partnership among stakeholders (public-private-community); strategies to achieve better involvement of the private sector in solid waste management (entrepreneurship in approach); simple and low-cost technology; and strategies to provide an effective interface among the complementing sectors are identified as important strategies for its replication.

  3. DNA Copy-Number Control through Inhibition of Replication Fork Progression

    Directory of Open Access Journals (Sweden)

    Jared T. Nordman

    2014-11-01

    Full Text Available Proper control of DNA replication is essential to ensure faithful transmission of genetic material and prevent chromosomal aberrations that can drive cancer progression and developmental disorders. DNA replication is regulated primarily at the level of initiation and is under strict cell-cycle regulation. Importantly, DNA replication is highly influenced by developmental cues. In Drosophila, specific regions of the genome are repressed for DNA replication during differentiation by the SNF2 domain-containing protein SUUR through an unknown mechanism. We demonstrate that SUUR is recruited to active replication forks and mediates the repression of DNA replication by directly inhibiting replication fork progression instead of functioning as a replication fork barrier. Mass spectrometry identification of SUUR-associated proteins identified the replicative helicase member CDC45 as a SUUR-associated protein, supporting a role for SUUR directly at replication forks. Our results reveal that control of eukaryotic DNA copy number can occur through the inhibition of replication fork progression.

  4. Mechanism of Archaeal MCM Helicase Recruitment to DNA Replication Origins

    Science.gov (United States)

    Samson, Rachel Y.; Abeyrathne, Priyanka D.; Bell, Stephen D.

    2015-01-01

    Summary Cellular DNA replication origins direct the recruitment of replicative helicases via the action of initiator proteins belonging to the AAA+ superfamily of ATPases. Archaea have a simplified subset of the eukaryotic DNA replication machinery proteins and possess initiators that appear ancestral to both eukaryotic Orc1 and Cdc6. We have reconstituted origin-dependent recruitment of the homohexameric archaeal MCM in vitro with purified recombinant proteins. Using this system, we reveal that archaeal Orc1-1 fulfills both Orc1 and Cdc6 functions by binding to a replication origin and directly recruiting MCM helicase. We identify the interaction interface between these proteins and reveal how ATP binding by Orc1-1 modulates recruitment of MCM. Additionally, we provide evidence that an open-ring form of the archaeal MCM homohexamer is loaded at origins. PMID:26725007

  5. Conflict Resolution in the Genome: How Transcription and Replication Make It Work.

    Science.gov (United States)

    Hamperl, Stephan; Cimprich, Karlene A

    2016-12-01

    The complex machineries involved in replication and transcription translocate along the same DNA template, often in opposing directions and at different rates. These processes routinely interfere with each other in prokaryotes, and mounting evidence now suggests that RNA polymerase complexes also encounter replication forks in higher eukaryotes. Indeed, cells rely on numerous mechanisms to avoid, tolerate, and resolve such transcription-replication conflicts, and the absence of these mechanisms can lead to catastrophic effects on genome stability and cell viability. In this article, we review the cellular responses to transcription-replication conflicts and highlight how these inevitable encounters shape the genome and impact diverse cellular processes. Copyright © 2016 Elsevier Inc. All rights reserved.

  6. Does unconscious thought outperform conscious thought on complex decisions? A further examination

    Directory of Open Access Journals (Sweden)

    Todd J. Thorsteinson

    2009-04-01

    Full Text Available Two experiments examined the benefits of unconscious thought on complex decisions (Dijksterhuis, 2004. Experiment 1 attempted to replicate and extend past research by examining the effect of providing reasons prior to rating the options. Results indicated no significant differences between the conditions. Experiment 2 attempted to replicate the findings of Dijksterhuis, Bos, Nordgren, and van Baaren (2006 and determine if a memory aid could overcome the limitations of conscious thought on complex tasks. Results revealed that a memory aid improved decisions compared to the conscious thought condition. Participants in the unconscious thought condition did not perform significantly better than did participants in the conscious thought condition.

  7. Addressing the "Replication Crisis": Using Original Studies to Design Replication Studies with Appropriate Statistical Power.

    Science.gov (United States)

    Anderson, Samantha F; Maxwell, Scott E

    2017-01-01

    Psychology is undergoing a replication crisis. The discussion surrounding this crisis has centered on mistrust of previous findings. Researchers planning replication studies often use the original study sample effect size as the basis for sample size planning. However, this strategy ignores uncertainty and publication bias in estimated effect sizes, resulting in overly optimistic calculations. A psychologist who intends to obtain power of .80 in the replication study, and performs calculations accordingly, may have an actual power lower than .80. We performed simulations to reveal the magnitude of the difference between actual and intended power based on common sample size planning strategies and assessed the performance of methods that aim to correct for effect size uncertainty and/or bias. Our results imply that even if original studies reflect actual phenomena and were conducted in the absence of questionable research practices, popular approaches to designing replication studies may result in a low success rate, especially if the original study is underpowered. Methods correcting for bias and/or uncertainty generally had higher actual power, but were not a panacea for an underpowered original study. Thus, it becomes imperative that 1) original studies are adequately powered and 2) replication studies are designed with methods that are more likely to yield the intended level of power.

  8. Morphological and biochemical characterization of the membranous hepatitis C virus replication compartment.

    Science.gov (United States)

    Paul, David; Hoppe, Simone; Saher, Gesine; Krijnse-Locker, Jacomine; Bartenschlager, Ralf

    2013-10-01

    Like all other positive-strand RNA viruses, hepatitis C virus (HCV) induces rearrangements of intracellular membranes that are thought to serve as a scaffold for the assembly of the viral replicase machinery. The most prominent membranous structures present in HCV-infected cells are double-membrane vesicles (DMVs). However, their composition and role in the HCV replication cycle are poorly understood. To gain further insights into the biochemcial properties of HCV-induced membrane alterations, we generated a functional replicon containing a hemagglutinin (HA) affinity tag in nonstructural protein 4B (NS4B), the supposed scaffold protein of the viral replication complex. By using HA-specific affinity purification we isolated NS4B-containing membranes from stable replicon cells. Complementing biochemical and electron microscopy analyses of purified membranes revealed predominantly DMVs, which contained viral proteins NS3 and NS5A as well as enzymatically active viral replicase capable of de novo synthesis of HCV RNA. In addition to viral factors, co-opted cellular proteins, such as vesicle-associated membrane protein-associated protein A (VAP-A) and VAP-B, that are crucial for viral RNA replication, as well as cholesterol, a major structural lipid of detergent-resistant membranes, are highly enriched in DMVs. Here we describe the first isolation and biochemical characterization of HCV-induced DMVs. The results obtained underline their central role in the HCV replication cycle and suggest that DMVs are sites of viral RNA replication. The experimental approach described here is a powerful tool to more precisely define the molecular composition of membranous replication factories induced by other positive-strand RNA viruses, such as picorna-, arteri- and coronaviruses.

  9. Viral hijacking of a replicative helicase loader and its implications for helicase loading control and phage replication

    Energy Technology Data Exchange (ETDEWEB)

    Hood, Iris V.; Berger, James M.

    2016-05-31

    Replisome assembly requires the loading of replicative hexameric helicases onto origins by AAA+ ATPases. How loader activity is appropriately controlled remains unclear. Here, we use structural and biochemical analyses to establish how an antimicrobial phage protein interferes with the function of theStaphylococcus aureusreplicative helicase loader, DnaI. The viral protein binds to the loader’s AAA+ ATPase domain, allowing binding of the host replicative helicase but impeding loader self-assembly and ATPase activity. Close inspection of the complex highlights an unexpected locus for the binding of an interdomain linker element in DnaI/DnaC-family proteins. We find that the inhibitor protein is genetically coupled to a phage-encoded homolog of the bacterial helicase loader, which we show binds to the host helicase but not to the inhibitor itself. These findings establish a new approach by which viruses can hijack host replication processes and explain how loader activity is internally regulated to prevent aberrant auto-association.

  10. Replication of linkage to quantitative trait loci: variation in location and magnitude of the lod score.

    Science.gov (United States)

    Hsueh, W C; Göring, H H; Blangero, J; Mitchell, B D

    2001-01-01

    Replication of linkage signals from independent samples is considered an important step toward verifying the significance of linkage signals in studies of complex traits. The purpose of this empirical investigation was to examine the variability in the precision of localizing a quantitative trait locus (QTL) by analyzing multiple replicates of a simulated data set with the use of variance components-based methods. Specifically, we evaluated across replicates the variation in both the magnitude and the location of the peak lod scores. We analyzed QTLs whose effects accounted for 10-37% of the phenotypic variance in the quantitative traits. Our analyses revealed that the precision of QTL localization was directly related to the magnitude of the QTL effect. For a QTL with effect accounting for > 20% of total phenotypic variation, > 90% of the linkage peaks fall within 10 cM from the true gene location. We found no evidence that, for a given magnitude of the lod score, the presence of interaction influenced the precision of QTL localization.

  11. Chromatin Constrains the Initiation and Elongation of DNA Replication.

    Science.gov (United States)

    Devbhandari, Sujan; Jiang, Jieqing; Kumar, Charanya; Whitehouse, Iestyn; Remus, Dirk

    2017-01-05

    Eukaryotic chromosomal DNA is faithfully replicated in a complex series of cell-cycle-regulated events that are incompletely understood. Here we report the reconstitution of DNA replication free in solution with purified proteins from the budding yeast Saccharomyces cerevisiae. The system recapitulates regulated bidirectional origin activation; synthesis of leading and lagging strands by the three replicative DNA polymerases Pol α, Pol δ, and Pol ε; and canonical maturation of Okazaki fragments into continuous daughter strands. We uncover a dual regulatory role for chromatin during DNA replication: promoting origin dependence and determining Okazaki fragment length by restricting Pol δ progression. This system thus provides a functional platform for the detailed mechanistic analysis of eukaryotic chromosome replication. Copyright © 2017 Elsevier Inc. All rights reserved.

  12. Dynamics of DNA replication loops reveal temporal control of lagging-strand synthesis

    NARCIS (Netherlands)

    Hamdan, Samir M.; Loparo, Joseph J.; Takahashi, Masateru; Richardson, Charles C.; Oijen, Antoine M. van

    2009-01-01

    In all organisms, the protein machinery responsible for the replication of DNA, the replisome, is faced with a directionality problem. The antiparallel nature of duplex DNA permits the leading-strand polymerase to advance in a continuous fashion, but forces the lagging-strand polymerase to

  13. GEMC1 is a TopBP1 interacting protein required for chromosomal DNA replication

    Science.gov (United States)

    Balestrini, Alessia; Cosentino, Claudia; Errico, Alessia; Garner, Elizabeth; Costanzo, Vincenzo

    2010-01-01

    Many factors required for chromosomal DNA replication have been identified in unicellular eukaryotes. However, DNA replication in complex multicellular organisms is poorly understood. Here, we report the identification of GEMC1, a novel vertebrate protein required for chromosomal DNA replication. GEMC1 is highly conserved in vertebrates and is preferentially expressed in proliferating cells. Using Xenopus egg extract we show that Xenopus GEMC1 (xGEMC1) binds to checkpoint and replication factor TopBP1, which promotes xGEMC1 binding to chromatin during pre-replication complex (pre-RC) formation. We demonstrate that xGEMC1 directly interacts with replication factors such as Cdc45 and Cdk2-CyclinE by which it is heavily phosphorylated. Phosphorylated xGEMC1 stimulates initiation of DNA replication whereas depletion of xGEMC1 prevents DNA replication onset due to impairment of Cdc45 loading onto chromatin. Likewise, inhibition of GEMC1 expression by morpholino and siRNA oligos prevents DNA replication in embryonic and somatic vertebrate cells. These data suggest that GEMC1 promotes initiation of chromosomal DNA replication in higher eukaryotes by mediating TopBP1 and Cdk2 dependent recruitment of Cdc45 onto replication origins. PMID:20383140

  14. Hsp90 interacts specifically with viral RNA and differentially regulates replication initiation of Bamboo mosaic virus and associated satellite RNA.

    Directory of Open Access Journals (Sweden)

    Ying Wen Huang

    Full Text Available Host factors play crucial roles in the replication of plus-strand RNA viruses. In this report, a heat shock protein 90 homologue of Nicotiana benthamiana, NbHsp90, was identified in association with partially purified replicase complexes from BaMV-infected tissue, and shown to specifically interact with the 3' untranslated region (3' UTR of BaMV genomic RNA, but not with the 3' UTR of BaMV-associated satellite RNA (satBaMV RNA or that of genomic RNA of other viruses, such as Potato virus X (PVX or Cucumber mosaic virus (CMV. Mutational analyses revealed that the interaction occurs between the middle domain of NbHsp90 and domain E of the BaMV 3' UTR. The knockdown or inhibition of NbHsp90 suppressed BaMV infectivity, but not that of satBaMV RNA, PVX, or CMV in N. benthamiana. Time-course analysis further revealed that the inhibitory effect of 17-AAG is significant only during the immediate early stages of BaMV replication. Moreover, yeast two-hybrid and GST pull-down assays demonstrated the existence of an interaction between NbHsp90 and the BaMV RNA-dependent RNA polymerase. These results reveal a novel role for NbHsp90 in the selective enhancement of BaMV replication, most likely through direct interaction with the 3' UTR of BaMV RNA during the initiation of BaMV RNA replication.

  15. Conjunctival lymphangioma in a 4-year-old girl revealed tuberous sclerosis complex

    Directory of Open Access Journals (Sweden)

    Freiberg, Florentina Joyce

    2016-09-01

    Full Text Available Background: To present a case of conjunctival lymphangioma in a girl with tuberous sclerosis complex.Methods/results: A 4-year-old girl presented with a relapsing cystic lesion of the bulbar conjunctiva in the right eye with string-of-pearl-like dilation of lymphatic vessels and right-sided facial swelling with mild pain. Best-corrected vision was not impaired. Examination of the skin revealed three hypomelanotic macules and a lumbal Shagreen patch. Magnetic resonance imaging (MRI findings displayed minimal enhancement of buccal fat on the right side. Cranial and orbital MRI showed signal enhancement in the right cortical and subcortical areas. Genetic analysis revealed a heterozygous deletion encompassing exon 1 and 2 of the gene (tuberous sclerosis complex 1 gene, confirming the diagnosis of tuberous sclerosis complex.Conclusion: In conjunctival lymphangioma, tuberous sclerosis complex should be considered as the primary disease.

  16. MMSET is dynamically regulated during cell-cycle progression and promotes normal DNA replication.

    Science.gov (United States)

    Evans, Debra L; Zhang, Haoxing; Ham, Hyoungjun; Pei, Huadong; Lee, SeungBaek; Kim, JungJin; Billadeau, Daniel D; Lou, Zhenkun

    2016-01-01

    The timely and precise duplication of cellular DNA is essential for maintaining genome integrity and is thus tightly-regulated. During mitosis and G1, the Origin Recognition Complex (ORC) binds to future replication origins, coordinating with multiple factors to load the minichromosome maintenance (MCM) complex onto future replication origins as part of the pre-replication complex (pre-RC). The pre-RC machinery, in turn, remains inactive until the subsequent S phase when it is required for replication fork formation, thereby initiating DNA replication. Multiple myeloma SET domain-containing protein (MMSET, a.k.a. WHSC1, NSD2) is a histone methyltransferase that is frequently overexpressed in aggressive cancers and is essential for normal human development. Several studies have suggested a role for MMSET in cell-cycle regulation; however, whether MMSET is itself regulated during cell-cycle progression has not been examined. In this study, we report that MMSET is degraded during S phase in a cullin-ring ligase 4-Cdt2 (CRL4(Cdt2)) and proteasome-dependent manner. Notably, we also report defects in DNA replication and a decreased association of pre-RC factors with chromatin in MMSET-depleted cells. Taken together, our results suggest a dynamic regulation of MMSET levels throughout the cell cycle, and further characterize the role of MMSET in DNA replication and cell-cycle progression.

  17. COPI is required for enterovirus 71 replication.

    Directory of Open Access Journals (Sweden)

    Jianmin Wang

    Full Text Available Enterovirus 71 (EV71, a member of the Picornaviridae family, is found in Asian countries where it causes a wide range of human diseases. No effective therapy is available for the treatment of these infections. Picornaviruses undergo RNA replication in association with membranes of infected cells. COPI and COPII have been shown to be involved in the formation of picornavirus-induced vesicles. Replication of several picornaviruses, including poliovirus and Echovirus 11 (EV11, is dependent on COPI or COPII. Here, we report that COPI, but not COPII, is required for EV71 replication. Replication of EV71 was inhibited by brefeldin A and golgicide A, inhibitors of COPI activity. Furthermore, we found EV71 2C protein interacted with COPI subunits by co-immunoprecipitation and GST pull-down assay, indicating that COPI coatomer might be directed to the viral replication complex through viral 2C protein. Additionally, because the pathway is conserved among different species of enteroviruses, it may represent a novel target for antiviral therapies.

  18. MCM Paradox: Abundance of Eukaryotic Replicative Helicases and Genomic Integrity.

    Science.gov (United States)

    Das, Mitali; Singh, Sunita; Pradhan, Satyajit; Narayan, Gopeshwar

    2014-01-01

    As a crucial component of DNA replication licensing system, minichromosome maintenance (MCM) 2-7 complex acts as the eukaryotic DNA replicative helicase. The six related MCM proteins form a heterohexamer and bind with ORC, CDC6, and Cdt1 to form the prereplication complex. Although the MCMs are well known as replicative helicases, their overabundance and distribution patterns on chromatin present a paradox called the "MCM paradox." Several approaches had been taken to solve the MCM paradox and describe the purpose of excess MCMs distributed beyond the replication origins. Alternative functions of these MCMs rather than a helicase had also been proposed. This review focuses on several models and concepts generated to solve the MCM paradox coinciding with their helicase function and provides insight into the concept that excess MCMs are meant for licensing dormant origins as a backup during replication stress. Finally, we extend our view towards the effect of alteration of MCM level. Though an excess MCM constituent is needed for normal cells to withstand stress, there must be a delineation of the threshold level in normal and malignant cells. This review also outlooks the future prospects to better understand the MCM biology.

  19. Autophagy Facilitates Salmonella Replication in HeLa Cells

    Science.gov (United States)

    Yu, Hong B.; Croxen, Matthew A.; Marchiando, Amanda M.; Ferreira, Rosana B. R.; Cadwell, Ken; Foster, Leonard J.; Finlay, B. Brett

    2014-01-01

    ABSTRACT Autophagy is a process whereby a double-membrane structure (autophagosome) engulfs unnecessary cytosolic proteins, organelles, and invading pathogens and delivers them to the lysosome for degradation. We examined the fate of cytosolic Salmonella targeted by autophagy and found that autophagy-targeted Salmonella present in the cytosol of HeLa cells correlates with intracellular bacterial replication. Real-time analyses revealed that a subset of cytosolic Salmonella extensively associates with autophagy components p62 and/or LC3 and replicates quickly, whereas intravacuolar Salmonella shows no or very limited association with p62 or LC3 and replicates much more slowly. Replication of cytosolic Salmonella in HeLa cells is significantly decreased when autophagy components are depleted. Eventually, hyperreplication of cytosolic Salmonella potentiates cell detachment, facilitating the dissemination of Salmonella to neighboring cells. We propose that Salmonella benefits from autophagy for its cytosolic replication in HeLa cells. PMID:24618251

  20. Chaotic interactions of self-replicating RNA.

    Science.gov (United States)

    Forst, C V

    1996-03-01

    A general system of high-order differential equations describing complex dynamics of replicating biomolecules is given. Symmetry relations and coordinate transformations of general replication systems leading to topologically equivalent systems are derived. Three chaotic attractors observed in Lotka-Volterra equations of dimension n = 3 are shown to represent three cross-sections of one and the same chaotic regime. Also a fractal torus in a generalized three-dimensional Lotka-Volterra Model has been linked to one of the chaotic attractors. The strange attractors are studied in the equivalent four-dimensional catalytic replicator network. The fractal torus has been examined in adapted Lotka-Volterra equations. Analytic expressions are derived for the Lyapunov exponents of the flow in the replicator system. Lyapunov spectra for different pathways into chaos has been calculated. In the generalized Lotka-Volterra system a second inner rest point--coexisting with (quasi)-periodic orbits--can be observed; with an abundance of different bifurcations. Pathways from chaotic tori, via quasi-periodic tori, via limit cycles, via multi-periodic orbits--emerging out of periodic doubling bifurcations--to "simple" chaotic attractors can be found.

  1. Gene organization inside replication domains in mammalian genomes

    Science.gov (United States)

    Zaghloul, Lamia; Baker, Antoine; Audit, Benjamin; Arneodo, Alain

    2012-11-01

    We investigate the large-scale organization of human genes with respect to "master" replication origins that were previously identified as bordering nucleotide compositional skew domains. We separate genes in two categories depending on their CpG enrichment at the promoter which can be considered as a marker of germline DNA methylation. Using expression data in mouse, we confirm that CpG-rich genes are highly expressed in germline whereas CpG-poor genes are in a silent state. We further show that, whether tissue-specific or broadly expressed (housekeeping genes), the CpG-rich genes are over-represented close to the replication skew domain borders suggesting some coordination of replication and transcription. We also reveal that the transcription of the longest CpG-rich genes is co-oriented with replication fork progression so that the promoter of these transcriptionally active genes be located into the accessible open chromatin environment surrounding the master replication origins that border the replication skew domains. The observation of a similar gene organization in the mouse genome confirms the interplay of replication, transcription and chromatin structure as the cornerstone of mammalian genome architecture.

  2. Temporal profiling of the chromatin proteome reveals system-wide responses to replication inhibition

    DEFF Research Database (Denmark)

    Khoudoli, Guennadi A; Gillespie, Peter J; Stewart, Graeme

    2008-01-01

    Although the replication, expression, and maintenance of DNA are well-studied processes, the way that they are coordinated is poorly understood. Here, we report an analysis of the changing association of proteins with chromatin (the chromatin proteome) during progression through interphase...... of the cell cycle. Sperm nuclei were incubated in Xenopus egg extracts, and chromatin-associated proteins were analyzed by mass spectrometry at different times. Approximately 75% of the proteins varied in abundance on chromatin by more than 15%, suggesting that the chromatin proteome is highly dynamic....... Proteins were then assigned to one of 12 different clusters on the basis of their pattern of chromatin association. Each cluster contained functional groups of proteins involved in different nuclear processes related to progression through interphase. We also blocked DNA replication by inhibiting either...

  3. The Replication Mechanism in a Romanian ERP System Environment

    Directory of Open Access Journals (Sweden)

    Iuliana SCORTA

    2008-01-01

    Full Text Available Modern Relational Database Management Systems have Replication technology. Large enterprises are often spread around the country, and although WANs can be very fast and reliable these days, it is often better for each location to have a local copy of data rather than have a single database at a central location. This usually means that replication is a requirement in order for each location to have the most up-to-date data. This paper reveals a replication mechanism implemented in a Romanian ERP system environment.

  4. Tombusviruses upregulate phospholipid biosynthesis via interaction between p33 replication protein and yeast lipid sensor proteins during virus replication in yeast

    International Nuclear Information System (INIS)

    Barajas, Daniel; Xu, Kai; Sharma, Monika; Wu, Cheng-Yu; Nagy, Peter D.

    2014-01-01

    Positive-stranded RNA viruses induce new membranous structures and promote membrane proliferation in infected cells to facilitate viral replication. In this paper, the authors show that a plant-infecting tombusvirus upregulates transcription of phospholipid biosynthesis genes, such as INO1, OPI3 and CHO1, and increases phospholipid levels in yeast model host. This is accomplished by the viral p33 replication protein, which interacts with Opi1p FFAT domain protein and Scs2p VAP protein. Opi1p and Scs2p are phospholipid sensor proteins and they repress the expression of phospholipid genes. Accordingly, deletion of OPI1 transcription repressor in yeast has a stimulatory effect on TBSV RNA accumulation and enhanced tombusvirus replicase activity in an in vitro assay. Altogether, the presented data convincingly demonstrate that de novo lipid biosynthesis is required for optimal TBSV replication. Overall, this work reveals that a (+)RNA virus reprograms the phospholipid biosynthesis pathway in a unique way to facilitate its replication in yeast cells. - Highlights: • Tombusvirus p33 replication protein interacts with FFAT-domain host protein. • Tombusvirus replication leads to upregulation of phospholipids. • Tombusvirus replication depends on de novo lipid synthesis. • Deletion of FFAT-domain host protein enhances TBSV replication. • TBSV rewires host phospholipid synthesis

  5. BRPF3-HBO1 regulates replication origin activation and histone H3K14 acetylation

    DEFF Research Database (Denmark)

    Feng, Yunpeng; Vlassis, Arsenios; Roques, Céline

    2016-01-01

    implicated in replication control by measuring RPA accumulation upon replication stress. We identify six factors required for normal rates of DNA replication and characterize a function of the bromodomain and PHD finger-containing protein 3 (BRPF3) in replication initiation. BRPF3 forms a complex with HBO1...

  6. Inhibition of herpesvirus and influenza virus replication by blocking polymerase subunit interactions.

    Science.gov (United States)

    Palù, Giorgio; Loregian, Arianna

    2013-09-01

    Protein-protein interactions (PPIs) play a key role in many biological processes, including virus replication in the host cell. Since most of the PPIs are functionally essential, a possible strategy to inhibit virus replication is based on the disruption of viral protein complexes by peptides or small molecules that interfere with subunit interactions. In particular, an attractive target for antiviral drugs is the binding between the subunits of essential viral enzymes. This review describes the development of new antiviral compounds that inhibit herpesvirus and influenza virus replication by blocking interactions between subunit proteins of their polymerase complexes. Copyright © 2013 Elsevier B.V. All rights reserved.

  7. The multi-replication protein A (RPA) system--a new perspective.

    Science.gov (United States)

    Sakaguchi, Kengo; Ishibashi, Toyotaka; Uchiyama, Yukinobu; Iwabata, Kazuki

    2009-02-01

    Replication protein A (RPA) complex has been shown, using both in vivo and in vitro approaches, to be required for most aspects of eukaryotic DNA metabolism: replication, repair, telomere maintenance and homologous recombination. Here, we review recent data concerning the function and biological importance of the multi-RPA complex. There are distinct complexes of RPA found in the biological kingdoms, although for a long time only one type of RPA complex was believed to be present in eukaryotes. Each complex probably serves a different role. In higher plants, three distinct large and medium subunits are present, but only one species of the smallest subunit. Each of these protein subunits forms stable complexes with their respective partners. They are paralogs as complex. Humans possess two paralogs and one analog of RPA. The multi-RPA system can be regarded as universal in eukaryotes. Among eukaryotic kingdoms, paralogs, orthologs, analogs and heterologs of many DNA synthesis-related factors, including RPA, are ubiquitous. Convergent evolution seems to be ubiquitous in these processes. Using recent findings, we review the composition and biological functions of RPA complexes.

  8. Mechanisms Governing DDK Regulation of the Initiation of DNA Replication

    Directory of Open Access Journals (Sweden)

    Larasati

    2016-12-01

    Full Text Available The budding yeast Dbf4-dependent kinase (DDK complex—comprised of cell division cycle (Cdc7 kinase and its regulatory subunit dumbbell former 4 (Dbf4—is required to trigger the initiation of DNA replication through the phosphorylation of multiple minichromosome maintenance complex subunits 2-7 (Mcm2-7. DDK is also a target of the radiation sensitive 53 (Rad53 checkpoint kinase in response to replication stress. Numerous investigations have determined mechanistic details, including the regions of Mcm2, Mcm4, and Mcm6 phosphorylated by DDK, and a number of DDK docking sites. Similarly, the way in which the Rad53 forkhead-associated 1 (FHA1 domain binds to DDK—involving both canonical and non-canonical interactions—has been elucidated. Recent work has revealed mutual promotion of DDK and synthetic lethal with dpb11-1 3 (Sld3 roles. While DDK phosphorylation of Mcm2-7 subunits facilitates their interaction with Sld3 at origins, Sld3 in turn stimulates DDK phosphorylation of Mcm2. Details of a mutually antagonistic relationship between DDK and Rap1-interacting factor 1 (Rif1 have also recently come to light. While Rif1 is able to reverse DDK-mediated Mcm2-7 complex phosphorylation by targeting the protein phosphatase glycogen 7 (Glc7 to origins, there is evidence to suggest that DDK can counteract this activity by binding to and phosphorylating Rif1.

  9. Commercial Building Partnerships Replication and Diffusion

    Energy Technology Data Exchange (ETDEWEB)

    Antonopoulos, Chrissi A.; Dillon, Heather E.; Baechler, Michael C.

    2013-09-16

    This study presents findings from survey and interview data investigating replication efforts of Commercial Building Partnership (CBP) partners that worked directly with the Pacific Northwest National Laboratory (PNNL). PNNL partnered directly with 12 organizations on new and retrofit construction projects, which represented approximately 28 percent of the entire U.S. Department of Energy (DOE) CBP program. Through a feedback survey mechanism, along with personal interviews, PNNL gathered quantitative and qualitative data relating to replication efforts by each organization. These data were analyzed to provide insight into two primary research areas: 1) CBP partners’ replication efforts of technologies and approaches used in the CBP project to the rest of the organization’s building portfolio (including replication verification), and, 2) the market potential for technology diffusion into the total U.S. commercial building stock, as a direct result of the CBP program. The first area of this research focused specifically on replication efforts underway or planned by each CBP program participant. Factors that impact replication include motivation, organizational structure and objectives firms have for implementation of energy efficient technologies. Comparing these factors between different CBP partners revealed patterns in motivation for constructing energy efficient buildings, along with better insight into market trends for green building practices. The second area of this research develops a diffusion of innovations model to analyze potential broad market impacts of the CBP program on the commercial building industry in the United States.

  10. DNA replication after mutagenic treatment in Hordeum vulgare.

    Science.gov (United States)

    Kwasniewska, Jolanta; Kus, Arita; Swoboda, Monika; Braszewska-Zalewska, Agnieszka

    2016-12-01

    The temporal and spatial properties of DNA replication in plants related to DNA damage and mutagenesis is poorly understood. Experiments were carried out to explore the relationships between DNA replication, chromatin structure and DNA damage in nuclei from barley root tips. We quantitavely analysed the topological organisation of replication foci using pulse EdU labelling during the S phase and its relationship with the DNA damage induced by mutagenic treatment with maleic hydrazide (MH), nitroso-N-methyl-urea (MNU) and gamma ray. Treatment with mutagens did not change the characteristic S-phase patterns in the nuclei; however, the frequencies of the S-phase-labelled cells after treatment differed from those observed in the control cells. The analyses of DNA replication in barley nuclei were extended to the micronuclei induced by mutagens. Replication in the chromatin of the micronuclei was rare. The results of simultanous TUNEL reaction to identify cells with DNA strand breaks and the labelling of the S-phase cells with EdU revealed the possibility of DNA replication occurring in damaged nuclei. For the first time, the intensity of EdU fluorescence to study the rate of DNA replication was analysed. Copyright © 2016 Elsevier B.V. All rights reserved.

  11. Diversity of the DNA Replication System in the Archaea Domain

    Directory of Open Access Journals (Sweden)

    Felipe Sarmiento

    2014-01-01

    Full Text Available The precise and timely duplication of the genome is essential for cellular life. It is achieved by DNA replication, a complex process that is conserved among the three domains of life. Even though the cellular structure of archaea closely resembles that of bacteria, the information processing machinery of archaea is evolutionarily more closely related to the eukaryotic system, especially for the proteins involved in the DNA replication process. While the general DNA replication mechanism is conserved among the different domains of life, modifications in functionality and in some of the specialized replication proteins are observed. Indeed, Archaea possess specific features unique to this domain. Moreover, even though the general pattern of the replicative system is the same in all archaea, a great deal of variation exists between specific groups.

  12. An integrated chemical biology approach reveals the mechanism of action of HIV replication inhibitors.

    Science.gov (United States)

    Pagano, Nicholas; Teriete, Peter; Mattmann, Margrith E; Yang, Li; Snyder, Beth A; Cai, Zhaohui; Heil, Marintha L; Cosford, Nicholas D P

    2017-12-01

    Continuous flow (microfluidic) chemistry was employed to prepare a small focused library of dihydropyrimidinone (DHPM) derivatives. Compounds in this class have been reported to exhibit activity against the human immunodeficiency virus (HIV), but their molecular target had not been identified. We tested the initial set of DHPMs in phenotypic assays providing a hit (1i) that inhibited the replication of the human immunodeficiency virus HIV in cells. Flow chemistry-driven optimization of 1i led to the identification of HIV replication inhibitors such as 1l with cellular potency comparable with the clinical drug nevirapine (NVP). Mechanism of action (MOA) studies using cellular and biochemical assays coupled with 3D fingerprinting and in silico modeling demonstrated that these drug-like probe compounds exert their effects by inhibiting the viral reverse transcriptase polymerase (RT). This led to the design and synthesis of the novel DHPM 1at that inhibits the replication of drug resistant strains of HIV. Our work demonstrates that combining flow chemistry-driven analogue refinement with phenotypic assays, in silico modeling and MOA studies is a highly effective strategy for hit-to-lead optimization applicable to the discovery of future therapeutic agents. Copyright © 2017. Published by Elsevier Ltd.

  13. Deficiency of the Arabidopsis helicase RTEL1 triggers a SOG1-dependent replication checkpoint in response to DNA cross-links.

    Science.gov (United States)

    Hu, Zhubing; Cools, Toon; Kalhorzadeh, Pooneh; Heyman, Jefri; De Veylder, Lieven

    2015-01-01

    To maintain genome integrity, DNA replication is executed and regulated by a complex molecular network of numerous proteins, including helicases and cell cycle checkpoint regulators. Through a systematic screening for putative replication mutants, we identified an Arabidopsis thaliana homolog of human Regulator of Telomere Length 1 (RTEL1), which functions in DNA replication, DNA repair, and recombination. RTEL1 deficiency retards plant growth, a phenotype including a prolonged S-phase duration and decreased cell proliferation. Genetic analysis revealed that rtel1 mutant plants show activated cell cycle checkpoints, specific sensitivity to DNA cross-linking agents, and increased homologous recombination, but a lack of progressive shortening of telomeres, indicating that RTEL1 functions have only been partially conserved between mammals and plants. Surprisingly, RTEL1 deficiency induces tolerance to the deoxynucleotide-depleting drug hydroxyurea, which could be mimicked by DNA cross-linking agents. This resistance does not rely on the essential replication checkpoint regulator WEE1 but could be blocked by a mutation in the SOG1 transcription factor. Taken together, our data indicate that RTEL1 is required for DNA replication and that its deficiency activates a SOG1-dependent replication checkpoint. © 2015 American Society of Plant Biologists. All rights reserved.

  14. Ultrastructural Characterization of Zika Virus Replication Factories

    Directory of Open Access Journals (Sweden)

    Mirko Cortese

    2017-02-01

    Full Text Available Summary: A global concern has emerged with the pandemic spread of Zika virus (ZIKV infections that can cause severe neurological symptoms in adults and newborns. ZIKV is a positive-strand RNA virus replicating in virus-induced membranous replication factories (RFs. Here we used various imaging techniques to investigate the ultrastructural details of ZIKV RFs and their relationship with host cell organelles. Analyses of human hepatic cells and neural progenitor cells infected with ZIKV revealed endoplasmic reticulum (ER membrane invaginations containing pore-like openings toward the cytosol, reminiscent to RFs in Dengue virus-infected cells. Both the MR766 African strain and the H/PF/2013 Asian strain, the latter linked to neurological diseases, induce RFs of similar architecture. Importantly, ZIKV infection causes a drastic reorganization of microtubules and intermediate filaments forming cage-like structures surrounding the viral RF. Consistently, ZIKV replication is suppressed by cytoskeleton-targeting drugs. Thus, ZIKV RFs are tightly linked to rearrangements of the host cell cytoskeleton. : Cortese et al. show that ZIKV infection in both human hepatoma and neuronal progenitor cells induces drastic structural modification of the cellular architecture. Microtubules and intermediate filaments surround the viral replication factory composed of vesicles corresponding to ER membrane invagination toward the ER lumen. Importantly, alteration of microtubule flexibility impairs ZIKV replication. Keywords: Zika virus, flavivirus, human neural progenitor cells, replication factories, replication organelles, microtubules, intermediate filaments, electron microscopy, electron tomography, live-cell imaging

  15. Replication stress interferes with histone recycling and predeposition marking of new histones

    DEFF Research Database (Denmark)

    Jasencakova, Zuzana; Scharf, Annette N D; Ask, Katrine

    2010-01-01

    To restore chromatin on new DNA during replication, recycling of histones evicted ahead of the fork is combined with new histone deposition. The Asf1 histone chaperone, which buffers excess histones under stress, is a key player in this process. Yet how histones handled by human Asf1 are modified...... remains unclear. Here we identify marks on histones H3-H4 bound to Asf1 and changes induced upon replication stress. In S phase, distinct cytosolic and nuclear Asf1b complexes show ubiquitous H4K5K12diAc and heterogeneous H3 marks, including K9me1, K14ac, K18ac, and K56ac. Upon acute replication arrest......, the predeposition mark H3K9me1 and modifications typical of chromatin accumulate in Asf1 complexes. In parallel, ssDNA is generated at replication sites, consistent with evicted histones being trapped with Asf1. During recovery, histones stored with Asf1 are rapidly used as replication resumes. This shows...

  16. Rv0004 is a new essential member of the mycobacterial DNA replication machinery.

    Science.gov (United States)

    Mann, Katherine M; Huang, Deborah L; Hooppaw, Anna J; Logsdon, Michelle M; Richardson, Kirill; Lee, Hark Joon; Kimmey, Jacqueline M; Aldridge, Bree B; Stallings, Christina L

    2017-11-01

    DNA replication is fundamental for life, yet a detailed understanding of bacterial DNA replication is limited outside the organisms Escherichia coli and Bacillus subtilis. Many bacteria, including mycobacteria, encode no identified homologs of helicase loaders or regulators of the initiator protein DnaA, despite these factors being essential for DNA replication in E. coli and B. subtilis. In this study we discover that a previously uncharacterized protein, Rv0004, from the human pathogen Mycobacterium tuberculosis is essential for bacterial viability and that depletion of Rv0004 leads to a block in cell cycle progression. Using a combination of genetic and biochemical approaches, we found that Rv0004 has a role in DNA replication, interacts with DNA and the replicative helicase DnaB, and affects DnaB-DnaA complex formation. We also identify a conserved domain in Rv0004 that is predicted to structurally resemble the N-terminal protein-protein interaction domain of DnaA. Mutation of a single conserved tryptophan within Rv0004's DnaA N-terminal-like domain leads to phenotypes similar to those observed upon Rv0004 depletion and can affect the association of Rv0004 with DnaB. In addition, using live cell imaging during depletion of Rv0004, we have uncovered a previously unappreciated role for DNA replication in coordinating mycobacterial cell division and cell size. Together, our data support that Rv0004 encodes a homolog of the recently identified DciA family of proteins found in most bacteria that lack the DnaC-DnaI helicase loaders in E. coli and B. subtilis. Therefore, the mechanisms of Rv0004 elucidated here likely apply to other DciA homologs and reveal insight into the diversity of bacterial strategies in even the most conserved biological processes.

  17. Chromatin replication and histone dynamics

    DEFF Research Database (Denmark)

    Alabert, Constance; Jasencakova, Zuzana; Groth, Anja

    2017-01-01

    Inheritance of the DNA sequence and its proper organization into chromatin is fundamental for genome stability and function. Therefore, how specific chromatin structures are restored on newly synthesized DNA and transmitted through cell division remains a central question to understand cell fate...... choices and self-renewal. Propagation of genetic information and chromatin-based information in cycling cells entails genome-wide disruption and restoration of chromatin, coupled with faithful replication of DNA. In this chapter, we describe how cells duplicate the genome while maintaining its proper...... organization into chromatin. We reveal how specialized replication-coupled mechanisms rapidly assemble newly synthesized DNA into nucleosomes, while the complete restoration of chromatin organization including histone marks is a continuous process taking place throughout the cell cycle. Because failure...

  18. Insulated hsp70B' promoter: stringent heat-inducible activity in replication-deficient, but not replication-competent adenoviruses.

    Science.gov (United States)

    Rohmer, Stanimira; Mainka, Astrid; Knippertz, Ilka; Hesse, Andrea; Nettelbeck, Dirk M

    2008-04-01

    Key to the realization of gene therapy is the development of efficient and targeted gene transfer vectors. Therapeutic gene transfer by replication-deficient or more recently by conditionally replication-competent/oncolytic adenoviruses has shown much promise. For specific applications, however, it will be advantageous to provide vectors that allow for external control of gene expression. The efficient cellular heat shock system in combination with available technology for focused and controlled hyperthermia suggests heat-regulated transcription control as a promising tool for this purpose. We investigated the feasibility of a short fragment of the human hsp70B' promoter, with and without upstream insulator elements, for the regulation of transgene expression by replication-deficient or oncolytic adenoviruses. Two novel adenoviral vectors with an insulated hsp70B' promoter were developed and showed stringent heat-inducible gene expression with induction ratios up to 8000-fold. In contrast, regulation of gene expression from the hsp70B' promoter without insulation was suboptimal. In replication-competent/oncolytic adenoviruses regulation of the hsp70B' promoter was lost specifically during late replication in permissive cells and could not be restored by the insulators. We developed novel adenovirus gene transfer vectors that feature improved and stringent regulation of transgene expression from the hsp70B' promoter using promoter insulation. These vectors have potential for gene therapy applications that benefit from external modulation of therapeutic gene expression or for combination therapy with hyperthermia. Furthermore, our study reveals that vector replication can deregulate inserted cellular promoters, an observation which is of relevance for the development of replication-competent/oncolytic gene transfer vectors. (c) 2008 John Wiley & Sons, Ltd.

  19. Combining complexity measures of EEG data: multiplying measures reveal previously hidden information.

    Science.gov (United States)

    Burns, Thomas; Rajan, Ramesh

    2015-01-01

    Many studies have noted significant differences among human electroencephalograph (EEG) results when participants or patients are exposed to different stimuli, undertaking different tasks, or being affected by conditions such as epilepsy or Alzheimer's disease. Such studies often use only one or two measures of complexity and do not regularly justify their choice of measure beyond the fact that it has been used in previous studies. If more measures were added to such studies, however, more complete information might be found about these reported differences. Such information might be useful in confirming the existence or extent of such differences, or in understanding their physiological bases. In this study we analysed publically-available EEG data using a range of complexity measures to determine how well the measures correlated with one another. The complexity measures did not all significantly correlate, suggesting that different measures were measuring unique features of the EEG signals and thus revealing information which other measures were unable to detect. Therefore, the results from this analysis suggests that combinations of complexity measures reveal unique information which is in addition to the information captured by other measures of complexity in EEG data. For this reason, researchers using individual complexity measures for EEG data should consider using combinations of measures to more completely account for any differences they observe and to ensure the robustness of any relationships identified.

  20. Distributional Replication

    OpenAIRE

    Beare, Brendan K.

    2009-01-01

    Suppose that X and Y are random variables. We define a replicating function to be a function f such that f(X) and Y have the same distribution. In general, the set of replicating functions for a given pair of random variables may be infinite. Suppose we have some objective function, or cost function, defined over the set of replicating functions, and we seek to estimate the replicating function with the lowest cost. We develop an approach to estimating the cheapest replicating function that i...

  1. Structural organization of poliovirus RNA replication is mediated by viral proteins of the P2 genomic region

    International Nuclear Information System (INIS)

    Bienz, K.; Egger, D.; Troxler, M.; Pasamontes, L.

    1990-01-01

    Transcriptionally active replication complexes bound to smooth membrane vesicles were isolated from poliovirus-infected cells. In electron microscopic, negatively stained preparations, the replication complex appeared as an irregularly shaped, oblong structure attached to several virus-induced vesicles of a rosettelike arrangement. Electron microscopic immunocytochemistry of such preparations demonstrated that the poliovirus replication complex contains the proteins coded by the P2 genomic region (P2 proteins) in a membrane-associated form. In addition, the P2 proteins are also associated with viral RNA, and they can be cross-linked to viral RNA by UV irradiation. Guanidine hydrochloride prevented the P2 proteins from becoming membrane bound but did not change their association with viral RNA. The findings allow the conclusion that the protein 2C or 2C-containing precursor(s) is responsible for the attachment of the viral RNA to the vesicular membrane and for the spatial organization of the replication complex necessary for its proper functioning in viral transcription. A model for the structure of the viral replication complex and for the function of the 2C-containing P2 protein(s) and the vesicular membranes is proposed

  2. Replication Catastrophe

    DEFF Research Database (Denmark)

    Toledo, Luis; Neelsen, Kai John; Lukas, Jiri

    2017-01-01

    Proliferating cells rely on the so-called DNA replication checkpoint to ensure orderly completion of genome duplication, and its malfunction may lead to catastrophic genome disruption, including unscheduled firing of replication origins, stalling and collapse of replication forks, massive DNA...... breakage, and, ultimately, cell death. Despite many years of intensive research into the molecular underpinnings of the eukaryotic replication checkpoint, the mechanisms underlying the dismal consequences of its failure remain enigmatic. A recent development offers a unifying model in which the replication...... checkpoint guards against global exhaustion of rate-limiting replication regulators. Here we discuss how such a mechanism can prevent catastrophic genome disruption and suggest how to harness this knowledge to advance therapeutic strategies to eliminate cancer cells that inherently proliferate under...

  3. Host ESCRT proteins are required for bromovirus RNA replication compartment assembly and function.

    Directory of Open Access Journals (Sweden)

    Arturo Diaz

    2015-03-01

    Full Text Available Positive-strand RNA viruses genome replication invariably is associated with vesicles or other rearranged cellular membranes. Brome mosaic virus (BMV RNA replication occurs on perinuclear endoplasmic reticulum (ER membranes in ~70 nm vesicular invaginations (spherules. BMV RNA replication vesicles show multiple parallels with membrane-enveloped, budding retrovirus virions, whose envelopment and release depend on the host ESCRT (endosomal sorting complexes required for transport membrane-remodeling machinery. We now find that deleting components of the ESCRT pathway results in at least two distinct BMV phenotypes. One group of genes regulate RNA replication and the frequency of viral replication complex formation, but had no effect on spherule size, while a second group of genes regulate RNA replication in a way or ways independent of spherule formation. In particular, deleting SNF7 inhibits BMV RNA replication > 25-fold and abolishes detectable BMV spherule formation, even though the BMV RNA replication proteins accumulate and localize normally on perinuclear ER membranes. Moreover, BMV ESCRT recruitment and spherule assembly depend on different sets of protein-protein interactions from those used by multivesicular body vesicles, HIV-1 virion budding, or tomato bushy stunt virus (TBSV spherule formation. These and other data demonstrate that BMV requires cellular ESCRT components for proper formation and function of its vesicular RNA replication compartments. The results highlight growing but diverse interactions of ESCRT factors with many viruses and viral processes, and potential value of the ESCRT pathway as a target for broad-spectrum antiviral resistance.

  4. Genes and sequences involved in the replication of cowpea mosaic virus RNAs

    NARCIS (Netherlands)

    Eggen, R.

    1989-01-01

    The aim of the studies described in this thesis was to gain more insight in the complex molecular mechanisms underlying the RNA replication of the cowpea mosaic virus genome. Previously the replication of CPMV RNA has been examined extensively with crude membrane fractions prepared from

  5. Production of bioactive soluble interleukin-15 in complex with interleukin-15 receptor alpha from a conditionally-replicating oncolytic HSV-1.

    Directory of Open Access Journals (Sweden)

    David C Gaston

    Full Text Available Oncolytic type-1 herpes simplex viruses (oHSVs lacking the γ134.5 neurovirulence gene are being evaluated for treatment of a variety of malignancies. oHSVs replicate within and directly kill permissive cancer cells. To augment their anti-tumor activity, oHSVs have been engineered to express immunostimulatory molecules, including cytokines, to elicit tumor-specific immune responses. Interleukin-15 (IL-15 holds potential as an immunotherapeutic cytokine because it has been demonstrated to promote both natural killer (NK cell-mediated and CD8(+ T cell-mediated cytotoxicity against cancer cells. The purpose of these studies was to engineer an oHSV producing bioactive IL-15. Two oHSVs were constructed encoding murine (mIL-15 alone (J100 or with the mIL-15 receptor α (mIL-15Rα, J100D to determine whether co-expression of these proteins is required for production of bioactive mIL-15 from oHSV. The following were demonstrated: i both oHSVs retain replication competence and cytotoxicity in permissive tumor cell lines. ii Enhanced production of mIL-15 was detected in cell lysates of neuro-2a cells following J100D infection as compared to J100 infection, suggesting that mIL-15Rα improved mIL-15 production. iii Soluble mIL-15 in complex with mIL-15Rα was detected in supernates from J100D-infected, but not J100-infected, neuro-2a, GL261, and CT-2A cells. These cell lines vary in permissiveness to oHSV replication and cytotoxicity, demonstrating soluble mIL-15/IL-15Rα complex production from J100D was independent of direct oHSV effects. iv The soluble mIL-15/IL-15Rα complex produced by J100D was bioactive, stimulating NK cells to proliferate and reduce the viability of syngeneic GL261 and CT-2A cells. v J100 and J100D were aneurovirulent inasmuch as no neuropathologic effects were documented following direct inoculation into brains of CBA/J mice at up to 1x10(7 plaque forming units. The production of mIL-15/mIL-15Rα from multiple tumor lines, as well

  6. Involvement of Autophagy in Coronavirus Replication

    Directory of Open Access Journals (Sweden)

    Paul Britton

    2012-11-01

    Full Text Available Coronaviruses are single stranded, positive sense RNA viruses, which induce the rearrangement of cellular membranes upon infection of a host cell. This provides the virus with a platform for the assembly of viral replication complexes, improving efficiency of RNA synthesis. The membranes observed in coronavirus infected cells include double membrane vesicles. By nature of their double membrane, these vesicles resemble cellular autophagosomes, generated during the cellular autophagy pathway. In addition, coronavirus infection has been demonstrated to induce autophagy. Here we review current knowledge of coronavirus induced membrane rearrangements and the involvement of autophagy or autophagy protein microtubule associated protein 1B light chain 3 (LC3 in coronavirus replication.

  7. p53 Maintains Genomic Stability by Preventing Interference between Transcription and Replication

    Directory of Open Access Journals (Sweden)

    Constance Qiao Xin Yeo

    2016-04-01

    Full Text Available p53 tumor suppressor maintains genomic stability, typically acting through cell-cycle arrest, senescence, and apoptosis. We discovered a function of p53 in preventing conflicts between transcription and replication, independent of its canonical roles. p53 deficiency sensitizes cells to Topoisomerase (Topo II inhibitors, resulting in DNA damage arising spontaneously during replication. Topoisomerase IIα (TOP2A-DNA complexes preferentially accumulate in isogenic p53 mutant or knockout cells, reflecting an increased recruitment of TOP2A to regulate DNA topology. We propose that p53 acts to prevent DNA topological stress originating from transcription during the S phase and, therefore, promotes normal replication fork progression. Consequently, replication fork progression is impaired in the absence of p53, which is reversed by transcription inhibition. Pharmacologic inhibition of transcription also attenuates DNA damage and decreases Topo-II-DNA complexes, restoring cell viability in p53-deficient cells. Together, our results demonstrate a function of p53 that may underlie its role in tumor suppression.

  8. Database Replication Prototype

    OpenAIRE

    Vandewall, R.

    2000-01-01

    This report describes the design of a Replication Framework that facilitates the implementation and com-parison of database replication techniques. Furthermore, it discusses the implementation of a Database Replication Prototype and compares the performance measurements of two replication techniques based on the Atomic Broadcast communication primitive: pessimistic active replication and optimistic active replication. The main contributions of this report can be split into four parts....

  9. UGGT1 enhances enterovirus 71 pathogenicity by promoting viral RNA synthesis and viral replication.

    Directory of Open Access Journals (Sweden)

    Peng-Nien Huang

    2017-05-01

    Full Text Available Positive-strand RNA virus infections can induce the stress-related unfolded protein response (UPR in host cells. This study found that enterovirus A71 (EVA71 utilizes host UDP-glucose glycoprotein glucosyltransferase 1 (UGGT1, a key endoplasmic reticulum protein (ER involved in UPR, to enhance viral replication and virulence. EVA71 forms replication complexes (RCs on cellular membranes that contain a mix of host and viral proteins to facilitate viral replication, but the components and processes involved in the assembly and function of RCs are not fully understood. Using EVA71 as a model, this study found that host UGGT1 and viral 3D polymerase co-precipitate along with other factors on membranous replication complexes to enhance viral replication. Increased UGGT1 levels elevated viral growth rates, while viral pathogenicity was observed to be lower in heterozygous knockout mice (Uggt1 +/- mice. These findings provide important insight on the role of UPR and host UGGT1 in regulating RNA virus replication and pathogenicity.

  10. Meta-replication reveals nonstationarity in multi-scale habitat selection of Mexican Spotted Owl

    Science.gov (United States)

    Ho Yi Wan; Kevin McGarigal; Joseph L. Ganey; Valentin Lauret; Brad C. Timm; Samuel A. Cushman

    2017-01-01

    Anthropogenic environmental changes are leading to habitat loss and degradation, driving many species to extinction. In this context, habitat models become increasingly important for effective species management and conservation. However, most habitat studies lack replicated study areas and do not properly address the role of nonstationarity and spatial scales in...

  11. A systematic analysis of host factors reveals a Med23-interferon-λ regulatory axis against herpes simplex virus type 1 replication.

    Science.gov (United States)

    Griffiths, Samantha J; Koegl, Manfred; Boutell, Chris; Zenner, Helen L; Crump, Colin M; Pica, Francesca; Gonzalez, Orland; Friedel, Caroline C; Barry, Gerald; Martin, Kim; Craigon, Marie H; Chen, Rui; Kaza, Lakshmi N; Fossum, Even; Fazakerley, John K; Efstathiou, Stacey; Volpi, Antonio; Zimmer, Ralf; Ghazal, Peter; Haas, Jürgen

    2013-01-01

    Herpes simplex virus type 1 (HSV-1) is a neurotropic virus causing vesicular oral or genital skin lesions, meningitis and other diseases particularly harmful in immunocompromised individuals. To comprehensively investigate the complex interaction between HSV-1 and its host we combined two genome-scale screens for host factors (HFs) involved in virus replication. A yeast two-hybrid screen for protein interactions and a RNA interference (RNAi) screen with a druggable genome small interfering RNA (siRNA) library confirmed existing and identified novel HFs which functionally influence HSV-1 infection. Bioinformatic analyses found the 358 HFs were enriched for several pathways and multi-protein complexes. Of particular interest was the identification of Med23 as a strongly anti-viral component of the largely pro-viral Mediator complex, which links specific transcription factors to RNA polymerase II. The anti-viral effect of Med23 on HSV-1 replication was confirmed in gain-of-function gene overexpression experiments, and this inhibitory effect was specific to HSV-1, as a range of other viruses including Vaccinia virus and Semliki Forest virus were unaffected by Med23 depletion. We found Med23 significantly upregulated expression of the type III interferon family (IFN-λ) at the mRNA and protein level by directly interacting with the transcription factor IRF7. The synergistic effect of Med23 and IRF7 on IFN-λ induction suggests this is the major transcription factor for IFN-λ expression. Genotypic analysis of patients suffering recurrent orofacial HSV-1 outbreaks, previously shown to be deficient in IFN-λ secretion, found a significant correlation with a single nucleotide polymorphism in the IFN-λ3 (IL28b) promoter strongly linked to Hepatitis C disease and treatment outcome. This paper describes a link between Med23 and IFN-λ, provides evidence for the crucial role of IFN-λ in HSV-1 immune control, and highlights the power of integrative genome-scale approaches to

  12. A systematic analysis of host factors reveals a Med23-interferon-λ regulatory axis against herpes simplex virus type 1 replication.

    Directory of Open Access Journals (Sweden)

    Samantha J Griffiths

    Full Text Available Herpes simplex virus type 1 (HSV-1 is a neurotropic virus causing vesicular oral or genital skin lesions, meningitis and other diseases particularly harmful in immunocompromised individuals. To comprehensively investigate the complex interaction between HSV-1 and its host we combined two genome-scale screens for host factors (HFs involved in virus replication. A yeast two-hybrid screen for protein interactions and a RNA interference (RNAi screen with a druggable genome small interfering RNA (siRNA library confirmed existing and identified novel HFs which functionally influence HSV-1 infection. Bioinformatic analyses found the 358 HFs were enriched for several pathways and multi-protein complexes. Of particular interest was the identification of Med23 as a strongly anti-viral component of the largely pro-viral Mediator complex, which links specific transcription factors to RNA polymerase II. The anti-viral effect of Med23 on HSV-1 replication was confirmed in gain-of-function gene overexpression experiments, and this inhibitory effect was specific to HSV-1, as a range of other viruses including Vaccinia virus and Semliki Forest virus were unaffected by Med23 depletion. We found Med23 significantly upregulated expression of the type III interferon family (IFN-λ at the mRNA and protein level by directly interacting with the transcription factor IRF7. The synergistic effect of Med23 and IRF7 on IFN-λ induction suggests this is the major transcription factor for IFN-λ expression. Genotypic analysis of patients suffering recurrent orofacial HSV-1 outbreaks, previously shown to be deficient in IFN-λ secretion, found a significant correlation with a single nucleotide polymorphism in the IFN-λ3 (IL28b promoter strongly linked to Hepatitis C disease and treatment outcome. This paper describes a link between Med23 and IFN-λ, provides evidence for the crucial role of IFN-λ in HSV-1 immune control, and highlights the power of integrative genome

  13. Two subunits of human ORC are dispensable for DNA replication and proliferation.

    Science.gov (United States)

    Shibata, Etsuko; Kiran, Manjari; Shibata, Yoshiyuki; Singh, Samarendra; Kiran, Shashi; Dutta, Anindya

    2016-12-01

    The six-subunit Origin Recognition Complex (ORC) is believed to be an essential eukaryotic ATPase that binds to origins of replication as a ring-shaped heterohexamer to load MCM2-7 and initiate DNA replication. We have discovered that human cell lines in culture proliferate with intact chromosomal origins of replication after disruption of both alleles of ORC2 or of the ATPase subunit, ORC1 . The ORC1 or ORC2 -depleted cells replicate with decreased chromatin loading of MCM2-7 and become critically dependent on another ATPase, CDC6, for survival and DNA replication. Thus, either the ORC ring lacking a subunit, even its ATPase subunit, can load enough MCM2-7 in partnership with CDC6 to initiate DNA replication, or cells have an ORC-independent, CDC6-dependent mechanism to load MCM2-7 on origins of replication.

  14. Quantum coherence spectroscopy reveals complex dynamics in bacterial light-harvesting complex 2 (LH2).

    Science.gov (United States)

    Harel, Elad; Engel, Gregory S

    2012-01-17

    Light-harvesting antenna complexes transfer energy from sunlight to photosynthetic reaction centers where charge separation drives cellular metabolism. The process through which pigments transfer excitation energy involves a complex choreography of coherent and incoherent processes mediated by the surrounding protein and solvent environment. The recent discovery of coherent dynamics in photosynthetic light-harvesting antennae has motivated many theoretical models exploring effects of interference in energy transfer phenomena. In this work, we provide experimental evidence of long-lived quantum coherence between the spectrally separated B800 and B850 rings of the light-harvesting complex 2 (LH2) of purple bacteria. Spectrally resolved maps of the detuning, dephasing, and the amplitude of electronic coupling between excitons reveal that different relaxation pathways act in concert for optimal transfer efficiency. Furthermore, maps of the phase of the signal suggest that quantum mechanical interference between different energy transfer pathways may be important even at ambient temperature. Such interference at a product state has already been shown to enhance the quantum efficiency of transfer in theoretical models of closed loop systems such as LH2.

  15. Replication termination and chromosome dimer resolution in the archaeon Sulfolobus solfataricus.

    Science.gov (United States)

    Duggin, Iain G; Dubarry, Nelly; Bell, Stephen D

    2011-01-05

    Archaea of the genus Sulfolobus have a single-circular chromosome with three replication origins. All three origins fire in every cell in every cell cycle. Thus, three pairs of replication forks converge and terminate in each replication cycle. Here, we report 2D gel analyses of the replication fork fusion zones located between origins. These indicate that replication termination involves stochastic fork collision. In bacteria, replication termination is linked to chromosome dimer resolution, a process that requires the XerC and D recombinases, FtsK and the chromosomal dif site. Sulfolobus encodes a single-Xer homologue and its deletion gave rise to cells with aberrant DNA contents and increased volumes. Identification of the chromosomal dif site that binds Xer in vivo, and biochemical characterization of Xer/dif recombination revealed that, in contrast to bacteria, dif is located outside the fork fusion zones. Therefore, it appears that replication termination and dimer resolution are temporally and spatially distinct processes in Sulfolobus.

  16. G-Quadruplexes in DNA Replication: A Problem or a Necessity?

    Science.gov (United States)

    Valton, Anne-Laure; Prioleau, Marie-Noëlle

    2016-11-01

    DNA replication is a highly regulated process that ensures the correct duplication of the genome at each cell cycle. A precise cell type-specific temporal program controls the duplication of complex vertebrate genomes in an orderly manner. This program is based on the regulation of both replication origin firing and replication fork progression. G-quadruplexes (G4s), DNA secondary structures displaying noncanonical Watson-Crick base pairing, have recently emerged as key controllers of genome duplication. Here we discuss the various means by which G4s affect this fundamental cellular process. Copyright © 2016 Elsevier Ltd. All rights reserved.

  17. Calcein represses human papillomavirus 16 E1-E2 mediated DNA replication via blocking their binding to the viral origin of replication.

    Science.gov (United States)

    Das, Dipon; Smith, Nathan W; Wang, Xu; Richardson, Stacie L; Hartman, Matthew C T; Morgan, Iain M

    2017-08-01

    Human papillomaviruses are causative agents in several human diseases ranging from genital warts to ano-genital and oropharyngeal cancers. Currently only symptoms of HPV induced disease are treated; there are no antivirals available that directly target the viral life cycle. Previously, we determined that the cellular protein TopBP1 interacts with the HPV16 replication/transcription factor E2. This E2-TopBP1 interaction is essential for optimal E1-E2 DNA replication and for the viral life cycle. The drug calcein disrupts the interaction of TopBP1 with itself and other host proteins to promote cell death. Here we demonstrate that calcein blocks HPV16 E1-E2 DNA replication via blocking the viral replication complex forming at the origin of replication. This occurs at non-toxic levels of calcein and demonstrates specificity as it does not block the ability of E2 to regulate transcription. We propose that calcein or derivatives could be developed as an anti-HPV therapeutic. Copyright © 2017 Elsevier Inc. All rights reserved.

  18. DNA replication at the single-molecule level

    NARCIS (Netherlands)

    Stratmann, S.A.; Oijen, A.M. van

    2014-01-01

    A cell can be thought of as a highly sophisticated micro factory: in a pool of billions of molecules – metabolites, structural proteins, enzymes, oligonucleotides – multi-subunit complexes assemble to perform a large number of basic cellular tasks, such as DNA replication, RNA/protein synthesis or

  19. Proteomic analysis of HIV-1 Nef cellular binding partners reveals a role for exocyst complex proteins in mediating enhancement of intercellular nanotube formation

    Directory of Open Access Journals (Sweden)

    Mukerji Joya

    2012-06-01

    Full Text Available Abstract Background HIV-1 Nef protein contributes to pathogenesis via multiple functions that include enhancement of viral replication and infectivity, alteration of intracellular trafficking, and modulation of cellular signaling pathways. Nef stimulates formation of tunneling nanotubes and virological synapses, and is transferred to bystander cells via these intercellular contacts and secreted microvesicles. Nef associates with and activates Pak2, a kinase that regulates T-cell signaling and actin cytoskeleton dynamics, but how Nef promotes nanotube formation is unknown. Results To identify Nef binding partners involved in Pak2-association dependent Nef functions, we employed tandem mass spectrometry analysis of Nef immunocomplexes from Jurkat cells expressing wild-type Nef or Nef mutants defective for the ability to associate with Pak2 (F85L, F89H, H191F and A72P, A75P in NL4-3. We report that wild-type, but not mutant Nef, was associated with 5 components of the exocyst complex (EXOC1, EXOC2, EXOC3, EXOC4, and EXOC6, an octameric complex that tethers vesicles at the plasma membrane, regulates polarized exocytosis, and recruits membranes and proteins required for nanotube formation. Additionally, Pak2 kinase was associated exclusively with wild-type Nef. Association of EXOC1, EXOC2, EXOC3, and EXOC4 with wild-type, but not mutant Nef, was verified by co-immunoprecipitation assays in Jurkat cells. Furthermore, shRNA-mediated depletion of EXOC2 in Jurkat cells abrogated Nef-mediated enhancement of nanotube formation. Using bioinformatic tools, we visualized protein interaction networks that reveal functional linkages between Nef, the exocyst complex, and the cellular endocytic and exocytic trafficking machinery. Conclusions Exocyst complex proteins are likely a key effector of Nef-mediated enhancement of nanotube formation, and possibly microvesicle secretion. Linkages revealed between Nef and the exocyst complex suggest a new paradigm of

  20. Insights into the Initiation of Eukaryotic DNA Replication.

    Science.gov (United States)

    Bruck, Irina; Perez-Arnaiz, Patricia; Colbert, Max K; Kaplan, Daniel L

    2015-01-01

    The initiation of DNA replication is a highly regulated event in eukaryotic cells to ensure that the entire genome is copied once and only once during S phase. The primary target of cellular regulation of eukaryotic DNA replication initiation is the assembly and activation of the replication fork helicase, the 11-subunit assembly that unwinds DNA at a replication fork. The replication fork helicase, called CMG for Cdc45-Mcm2-7, and GINS, assembles in S phase from the constituent Cdc45, Mcm2-7, and GINS proteins. The assembly and activation of the CMG replication fork helicase during S phase is governed by 2 S-phase specific kinases, CDK and DDK. CDK stimulates the interaction between Sld2, Sld3, and Dpb11, 3 initiation factors that are each required for the initiation of DNA replication. DDK, on the other hand, phosphorylates the Mcm2, Mcm4, and Mcm6 subunits of the Mcm2-7 complex. Sld3 recruits Cdc45 to Mcm2-7 in a manner that depends on DDK, and recent work suggests that Sld3 binds directly to Mcm2-7 and also to single-stranded DNA. Furthermore, recent work demonstrates that Sld3 and its human homolog Treslin substantially stimulate DDK phosphorylation of Mcm2. These data suggest that the initiation factor Sld3/Treslin coordinates the assembly and activation of the eukaryotic replication fork helicase by recruiting Cdc45 to Mcm2-7, stimulating DDK phosphorylation of Mcm2, and binding directly to single-stranded DNA as the origin is melted.

  1. The Replication Recipe: What makes for a convincing replication?

    NARCIS (Netherlands)

    Brandt, M.J.; IJzerman, H.; Dijksterhuis, A.J.; Farach, F.J.; Geller, J.; Giner-Sorolla, R.; Grange, J.A.; Perugini, M.; Spies, J.R.; Veer, A. van 't

    2014-01-01

    Psychological scientists have recently started to reconsider the importance of close replications in building a cumulative knowledge base; however, there is no consensus about what constitutes a convincing close replication study. To facilitate convincing close replication attempts we have developed

  2. The replication recipe : What makes for a convincing replication?

    NARCIS (Netherlands)

    Brandt, M.J.; IJzerman, H.; Dijksterhuis, Ap; Farach, Frank J.; Geller, Jason; Giner-Sorolla, Roger; Grange, James A.; Perugini, Marco; Spies, Jeffrey R.; van 't Veer, Anna

    Psychological scientists have recently started to reconsider the importance of close replications in building a cumulative knowledge base; however, there is no consensus about what constitutes a convincing close replication study. To facilitate convincing close replication attempts we have developed

  3. Transition-state destabilization reveals how human DNA polymerase β proceeds across the chemically unstable lesion N7-methylguanine

    Science.gov (United States)

    Ouzon-Shubeita, Hala; Lee, Seongmin

    2014-01-01

    N7-Methyl-2′-deoxyguanosine (m7dG) is the predominant lesion formed by methylating agents. A systematic investigation on the effect of m7dG on DNA replication has been difficult due to the chemical instability of m7dG. To gain insights into the m7dG effect, we employed a 2′-fluorine-mediated transition-state destabilzation strategy. Specifically, we determined kinetic parameters for dCTP insertion opposite a chemically stable m7dG analogue, 2′-fluoro-m7dG (Fm7dG), by human DNA polymerase β (polβ) and solved three X-ray structures of polβ in complex with the templating Fm7dG paired with incoming dCTP or dTTP analogues. The kinetic studies reveal that the templating Fm7dG slows polβ catalysis ∼300-fold, suggesting that m7dG in genomic DNA may impede replication by some DNA polymerases. The structural analysis reveals that Fm7dG forms a canonical Watson–Crick base pair with dCTP, but metal ion coordination is suboptimal for catalysis in the polβ-Fm7dG:dCTP complex, which partially explains the slow insertion of dCTP opposite Fm7dG by polβ. In addition, the polβ-Fm7dG:dTTP structure shows open protein conformations and staggered base pair conformations, indicating that N7-methylation of dG does not promote a promutagenic replication. Overall, the first systematic studies on the effect of m7dG on DNA replication reveal that polβ catalysis across m7dG is slow, yet highly accurate. PMID:24966350

  4. Inhibition of Human Cytomegalovirus pUL89 Terminase Subunit Blocks Virus Replication and Genome Cleavage.

    Science.gov (United States)

    Wang, Yan; Mao, Lili; Kankanala, Jayakanth; Wang, Zhengqiang; Geraghty, Robert J

    2017-02-01

    The human cytomegalovirus terminase complex cleaves concatemeric genomic DNA into unit lengths during genome packaging and particle assembly. This process is an attractive drug target because cleavage of concatemeric DNA is not required in mammalian cell DNA replication, indicating that drugs targeting the terminase complex could be safe and selective. One component of the human cytomegalovirus terminase complex, pUL89, provides the endonucleolytic activity for genome cleavage, and the domain responsible is reported to have an RNase H-like fold. We hypothesize that the pUL89 endonuclease activity is inhibited by known RNase H inhibitors. Using a novel enzyme-linked immunosorbent assay (ELISA) format as a screening assay, we found that a hydroxypyridonecarboxylic acid compound, previously reported to be an inhibitor of human immunodeficiency virus RNase H, inhibited pUL89 endonuclease activity at low-micromolar concentrations. Further characterization revealed that this pUL89 endonuclease inhibitor blocked human cytomegalovirus replication at a relatively late time point, similarly to other reported terminase complex inhibitors. Importantly, this inhibitor also prevented the cleavage of viral genomic DNA in infected cells. Taken together, these results substantiate our pharmacophore hypothesis and validate our ligand-based approach toward identifying novel inhibitors of pUL89 endonuclease. Human cytomegalovirus infection in individuals lacking a fully functioning immune system, such as newborns and transplant patients, can have severe and debilitating consequences. The U.S. Food and Drug Administration-approved anti-human cytomegalovirus drugs mainly target the viral polymerase, and resistance to these drugs has appeared. Therefore, anti-human cytomegalovirus drugs from novel targets are needed for use instead of, or in combination with, current polymerase inhibitors. pUL89 is a viral ATPase and endonuclease and is an attractive target for anti-human cytomegalovirus

  5. Mutational analysis of the hypervariable region of hepatitis e virus reveals its involvement in the efficiency of viral RNA replication.

    Science.gov (United States)

    Pudupakam, R S; Kenney, Scott P; Córdoba, Laura; Huang, Yao-Wei; Dryman, Barbara A; Leroith, Tanya; Pierson, F William; Meng, Xiang-Jin

    2011-10-01

    The RNA genome of the hepatitis E virus (HEV) contains a hypervariable region (HVR) in ORF1 that tolerates small deletions with respect to infectivity. To further investigate the role of the HVR in HEV replication, we constructed a panel of mutants with overlapping deletions in the N-terminal, central, and C-terminal regions of the HVR by using a genotype 1 human HEV luciferase replicon and analyzed the effects of deletions on viral RNA replication in Huh7 cells. We found that the replication levels of the HVR deletion mutants were markedly reduced in Huh7 cells, suggesting a role of the HVR in viral replication efficiency. To further verify the results, we constructed HVR deletion mutants by using a genetically divergent, nonmammalian avian HEV, and similar effects on viral replication efficiency were observed when the avian HEV mutants were tested in LMH cells. Furthermore, the impact of complete HVR deletion on virus infectivity was tested in chickens, using an avian HEV mutant with a complete HVR deletion. Although the deletion mutant was still replication competent in LMH cells, the complete HVR deletion resulted in a loss of avian HEV infectivity in chickens. Since the HVR exhibits extensive variations in sequence and length among different HEV genotypes, we further examined the interchangeability of HVRs and demonstrated that HVR sequences are functionally exchangeable between HEV genotypes with regard to viral replication and infectivity in vitro, although genotype-specific HVR differences in replication efficiency were observed. The results showed that although the HVR tolerates small deletions with regard to infectivity, it may interact with viral and host factors to modulate the efficiency of HEV replication.

  6. Protein-carbohydrate complex reveals circulating metastatic cells in a microfluidic assay

    KAUST Repository

    Simone, Giuseppina

    2013-02-11

    Advances in carbohydrate sequencing technologies reveal the tremendous complexity of the glycome and the role that glycomics might have to bring insight into the biological functions. Carbohydrate-protein interactions, in particular, are known to be crucial to most mammalian physiological processes as mediators of cell adhesion and metastasis, signal transducers, and organizers of protein interactions. An assay is developed here to mimic the multivalency of biological complexes that selectively and sensitively detect carbohydrate-protein interactions. The binding of β-galactosides and galectin-3 - a protein that is correlated to the progress of tumor and metastasis - is examined. The efficiency of the assay is related to the expression of the receptor while anchoring to the interaction\\'s strength. Comparative binding experiments reveal molecular binding preferences. This study establishes that the assay is robust to isolate metastatic cells from colon affected patients and paves the way to personalized medicine. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  7. Protein-carbohydrate complex reveals circulating metastatic cells in a microfluidic assay

    KAUST Repository

    Simone, Giuseppina; Malara, Natalia Maria; Trunzo, Valentina; Perozziello, Gerardo; Neužil, Pavel; Francardi, Marco; Roveda, Laura; Renne, Maria; Prati, Ubaldo; Mollace, Vincenzo; Manz, Andreas; Di Fabrizio, Enzo M.

    2013-01-01

    Advances in carbohydrate sequencing technologies reveal the tremendous complexity of the glycome and the role that glycomics might have to bring insight into the biological functions. Carbohydrate-protein interactions, in particular, are known to be crucial to most mammalian physiological processes as mediators of cell adhesion and metastasis, signal transducers, and organizers of protein interactions. An assay is developed here to mimic the multivalency of biological complexes that selectively and sensitively detect carbohydrate-protein interactions. The binding of β-galactosides and galectin-3 - a protein that is correlated to the progress of tumor and metastasis - is examined. The efficiency of the assay is related to the expression of the receptor while anchoring to the interaction's strength. Comparative binding experiments reveal molecular binding preferences. This study establishes that the assay is robust to isolate metastatic cells from colon affected patients and paves the way to personalized medicine. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  8. Symmetry of interactions rules in incompletely connected random replicator ecosystems.

    Science.gov (United States)

    Kärenlampi, Petri P

    2014-06-01

    The evolution of an incompletely connected system of species with speciation and extinction is investigated in terms of random replicators. It is found that evolving random replicator systems with speciation do become large and complex, depending on speciation parameters. Antisymmetric interactions result in large systems, whereas systems with symmetric interactions remain small. A co-dominating feature is within-species interaction pressure: large within-species interaction increases species diversity. Average fitness evolves in all systems, however symmetry and connectivity evolve in small systems only. Newcomers get extinct almost immediately in symmetric systems. The distribution in species lifetimes is determined for antisymmetric systems. The replicator systems investigated do not show any sign of self-organized criticality. The generalized Lotka-Volterra system is shown to be a tedious way of implementing the replicator system.

  9. Characterization of RyDEN (C19orf66 as an Interferon-Stimulated Cellular Inhibitor against Dengue Virus Replication.

    Directory of Open Access Journals (Sweden)

    Youichi Suzuki

    2016-01-01

    Full Text Available Dengue virus (DENV is one of the most important arthropod-borne pathogens that cause life-threatening diseases in humans. However, no vaccine or specific antiviral is available for dengue. As seen in other RNA viruses, the innate immune system plays a key role in controlling DENV infection and disease outcome. Although the interferon (IFN response, which is central to host protective immunity, has been reported to limit DENV replication, the molecular details of how DENV infection is modulated by IFN treatment are elusive. In this study, by employing a gain-of-function screen using a type I IFN-treated cell-derived cDNA library, we identified a previously uncharacterized gene, C19orf66, as an IFN-stimulated gene (ISG that inhibits DENV replication, which we named Repressor of yield of DENV (RyDEN. Overexpression and gene knockdown experiments revealed that expression of RyDEN confers resistance to all serotypes of DENV in human cells. RyDEN expression also limited the replication of hepatitis C virus, Kunjin virus, Chikungunya virus, herpes simplex virus type 1, and human adenovirus. Importantly, RyDEN was considered to be a crucial effector molecule in the IFN-mediated anti-DENV response. When affinity purification-mass spectrometry analysis was performed, RyDEN was revealed to form a complex with cellular mRNA-binding proteins, poly(A-binding protein cytoplasmic 1 (PABPC1, and La motif-related protein 1 (LARP1. Interestingly, PABPC1 and LARP1 were found to be positive modulators of DENV replication. Since RyDEN influenced intracellular events on DENV replication and, suppression of protein synthesis from DENV-based reporter construct RNA was also observed in RyDEN-expressing cells, our data suggest that RyDEN is likely to interfere with the translation of DENV via interaction with viral RNA and cellular mRNA-binding proteins, resulting in the inhibition of virus replication in infected cells.

  10. High-fidelity DNA replication in Mycobacterium tuberculosis relies on a trinuclear zinc center.

    Science.gov (United States)

    Baños-Mateos, Soledad; van Roon, Anne-Marie M; Lang, Ulla F; Maslen, Sarah L; Skehel, J Mark; Lamers, Meindert H

    2017-10-11

    High-fidelity DNA replication depends on a proofreading 3'-5' exonuclease that is associated with the replicative DNA polymerase. The replicative DNA polymerase DnaE1 from the major pathogen Mycobacterium tuberculosis (Mtb) uses its intrinsic PHP-exonuclease that is distinct from the canonical DEDD exonucleases found in the Escherichia coli and eukaryotic replisomes. The mechanism of the PHP-exonuclease is not known. Here, we present the crystal structure of the Mtb DnaE1 polymerase. The PHP-exonuclease has a trinuclear zinc center, coordinated by nine conserved residues. Cryo-EM analysis reveals the entry path of the primer strand in the PHP-exonuclease active site. Furthermore, the PHP-exonuclease shows a striking similarity to E. coli endonuclease IV, which provides clues regarding the mechanism of action. Altogether, this work provides important insights into the PHP-exonuclease and reveals unique properties that make it an attractive target for novel anti-mycobacterial drugs.The polymerase and histidinol phosphatase (PHP) domain in the DNA polymerase DnaE1 is essential for mycobacterial high-fidelity DNA replication. Here, the authors determine the DnaE1 crystal structure, which reveals the PHP-exonuclease mechanism that can be exploited for antibiotic development.

  11. Initiation of DNA replication requires actin dynamics and formin activity.

    Science.gov (United States)

    Parisis, Nikolaos; Krasinska, Liliana; Harker, Bethany; Urbach, Serge; Rossignol, Michel; Camasses, Alain; Dewar, James; Morin, Nathalie; Fisher, Daniel

    2017-11-02

    Nuclear actin regulates transcriptional programmes in a manner dependent on its levels and polymerisation state. This dynamics is determined by the balance of nucleocytoplasmic shuttling, formin- and redox-dependent filament polymerisation. Here, using Xenopus egg extracts and human somatic cells, we show that actin dynamics and formins are essential for DNA replication. In proliferating cells, formin inhibition abolishes nuclear transport and initiation of DNA replication, as well as general transcription. In replicating nuclei from transcriptionally silent Xenopus egg extracts, we identified numerous actin regulators, and disruption of actin dynamics abrogates nuclear transport, preventing NLS (nuclear localisation signal)-cargo release from RanGTP-importin complexes. Nuclear formin activity is further required to promote loading of cyclin-dependent kinase (CDK) and proliferating cell nuclear antigen (PCNA) onto chromatin, as well as initiation and elongation of DNA replication. Therefore, actin dynamics and formins control DNA replication by multiple direct and indirect mechanisms. © 2017 The Authors.

  12. Mouse Norovirus infection promotes autophagy induction to facilitate replication but prevents final autophagosome maturation

    Energy Technology Data Exchange (ETDEWEB)

    O’Donnell, Tanya B. [Department of Microbiology and Immunology, at the Peter Doherty Institute for Infection and Immunity, University of Melbourne, Melbourne 3010 (Australia); Hyde, Jennifer L. [School of Chemical and Biological Sciences, University of Queensland, St. Lucia, Brisbane, Queensland 4072 (Australia); Mintern, Justine D. [Department of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Melbourne 3010 (Australia); Mackenzie, Jason M., E-mail: jason.mackenzie@unimelb.edu.au [Department of Microbiology and Immunology, at the Peter Doherty Institute for Infection and Immunity, University of Melbourne, Melbourne 3010 (Australia)

    2016-05-15

    Autophagy is a cellular process used to eliminate intracellular pathogens. Many viruses however are able to manipulate this cellular process for their own advantage. Here we demonstrate that Mouse Norovirus (MNV) infection induces autophagy but does not appear to utilise the autophagosomal membrane for establishment and formation of the viral replication complex. We have observed that MNV infection results in lipidation and recruitment of LC3 to the autophagosome membrane but prevents subsequent fusion of the autophagosomes with lysosomes, as SQSTM1 (an autophagy receptor) accumulates and Lysosome-Associated Membrane Protein1 is sequestered to the MNV replication complex (RC) rather than to autophagosomes. We have additionally observed that chemical modulation of autophagy differentially affects MNV replication. From this study we can conclude that MNV infection induces autophagy, however suppresses the final maturation step of this response, indicating that autophagy induction contributes to MNV replication independently of RC biogenesis. - Highlights: • MNV induces autophagy in infected murine macrophages. • MNV does not utilise autophagosomal membranes for replication. • The MNV-induced autophagosomes do not fuse with lysosomes. • MNV sequesters SQSTM1 to prevent autophagy degradation and turnover. • Chemical modulation of autophagy enhances MNV replication.

  13. Mouse Norovirus infection promotes autophagy induction to facilitate replication but prevents final autophagosome maturation

    International Nuclear Information System (INIS)

    O’Donnell, Tanya B.; Hyde, Jennifer L.; Mintern, Justine D.; Mackenzie, Jason M.

    2016-01-01

    Autophagy is a cellular process used to eliminate intracellular pathogens. Many viruses however are able to manipulate this cellular process for their own advantage. Here we demonstrate that Mouse Norovirus (MNV) infection induces autophagy but does not appear to utilise the autophagosomal membrane for establishment and formation of the viral replication complex. We have observed that MNV infection results in lipidation and recruitment of LC3 to the autophagosome membrane but prevents subsequent fusion of the autophagosomes with lysosomes, as SQSTM1 (an autophagy receptor) accumulates and Lysosome-Associated Membrane Protein1 is sequestered to the MNV replication complex (RC) rather than to autophagosomes. We have additionally observed that chemical modulation of autophagy differentially affects MNV replication. From this study we can conclude that MNV infection induces autophagy, however suppresses the final maturation step of this response, indicating that autophagy induction contributes to MNV replication independently of RC biogenesis. - Highlights: • MNV induces autophagy in infected murine macrophages. • MNV does not utilise autophagosomal membranes for replication. • The MNV-induced autophagosomes do not fuse with lysosomes. • MNV sequesters SQSTM1 to prevent autophagy degradation and turnover. • Chemical modulation of autophagy enhances MNV replication.

  14. The mitochondrial outer membrane protein MDI promotes local protein synthesis and mtDNA replication.

    Science.gov (United States)

    Zhang, Yi; Chen, Yong; Gucek, Marjan; Xu, Hong

    2016-05-17

    Early embryonic development features rapid nuclear DNA replication cycles, but lacks mtDNA replication. To meet the high-energy demands of embryogenesis, mature oocytes are furnished with vast amounts of mitochondria and mtDNA However, the cellular machinery driving massive mtDNA replication in ovaries remains unknown. Here, we describe a Drosophila AKAP protein, MDI that recruits a translation stimulator, La-related protein (Larp), to the mitochondrial outer membrane in ovaries. The MDI-Larp complex promotes the synthesis of a subset of nuclear-encoded mitochondrial proteins by cytosolic ribosomes on the mitochondrial surface. MDI-Larp's targets include mtDNA replication factors, mitochondrial ribosomal proteins, and electron-transport chain subunits. Lack of MDI abolishes mtDNA replication in ovaries, which leads to mtDNA deficiency in mature eggs. Targeting Larp to the mitochondrial outer membrane independently of MDI restores local protein synthesis and rescues the phenotypes of mdi mutant flies. Our work suggests that a selective translational boost by the MDI-Larp complex on the outer mitochondrial membrane might be essential for mtDNA replication and mitochondrial biogenesis during oogenesis. Published 2016. This article is a U.S. Government work and is in the public domain in the USA.

  15. A transgenic mouse marking live replicating cells reveals in vivo transcriptional program of proliferation

    DEFF Research Database (Denmark)

    Klochendler, Agnes; Weinberg-Corem, Noa; Moran, Maya

    2012-01-01

    Most adult mammalian tissues are quiescent, with rare cell divisions serving to maintain homeostasis. At present, the isolation and study of replicating cells from their in vivo niche typically involves immunostaining for intracellular markers of proliferation, causing the loss of sensitive biolo...

  16. Rapid transient production in plants by replicating and non-replicating vectors yields high quality functional anti-HIV antibody.

    Directory of Open Access Journals (Sweden)

    Frank Sainsbury

    2010-11-01

    Full Text Available The capacity of plants and plant cells to produce large amounts of recombinant protein has been well established. Due to advantages in terms of speed and yield, attention has recently turned towards the use of transient expression systems, including viral vectors, to produce proteins of pharmaceutical interest in plants. However, the effects of such high level expression from viral vectors and concomitant effects on host cells may affect the quality of the recombinant product.To assess the quality of antibodies transiently expressed to high levels in plants, we have expressed and characterised the human anti-HIV monoclonal antibody, 2G12, using both replicating and non-replicating systems based on deleted versions of Cowpea mosaic virus (CPMV RNA-2. The highest yield (approximately 100 mg/kg wet weight leaf tissue of affinity purified 2G12 was obtained when the non-replicating CPMV-HT system was used and the antibody was retained in the endoplasmic reticulum (ER. Glycan analysis by mass-spectrometry showed that the glycosylation pattern was determined exclusively by whether the antibody was retained in the ER and did not depend on whether a replicating or non-replicating system was used. Characterisation of the binding and neutralisation properties of all the purified 2G12 variants from plants showed that these were generally similar to those of the Chinese hamster ovary (CHO cell-produced 2G12.Overall, the results demonstrate that replicating and non-replicating CPMV-based vectors are able to direct the production of a recombinant IgG similar in activity to the CHO-produced control. Thus, a complex recombinant protein was produced with no apparent effect on its biochemical properties using either high-level expression or viral replication. The speed with which a recombinant pharmaceutical with excellent biochemical characteristics can be produced transiently in plants makes CPMV-based expression vectors an attractive option for

  17. Cdt1 revisited: complex and tight regulation during the cell cycle and consequences of deregulation in mammalian cells

    Directory of Open Access Journals (Sweden)

    Fujita Masatoshi

    2006-10-01

    Full Text Available Abstract In eukaryotic cells, replication of genomic DNA initiates from multiple replication origins distributed on multiple chromosomes. To ensure that each origin is activated precisely only once during each S phase, a system has evolved which features periodic assembly and disassembly of essential pre-replication complexes (pre-RCs at replication origins. The pre-RC assembly reaction involves the loading of a presumptive replicative helicase, the MCM2-7 complexes, onto chromatin by the origin recognition complex (ORC and two essential factors, CDC6 and Cdt1. The eukaryotic cell cycle is driven by the periodic activation and inactivation of cyclin-dependent kinases (Cdks and assembly of pre-RCs can only occur during the low Cdk activity period from late mitosis through G1 phase, with inappropriate re-assembly suppressed during S, G2, and M phases. It was originally suggested that inhibition of Cdt1 function after S phase in vertebrate cells is due to geminin binding and that Cdt1 hyperfunction resulting from Cdt1-geminin imbalance induces re-replication. However, recent progress has revealed that Cdt1 activity is more strictly regulated by two other mechanisms in addition to geminin: (1 functional and SCFSkp2-mediated proteolytic regulation through phosphorylation by Cdks; and (2 replication-coupled proteolysis mediated by the Cullin4-DDB1Cdt2 ubiquitin ligase and PCNA, an eukaryotic sliding clamp stimulating replicative DNA polymerases. The tight regulation implies that Cdt1 control is especially critical for the regulation of DNA replication in mammalian cells. Indeed, Cdt1 overexpression evokes chromosomal damage even without re-replication. Furthermore, deregulated Cdt1 induces chromosomal instability in normal human cells. Since Cdt1 is overexpressed in cancer cells, this could be a new molecular mechanism leading to carcinogenesis. In this review, recent insights into Cdt1 function and regulation in mammalian cells are discussed.

  18. DNA replication restart and cellular dynamics of Hef helicase/nuclease protein in Haloferax volcanii.

    Science.gov (United States)

    Lestini, Roxane; Delpech, Floriane; Myllykallio, Hannu

    2015-11-01

    Understanding how frequently spontaneous replication arrests occur and how archaea deal with these arrests are very interesting and challenging research topics. Here we will described how genetic and imaging studies have revealed the central role of the archaeal helicase/nuclease Hef belonging to the XPF/MUS81/FANCM family of endonucleases in repair of arrested replication forks. Special focus will be on description of a recently developed combination of genetic and imaging tools to study the dynamic localization of a functional Hef::GFP (Green Fluorescent Protein) fusion protein in the living cells of halophilic archaea Haloferax volcanii. As Archaea provide an excellent and unique model for understanding how DNA replication is regulated to allow replication of a circular DNA molecule either from single or multiple replication origins, we will also summarize recent studies that have revealed peculiar features regarding DNA replication, particularly in halophilic archaea. We strongly believe that fundamental knowledge of our on-going studies will shed light on the evolutionary history of the DNA replication machinery and will help to establish general rules concerning replication restart and the key role of recombination proteins not only in bacteria, yeast and higher eukaryotes but also in archaea. Copyright © 2015 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

  19. Arranging eukaryotic nuclear DNA polymerases for replication: Specific interactions with accessory proteins arrange Pols α, δ, and ϵ in the replisome for leading-strand and lagging-strand DNA replication.

    Science.gov (United States)

    Kunkel, Thomas A; Burgers, Peter M J

    2017-08-01

    Biochemical and cryo-electron microscopy studies have just been published revealing interactions among proteins of the yeast replisome that are important for highly coordinated synthesis of the two DNA strands of the nuclear genome. These studies reveal key interactions important for arranging DNA polymerases α, δ, and ϵ for leading and lagging strand replication. The CMG (Mcm2-7, Cdc45, GINS) helicase is central to this interaction network. These are but the latest examples of elegant studies performed in the recent past that lead to a much better understanding of how the eukaryotic replication fork achieves efficient DNA replication that is accurate enough to prevent diseases yet allows evolution. This article has been contributed to by US Government employees and their work is in the public domain in the USA.

  20. DNA replication stress restricts ribosomal DNA copy number.

    Science.gov (United States)

    Salim, Devika; Bradford, William D; Freeland, Amy; Cady, Gillian; Wang, Jianmin; Pruitt, Steven C; Gerton, Jennifer L

    2017-09-01

    Ribosomal RNAs (rRNAs) in budding yeast are encoded by ~100-200 repeats of a 9.1kb sequence arranged in tandem on chromosome XII, the ribosomal DNA (rDNA) locus. Copy number of rDNA repeat units in eukaryotic cells is maintained far in excess of the requirement for ribosome biogenesis. Despite the importance of the repeats for both ribosomal and non-ribosomal functions, it is currently not known how "normal" copy number is determined or maintained. To identify essential genes involved in the maintenance of rDNA copy number, we developed a droplet digital PCR based assay to measure rDNA copy number in yeast and used it to screen a yeast conditional temperature-sensitive mutant collection of essential genes. Our screen revealed that low rDNA copy number is associated with compromised DNA replication. Further, subculturing yeast under two separate conditions of DNA replication stress selected for a contraction of the rDNA array independent of the replication fork blocking protein, Fob1. Interestingly, cells with a contracted array grew better than their counterparts with normal copy number under conditions of DNA replication stress. Our data indicate that DNA replication stresses select for a smaller rDNA array. We speculate that this liberates scarce replication factors for use by the rest of the genome, which in turn helps cells complete DNA replication and continue to propagate. Interestingly, tumors from mini chromosome maintenance 2 (MCM2)-deficient mice also show a loss of rDNA repeats. Our data suggest that a reduction in rDNA copy number may indicate a history of DNA replication stress, and that rDNA array size could serve as a diagnostic marker for replication stress. Taken together, these data begin to suggest the selective pressures that combine to yield a "normal" rDNA copy number.

  1. DNA replication stress restricts ribosomal DNA copy number

    Science.gov (United States)

    Salim, Devika; Bradford, William D.; Freeland, Amy; Cady, Gillian; Wang, Jianmin

    2017-01-01

    Ribosomal RNAs (rRNAs) in budding yeast are encoded by ~100–200 repeats of a 9.1kb sequence arranged in tandem on chromosome XII, the ribosomal DNA (rDNA) locus. Copy number of rDNA repeat units in eukaryotic cells is maintained far in excess of the requirement for ribosome biogenesis. Despite the importance of the repeats for both ribosomal and non-ribosomal functions, it is currently not known how “normal” copy number is determined or maintained. To identify essential genes involved in the maintenance of rDNA copy number, we developed a droplet digital PCR based assay to measure rDNA copy number in yeast and used it to screen a yeast conditional temperature-sensitive mutant collection of essential genes. Our screen revealed that low rDNA copy number is associated with compromised DNA replication. Further, subculturing yeast under two separate conditions of DNA replication stress selected for a contraction of the rDNA array independent of the replication fork blocking protein, Fob1. Interestingly, cells with a contracted array grew better than their counterparts with normal copy number under conditions of DNA replication stress. Our data indicate that DNA replication stresses select for a smaller rDNA array. We speculate that this liberates scarce replication factors for use by the rest of the genome, which in turn helps cells complete DNA replication and continue to propagate. Interestingly, tumors from mini chromosome maintenance 2 (MCM2)-deficient mice also show a loss of rDNA repeats. Our data suggest that a reduction in rDNA copy number may indicate a history of DNA replication stress, and that rDNA array size could serve as a diagnostic marker for replication stress. Taken together, these data begin to suggest the selective pressures that combine to yield a “normal” rDNA copy number. PMID:28915237

  2. DNA replication stress restricts ribosomal DNA copy number.

    Directory of Open Access Journals (Sweden)

    Devika Salim

    2017-09-01

    Full Text Available Ribosomal RNAs (rRNAs in budding yeast are encoded by ~100-200 repeats of a 9.1kb sequence arranged in tandem on chromosome XII, the ribosomal DNA (rDNA locus. Copy number of rDNA repeat units in eukaryotic cells is maintained far in excess of the requirement for ribosome biogenesis. Despite the importance of the repeats for both ribosomal and non-ribosomal functions, it is currently not known how "normal" copy number is determined or maintained. To identify essential genes involved in the maintenance of rDNA copy number, we developed a droplet digital PCR based assay to measure rDNA copy number in yeast and used it to screen a yeast conditional temperature-sensitive mutant collection of essential genes. Our screen revealed that low rDNA copy number is associated with compromised DNA replication. Further, subculturing yeast under two separate conditions of DNA replication stress selected for a contraction of the rDNA array independent of the replication fork blocking protein, Fob1. Interestingly, cells with a contracted array grew better than their counterparts with normal copy number under conditions of DNA replication stress. Our data indicate that DNA replication stresses select for a smaller rDNA array. We speculate that this liberates scarce replication factors for use by the rest of the genome, which in turn helps cells complete DNA replication and continue to propagate. Interestingly, tumors from mini chromosome maintenance 2 (MCM2-deficient mice also show a loss of rDNA repeats. Our data suggest that a reduction in rDNA copy number may indicate a history of DNA replication stress, and that rDNA array size could serve as a diagnostic marker for replication stress. Taken together, these data begin to suggest the selective pressures that combine to yield a "normal" rDNA copy number.

  3. The spontaneous replication error and the mismatch discrimination mechanisms of human DNA polymerase β

    Science.gov (United States)

    Koag, Myong-Chul; Nam, Kwangho; Lee, Seongmin

    2014-01-01

    To provide molecular-level insights into the spontaneous replication error and the mismatch discrimination mechanisms of human DNA polymerase β (polβ), we report four crystal structures of polβ complexed with dG•dTTP and dA•dCTP mismatches in the presence of Mg2+ or Mn2+. The Mg2+-bound ground-state structures show that the dA•dCTP-Mg2+ complex adopts an ‘intermediate’ protein conformation while the dG•dTTP-Mg2+ complex adopts an open protein conformation. The Mn2+-bound ‘pre-chemistry-state’ structures show that the dA•dCTP-Mn2+ complex is structurally very similar to the dA•dCTP-Mg2+ complex, whereas the dG•dTTP-Mn2+ complex undergoes a large-scale conformational change to adopt a Watson–Crick-like dG•dTTP base pair and a closed protein conformation. These structural differences, together with our molecular dynamics simulation studies, suggest that polβ increases replication fidelity via a two-stage mismatch discrimination mechanism, where one is in the ground state and the other in the closed conformation state. In the closed conformation state, polβ appears to allow only a Watson–Crick-like conformation for purine•pyrimidine base pairs, thereby discriminating the mismatched base pairs based on their ability to form the Watson–Crick-like conformation. Overall, the present studies provide new insights into the spontaneous replication error and the replication fidelity mechanisms of polβ. PMID:25200079

  4. Susceptibility to bystander DNA damage is influenced by replication and transcriptional activity

    Science.gov (United States)

    Dickey, Jennifer S.; Baird, Brandon J.; Redon, Christophe E.; Avdoshina, Valeriya; Palchik, Guillermo; Wu, Junfang; Kondratyev, Alexei; Bonner, William M.; Martin, Olga A.

    2012-01-01

    Direct cellular DNA damage may lead to genome destabilization in unexposed, bystander, cells sharing the same milieu with directly damaged cells by means of the bystander effect. One proposed mechanism involves double strand break (DSB) formation in S phase cells at sites of single strand lesions in the DNA of replication complexes, which has a more open structure compared with neighboring DNA. The DNA in transcription complexes also has a more open structure, and hence may be susceptible to bystander DSB formation from single strand lesions. To examine whether transcription predisposes non-replicating cells to bystander effect-induced DNA DSBs, we examined two types of primary cells that exhibit high levels of transcription in the absence of replication, rat neurons and human lymphocytes. We found that non-replicating bystander cells with high transcription rates exhibited substantial levels of DNA DSBs, as monitored by γ-H2AX foci formation. Additionally, as reported in proliferating cells, TGF-β and NO were found to mimic bystander effects in cell populations lacking DNA synthesis. These results indicate that cell vulnerability to bystander DSB damage may result from transcription as well as replication. The findings offer insights into which tissues may be vulnerable to bystander genomic destabilization in vivo. PMID:22941641

  5. How MCM loading and spreading specify eukaryotic DNA replication initiation sites.

    Science.gov (United States)

    Hyrien, Olivier

    2016-01-01

    DNA replication origins strikingly differ between eukaryotic species and cell types. Origins are localized and can be highly efficient in budding yeast, are randomly located in early fly and frog embryos, which do not transcribe their genomes, and are clustered in broad (10-100 kb) non-transcribed zones, frequently abutting transcribed genes, in mammalian cells. Nonetheless, in all cases, origins are established during the G1-phase of the cell cycle by the loading of double hexamers of the Mcm 2-7 proteins (MCM DHs), the core of the replicative helicase. MCM DH activation in S-phase leads to origin unwinding, polymerase recruitment, and initiation of bidirectional DNA synthesis. Although MCM DHs are initially loaded at sites defined by the binding of the origin recognition complex (ORC), they ultimately bind chromatin in much greater numbers than ORC and only a fraction are activated in any one S-phase. Data suggest that the multiplicity and functional redundancy of MCM DHs provide robustness to the replication process and affect replication time and that MCM DHs can slide along the DNA and spread over large distances around the ORC. Recent studies further show that MCM DHs are displaced along the DNA by collision with transcription complexes but remain functional for initiation after displacement. Therefore, eukaryotic DNA replication relies on intrinsically mobile and flexible origins, a strategy fundamentally different from bacteria but conserved from yeast to human. These properties of MCM DHs likely contribute to the establishment of broad, intergenic replication initiation zones in higher eukaryotes.

  6. Dynamics of DNA replication during premeiosis and early meiosis in wheat.

    Science.gov (United States)

    Rey, María-Dolores; Prieto, Pilar

    2014-01-01

    Meiosis is a specialised cell division that involves chromosome replication, two rounds of chromosome segregation and results in the formation of the gametes. Meiotic DNA replication generally precedes chromosome pairing, recombination and synapsis in sexually developing eukaryotes. In this work, replication has been studied during premeiosis and early meiosis in wheat using flow cytometry, which has allowed the quantification of the amount of DNA in wheat anther in each phase of the cell cycle during premeiosis and each stage of early meiosis. Flow cytometry has been revealed as a suitable and user-friendly tool to detect and quantify DNA replication during early meiosis in wheat. Chromosome replication was detected in wheat during premeiosis and early meiosis until the stage of pachytene, when chromosomes are associated in pairs to further recombine and correctly segregate in the gametes. In addition, the effect of the Ph1 locus, which controls chromosome pairing and affects replication in wheat, was also studied by flow cytometry. Here we showed that the Ph1 locus plays an important role on the length of meiotic DNA replication in wheat, particularly affecting the rate of replication during early meiosis in wheat.

  7. Modeling HIV-1 intracellular replication: two simulation approaches

    NARCIS (Netherlands)

    Zarrabi, N.; Mancini, E.; Tay, J.; Shahand, S.; Sloot, P.M.A.

    2010-01-01

    Many mathematical and computational models have been developed to investigate the complexity of HIV dynamics, immune response and drug therapy. However, there are not many models which consider the dynamics of virus intracellular replication at a single level. We propose a model of HIV intracellular

  8. West Nile virus replication requires fatty acid synthesis but is independent on phosphatidylinositol-4-phosphate lipids.

    Directory of Open Access Journals (Sweden)

    Miguel A Martín-Acebes

    Full Text Available West Nile virus (WNV is a neurovirulent mosquito-borne flavivirus, which main natural hosts are birds but it also infects equines and humans, among other mammals. As in the case of other plus-stranded RNA viruses, WNV replication is associated to intracellular membrane rearrangements. Based on results obtained with a variety of viruses, different cellular processes have been shown to play important roles on these membrane rearrangements for efficient viral replication. As these processes are related to lipid metabolism, fatty acid synthesis, as well as generation of a specific lipid microenvironment enriched in phosphatidylinositol-4-phosphate (PI4P, has been associated to it in other viral models. In this study, intracellular membrane rearrangements following infection with a highly neurovirulent strain of WNV were addressed by means of electron and confocal microscopy. Infection of WNV, and specifically viral RNA replication, were dependent on fatty acid synthesis, as revealed by the inhibitory effect of cerulenin and C75, two pharmacological inhibitors of fatty acid synthase, a key enzyme of this process. However, WNV infection did not induce redistribution of PI4P lipids, and PI4P did not localize at viral replication complex. Even more, WNV multiplication was not inhibited by the use of the phosphatidylinositol-4-kinase inhibitor PIK93, while infection by the enterovirus Coxsackievirus B5 was reduced. Similar features were found when infection by other flavivirus, the Usutu virus (USUV, was analyzed. These features of WNV replication could help to design specific antiviral approaches against WNV and other related flaviviruses.

  9. Archaeal Genome Guardians Give Insights into Eukaryotic DNA Replication and Damage Response Proteins

    Directory of Open Access Journals (Sweden)

    David S. Shin

    2014-01-01

    Full Text Available As the third domain of life, archaea, like the eukarya and bacteria, must have robust DNA replication and repair complexes to ensure genome fidelity. Archaea moreover display a breadth of unique habitats and characteristics, and structural biologists increasingly appreciate these features. As archaea include extremophiles that can withstand diverse environmental stresses, they provide fundamental systems for understanding enzymes and pathways critical to genome integrity and stress responses. Such archaeal extremophiles provide critical data on the periodic table for life as well as on the biochemical, geochemical, and physical limitations to adaptive strategies allowing organisms to thrive under environmental stress relevant to determining the boundaries for life as we know it. Specifically, archaeal enzyme structures have informed the architecture and mechanisms of key DNA repair proteins and complexes. With added abilities to temperature-trap flexible complexes and reveal core domains of transient and dynamic complexes, these structures provide insights into mechanisms of maintaining genome integrity despite extreme environmental stress. The DNA damage response protein structures noted in this review therefore inform the basis for genome integrity in the face of environmental stress, with implications for all domains of life as well as for biomanufacturing, astrobiology, and medicine.

  10. Phosphatidic acid produced by phospholipase D promotes RNA replication of a plant RNA virus.

    Directory of Open Access Journals (Sweden)

    Kiwamu Hyodo

    2015-05-01

    Full Text Available Eukaryotic positive-strand RNA [(+RNA] viruses are intracellular obligate parasites replicate using the membrane-bound replicase complexes that contain multiple viral and host components. To replicate, (+RNA viruses exploit host resources and modify host metabolism and membrane organization. Phospholipase D (PLD is a phosphatidylcholine- and phosphatidylethanolamine-hydrolyzing enzyme that catalyzes the production of phosphatidic acid (PA, a lipid second messenger that modulates diverse intracellular signaling in various organisms. PA is normally present in small amounts (less than 1% of total phospholipids, but rapidly and transiently accumulates in lipid bilayers in response to different environmental cues such as biotic and abiotic stresses in plants. However, the precise functions of PLD and PA remain unknown. Here, we report the roles of PLD and PA in genomic RNA replication of a plant (+RNA virus, Red clover necrotic mosaic virus (RCNMV. We found that RCNMV RNA replication complexes formed in Nicotiana benthamiana contained PLDα and PLDβ. Gene-silencing and pharmacological inhibition approaches showed that PLDs and PLDs-derived PA are required for viral RNA replication. Consistent with this, exogenous application of PA enhanced viral RNA replication in plant cells and plant-derived cell-free extracts. We also found that a viral auxiliary replication protein bound to PA in vitro, and that the amount of PA increased in RCNMV-infected plant leaves. Together, our findings suggest that RCNMV hijacks host PA-producing enzymes to replicate.

  11. USP37 deubiquitinates Cdt1 and contributes to regulate DNA replication.

    Science.gov (United States)

    Hernández-Pérez, Santiago; Cabrera, Elisa; Amoedo, Hugo; Rodríguez-Acebes, Sara; Koundrioukoff, Stephane; Debatisse, Michelle; Méndez, Juan; Freire, Raimundo

    2016-10-01

    DNA replication control is a key process in maintaining genomic integrity. Monitoring DNA replication initiation is particularly important as it needs to be coordinated with other cellular events and should occur only once per cell cycle. Crucial players in the initiation of DNA replication are the ORC protein complex, marking the origin of replication, and the Cdt1 and Cdc6 proteins, that license these origins to replicate by recruiting the MCM2-7 helicase. To accurately achieve its functions, Cdt1 is tightly regulated. Cdt1 levels are high from metaphase and during G1 and low in S/G2 phases of the cell cycle. This control is achieved, among other processes, by ubiquitination and proteasomal degradation. In an overexpression screen for Cdt1 deubiquitinating enzymes, we isolated USP37, to date the first ubiquitin hydrolase controlling Cdt1. USP37 overexpression stabilizes Cdt1, most likely a phosphorylated form of the protein. In contrast, USP37 knock down destabilizes Cdt1, predominantly during G1 and G1/S phases of the cell cycle. USP37 interacts with Cdt1 and is able to de-ubiquitinate Cdt1 in vivo and, USP37 is able to regulate the loading of MCM complexes onto the chromatin. In addition, downregulation of USP37 reduces DNA replication fork speed. Taken together, here we show that the deubiquitinase USP37 plays an important role in the regulation of DNA replication. Whether this is achieved via Cdt1, a central protein in this process, which we have shown to be stabilized by USP37, or via additional factors, remains to be tested. Copyright © 2016 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  12. Improved understanding of protein complex offers insight into DNA

    Science.gov (United States)

    Summer Science Writing Internship Improved understanding of protein complex offers insight into DNA clearer understanding of the origin recognition complex (ORC) - a protein complex that directs DNA replication - through its crystal structure offers new insight into fundamental mechanisms of DNA replication

  13. Dynamic Architecture of Eukaryotic DNA Replication Forks In Vivo, Visualized by Electron Microscopy.

    Science.gov (United States)

    Zellweger, Ralph; Lopes, Massimo

    2018-01-01

    The DNA replication process can be heavily perturbed by several different conditions of genotoxic stress, particularly relevant for cancer onset and therapy. The combination of psoralen crosslinking and electron microscopy has proven instrumental to reveal the fine architecture of in vivo DNA replication intermediates and to uncover their remodeling upon specific conditions of genotoxic stress. The replication structures are stabilized in vivo (by psoralen crosslinking) prior to extraction and enrichment procedures, allowing their visualization at the transmission electron microscope. This chapter outlines the procedures required to visualize and interpret in vivo replication intermediates of eukaryotic genomic DNA, and includes an improved method for enrichment of replication intermediates, compared to previously used BND-cellulose columns.

  14. Both cis and trans Activities of Foot-and-Mouth Disease Virus 3D Polymerase Are Essential for Viral RNA Replication.

    Science.gov (United States)

    Herod, Morgan R; Ferrer-Orta, Cristina; Loundras, Eleni-Anna; Ward, Joseph C; Verdaguer, Nuria; Rowlands, David J; Stonehouse, Nicola J

    2016-08-01

    The Picornaviridae is a large family of positive-sense RNA viruses that contains numerous human and animal pathogens, including foot-and-mouth disease virus (FMDV). The picornavirus replication complex comprises a coordinated network of protein-protein and protein-RNA interactions involving multiple viral and host-cellular factors. Many of the proteins within the complex possess multiple roles in viral RNA replication, some of which can be provided in trans (i.e., via expression from a separate RNA molecule), while others are required in cis (i.e., expressed from the template RNA molecule). In vitro studies have suggested that multiple copies of the RNA-dependent RNA polymerase (RdRp) 3D are involved in the viral replication complex. However, it is not clear whether all these molecules are catalytically active or what other function(s) they provide. In this study, we aimed to distinguish between catalytically active 3D molecules and those that build a replication complex. We report a novel nonenzymatic cis-acting function of 3D that is essential for viral-genome replication. Using an FMDV replicon in complementation experiments, our data demonstrate that this cis-acting role of 3D is distinct from the catalytic activity, which is predominantly trans acting. Immunofluorescence studies suggest that both cis- and trans-acting 3D molecules localize to the same cellular compartment. However, our genetic and structural data suggest that 3D interacts in cis with RNA stem-loops that are essential for viral RNA replication. This study identifies a previously undescribed aspect of picornavirus replication complex structure-function and an important methodology for probing such interactions further. Foot-and-mouth disease virus (FMDV) is an important animal pathogen responsible for foot-and-mouth disease. The disease is endemic in many parts of the world with outbreaks within livestock resulting in major economic losses. Propagation of the viral genome occurs within

  15. Genome-wide alterations of the DNA replication program during tumor progression

    Science.gov (United States)

    Arneodo, A.; Goldar, A.; Argoul, F.; Hyrien, O.; Audit, B.

    2016-08-01

    Oncogenic stress is a major driving force in the early stages of cancer development. Recent experimental findings reveal that, in precancerous lesions and cancers, activated oncogenes may induce stalling and dissociation of DNA replication forks resulting in DNA damage. Replication timing is emerging as an important epigenetic feature that recapitulates several genomic, epigenetic and functional specificities of even closely related cell types. There is increasing evidence that chromosome rearrangements, the hallmark of many cancer genomes, are intimately associated with the DNA replication program and that epigenetic replication timing changes often precede chromosomic rearrangements. The recent development of a novel methodology to map replication fork polarity using deep sequencing of Okazaki fragments has provided new and complementary genome-wide replication profiling data. We review the results of a wavelet-based multi-scale analysis of genomic and epigenetic data including replication profiles along human chromosomes. These results provide new insight into the spatio-temporal replication program and its dynamics during differentiation. Here our goal is to bring to cancer research, the experimental protocols and computational methodologies for replication program profiling, and also the modeling of the spatio-temporal replication program. To illustrate our purpose, we report very preliminary results obtained for the chronic myelogeneous leukemia, the archetype model of cancer. Finally, we discuss promising perspectives on using genome-wide DNA replication profiling as a novel efficient tool for cancer diagnosis, prognosis and personalized treatment.

  16. 3D replicon distributions arise from stochastic initiation and domino-like DNA replication progression.

    Science.gov (United States)

    Löb, D; Lengert, N; Chagin, V O; Reinhart, M; Casas-Delucchi, C S; Cardoso, M C; Drossel, B

    2016-04-07

    DNA replication dynamics in cells from higher eukaryotes follows very complex but highly efficient mechanisms. However, the principles behind initiation of potential replication origins and emergence of typical patterns of nuclear replication sites remain unclear. Here, we propose a comprehensive model of DNA replication in human cells that is based on stochastic, proximity-induced replication initiation. Critical model features are: spontaneous stochastic firing of individual origins in euchromatin and facultative heterochromatin, inhibition of firing at distances below the size of chromatin loops and a domino-like effect by which replication forks induce firing of nearby origins. The model reproduces the empirical temporal and chromatin-related properties of DNA replication in human cells. We advance the one-dimensional DNA replication model to a spatial model by taking into account chromatin folding in the nucleus, and we are able to reproduce the spatial and temporal characteristics of the replication foci distribution throughout S-phase.

  17. Mutational Analysis of the Hypervariable Region of Hepatitis E Virus Reveals Its Involvement in the Efficiency of Viral RNA Replication

    Science.gov (United States)

    Pudupakam, R. S.; Kenney, Scott P.; Córdoba, Laura; Huang, Yao-Wei; Dryman, Barbara A.; LeRoith, Tanya; Pierson, F. William; Meng, Xiang-Jin

    2011-01-01

    The RNA genome of the hepatitis E virus (HEV) contains a hypervariable region (HVR) in ORF1 that tolerates small deletions with respect to infectivity. To further investigate the role of the HVR in HEV replication, we constructed a panel of mutants with overlapping deletions in the N-terminal, central, and C-terminal regions of the HVR by using a genotype 1 human HEV luciferase replicon and analyzed the effects of deletions on viral RNA replication in Huh7 cells. We found that the replication levels of the HVR deletion mutants were markedly reduced in Huh7 cells, suggesting a role of the HVR in viral replication efficiency. To further verify the results, we constructed HVR deletion mutants by using a genetically divergent, nonmammalian avian HEV, and similar effects on viral replication efficiency were observed when the avian HEV mutants were tested in LMH cells. Furthermore, the impact of complete HVR deletion on virus infectivity was tested in chickens, using an avian HEV mutant with a complete HVR deletion. Although the deletion mutant was still replication competent in LMH cells, the complete HVR deletion resulted in a loss of avian HEV infectivity in chickens. Since the HVR exhibits extensive variations in sequence and length among different HEV genotypes, we further examined the interchangeability of HVRs and demonstrated that HVR sequences are functionally exchangeable between HEV genotypes with regard to viral replication and infectivity in vitro, although genotype-specific HVR differences in replication efficiency were observed. The results showed that although the HVR tolerates small deletions with regard to infectivity, it may interact with viral and host factors to modulate the efficiency of HEV replication. PMID:21775444

  18. A Role of hIPI3 in DNA Replication Licensing in Human Cells.

    Science.gov (United States)

    Huang, Yining; Amin, Aftab; Qin, Yan; Wang, Ziyi; Jiang, Huadong; Liang, Lu; Shi, Linjing; Liang, Chun

    2016-01-01

    The yeast Ipi3p is required for DNA replication and cell viability in Sacharomyces cerevisiae. It is an essential component of the Rix1 complex (Rix1p/Ipi2p-Ipi1p-Ipi3p) that is required for the processing of 35S pre-rRNA in pre-60S ribosomal particles and for the initiation of DNA replication. The human IPI3 homolog is WDR18 (WD repeat domain 18), which shares significant homology with yIpi3p. Here we report that knockdown of hIPI3 resulted in substantial defects in the chromatin association of the MCM complex, DNA replication, cell cycle progression and cell proliferation. Importantly, hIPI3 silencing did not result in a reduction of the protein level of hCDC6, hMCM7, or the ectopically expressed GFP protein, indicating that protein synthesis was not defective in the same time frame of the DNA replication and cell cycle defects. Furthermore, the mRNA and protein levels of hIPI3 fluctuate in the cell cycle, with the highest levels from M phase to early G1 phase, similar to other pre-replicative (pre-RC) proteins. Moreover, hIPI3 interacts with other replication-initiation proteins, co-localizes with hMCM7 in the nucleus, and is important for the nuclear localization of hMCM7. We also found that hIPI3 preferentially binds to the origins of DNA replication including those at the c-Myc, Lamin-B2 and β-Globin loci. These results indicate that hIPI3 is involved in human DNA replication licensing independent of its role in ribosome biogenesis.

  19. How and why multiple MCMs are loaded at origins of DNA replication.

    Science.gov (United States)

    Das, Shankar P; Rhind, Nicholas

    2016-07-01

    Recent work suggests that DNA replication origins are regulated by the number of multiple mini-chromosome maintenance (MCM) complexes loaded. Origins are defined by the loading of MCM - the replicative helicase which initiates DNA replication and replication kinetics determined by origin's location and firing times. However, activation of MCM is heterogeneous; different origins firing at different times in different cells. Also, more MCMs are loaded in G1 than are used in S phase. These aspects of MCM biology are explained by the observation that multiple MCMs are loaded at origins. Having more MCMs at early origins makes them more likely to fire, effecting differences in origin efficiency that define replication timing. Nonetheless, multiple MCM loading raises new questions, such as how they are loaded, where these MCMs reside at origins, and how their presence affects replication timing. In this review, we address these questions and discuss future avenues of research. © 2016 WILEY Periodicals, Inc.

  20. Replicative Intermediates of Human Papillomavirus Type 11 in Laryngeal Papillomas: Site of Replication Initiation and Direction of Replication

    Science.gov (United States)

    Auborn, K. J.; Little, R. D.; Platt, T. H. K.; Vaccariello, M. A.; Schildkraut, C. L.

    1994-07-01

    We have examined the structures of replication intermediates from the human papillomavirus type 11 genome in DNA extracted from papilloma lesions (laryngeal papillomas). The sites of replication initiation and termination utilized in vivo were mapped by using neutral/neutral and neutral/alkaline two-dimensional agarose gel electrophoresis methods. Initiation of replication was detected in or very close to the upstream regulatory region (URR; the noncoding, regulatory sequences upstream of the open reading frames in the papillomavirus genome). We also show that replication forks proceed bidirectionally from the origin and converge 180circ opposite the URR. These results demonstrate the feasibility of analysis of replication of viral genomes directly from infected tissue.

  1. DNA replication and cancer: From dysfunctional replication origin activities to therapeutic opportunities.

    Science.gov (United States)

    Boyer, Anne-Sophie; Walter, David; Sørensen, Claus Storgaard

    2016-06-01

    A dividing cell has to duplicate its DNA precisely once during the cell cycle to preserve genome integrity avoiding the accumulation of genetic aberrations that promote diseases such as cancer. A large number of endogenous impacts can challenge DNA replication and cells harbor a battery of pathways to promote genome integrity during DNA replication. This includes suppressing new replication origin firing, stabilization of replicating forks, and the safe restart of forks to prevent any loss of genetic information. Here, we describe mechanisms by which oncogenes can interfere with DNA replication thereby causing DNA replication stress and genome instability. Further, we describe cellular and systemic responses to these insults with a focus on DNA replication restart pathways. Finally, we discuss the therapeutic potential of exploiting intrinsic replicative stress in cancer cells for targeted therapy. Copyright © 2016 Elsevier Ltd. All rights reserved.

  2. SMC1-Mediated Intra-S-Phase Arrest Facilitates Bocavirus DNA Replication

    Science.gov (United States)

    Luo, Yong; Deng, Xuefeng; Cheng, Fang; Li, Yi

    2013-01-01

    Activation of a host DNA damage response (DDR) is essential for DNA replication of minute virus of canines (MVC), a member of the genus Bocavirus of the Parvoviridae family; however, the mechanism by which DDR contributes to viral DNA replication is unknown. In the current study, we demonstrate that MVC infection triggers the intra-S-phase arrest to slow down host cellular DNA replication and to recruit cellular DNA replication factors for viral DNA replication. The intra-S-phase arrest is regulated by ATM (ataxia telangiectasia-mutated kinase) signaling in a p53-independent manner. Moreover, we demonstrate that SMC1 (structural maintenance of chromosomes 1) is the key regulator of the intra-S-phase arrest induced during infection. Either knockdown of SMC1 or complementation with a dominant negative SMC1 mutant blocks both the intra-S-phase arrest and viral DNA replication. Finally, we show that the intra-S-phase arrest induced during MVC infection was caused neither by damaged host cellular DNA nor by viral proteins but by replicating viral genomes physically associated with the DNA damage sensor, the Mre11-Rad50-Nbs1 (MRN) complex. In conclusion, the feedback loop between MVC DNA replication and the intra-S-phase arrest is mediated by ATM-SMC1 signaling and plays a critical role in MVC DNA replication. Thus, our findings unravel the mechanism underlying DDR signaling-facilitated MVC DNA replication and demonstrate a novel strategy of DNA virus-host interaction. PMID:23365434

  3. Role of DNA Repair Factor Xeroderma Pigmentosum Protein Group C in Response to Replication Stress As Revealed by DNA Fragile Site Affinity Chromatography and Quantitative Proteomics.

    Science.gov (United States)

    Beresova, Lucie; Vesela, Eva; Chamrad, Ivo; Voller, Jiri; Yamada, Masayuki; Furst, Tomas; Lenobel, Rene; Chroma, Katarina; Gursky, Jan; Krizova, Katerina; Mistrik, Martin; Bartek, Jiri

    2016-12-02

    Replication stress (RS) fuels genomic instability and cancer development and may contribute to aging, raising the need to identify factors involved in cellular responses to such stress. Here, we present a strategy for identification of factors affecting the maintenance of common fragile sites (CFSs), which are genomic loci that are particularly sensitive to RS and suffer from increased breakage and rearrangements in tumors. A DNA probe designed to match the high flexibility island sequence typical for the commonly expressed CFS (FRA16D) was used as specific DNA affinity bait. Proteins significantly enriched at the FRA16D fragment under normal and replication stress conditions were identified using stable isotope labeling of amino acids in cell culture-based quantitative mass spectrometry. The identified proteins interacting with the FRA16D fragment included some known CFS stabilizers, thereby validating this screening approach. Among the hits from our screen so far not implicated in CFS maintenance, we chose Xeroderma pigmentosum protein group C (XPC) for further characterization. XPC is a key factor in the DNA repair pathway known as global genomic nucleotide excision repair (GG-NER), a mechanism whose several components were enriched at the FRA16D fragment in our screen. Functional experiments revealed defective checkpoint signaling and escape of DNA replication intermediates into mitosis and the next generation of XPC-depleted cells exposed to RS. Overall, our results provide insights into an unexpected biological role of XPC in response to replication stress and document the power of proteomics-based screening strategies to elucidate mechanisms of pathophysiological significance.

  4. DNA Replication Is Required for Circadian Clock Function by Regulating Rhythmic Nucleosome Composition.

    Science.gov (United States)

    Liu, Xiao; Dang, Yunkun; Matsu-Ura, Toru; He, Yubo; He, Qun; Hong, Christian I; Liu, Yi

    2017-07-20

    Although the coupling between circadian and cell cycles allows circadian clocks to gate cell division and DNA replication in many organisms, circadian clocks were thought to function independently of cell cycle. Here, we show that DNA replication is required for circadian clock function in Neurospora. Genetic and pharmacological inhibition of DNA replication abolished both overt and molecular rhythmicities by repressing frequency (frq) gene transcription. DNA replication is essential for the rhythmic changes of nucleosome composition at the frq promoter. The FACT complex, known to be involved in histone disassembly/reassembly, is required for clock function and is recruited to the frq promoter in a replication-dependent manner to promote replacement of histone H2A.Z by H2A. Finally, deletion of H2A.Z uncoupled the dependence of the circadian clock on DNA replication. Together, these results establish circadian clock and cell cycle as interdependent coupled oscillators and identify DNA replication as a critical process in the circadian mechanism. Published by Elsevier Inc.

  5. Controlled initiation of chromosomal replication in Escherichia coli requires functional Hda protein.

    Science.gov (United States)

    Camara, Johanna Eltz; Skarstad, Kirsten; Crooke, Elliott

    2003-05-01

    Regulatory inactivation of DnaA helps ensure that the Escherichia coli chromosome is replicated only once per cell cycle, through accelerated hydrolysis of active replication initiator ATP-DnaA to inactive ADP-DnaA. Analysis of deltahda strains revealed that the regulatory inactivation of DnaA component Hda is necessary for maintaining controlled initiation but not for cell growth or viability.

  6. Expression profiling of colorectal cancer cells reveals inhibition of DNA replication licensing by extracellular calcium.

    Science.gov (United States)

    Aggarwal, Abhishek; Schulz, Herbert; Manhardt, Teresa; Bilban, Martin; Thakker, Rajesh V; Kallay, Enikö

    2017-06-01

    Colorectal cancer is one of the most common cancers in industrialised societies. Epidemiological studies, animal experiments, and randomized clinical trials have shown that dietary factors can influence all stages of colorectal carcinogenesis, from initiation through promotion to progression. Calcium is one of the factors with a chemoprophylactic effect in colorectal cancer. The aim of this study was to understand the molecular mechanisms of the anti-tumorigenic effects of extracellular calcium ([Ca 2+ ] o ) in colon cancer cells. Gene expression microarray analysis of colon cancer cells treated for 1, 4, and 24h with 2mM [Ca 2+ ] o identified significant changes in expression of 1571 probe sets (ANOVA, pcalcium-sensing receptor (CaSR), a G protein-coupled receptor is a mediator involved in this process. To test whether these results were physiologically relevant, we fed mice with a standard diet containing low (0.04%), intermediate (0.1%), or high (0.9%) levels of dietary calcium. The main molecules regulating replication licensing were inhibited also in vivo, in the colon of mice fed high calcium diet. We show that among the mechanisms behind the chemopreventive effect of [Ca 2+ ] o is inhibition of replication licensing, a process often deregulated in neoplastic transformation. Our data suggest that dietary calcium is effective in preventing replicative stress, one of the main drivers of cancer and this process is mediated by the calcium-sensing receptor. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

  7. RAD51 interconnects between DNA replication, DNA repair and immunity.

    Science.gov (United States)

    Bhattacharya, Souparno; Srinivasan, Kalayarasan; Abdisalaam, Salim; Su, Fengtao; Raj, Prithvi; Dozmorov, Igor; Mishra, Ritu; Wakeland, Edward K; Ghose, Subroto; Mukherjee, Shibani; Asaithamby, Aroumougame

    2017-05-05

    RAD51, a multifunctional protein, plays a central role in DNA replication and homologous recombination repair, and is known to be involved in cancer development. We identified a novel role for RAD51 in innate immune response signaling. Defects in RAD51 lead to the accumulation of self-DNA in the cytoplasm, triggering a STING-mediated innate immune response after replication stress and DNA damage. In the absence of RAD51, the unprotected newly replicated genome is degraded by the exonuclease activity of MRE11, and the fragmented nascent DNA accumulates in the cytosol, initiating an innate immune response. Our data suggest that in addition to playing roles in homologous recombination-mediated DNA double-strand break repair and replication fork processing, RAD51 is also implicated in the suppression of innate immunity. Thus, our study reveals a previously uncharacterized role of RAD51 in initiating immune signaling, placing it at the hub of new interconnections between DNA replication, DNA repair, and immunity. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  8. Promotion of Hendra Virus Replication by MicroRNA 146a

    Science.gov (United States)

    Marsh, Glenn A.; Jenkins, Kristie A.; Gantier, Michael P.; Tizard, Mark L.; Middleton, Deborah; Lowenthal, John W.; Haining, Jessica; Izzard, Leonard; Gough, Tamara J.; Deffrasnes, Celine; Stambas, John; Robinson, Rachel; Heine, Hans G.; Pallister, Jackie A.; Foord, Adam J.; Bean, Andrew G.; Wang, Lin-Fa

    2013-01-01

    Hendra virus is a highly pathogenic zoonotic paramyxovirus in the genus Henipavirus. Thirty-nine outbreaks of Hendra virus have been reported since its initial identification in Queensland, Australia, resulting in seven human infections and four fatalities. Little is known about cellular host factors impacting Hendra virus replication. In this work, we demonstrate that Hendra virus makes use of a microRNA (miRNA) designated miR-146a, an NF-κB-responsive miRNA upregulated by several innate immune ligands, to favor its replication. miR-146a is elevated in the blood of ferrets and horses infected with Hendra virus and is upregulated by Hendra virus in human cells in vitro. Blocking miR-146a reduces Hendra virus replication in vitro, suggesting a role for this miRNA in Hendra virus replication. In silico analysis of miR-146a targets identified ring finger protein (RNF)11, a member of the A20 ubiquitin editing complex that negatively regulates NF-κB activity, as a novel component of Hendra virus replication. RNA interference-mediated silencing of RNF11 promotes Hendra virus replication in vitro, suggesting that increased NF-κB activity aids Hendra virus replication. Furthermore, overexpression of the IκB superrepressor inhibits Hendra virus replication. These studies are the first to demonstrate a host miRNA response to Hendra virus infection and suggest an important role for host miRNAs in Hendra virus disease. PMID:23345523

  9. In vivo dynamics of EBNA1-oriP interaction during latent and lytic replication of Epstein-Barr virus.

    Science.gov (United States)

    Daikoku, Tohru; Kudoh, Ayumi; Fujita, Masatoshi; Sugaya, Yutaka; Isomura, Hiroki; Tsurumi, Tatsuya

    2004-12-24

    The Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA1) is required for maintenance of the viral genome DNA during the latent phase of EBV replication but continues to be synthesized after the induction of viral productive replication. An EBV genome-wide chromatin immunoprecipitation assay revealed that EBNA1 constantly binds to oriP of the EBV genome during not only latent but also lytic infection. Although the total levels of EBNA1 proved constant throughout the latter, the levels of the oriP-bound form were increased as lytic infection proceeded. EBV productive DNA replication occurs at discrete sites in nuclei, called replication compartments, where viral replication proteins are clustered. Confocal laser microscopic analyses revealed that whereas EBNA1 was distributed broadly in nuclei as fine punctate dots during the latent phase of infection, the protein became redistributed to the viral replication compartments and localized as distinct spots within and/or nearby the compartments after the induction of lytic replication. Taking these findings into consideration, oriP regions of the EBV genome might be organized by EBNA1 into replication domains that may set up scaffolding for lytic replication and transcription.

  10. Highly stable loading of Mcm proteins onto chromatin in living cells requires replication to unload

    Science.gov (United States)

    Kuipers, Marjorie A.; Stasevich, Timothy J.; Sasaki, Takayo; Wilson, Korey A.; Hazelwood, Kristin L.; McNally, James G.; Davidson, Michael W.

    2011-01-01

    The heterohexameric minichromosome maintenance protein complex (Mcm2-7) functions as the eukaryotic helicase during DNA replication. Mcm2-7 loads onto chromatin during early G1 phase but is not converted into an active helicase until much later during S phase. Hence, inactive Mcm complexes are presumed to remain stably bound from early G1 through the completion of S phase. Here, we investigated Mcm protein dynamics in live mammalian cells. We demonstrate that Mcm proteins are irreversibly loaded onto chromatin cumulatively throughout G1 phase, showing no detectable exchange with a gradually diminishing soluble pool. Eviction of Mcm requires replication; during replication arrest, Mcm proteins remained bound indefinitely. Moreover, the density of immobile Mcms is reduced together with chromatin decondensation within sites of active replication, which provides an explanation for the lack of colocalization of Mcm with replication fork proteins. These results provide in vivo evidence for an exceptionally stable lockdown mechanism to retain all loaded Mcm proteins on chromatin throughout prolonged cell cycles. PMID:21220507

  11. A microhomology-mediated break-induced replication model for the origin of human copy number variation.

    Directory of Open Access Journals (Sweden)

    P J Hastings

    2009-01-01

    Full Text Available Chromosome structural changes with nonrecurrent endpoints associated with genomic disorders offer windows into the mechanism of origin of copy number variation (CNV. A recent report of nonrecurrent duplications associated with Pelizaeus-Merzbacher disease identified three distinctive characteristics. First, the majority of events can be seen to be complex, showing discontinuous duplications mixed with deletions, inverted duplications, and triplications. Second, junctions at endpoints show microhomology of 2-5 base pairs (bp. Third, endpoints occur near pre-existing low copy repeats (LCRs. Using these observations and evidence from DNA repair in other organisms, we derive a model of microhomology-mediated break-induced replication (MMBIR for the origin of CNV and, ultimately, of LCRs. We propose that breakage of replication forks in stressed cells that are deficient in homologous recombination induces an aberrant repair process with features of break-induced replication (BIR. Under these circumstances, single-strand 3' tails from broken replication forks will anneal with microhomology on any single-stranded DNA nearby, priming low-processivity polymerization with multiple template switches generating complex rearrangements, and eventual re-establishment of processive replication.

  12. In Vitro Replication of Chelonid Herpesvirus 5 in Organotypic Skin Cultures from Hawaiian Green Turtles (Chelonia mydas).

    Science.gov (United States)

    Work, Thierry M; Dagenais, Julie; Weatherby, Tina M; Balazs, George H; Ackermann, Mathias

    2017-09-01

    endangered marine turtles. Pathological examination shows that ChHV5 is shed in skin. Here we show that ChHV5 will grow in vitro if we replicate the complex three-dimensional structure of turtle skin. Moreover, lytic virus growth requires a close interplay between fibroblasts and keratinocytes. Finally, the morphogenesis of herpesviral growth in three-dimensional cultures reveals a far richer, and likely more realistic, array of capsid morphologies than that encountered in traditional monolayer cell cultures. Our findings have applications to other viruses, including those of humans. Copyright © 2017 American Society for Microbiology.

  13. Identification, replication, and fine-mapping of Loci associated with adult height in individuals of african ancestry.

    Directory of Open Access Journals (Sweden)

    Amidou N'Diaye

    2011-10-01

    Full Text Available Adult height is a classic polygenic trait of high heritability (h(2 approximately 0.8. More than 180 single nucleotide polymorphisms (SNPs, identified mostly in populations of European descent, are associated with height. These variants convey modest effects and explain approximately10% of the variance in height. Discovery efforts in other populations, while limited, have revealed loci for height not previously implicated in individuals of European ancestry. Here, we performed a meta-analysis of genome-wide association (GWA results for adult height in 20,427 individuals of African ancestry with replication in up to 16,436 African Americans. We found two novel height loci (Xp22-rs12393627, P = 3.4×10(-12 and 2p14-rs4315565, P = 1.2×10(-8. As a group, height associations discovered in European-ancestry samples replicate in individuals of African ancestry (P = 1.7×10(-4 for overall replication. Fine-mapping of the European height loci in African-ancestry individuals showed an enrichment of SNPs that are associated with expression of nearby genes when compared to the index European height SNPs (P<0.01. Our results highlight the utility of genetic studies in non-European populations to understand the etiology of complex human diseases and traits.

  14. Cell lethality after selective irradiation of the DNA replication fork

    International Nuclear Information System (INIS)

    Hofer, K.G.; Warters, R.L.

    1985-01-01

    It has been suggested that nascent DNA located at the DNA replication fork may exhibit enhanced sensitivity to radiation damage. To evaluate this hypothesis, Chinese hamster ovary cells (CHO) were labeled with 125 I-iododeoxyuridine ( 125 IUdR) either in the presence or absence of aphidicolin. Aphidicolin (5 μg/ml) reduced cellular 125 IUdR incorporation to 3-5% of the control value. The residual 125 I incorporation appeared to be restricted to low molecular weight (sub-replicon sized) fragments of DNA which were more sensitive to micrococcal nuclease attack and less sensitive to high salt DNase I digestion than randomly labeled DNA. These findings suggest that DNA replicated in the presence of aphidicolin remains localized at the replication fork adjacent to the nuclear matrix. Based on these observations an attempt was made to compare the lethal consequences of 125 I decays at the replication fork to that of 125 I decays randomly distributed over the entire genome. Regardless of the distribution of decay events, all treatment groups exhibited identical dose-response curves (D 0 : 101 125 I decays/cell). Since differential irradiation of the replication complex did not result in enhanced cell lethality, it can be concluded that neither the nascent DNA nor the protein components (replicative enzymes, nuclear protein matrix) associated with the DNA replication site constitute key radiosensitive targets within the cellular genome. (orig.)

  15. Analysis of JC virus DNA replication using a quantitative and high-throughput assay

    Science.gov (United States)

    Shin, Jong; Phelan, Paul J.; Chhum, Panharith; Bashkenova, Nazym; Yim, Sung; Parker, Robert; Gagnon, David; Gjoerup, Ole; Archambault, Jacques; Bullock, Peter A.

    2015-01-01

    Progressive Multifocal Leukoencephalopathy (PML) is caused by lytic replication of JC virus (JCV) in specific cells of the central nervous system. Like other polyomaviruses, JCV encodes a large T-antigen helicase needed for replication of the viral DNA. Here, we report the development of a luciferase-based, quantitative and high-throughput assay of JCV DNA replication in C33A cells, which, unlike the glial cell lines Hs 683 and U87, accumulate high levels of nuclear T-ag needed for robust replication. Using this assay, we investigated the requirement for different domains of T-ag, and for specific sequences within and flanking the viral origin, in JCV DNA replication. Beyond providing validation of the assay, these studies revealed an important stimulatory role of the transcription factor NF1 in JCV DNA replication. Finally, we show that the assay can be used for inhibitor testing, highlighting its value for the identification of antiviral drugs targeting JCV DNA replication. PMID:25155200

  16. Nuclear DNA Replication in Trypanosomatids: There Are No Easy Methods for Solving Difficult Problems.

    Science.gov (United States)

    da Silva, Marcelo S; Pavani, Raphael S; Damasceno, Jeziel D; Marques, Catarina A; McCulloch, Richard; Tosi, Luiz Ricardo Orsini; Elias, Maria Carolina

    2017-11-01

    In trypanosomatids, etiological agents of devastating diseases, replication is robust and finely controlled to maintain genome stability and function in stressful environments. However, these parasites encode several replication protein components and complexes that show potentially variant composition compared with model eukaryotes. This review focuses on the advances made in recent years regarding the differences and peculiarities of the replication machinery in trypanosomatids, including how such divergence might affect DNA replication dynamics and the replication stress response. Comparing the DNA replication machinery and processes of parasites and their hosts may provide a foundation for the identification of targets that can be used in the development of chemotherapies to assist in the eradication of diseases caused by these pathogens. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

  17. The Green Revolution in Indonesia: A Replicable Success?

    NARCIS (Netherlands)

    Frankema, E.H.P.

    2015-01-01

    This chapter aims to disentangle the causal complex underpinning Indonesia’s ‘green revolution’ in order to assess which aspects of it are, in principle, replicable in other parts of the world and which aspects are not. More in particular this study focuses on the question which elements of the

  18. Identification of Poxvirus Genome Uncoating and DNA Replication Factors with Mutually Redundant Roles.

    Science.gov (United States)

    Liu, Baoming; Panda, Debasis; Mendez-Rios, Jorge D; Ganesan, Sundar; Wyatt, Linda S; Moss, Bernard

    2018-04-01

    Genome uncoating is essential for replication of most viruses. For poxviruses, the process is divided into two stages: removal of the envelope, allowing early gene expression, and breaching of the core wall, allowing DNA release, replication, and late gene expression. Subsequent studies showed that the host proteasome and the viral D5 protein, which has an essential role in DNA replication, are required for vaccinia virus (VACV) genome uncoating. In a search for additional VACV uncoating proteins, we noted a report that described a defect in DNA replication and late expression when the gene encoding a 68-kDa ankyrin repeat/F-box protein (68k-ank), associated with the cellular SCF (Skp1, cullin1, F-box-containing complex) ubiquitin ligase complex, was deleted from the attenuated modified vaccinia virus Ankara (MVA). Here we showed that the 68k-ank deletion mutant exhibited diminished genome uncoating, formation of DNA prereplication sites, and degradation of viral cores as well as an additional, independent defect in DNA synthesis. Deletion of the 68k-ank homolog of VACV strain WR, however, was without effect, suggesting the existence of compensating genes. By inserting VACV genes into an MVA 68k-ank deletion mutant, we discovered that M2, a member of the poxvirus immune evasion (PIE) domain superfamily and a regulator of NF-κB, and C5, a member of the BTB/Kelch superfamily associated with cullin-3-based ligase complexes, independently rescued the 68k-ank deletion phenotype. Thus, poxvirus uncoating and DNA replication are intertwined processes involving at least three viral proteins with mutually redundant functions in addition to D5. IMPORTANCE Poxviruses comprise a family of large DNA viruses that infect vertebrates and invertebrates and cause diseases of medical and zoological importance. Poxviruses, unlike most other DNA viruses, replicate in the cytoplasm, and their large genomes usually encode 200 or more proteins with diverse functions. About 90 genes may

  19. HCV-induced autophagosomes are generated via homotypic fusion of phagophores that mediate HCV RNA replication.

    Directory of Open Access Journals (Sweden)

    Linya Wang

    2017-09-01

    Full Text Available Hepatitis C virus (HCV induces autophagy to promote its replication, including its RNA replication, which can take place on double-membrane vesicles known as autophagosomes. However, how HCV induces the biogenesis of autophagosomes and how HCV RNA replication complex may be assembled on autophagosomes were largely unknown. During autophagy, crescent membrane structures known as phagophores first appear in the cytoplasm, which then progress to become autophagosomes. By conducting electron microscopy and in vitro membrane fusion assay, we found that phagophores induced by HCV underwent homotypic fusion to generate autophagosomes in a process dependent on the SNARE protein syntaxin 7 (STX7. Further analyses by live-cell imaging and fluorescence microscopy indicated that HCV-induced phagophores originated from the endoplasmic reticulum (ER. Interestingly, comparing with autophagy induced by nutrient starvation, the progression of phagophores to autophagosomes induced by HCV took significantly longer time, indicating fundamental differences in the biogenesis of autophagosomes induced by these two different stimuli. As the knockdown of STX7 to inhibit the formation of autophagosomes did not affect HCV RNA replication, and purified phagophores could mediate HCV RNA replication, the assembly of the HCV RNA replication complex on autophagosomes apparently took place during the formative stage of phagophores. These findings provided important information for understanding how HCV controlled and modified this important cellular pathway for its own replication.

  20. Isolation of proteins involved in the replication of adenoviral DNA in vitro

    International Nuclear Information System (INIS)

    Lichy, J.H.; Nagata, K.; Friefeld, B.R.; Enomoto, T.; Field, J.; Guggenheimer, R.A.; Ikeda, J.E.; Horwitz, M.S.; Hurwitz, J.

    1983-01-01

    The simple mechanism of replication of adenoviral DNA has made adenovirus an especially useful model system for studies of eukaryotic replication mechanisms. The availability of this in vitro system that replicates exogenously added Ad DNA-pro has made it possible to characterize the factors involved in replication. The results presented in this paper summarize our further fractionation of the in vitro system. First, the properties of two factors purified from the uninfected nuclear extract are described. Second, the separation of the pTP/Ad Pol complex into subunits and the properties of the isolated subunits are presented. The 140K protein is shown to possess the Ad DNA polymerase activity. The results suggest that the only DNA polymerase required for adenoviral DNA replication in vitro is the 140K Ad DNA polymerase and that this enzyme is probably a viral gene product. 50 references, 10 figures, 3 tables

  1. Repair replication in replicating and nonreplicating DNA after irradiation with uv light

    Energy Technology Data Exchange (ETDEWEB)

    Slor, H.; Cleaver, J.E.

    1978-06-01

    Ultraviolet light induces more pyrimidine dimers and more repair replication in DNA that replicates within 2 to 3 h of irradiation than in DNA that does not replicate during this period. This difference may be due to special conformational changes in DNA and chromatin that might be associated with semiconservative DNA replication.

  2. Fear of AIDS : are there replicable, invariant questionnaire dimensions?

    NARCIS (Netherlands)

    Arrindell, W.A.; Ross, M.W.; Bridges, K.Robert; van Hout, W.; Hofman, A.; Sanderman, R.

    1989-01-01

    Explored the dimensional structure of the 38-item Fear of acquired immune deficiency syndrome (AIDS) Schedule with 684 American students. Principal components analysis with VARIMAX rotation revealed 2 separate but related, internally consistent, and replicable dimensions of AIDS fear. These were (1)

  3. Reproducibility and replicability of rodent phenotyping in preclinical studies

    NARCIS (Netherlands)

    Kafkafi, Neri; Agassi, Joseph; Chesler, Elissa J.; Crabbe, John C.; Crusio, Wim E.; Eilam, David; Gerlai, Robert; Golani, Ilan; Gomez-Marin, Alex; Heller, Ruth; Iraqi, Fuad; Jaljuli, Iman; Karp, Natasha A.; Morgan, Hugh; Nicholson, George; Pfaff, Donald W.; Richter, S. Helene; Stark, Philip B.; Stiedl, Oliver; Stodden, Victoria; Tarantino, Lisa M.; Tucci, Valter; Valdar, William; Williams, Robert W.; Würbel, Hanno; Benjamini, Yoav

    The scientific community is increasingly concerned with the proportion of published “discoveries” that are not replicated in subsequent studies. The field of rodent behavioral phenotyping was one of the first to raise this concern, and to relate it to other methodological issues: the complex

  4. Interplay between Selenium Levels and Replicative Senescence in WI-38 Human Fibroblasts: A Proteomic Approach

    Directory of Open Access Journals (Sweden)

    Ghania Hammad

    2018-01-01

    Full Text Available Selenoproteins are essential components of antioxidant defense, redox homeostasis, and cell signaling in mammals, where selenium is found in the form of a rare amino acid, selenocysteine. Selenium, which is often limited both in food intake and cell culture media, is a strong regulator of selenoprotein expression and selenoenzyme activity. Aging is a slow, complex, and multifactorial process, resulting in a gradual and irreversible decline of various functions of the body. Several cellular aspects of organismal aging are recapitulated in the replicative senescence of cultured human diploid fibroblasts, such as embryonic lung fibroblast WI-38 cells. We previously reported that the long-term growth of young WI-38 cells with high (supplemented, moderate (control, or low (depleted concentrations of selenium in the culture medium impacts their replicative lifespan, due to rapid changes in replicative senescence-associated markers and signaling pathways. In order to gain insight into the molecular link between selenium levels and replicative senescence, in the present work, we have applied a quantitative proteomic approach based on 2-Dimensional Differential in-Gel Electrophoresis (2D-DIGE to the study of young and presenescent cells grown in selenium-supplemented, control, or depleted media. Applying a restrictive cut-off (spot intensity ±50% and a p value < 0.05 to the 2D-DIGE analyses revealed 81 differentially expressed protein spots, from which 123 proteins of interest were identified by mass spectrometry. We compared the changes in protein abundance for three different conditions: (i spots varying between young and presenescent cells, (ii spots varying in response to selenium concentration in young cells, and (iii spots varying in response to selenium concentration in presenescent cells. Interestingly, a 72% overlap between the impact of senescence and selenium was observed in our proteomic results, demonstrating a strong interplay between

  5. From the chromatin interaction network to the organization of the human genome into replication N/U-domains

    International Nuclear Information System (INIS)

    Boulos, Rasha E; Julienne, Hanna; Baker, Antoine; Jensen, Pablo; Arneodo, Alain; Audit, Benjamin; Chen, Chun-Long; D'Aubenton-Carafa, Yves; Thermes, Claude; Petryk, Nataliya; Kahli, Malik; Hyrien, Olivier; Goldar, Arach

    2014-01-01

    The three-dimensional (3D) architecture of the mammalian nucleus is now being unraveled thanks to the recent development of chromatin conformation capture (3C) technologies. Here we report the results of a combined multiscale analysis of genome-wide mean replication timing and chromatin conformation data that reveal some intimate relationships between chromatin folding and human DNA replication. We previously described megabase replication N/U-domains as mammalian multiorigin replication units, and showed that their borders are ‘master’ replication initiation zones that likely initiate cascades of origin firing responsible for the stereotypic replication of these domains. Here, we demonstrate that replication N/U-domains correspond to the structural domains of self-interacting chromatin, and that their borders act as insulating regions both in high-throughput 3C (Hi-C) data and high-resolution 3C (4C) experiments. Further analyses of Hi-C data using a graph-theoretical approach reveal that N/U-domain borders are long-distance, interconnected hubs of the chromatin interaction network. Overall, these results and the observation that a well-defined ordering of chromatin states exists from N/U-domain borders to centers suggest that ‘master’ replication initiation zones are at the heart of a high-order, epigenetically controlled 3D organization of the human genome. (paper)

  6. MOF Suppresses Replication Stress and Contributes to Resolution of Stalled Replication Forks.

    Science.gov (United States)

    Singh, Dharmendra Kumar; Pandita, Raj K; Singh, Mayank; Chakraborty, Sharmistha; Hambarde, Shashank; Ramnarain, Deepti; Charaka, Vijaya; Ahmed, Kazi Mokim; Hunt, Clayton R; Pandita, Tej K

    2018-03-15

    The human MOF (hMOF) protein belongs to the MYST family of histone acetyltransferases and plays a critical role in transcription and the DNA damage response. MOF is essential for cell proliferation; however, its role during replication and replicative stress is unknown. Here we demonstrate that cells depleted of MOF and under replicative stress induced by cisplatin, hydroxyurea, or camptothecin have reduced survival, a higher frequency of S-phase-specific chromosome damage, and increased R-loop formation. MOF depletion decreased replication fork speed and, when combined with replicative stress, also increased stalled replication forks as well as new origin firing. MOF interacted with PCNA, a key coordinator of replication and repair machinery at replication forks, and affected its ubiquitination and recruitment to the DNA damage site. Depletion of MOF, therefore, compromised the DNA damage repair response as evidenced by decreased Mre11, RPA70, Rad51, and PCNA focus formation, reduced DNA end resection, and decreased CHK1 phosphorylation in cells after exposure to hydroxyurea or cisplatin. These results support the argument that MOF plays an important role in suppressing replication stress induced by genotoxic agents at several stages during the DNA damage response. Copyright © 2018 American Society for Microbiology.

  7. The actin-like MreB cytoskeleton organizes viral DNA replication in bacteria.

    Science.gov (United States)

    Muñoz-Espín, Daniel; Daniel, Richard; Kawai, Yoshikazu; Carballido-López, Rut; Castilla-Llorente, Virginia; Errington, Jeff; Meijer, Wilfried J J; Salas, Margarita

    2009-08-11

    Little is known about the organization or proteins involved in membrane-associated replication of prokaryotic genomes. Here we show that the actin-like MreB cytoskeleton of the distantly related bacteria Escherichia coli and Bacillus subtilis is required for efficient viral DNA replication. Detailed analyses of B. subtilis phage ϕ29 showed that the MreB cytoskeleton plays a crucial role in organizing phage DNA replication at the membrane. Thus, phage double-stranded DNA and components of the ϕ29 replication machinery localize in peripheral helix-like structures in a cytoskeleton-dependent way. Importantly, we show that MreB interacts directly with the ϕ29 membrane-protein p16.7, responsible for attaching viral DNA at the cell membrane. Altogether, the results reveal another function for the MreB cytoskeleton and describe a mechanism by which viral DNA replication is organized at the bacterial membrane.

  8. Dynamic assembly, localization and proteolysis of the Bacillus subtilis SMC complex

    Directory of Open Access Journals (Sweden)

    Rinn Cornelia

    2005-06-01

    Full Text Available Abstract Background SMC proteins are key components of several protein complexes that perform vital tasks in different chromosome dynamics. Bacterial SMC forms a complex with ScpA and ScpB that is essential for chromosome arrangement and segregation. The complex localizes to discrete centres on the nucleoids that during most of the time of the cell cycle localize in a bipolar manner. The complex binds to DNA and condenses DNA in an as yet unknown manner. Results We show that in vitro, ScpA and ScpB form different complexes with each other, among which the level of the putative 2 ScpA/4 ScpB complex showed a pronounced decrease in level upon addition of SMC protein. Different mutations of the ATPase-binding pocket of SMC reduced, but did not abolish interaction of mutant SMC with ScpA and ScpB. The loss of SMC ATPase activity led to a loss of function in vivo, and abolished proper localization of the SMC complex. The formation of bipolar SMC centres was also lost after repression of gyrase activity, and was abnormal during inhibition of replication, resulting in single central clusters. Resumption of replication quickly re-established bipolar SMC centres, showing that proper localization depends on ongoing replication. We also found that the SMC protein is subject to induced proteolysis, most strikingly as cells enter stationary phase, which is partly achieved by ClpX and LonA proteases. Atomic force microscopy revealed the existence of high order rosette-like SMC structures in vitro, which might explain the formation of the SMC centres in vivo. Conclusion Our data suggest that a ScpA/ScpB sub-complex is directly recruited into the SMC complex. This process does not require SMC ATPase activity, which, however, appears to facilitate loading of ScpA and ScpB. Thus, the activity of SMC could be regulated through binding and release of ScpA and ScpB, which has been shown to affect SMC ATPase activity. The proper bipolar localization of the SMC

  9. Physical interactions between bacteriophage and Escherichia coli proteins required for initiation of lambda DNA replication.

    Science.gov (United States)

    Liberek, K; Osipiuk, J; Zylicz, M; Ang, D; Skorko, J; Georgopoulos, C

    1990-02-25

    The process of initiation of lambda DNA replication requires the assembly of the proper nucleoprotein complex at the origin of replication, ori lambda. The complex is composed of both phage and host-coded proteins. The lambda O initiator protein binds specifically to ori lambda. The lambda P initiator protein binds to both lambda O and the host-coded dnaB helicase, giving rise to an ori lambda DNA.lambda O.lambda P.dnaB structure. The dnaK and dnaJ heat shock proteins have been shown capable of dissociating this complex. The thus freed dnaB helicase unwinds the duplex DNA template at the replication fork. In this report, through cross-linking, size chromatography, and protein affinity chromatography, we document some of the protein-protein interactions occurring at ori lambda. Our results show that the dnaK protein specifically interacts with both lambda O and lambda P, and that the dnaJ protein specifically interacts with the dnaB helicase.

  10. Database Replication

    CERN Document Server

    Kemme, Bettina

    2010-01-01

    Database replication is widely used for fault-tolerance, scalability and performance. The failure of one database replica does not stop the system from working as available replicas can take over the tasks of the failed replica. Scalability can be achieved by distributing the load across all replicas, and adding new replicas should the load increase. Finally, database replication can provide fast local access, even if clients are geographically distributed clients, if data copies are located close to clients. Despite its advantages, replication is not a straightforward technique to apply, and

  11. A Scalable Permutation Approach Reveals Replication and Preservation Patterns of Network Modules in Large Datasets.

    Science.gov (United States)

    Ritchie, Scott C; Watts, Stephen; Fearnley, Liam G; Holt, Kathryn E; Abraham, Gad; Inouye, Michael

    2016-07-01

    Network modules-topologically distinct groups of edges and nodes-that are preserved across datasets can reveal common features of organisms, tissues, cell types, and molecules. Many statistics to identify such modules have been developed, but testing their significance requires heuristics. Here, we demonstrate that current methods for assessing module preservation are systematically biased and produce skewed p values. We introduce NetRep, a rapid and computationally efficient method that uses a permutation approach to score module preservation without assuming data are normally distributed. NetRep produces unbiased p values and can distinguish between true and false positives during multiple hypothesis testing. We use NetRep to quantify preservation of gene coexpression modules across murine brain, liver, adipose, and muscle tissues. Complex patterns of multi-tissue preservation were revealed, including a liver-derived housekeeping module that displayed adipose- and muscle-specific association with body weight. Finally, we demonstrate the broader applicability of NetRep by quantifying preservation of bacterial networks in gut microbiota between men and women. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

  12. Stalled replication forks generate a distinct mutational signature in yeast

    DEFF Research Database (Denmark)

    Larsen, Nicolai B.; Liberti, Sascha E.; Vogel, Ivan

    2017-01-01

    Proliferating cells acquire genome alterations during the act of DNA replication. This leads to mutation accumulation and somatic cell mosaicism in multicellular organisms, and is also implicated as an underlying cause of aging and tumorigenesis. The molecular mechanisms of DNA replication...... Escherichia coli Tus/Ter complex) engineered into the yeast genome. We demonstrate that transient stalling at this barrier induces a distinct pattern of genome rearrangements in the newly replicated region behind the stalled fork, which primarily consist of localized losses and duplications of DNA sequences....... These genetic alterations arise through the aberrant repair of a single-stranded DNA gap, in a process that is dependent on Exo1- and Shu1-dependent homologous recombination repair (HRR). Furthermore, aberrant processing of HRR intermediates, and elevated HRR-associated mutagenesis, is detectable in a yeast...

  13. Substrate recognition by complement convertases revealed in the C5-cobra venom factor complex

    DEFF Research Database (Denmark)

    Laursen, Nick Stub; Andersen, Kasper Røjkjær; Braren, Ingke

    2011-01-01

    with a protease subunit (Bb or C2a). We determined the crystal structures of the C3b homologue cobra venom factor (CVF) in complex with C5, and in complex with C5 and the inhibitor SSL7 at 4.3 Å resolution. The structures reveal a parallel two-point attachment between C5 and CVF, where the presence of SSL7 only...

  14. X-irradiation affects all DNA replication intermediates when inhibiting replication initiation

    International Nuclear Information System (INIS)

    Loenn, U.; Karolinska Hospital, Stockholm

    1982-01-01

    When a human melanoma line was irradiated with 10 Gy, there was, after 30 to 60 min, a gradual reduction in the DNA replication rate. Ten to twelve hours after the irradiation, the DNA replication had returned to near normal rate. The results showed tht low dose-rate X-irradiation inhibits preferentially the formation of small DNA replication intermediates. There is no difference between the inhibition of these replication intermediates formed only in the irradiated cells and those formed also in untreated cells. (U.K.)

  15. Chromatin Structure and Replication Origins: Determinants Of Chromosome Replication And Nuclear Organization

    Science.gov (United States)

    Smith, Owen K.; Aladjem, Mirit I.

    2014-01-01

    The DNA replication program is, in part, determined by the epigenetic landscape that governs local chromosome architecture and directs chromosome duplication. Replication must coordinate with other biochemical processes occurring concomitantly on chromatin, such as transcription and remodeling, to insure accurate duplication of both genetic and epigenetic features and to preserve genomic stability. The importance of genome architecture and chromatin looping in coordinating cellular processes on chromatin is illustrated by two recent sets of discoveries. First, chromatin-associated proteins that are not part of the core replication machinery were shown to affect the timing of DNA replication. These chromatin-associated proteins could be working in concert, or perhaps in competition, with the transcriptional machinery and with chromatin modifiers to determine the spatial and temporal organization of replication initiation events. Second, epigenetic interactions are mediated by DNA sequences that determine chromosomal replication. In this review we summarize recent findings and current models linking spatial and temporal regulation of the replication program with epigenetic signaling. We discuss these issues in the context of the genome’s three-dimensional structure with an emphasis on events occurring during the initiation of DNA replication. PMID:24905010

  16. RADX interacts with single-stranded DNA to promote replication fork stability

    DEFF Research Database (Denmark)

    Schubert, Lisa; Ho, Teresa; Hoffmann, Saskia

    2017-01-01

    Single-stranded DNA (ssDNA) regions form as an intermediate in many DNA-associated transactions. Multiple cellular proteins interact with ssDNA via the oligonucleotide/oligosaccharide-binding (OB) fold domain. The heterotrimeric, multi-OB fold domain-containing Replication Protein A (RPA) complex...... ssDNA-binding activities is critical for avoiding these defects. Our findings establish RADX as an important component of cellular pathways that promote DNA replication integrity under basal and stressful conditions by means of multiple ssDNA-binding proteins....

  17. Segment-specific terminal sequences of Bunyamwera bunyavirus regulate genome replication

    International Nuclear Information System (INIS)

    Barr, John N.; Elliott, Richard M.; Dunn, Ewan F.; Wertz, Gail W.

    2003-01-01

    Bunyamwera virus (BUNV) is the prototype of both the Orthobunyavirus genus and the Bunyaviridae family of segmented negative sense RNA viruses. The tripartite BUNV genome consists of small (S), medium (M), and large (L) segments that are transcribed to give a single mRNA and replicated to generate an antigenome that is the template for synthesis of further genomic RNA strands. We modified an existing cDNA-derived RNA synthesis system to allow identification of BUNV RNA replication and transcription products by direct metabolic labeling. Direct RNA analysis allowed us to distinguish between template activities that affected either RNA replication or mRNA transcription, an ability that was not possible using previous reporter gene expression assays. We generated genome analogs containing the entire nontranslated terminal sequences of the S, M, and L BUNV segments surrounding a common sequence. Analysis of RNAs synthesized from these templates revealed that the relative abilities of BUNV segments to perform RNA replication was M > L > S. Exchange of segment-specific terminal nucleotides identified a 12-nt region located within both the 3' and 5' termini of the M segment that correlated with its high replication ability

  18. APOBEC3 Interference during Replication of Viral Genomes

    Directory of Open Access Journals (Sweden)

    Luc Willems

    2015-06-01

    Full Text Available Co-evolution of viruses and their hosts has reached a fragile and dynamic equilibrium that allows viral persistence, replication and transmission. In response, infected hosts have developed strategies of defense that counteract the deleterious effects of viral infections. In particular, single-strand DNA editing by Apolipoprotein B Editing Catalytic subunits proteins 3 (APOBEC3s is a well-conserved mechanism of mammalian innate immunity that mutates and inactivates viral genomes. In this review, we describe the mechanisms of APOBEC3 editing during viral replication, the viral strategies that prevent APOBEC3 activity and the consequences of APOBEC3 modulation on viral fitness and host genome integrity. Understanding the mechanisms involved reveals new prospects for therapeutic intervention.

  19. A unique binding mode enables MCM2 to chaperone histones H3-H4 at replication forks.

    Science.gov (United States)

    Huang, Hongda; Strømme, Caroline B; Saredi, Giulia; Hödl, Martina; Strandsby, Anne; González-Aguilera, Cristina; Chen, Shoudeng; Groth, Anja; Patel, Dinshaw J

    2015-08-01

    During DNA replication, chromatin is reassembled by recycling of modified old histones and deposition of new ones. How histone dynamics integrates with DNA replication to maintain genome and epigenome information remains unclear. Here, we reveal how human MCM2, part of the replicative helicase, chaperones histones H3-H4. Our first structure shows an H3-H4 tetramer bound by two MCM2 histone-binding domains (HBDs), which hijack interaction sites used by nucleosomal DNA. Our second structure reveals MCM2 and ASF1 cochaperoning an H3-H4 dimer. Mutational analyses show that the MCM2 HBD is required for MCM2-7 histone-chaperone function and normal cell proliferation. Further, we show that MCM2 can chaperone both new and old canonical histones H3-H4 as well as H3.3 and CENPA variants. The unique histone-binding mode of MCM2 thus endows the replicative helicase with ideal properties for recycling histones genome wide during DNA replication.

  20. Prelife catalysts and replicators

    OpenAIRE

    Ohtsuki, Hisashi; Nowak, Martin A.

    2009-01-01

    Life is based on replication and evolution. But replication cannot be taken for granted. We must ask what there was prior to replication and evolution. How does evolution begin? We have proposed prelife as a generative system that produces information and diversity in the absence of replication. We model prelife as a binary soup of active monomers that form random polymers. ‘Prevolutionary’ dynamics can have mutation and selection prior to replication. Some sequences might have catalytic acti...

  1. Timeless links replication termination to mitotic kinase activation.

    Directory of Open Access Journals (Sweden)

    Jayaraju Dheekollu

    2011-05-01

    Full Text Available The mechanisms that coordinate the termination of DNA replication with progression through mitosis are not completely understood. The human Timeless protein (Tim associates with S phase replication checkpoint proteins Claspin and Tipin, and plays an important role in maintaining replication fork stability at physical barriers, like centromeres, telomeres and ribosomal DNA repeats, as well as at termination sites. We show here that human Tim can be isolated in a complex with mitotic entry kinases CDK1, Auroras A and B, and Polo-like kinase (Plk1. Plk1 bound Tim directly and colocalized with Tim at a subset of mitotic structures in M phase. Tim depletion caused multiple mitotic defects, including the loss of sister-chromatid cohesion, loss of mitotic spindle architecture, and a failure to exit mitosis. Tim depletion caused a delay in mitotic kinase activity in vivo and in vitro, as well as a reduction in global histone H3 S10 phosphorylation during G2/M phase. Tim was also required for the recruitment of Plk1 to centromeric DNA and formation of catenated DNA structures at human centromere alpha satellite repeats. Taken together, these findings suggest that Tim coordinates mitotic kinase activation with termination of DNA replication.

  2. Loss of Hda activity stimulates replication initiation from I-box, but not R4 mutant origins in Escherichia coli.

    Science.gov (United States)

    Riber, Leise; Fujimitsu, Kazuyuki; Katayama, Tsutomu; Løbner-Olesen, Anders

    2009-01-01

    Initiation of chromosome replication in Escherichia coli is limited by the initiator protein DnaA associated with ATP. Within the replication origin, binding sites for DnaA associated with ATP or ADP (R boxes) and the DnaA(ATP) specific sites (I-boxes, tau-boxes and 6-mer sites) are found. We analysed chromosome replication of cells carrying mutations in conserved regions of oriC. Cells carrying mutations in DnaA-boxes I2, I3, R2, R3 and R5 as well as FIS and IHF binding sites resembled wild-type cells with respect to origin concentration. Initiation of replication in these mutants occurred in synchrony or with slight asynchrony only. Furthermore, lack of Hda stimulated initiation in all these mutants. The DnaA(ATP) containing complex that leads to initiation can therefore be formed in the absence of several of the origin DnaA binding sites including both DnaA(ATP) specific I-boxes. However, competition between I-box mutant and wild-type origins, revealed a positive role of I-boxes on initiation. On the other hand, mutations affecting DnaA-box R4 were found to be compromised for initiation and could not be augmented by an increase in cellular DnaA(ATP)/DnaA(ADP) ratio. Compared with the sites tested here, R4 therefore seems to contribute to initiation most critically.

  3. Novel host restriction factors implicated in HIV-1 replication.

    Science.gov (United States)

    Ghimire, Dibya; Rai, Madhu; Gaur, Ritu

    2018-04-01

    Human immunodeficiency virus-1 (HIV-1) is known to interact with multiple host cellular proteins during its replication in the target cell. While many of these host cellular proteins facilitate viral replication, a number of them are reported to inhibit HIV-1 replication at various stages of its life cycle. These host cellular proteins, which are known as restriction factors, constitute an integral part of the host's first line of defence against the viral pathogen. Since the discovery of apolipoprotein B mRNA-editing enzyme 3G (APOBEC3G) as an HIV-1 restriction factor, several human proteins have been identified that exhibit anti-HIV-1 restriction. While each restriction factor employs a distinct mechanism of inhibition, the HIV-1 virus has equally evolved complex counter strategies to neutralize their inhibitory effect. APOBEC3G, tetherin, sterile alpha motif and histidine-aspartate domain 1 (SAMHD1), and trim-5α are some of the best known HIV-1 restriction factors that have been studied in great detail. Recently, six novel restriction factors were discovered that exhibit significant antiviral activity: endoplasmic reticulum α1,2-mannosidase I (ERManI), translocator protein (TSPO), guanylate-binding protein 5 (GBP5), serine incorporator (SERINC3/5) and zinc-finger antiviral protein (ZAP). The focus of this review is to discuss the antiviral mechanism of action of these six restriction factors and provide insights into the probable counter-evasion strategies employed by the HIV-1 virus. The recent discovery of new restriction factors substantiates the complex host-pathogen interactions occurring during HIV-1 pathogenesis and makes it imperative that further investigations are conducted to elucidate the molecular basis of HIV-1 replication.

  4. Sustainability of a Compartmentalized Host-Parasite Replicator System under Periodic Washout-Mixing Cycles

    Directory of Open Access Journals (Sweden)

    Taro Furubayashi

    2018-01-01

    Full Text Available The emergence and dominance of parasitic replicators are among the major hurdles for the proliferation of primitive replicators. Compartmentalization of replicators is proposed to relieve the parasite dominance; however, it remains unclear under what conditions simple compartmentalization uncoupled with internal reaction secures the long-term survival of a population of primitive replicators against incessant parasite emergence. Here, we investigate the sustainability of a compartmentalized host-parasite replicator (CHPR system undergoing periodic washout-mixing cycles, by constructing a mathematical model and performing extensive simulations. We describe sustainable landscapes of the CHPR system in the parameter space and elucidate the mechanism of phase transitions between sustainable and extinct regions. Our findings revealed that a large population size of compartments, a high mixing intensity, and a modest amount of nutrients are important factors for the robust survival of replicators. We also found two distinctive sustainable phases with different mixing intensities. These results suggest that a population of simple host–parasite replicators assumed before the origin of life can be sustained by a simple compartmentalization with periodic washout-mixing processes.

  5. GEMC1 is a TopBP1-interacting protein required for chromosomal DNA replication.

    Science.gov (United States)

    Balestrini, Alessia; Cosentino, Claudia; Errico, Alessia; Garner, Elizabeth; Costanzo, Vincenzo

    2010-05-01

    Many of the factors required for chromosomal DNA replication have been identified in unicellular eukaryotes. However, DNA replication is poorly understood in multicellular organisms. Here, we report the identification of GEMC1 (geminin coiled-coil containing protein 1), a novel vertebrate protein required for chromosomal DNA replication. GEMC1 is highly conserved in vertebrates and is preferentially expressed in proliferating cells. Using Xenopus laevis egg extract we show that Xenopus GEMC1 (xGEMC1) binds to the checkpoint and replication factor TopBP1, which promotes binding of xGEMC1 to chromatin during pre-replication complex (pre-RC) formation. We demonstrate that xGEMC1 interacts directly with replication factors such as Cdc45 and the kinase Cdk2-CyclinE, through which it is heavily phosphorylated. Phosphorylated xGEMC1 stimulates initiation of DNA replication, whereas depletion of xGEMC1 prevents the onset of DNA replication owing to the impairment of Cdc45 loading onto chromatin. Similarly, inhibition of GEMC1 expression with morpholino and siRNA oligos prevents DNA replication in embryonic and somatic vertebrate cells. These data suggest that GEMC1 promotes initiation of chromosomal DNA replication in multicellular organisms by mediating TopBP1- and Cdk2-dependent recruitment of Cdc45 onto replication origins.

  6. Data from Investigating Variation in Replicability: A “Many Labs” Replication Project

    Directory of Open Access Journals (Sweden)

    Richard A. Klein

    2014-04-01

    Full Text Available This dataset is from the Many Labs Replication Project in which 13 effects were replicated across 36 samples and over 6,000 participants. Data from the replications are included, along with demographic variables about the participants and contextual information about the environment in which the replication was conducted. Data were collected in-lab and online through a standardized procedure administered via an online link. The dataset is stored on the Open Science Framework website. These data could be used to further investigate the results of the included 13 effects or to study replication and generalizability more broadly.

  7. Suppression of the Escherichia coli dnaA46 mutation by changes in the activities of the pyruvate-acetate node links DNA replication regulation to central carbon metabolism.

    Science.gov (United States)

    Tymecka-Mulik, Joanna; Boss, Lidia; Maciąg-Dorszyńska, Monika; Matias Rodrigues, João F; Gaffke, Lidia; Wosinski, Anna; Cech, Grzegorz M; Szalewska-Pałasz, Agnieszka; Węgrzyn, Grzegorz; Glinkowska, Monika

    2017-01-01

    To ensure faithful transmission of genetic material to progeny cells, DNA replication is tightly regulated, mainly at the initiation step. Escherichia coli cells regulate the frequency of initiation according to growth conditions. Results of the classical, as well as the latest studies, suggest that the DNA replication in E. coli starts at a predefined, constant cell volume per chromosome but the mechanisms coordinating DNA replication with cell growth are still not fully understood. Results of recent investigations have revealed a role of metabolic pathway proteins in the control of cell division and a direct link between metabolism and DNA replication has also been suggested both in Bacillus subtilis and E. coli cells. In this work we show that defects in the acetate overflow pathway suppress the temperature-sensitivity of a defective replication initiator-DnaA under acetogenic growth conditions. Transcriptomic and metabolic analyses imply that this suppression is correlated with pyruvate accumulation, resulting from alterations in the pyruvate dehydrogenase (PDH) activity. Consequently, deletion of genes encoding the pyruvate dehydrogenase subunits likewise resulted in suppression of the thermal-sensitive growth of the dnaA46 strain. We propose that the suppressor effect may be directly related to the PDH complex activity, providing a link between an enzyme of the central carbon metabolism and DNA replication.

  8. Replicating Health Economic Models: Firm Foundations or a House of Cards?

    Science.gov (United States)

    Bermejo, Inigo; Tappenden, Paul; Youn, Ji-Hee

    2017-11-01

    Health economic evaluation is a framework for the comparative analysis of the incremental health gains and costs associated with competing decision alternatives. The process of developing health economic models is usually complex, financially expensive and time-consuming. For these reasons, model development is sometimes based on previous model-based analyses; this endeavour is usually referred to as model replication. Such model replication activity may involve the comprehensive reproduction of an existing model or 'borrowing' all or part of a previously developed model structure. Generally speaking, the replication of an existing model may require substantially less effort than developing a new de novo model by bypassing, or undertaking in only a perfunctory manner, certain aspects of model development such as the development of a complete conceptual model and/or comprehensive literature searching for model parameters. A further motivation for model replication may be to draw on the credibility or prestige of previous analyses that have been published and/or used to inform decision making. The acceptability and appropriateness of replicating models depends on the decision-making context: there exists a trade-off between the 'savings' afforded by model replication and the potential 'costs' associated with reduced model credibility due to the omission of certain stages of model development. This paper provides an overview of the different levels of, and motivations for, replicating health economic models, and discusses the advantages, disadvantages and caveats associated with this type of modelling activity. Irrespective of whether replicated models should be considered appropriate or not, complete replicability is generally accepted as a desirable property of health economic models, as reflected in critical appraisal checklists and good practice guidelines. To this end, the feasibility of comprehensive model replication is explored empirically across a small

  9. De novo identification of replication-timing domains in the human genome by deep learning.

    Science.gov (United States)

    Liu, Feng; Ren, Chao; Li, Hao; Zhou, Pingkun; Bo, Xiaochen; Shu, Wenjie

    2016-03-01

    The de novo identification of the initiation and termination zones-regions that replicate earlier or later than their upstream and downstream neighbours, respectively-remains a key challenge in DNA replication. Building on advances in deep learning, we developed a novel hybrid architecture combining a pre-trained, deep neural network and a hidden Markov model (DNN-HMM) for the de novo identification of replication domains using replication timing profiles. Our results demonstrate that DNN-HMM can significantly outperform strong, discriminatively trained Gaussian mixture model-HMM (GMM-HMM) systems and other six reported methods that can be applied to this challenge. We applied our trained DNN-HMM to identify distinct replication domain types, namely the early replication domain (ERD), the down transition zone (DTZ), the late replication domain (LRD) and the up transition zone (UTZ), using newly replicated DNA sequencing (Repli-Seq) data across 15 human cells. A subsequent integrative analysis revealed that these replication domains harbour unique genomic and epigenetic patterns, transcriptional activity and higher-order chromosomal structure. Our findings support the 'replication-domain' model, which states (1) that ERDs and LRDs, connected by UTZs and DTZs, are spatially compartmentalized structural and functional units of higher-order chromosomal structure, (2) that the adjacent DTZ-UTZ pairs form chromatin loops and (3) that intra-interactions within ERDs and LRDs tend to be short-range and long-range, respectively. Our model reveals an important chromatin organizational principle of the human genome and represents a critical step towards understanding the mechanisms regulating replication timing. Our DNN-HMM method and three additional algorithms can be freely accessed at https://github.com/wenjiegroup/DNN-HMM The replication domain regions identified in this study are available in GEO under the accession ID GSE53984. shuwj@bmi.ac.cn or boxc

  10. Cooperative working of bacterial chromosome replication proteins generated by a reconstituted protein expression system

    Science.gov (United States)

    Fujiwara, Kei; Katayama, Tsutomu; Nomura, Shin-ichiro M.

    2013-01-01

    Replication of all living cells relies on the multirounds flow of the central dogma. Especially, expression of DNA replication proteins is a key step to circulate the processes of the central dogma. Here we achieved the entire sequential transcription–translation–replication process by autonomous expression of chromosomal DNA replication machineries from a reconstituted transcription–translation system (PURE system). We found that low temperature is essential to express a complex protein, DNA polymerase III, in a single tube using the PURE system. Addition of the 13 genes, encoding initiator, DNA helicase, helicase loader, RNA primase and DNA polymerase III to the PURE system gave rise to a DNA replication system by a coupling manner. An artificial genetic circuit demonstrated that the DNA produced as a result of the replication is able to provide genetic information for proteins, indicating the in vitro central dogma can sequentially undergo two rounds. PMID:23737447

  11. Tus-Ter as a tool to study site-specific DNA replication perturbation in eukaryotes

    DEFF Research Database (Denmark)

    Larsen, Nicolai B; Hickson, Ian D; Mankouri, Hocine W

    2014-01-01

    The high-affinity binding of the Tus protein to specific 21-bp sequences, called Ter, causes site-specific, and polar, DNA replication fork arrest in E coli. The Tus-Ter complex serves to coordinate DNA replication with chromosome segregation in this organism. A number of recent and ongoing studies...... have demonstrated that Tus-Ter can be used as a heterologous tool to generate site-specific perturbation of DNA replication when reconstituted in eukaryotes. Here, we review these recent findings and explore the molecular mechanism by which Tus-Ter mediates replication fork (RF) arrest in the budding...... yeast, S. cerevisiae. We propose that Tus-Ter is a versatile, genetically tractable, and regulatable RF blocking system that can be utilized for disrupting DNA replication in a diverse range of host cells....

  12. Tus-Ter as a tool to study site-specific DNA replication perturbation in eukaryotes.

    Science.gov (United States)

    Larsen, Nicolai B; Hickson, Ian D; Mankouri, Hocine W

    2014-01-01

    The high-affinity binding of the Tus protein to specific 21-bp sequences, called Ter, causes site-specific, and polar, DNA replication fork arrest in E coli. The Tus-Ter complex serves to coordinate DNA replication with chromosome segregation in this organism. A number of recent and ongoing studies have demonstrated that Tus-Ter can be used as a heterologous tool to generate site-specific perturbation of DNA replication when reconstituted in eukaryotes. Here, we review these recent findings and explore the molecular mechanism by which Tus-Ter mediates replication fork (RF) arrest in the budding yeast, S. cerevisiae. We propose that Tus-Ter is a versatile, genetically tractable, and regulatable RF blocking system that can be utilized for disrupting DNA replication in a diverse range of host cells.

  13. Definition of herpes simplex virus type 1 helper activities for adeno-associated virus early replication events.

    Directory of Open Access Journals (Sweden)

    Nathalie Alazard-Dany

    2009-03-01

    Full Text Available The human parvovirus Adeno-Associated Virus (AAV type 2 can only replicate in cells co-infected with a helper virus, such as Adenovirus or Herpes Simplex Virus type 1 (HSV-1; whereas, in the absence of a helper virus, it establishes a latent infection. Previous studies demonstrated that the ternary HSV-1 helicase/primase (HP complex (UL5/8/52 and the single-stranded DNA-Binding Protein (ICP8 were sufficient to induce AAV-2 replication in transfected cells. We independently showed that, in the context of a latent AAV-2 infection, the HSV-1 ICP0 protein was able to activate rep gene expression. The present study was conducted to integrate these observations and to further explore the requirement of other HSV-1 proteins during early AAV replication steps, i.e. rep gene expression and AAV DNA replication. Using a cellular model that mimics AAV latency and composite constructs coding for various sets of HSV-1 genes, we first confirmed the role of ICP0 for rep gene expression and demonstrated a synergistic effect of ICP4 and, to a lesser extent, ICP22. Conversely, ICP27 displayed an inhibitory effect. Second, our analyses showed that the effect of ICP0, ICP4, and ICP22 on rep gene expression was essential for the onset of AAV DNA replication in conjunction with the HP complex and ICP8. Third, and most importantly, we demonstrated that the HSV-1 DNA polymerase complex (UL30/UL42 was critical to enhance AAV DNA replication to a significant level in transfected cells and that its catalytic activity was involved in this process. Altogether, this work represents the first comprehensive study recapitulating the series of early events taking place during HSV-1-induced AAV replication.

  14. Phosphorylation of Minichromosome Maintenance 3 (MCM3) by Checkpoint Kinase 1 (Chk1) Negatively Regulates DNA Replication and Checkpoint Activation.

    Science.gov (United States)

    Han, Xiangzi; Mayca Pozo, Franklin; Wisotsky, Jacob N; Wang, Benlian; Jacobberger, James W; Zhang, Youwei

    2015-05-08

    Mechanisms controlling DNA replication and replication checkpoint are critical for the maintenance of genome stability and the prevention or treatment of human cancers. Checkpoint kinase 1 (Chk1) is a key effector protein kinase that regulates the DNA damage response and replication checkpoint. The heterohexameric minichromosome maintenance (MCM) complex is the core component of mammalian DNA helicase and has been implicated in replication checkpoint activation. Here we report that Chk1 phosphorylates the MCM3 subunit of the MCM complex at Ser-205 under normal growth conditions. Mutating the Ser-205 of MCM3 to Ala increased the length of DNA replication track and shortened the S phase duration, indicating that Ser-205 phosphorylation negatively controls normal DNA replication. Upon replicative stress treatment, the inhibitory phosphorylation of MCM3 at Ser-205 was reduced, and this reduction was accompanied with the generation of single strand DNA, the key platform for ataxia telangiectasia mutated and Rad3-related (ATR) activation. As a result, the replication checkpoint is activated. Together, these data provide significant insights into the regulation of both normal DNA replication and replication checkpoint activation through the novel phosphorylation of MCM3 by Chk1. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  15. Functions of Ubiquitin and SUMO in DNA Replication and Replication Stress

    Science.gov (United States)

    García-Rodríguez, Néstor; Wong, Ronald P.; Ulrich, Helle D.

    2016-01-01

    Complete and faithful duplication of its entire genetic material is one of the essential prerequisites for a proliferating cell to maintain genome stability. Yet, during replication DNA is particularly vulnerable to insults. On the one hand, lesions in replicating DNA frequently cause a stalling of the replication machinery, as most DNA polymerases cannot cope with defective templates. This situation is aggravated by the fact that strand separation in preparation for DNA synthesis prevents common repair mechanisms relying on strand complementarity, such as base and nucleotide excision repair, from working properly. On the other hand, the replication process itself subjects the DNA to a series of hazardous transformations, ranging from the exposure of single-stranded DNA to topological contortions and the generation of nicks and fragments, which all bear the risk of inducing genomic instability. Dealing with these problems requires rapid and flexible responses, for which posttranslational protein modifications that act independently of protein synthesis are particularly well suited. Hence, it is not surprising that members of the ubiquitin family, particularly ubiquitin itself and SUMO, feature prominently in controlling many of the defensive and restorative measures involved in the protection of DNA during replication. In this review we will discuss the contributions of ubiquitin and SUMO to genome maintenance specifically as they relate to DNA replication. We will consider cases where the modifiers act during regular, i.e., unperturbed stages of replication, such as initiation, fork progression, and termination, but also give an account of their functions in dealing with lesions, replication stalling and fork collapse. PMID:27242895

  16. Apoptosis-like yeast cell death in response to DNA damage and replication defects

    Energy Technology Data Exchange (ETDEWEB)

    Burhans, William C.; Weinberger, Martin; Marchetti, Maria A.; Ramachandran, Lakshmi; D' Urso, Gennaro; Huberman, Joel A

    2003-11-27

    In budding (Saccharomyces cerevisiae) and fission (Schizosaccharomyces pombe) yeast and other unicellular organisms, DNA damage and other stimuli can induce cell death resembling apoptosis in metazoans, including the activation of a recently discovered caspase-like molecule in budding yeast. Induction of apoptotic-like cell death in yeasts requires homologues of cell cycle checkpoint proteins that are often required for apoptosis in metazoan cells. Here, we summarize these findings and our unpublished results which show that an important component of metazoan apoptosis recently detected in budding yeast - reactive oxygen species (ROS) - can also be detected in fission yeast undergoing an apoptotic-like cell death. ROS were detected in fission and budding yeast cells bearing conditional mutations in genes encoding DNA replication initiation proteins and in fission yeast cells with mutations that deregulate cyclin-dependent kinases (CDKs). These mutations may cause DNA damage by permitting entry of cells into S phase with a reduced number of replication forks and/or passage through mitosis with incompletely replicated chromosomes. This may be relevant to the frequent requirement for elevated CDK activity in mammalian apoptosis, and to the recent discovery that the initiation protein Cdc6 is destroyed during apoptosis in mammals and in budding yeast cells exposed to lethal levels of DNA damage. Our data indicate that connections between apoptosis-like cell death and DNA replication or CDK activity are complex. Some apoptosis-like pathways require checkpoint proteins, others are inhibited by them, and others are independent of them. This complexity resembles that of apoptotic pathways in mammalian cells, which are frequently deregulated in cancer. The greater genetic tractability of yeasts should help to delineate these complex pathways and their relationships to cancer and to the effects of apoptosis-inducing drugs that inhibit DNA replication.

  17. Apoptosis-like yeast cell death in response to DNA damage and replication defects

    International Nuclear Information System (INIS)

    Burhans, William C.; Weinberger, Martin; Marchetti, Maria A.; Ramachandran, Lakshmi; D'Urso, Gennaro; Huberman, Joel A.

    2003-01-01

    In budding (Saccharomyces cerevisiae) and fission (Schizosaccharomyces pombe) yeast and other unicellular organisms, DNA damage and other stimuli can induce cell death resembling apoptosis in metazoans, including the activation of a recently discovered caspase-like molecule in budding yeast. Induction of apoptotic-like cell death in yeasts requires homologues of cell cycle checkpoint proteins that are often required for apoptosis in metazoan cells. Here, we summarize these findings and our unpublished results which show that an important component of metazoan apoptosis recently detected in budding yeast - reactive oxygen species (ROS) - can also be detected in fission yeast undergoing an apoptotic-like cell death. ROS were detected in fission and budding yeast cells bearing conditional mutations in genes encoding DNA replication initiation proteins and in fission yeast cells with mutations that deregulate cyclin-dependent kinases (CDKs). These mutations may cause DNA damage by permitting entry of cells into S phase with a reduced number of replication forks and/or passage through mitosis with incompletely replicated chromosomes. This may be relevant to the frequent requirement for elevated CDK activity in mammalian apoptosis, and to the recent discovery that the initiation protein Cdc6 is destroyed during apoptosis in mammals and in budding yeast cells exposed to lethal levels of DNA damage. Our data indicate that connections between apoptosis-like cell death and DNA replication or CDK activity are complex. Some apoptosis-like pathways require checkpoint proteins, others are inhibited by them, and others are independent of them. This complexity resembles that of apoptotic pathways in mammalian cells, which are frequently deregulated in cancer. The greater genetic tractability of yeasts should help to delineate these complex pathways and their relationships to cancer and to the effects of apoptosis-inducing drugs that inhibit DNA replication

  18. Interplay between human high mobility group protein 1 and replication protein A on psoralen-cross-linked DNA

    DEFF Research Database (Denmark)

    Reddy, Madhava C; Christensen, Jesper; Vasquez, Karen M

    2005-01-01

    -DNA interstrand cross-link (ICL) to a specific site to determine the effect of HMGB proteins on recognition of these lesions. Our results reveal that human HMGB1 (but not HMGB2) binds with high affinity and specificity to psoralen ICLs, and interacts with the essential NER protein, replication protein A (RPA......), at these lesions. RPA, shown previously to bind tightly to these lesions, also binds in the presence of HMGB1, without displacing HMGB1. A discrete ternary complex is formed, containing HMGB1, RPA, and psoralen-damaged DNA. Thus, HMGB1 has the ability to recognize ICLs, can cooperate with RPA in doing so...

  19. Analysis of JC virus DNA replication using a quantitative and high-throughput assay

    International Nuclear Information System (INIS)

    Shin, Jong; Phelan, Paul J.; Chhum, Panharith; Bashkenova, Nazym; Yim, Sung; Parker, Robert; Gagnon, David; Gjoerup, Ole; Archambault, Jacques; Bullock, Peter A.

    2014-01-01

    Progressive Multifocal Leukoencephalopathy (PML) is caused by lytic replication of JC virus (JCV) in specific cells of the central nervous system. Like other polyomaviruses, JCV encodes a large T-antigen helicase needed for replication of the viral DNA. Here, we report the development of a luciferase-based, quantitative and high-throughput assay of JCV DNA replication in C33A cells, which, unlike the glial cell lines Hs 683 and U87, accumulate high levels of nuclear T-ag needed for robust replication. Using this assay, we investigated the requirement for different domains of T-ag, and for specific sequences within and flanking the viral origin, in JCV DNA replication. Beyond providing validation of the assay, these studies revealed an important stimulatory role of the transcription factor NF1 in JCV DNA replication. Finally, we show that the assay can be used for inhibitor testing, highlighting its value for the identification of antiviral drugs targeting JCV DNA replication. - Highlights: • Development of a high-throughput screening assay for JCV DNA replication using C33A cells. • Evidence that T-ag fails to accumulate in the nuclei of established glioma cell lines. • Evidence that NF-1 directly promotes JCV DNA replication in C33A cells. • Proof-of-concept that the HTS assay can be used to identify pharmacological inhibitor of JCV DNA replication

  20. Analysis of JC virus DNA replication using a quantitative and high-throughput assay

    Energy Technology Data Exchange (ETDEWEB)

    Shin, Jong; Phelan, Paul J.; Chhum, Panharith; Bashkenova, Nazym; Yim, Sung; Parker, Robert [Department of Developmental, Molecular and Chemical Biology, Tufts University School of Medicine, Boston, MA 02111 (United States); Gagnon, David [Institut de Recherches Cliniques de Montreal (IRCM), 110 Pine Avenue West, Montreal, Quebec, Canada H2W 1R7 (Canada); Department of Biochemistry and Molecular Medicine, Université de Montréal, Montréal, Quebec (Canada); Gjoerup, Ole [Molecular Oncology Research Institute, Tufts Medical Center, Boston, MA 02111 (United States); Archambault, Jacques [Institut de Recherches Cliniques de Montreal (IRCM), 110 Pine Avenue West, Montreal, Quebec, Canada H2W 1R7 (Canada); Department of Biochemistry and Molecular Medicine, Université de Montréal, Montréal, Quebec (Canada); Bullock, Peter A., E-mail: Peter.Bullock@tufts.edu [Department of Developmental, Molecular and Chemical Biology, Tufts University School of Medicine, Boston, MA 02111 (United States)

    2014-11-15

    Progressive Multifocal Leukoencephalopathy (PML) is caused by lytic replication of JC virus (JCV) in specific cells of the central nervous system. Like other polyomaviruses, JCV encodes a large T-antigen helicase needed for replication of the viral DNA. Here, we report the development of a luciferase-based, quantitative and high-throughput assay of JCV DNA replication in C33A cells, which, unlike the glial cell lines Hs 683 and U87, accumulate high levels of nuclear T-ag needed for robust replication. Using this assay, we investigated the requirement for different domains of T-ag, and for specific sequences within and flanking the viral origin, in JCV DNA replication. Beyond providing validation of the assay, these studies revealed an important stimulatory role of the transcription factor NF1 in JCV DNA replication. Finally, we show that the assay can be used for inhibitor testing, highlighting its value for the identification of antiviral drugs targeting JCV DNA replication. - Highlights: • Development of a high-throughput screening assay for JCV DNA replication using C33A cells. • Evidence that T-ag fails to accumulate in the nuclei of established glioma cell lines. • Evidence that NF-1 directly promotes JCV DNA replication in C33A cells. • Proof-of-concept that the HTS assay can be used to identify pharmacological inhibitor of JCV DNA replication.

  1. DNA replication: stalling a fork for imprinting and switching

    DEFF Research Database (Denmark)

    Egel, Richard

    2004-01-01

    Mating-type switching in fission yeast has long been known to be directed by a DNA 'imprint'. This imprint has now been firmly characterized as a protected site-specific and strand-specific nick. New work also links the widely conserved Swi1-Swi3 complex to the protection of stalled replication...

  2. Inhibition of in vitro SV40 DNA replication by ultraviolet light

    International Nuclear Information System (INIS)

    Gough, G.; Wood, R.W.

    1989-01-01

    Ultraviolet light-induced DNA damage was found to inhibit SV40 origin-dependent DNA synthesis carried out by soluble humancell extracts. Replication of SV40-based plasmids was reduced to approx. 35% of that in unirradiated controls after irradiation with 50-100 J/m 2 germicidal ultraviolet light, where an average of 3-6 pyrimidine dimer photoproducts were formed per plasmid circle. Inhibition of the DNA helicase activity of T antigen (required for initiation of replication in the in vitro system) was also investigated, and was only significant after much higher fluences, 1000-5000 J/m 2 . The data indicate that DNA damage by ultraviolet light inhibits DNA synthesis in cell-free extracts principally by affecting components of the replication complex other than the DNA helicase activity of T antigen. The soluble system could be used to biochemically investigate the possible bypass or tolerance of DNA damage during replication (author). 21 refs.; 2 figs

  3. NACSA Charter School Replication Guide: The Spectrum of Replication Options. Authorizing Matters. Replication Brief 1

    Science.gov (United States)

    O'Neill, Paul

    2010-01-01

    One of the most important and high-profile issues in public education reform today is the replication of successful public charter school programs. With more than 5,000 failing public schools in the United States, there is a tremendous need for strong alternatives for parents and students. Replicating successful charter school models is an…

  4. Mutational Analysis of the Hypervariable Region of Hepatitis E Virus Reveals Its Involvement in the Efficiency of Viral RNA Replication

    OpenAIRE

    Pudupakam, R. S.; Kenney, Scott P.; Córdoba, Laura; Huang, Yao-Wei; Dryman, Barbara A.; LeRoith, Tanya; Pierson, F. William; Meng, Xiang-Jin

    2011-01-01

    The RNA genome of the hepatitis E virus (HEV) contains a hypervariable region (HVR) in ORF1 that tolerates small deletions with respect to infectivity. To further investigate the role of the HVR in HEV replication, we constructed a panel of mutants with overlapping deletions in the N-terminal, central, and C-terminal regions of the HVR by using a genotype 1 human HEV luciferase replicon and analyzed the effects of deletions on viral RNA replication in Huh7 cells. We found that the replication...

  5. In vitro analysis of the role of replication protein A (RPA) and RPA phosphorylation in ATR-mediated checkpoint signaling.

    Science.gov (United States)

    Lindsey-Boltz, Laura A; Reardon, Joyce T; Wold, Marc S; Sancar, Aziz

    2012-10-19

    Replication protein A (RPA) plays essential roles in DNA metabolism, including replication, checkpoint, and repair. Recently, we described an in vitro system in which the phosphorylation of human Chk1 kinase by ATR (ataxia telangiectasia mutated and Rad3-related) is dependent on RPA bound to single-stranded DNA. Here, we report that phosphorylation of other ATR targets, p53 and Rad17, has the same requirements and that RPA is also phosphorylated in this system. At high p53 or Rad17 concentrations, RPA phosphorylation is inhibited and, in this system, RPA with phosphomimetic mutations cannot support ATR kinase function, whereas a non-phosphorylatable RPA mutant exhibits full activity. Phosphorylation of these ATR substrates depends on the recruitment of ATR and the substrates by RPA to the RPA-ssDNA complex. Finally, mutant RPAs lacking checkpoint function exhibit essentially normal activity in nucleotide excision repair, revealing RPA separation of function for checkpoint and excision repair.

  6. In Vitro Analysis of the Role of Replication Protein A (RPA) and RPA Phosphorylation in ATR-mediated Checkpoint Signaling*

    Science.gov (United States)

    Lindsey-Boltz, Laura A.; Reardon, Joyce T.; Wold, Marc S.; Sancar, Aziz

    2012-01-01

    Replication protein A (RPA) plays essential roles in DNA metabolism, including replication, checkpoint, and repair. Recently, we described an in vitro system in which the phosphorylation of human Chk1 kinase by ATR (ataxia telangiectasia mutated and Rad3-related) is dependent on RPA bound to single-stranded DNA. Here, we report that phosphorylation of other ATR targets, p53 and Rad17, has the same requirements and that RPA is also phosphorylated in this system. At high p53 or Rad17 concentrations, RPA phosphorylation is inhibited and, in this system, RPA with phosphomimetic mutations cannot support ATR kinase function, whereas a non-phosphorylatable RPA mutant exhibits full activity. Phosphorylation of these ATR substrates depends on the recruitment of ATR and the substrates by RPA to the RPA-ssDNA complex. Finally, mutant RPAs lacking checkpoint function exhibit essentially normal activity in nucleotide excision repair, revealing RPA separation of function for checkpoint and excision repair. PMID:22948311

  7. Internal Disequilibria and Phenotypic Diversification during Replication of Hepatitis C Virus in a Noncoevolving Cellular Environment.

    Science.gov (United States)

    Moreno, Elena; Gallego, Isabel; Gregori, Josep; Lucía-Sanz, Adriana; Soria, María Eugenia; Castro, Victoria; Beach, Nathan M; Manrubia, Susanna; Quer, Josep; Esteban, Juan Ignacio; Rice, Charles M; Gómez, Jordi; Gastaminza, Pablo; Domingo, Esteban; Perales, Celia

    2017-05-15

    Viral quasispecies evolution upon long-term virus replication in a noncoevolving cellular environment raises relevant general issues, such as the attainment of population equilibrium, compliance with the molecular-clock hypothesis, or stability of the phenotypic profile. Here, we evaluate the adaptation, mutant spectrum dynamics, and phenotypic diversification of hepatitis C virus (HCV) in the course of 200 passages in human hepatoma cells in an experimental design that precluded coevolution of the cells with the virus. Adaptation to the cells was evidenced by increase in progeny production. The rate of accumulation of mutations in the genomic consensus sequence deviated slightly from linearity, and mutant spectrum analyses revealed a complex dynamic of mutational waves, which was sustained beyond passage 100. The virus underwent several phenotypic changes, some of which impacted the virus-host relationship, such as enhanced cell killing, a shift toward higher virion density, and increased shutoff of host cell protein synthesis. Fluctuations in progeny production and failure to reach population equilibrium at the genomic level suggest internal instabilities that anticipate an unpredictable HCV evolution in the complex liver environment. IMPORTANCE Long-term virus evolution in an unperturbed cellular environment can reveal features of virus evolution that cannot be explained by comparing natural viral isolates. In the present study, we investigate genetic and phenotypic changes that occur upon prolonged passage of hepatitis C virus (HCV) in human hepatoma cells in an experimental design in which host cell evolutionary change is prevented. Despite replication in a noncoevolving cellular environment, the virus exhibited internal population disequilibria that did not decline with increased adaptation to the host cells. The diversification of phenotypic traits suggests that disequilibria inherent to viral populations may provide a selective advantage to viruses that can

  8. Transcription-Replication Conflict Orientation Modulates R-Loop Levels and Activates Distinct DNA Damage Responses.

    Science.gov (United States)

    Hamperl, Stephan; Bocek, Michael J; Saldivar, Joshua C; Swigut, Tomek; Cimprich, Karlene A

    2017-08-10

    Conflicts between transcription and replication are a potent source of DNA damage. Co-transcriptional R-loops could aggravate such conflicts by creating an additional barrier to replication fork progression. Here, we use a defined episomal system to investigate how conflict orientation and R-loop formation influence genome stability in human cells. R-loops, but not normal transcription complexes, induce DNA breaks and orientation-specific DNA damage responses during conflicts with replication forks. Unexpectedly, the replisome acts as an orientation-dependent regulator of R-loop levels, reducing R-loops in the co-directional (CD) orientation but promoting their formation in the head-on (HO) orientation. Replication stress and deregulated origin firing increase the number of HO collisions leading to genome-destabilizing R-loops. Our findings connect DNA replication to R-loop homeostasis and suggest a mechanistic basis for genome instability resulting from deregulated DNA replication, observed in cancer and other disease states. Copyright © 2017 Elsevier Inc. All rights reserved.

  9. Mms1 binds to G-rich regions in Saccharomyces cerevisiae and influences replication and genome stability

    NARCIS (Netherlands)

    Wanzek, Katharina; Schwindt, Eike; Capra, John A.; Paeschke, Katrin

    2017-01-01

    The regulation of replication is essential to preserve genome integrity. Mms1 is part of the E3 ubiquitin ligase complex that is linked to replication fork progression. By identifying Mms1 binding sites genome-wide in Saccharomyces cerevisiae we connected Mms1 function to genome integrity and

  10. RPA binds histone H3-H4 and functions in DNA replication-coupled nucleosome assembly.

    Science.gov (United States)

    Liu, Shaofeng; Xu, Zhiyun; Leng, He; Zheng, Pu; Yang, Jiayi; Chen, Kaifu; Feng, Jianxun; Li, Qing

    2017-01-27

    DNA replication-coupled nucleosome assembly is essential to maintain genome integrity and retain epigenetic information. Multiple involved histone chaperones have been identified, but how nucleosome assembly is coupled to DNA replication remains elusive. Here we show that replication protein A (RPA), an essential replisome component that binds single-stranded DNA, has a role in replication-coupled nucleosome assembly. RPA directly binds free H3-H4. Assays using a synthetic sequence that mimics freshly unwound single-stranded DNA at replication fork showed that RPA promotes DNA-(H3-H4) complex formation immediately adjacent to double-stranded DNA. Further, an RPA mutant defective in H3-H4 binding exhibited attenuated nucleosome assembly on nascent chromatin. Thus, we propose that RPA functions as a platform for targeting histone deposition to replication fork, through which RPA couples nucleosome assembly with ongoing DNA replication. Copyright © 2017, American Association for the Advancement of Science.

  11. P-body proteins regulate transcriptional rewiring to promote DNA replication stress resistance.

    Science.gov (United States)

    Loll-Krippleber, Raphael; Brown, Grant W

    2017-09-15

    mRNA-processing (P-) bodies are cytoplasmic granules that form in eukaryotic cells in response to numerous stresses to serve as sites of degradation and storage of mRNAs. Functional P-bodies are critical for the DNA replication stress response in yeast, yet the repertoire of P-body targets and the mechanisms by which P-bodies promote replication stress resistance are unknown. In this study we identify the complete complement of mRNA targets of P-bodies during replication stress induced by hydroxyurea treatment. The key P-body protein Lsm1 controls the abundance of HHT1, ACF4, ARL3, TMA16, RRS1 and YOX1 mRNAs to prevent their toxic accumulation during replication stress. Accumulation of YOX1 mRNA causes aberrant downregulation of a network of genes critical for DNA replication stress resistance and leads to toxic acetaldehyde accumulation. Our data reveal the scope and the targets of regulation by P-body proteins during the DNA replication stress response.P-bodies form in response to stress and act as sites of mRNA storage and degradation. Here the authors identify the mRNA targets of P-bodies during DNA replication stress, and show that P-body proteins act to prevent toxic accumulation of these target transcripts.

  12. Environmental stress speeds up DNA replication in Pseudomonas putida in chemostat cultivations.

    Science.gov (United States)

    Lieder, Sarah; Jahn, Michael; Koepff, Joachim; Müller, Susann; Takors, Ralf

    2016-01-01

    Cellular response to different types of stress is the hallmark of the cell's strategy for survival. How organisms adjust their cell cycle dynamics to compensate for changes in environmental conditions is an important unanswered question in bacterial physiology. A cell using binary fission for reproduction passes through three stages during its cell cycle: a stage from cell birth to initiation of replication, a DNA replication phase and a period of cell division. We present a detailed analysis of durations of cell cycle phases, investigating their dynamics under environmental stress conditions. Applying continuous steady state cultivations (chemostats), the DNA content of a Pseudomonas putida KT2440 population was quantified with flow cytometry at distinct growth rates. Data-driven modeling revealed that under stress conditions, such as oxygen deprivation, solvent exposure and decreased iron availability, DNA replication was accelerated correlated to the severity of the imposed stress (up to 1.9-fold). Cells maintained constant growth rates by balancing the shortened replication phase with extended cell cycle phases before and after replication. Transcriptome data underpin the transcriptional upregulation of crucial genes of the replication machinery. Hence adaption of DNA replication speed appears to be an important strategy to withstand environmental stress. Copyright © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  13. Molecular basis for PrimPol recruitment to replication forks by RPA.

    Science.gov (United States)

    Guilliam, Thomas A; Brissett, Nigel C; Ehlinger, Aaron; Keen, Benjamin A; Kolesar, Peter; Taylor, Elaine M; Bailey, Laura J; Lindsay, Howard D; Chazin, Walter J; Doherty, Aidan J

    2017-05-23

    DNA damage and secondary structures can stall the replication machinery. Cells possess numerous tolerance mechanisms to complete genome duplication in the presence of such impediments. In addition to translesion synthesis (TLS) polymerases, most eukaryotic cells contain a multifunctional replicative enzyme called primase-polymerase (PrimPol) that is capable of directly bypassing DNA damage by TLS, as well as repriming replication downstream of impediments. Here, we report that PrimPol is recruited to reprime through its interaction with RPA. Using biophysical and crystallographic approaches, we identify that PrimPol possesses two RPA-binding motifs and ascertained the key residues required for these interactions. We demonstrate that one of these motifs is critical for PrimPol's recruitment to stalled replication forks in vivo. In addition, biochemical analysis reveals that RPA serves to stimulate the primase activity of PrimPol. Together, these findings provide significant molecular insights into PrimPol's mode of recruitment to stalled forks to facilitate repriming and restart.

  14. Hydroxyurea-Induced Replication Stress

    Directory of Open Access Journals (Sweden)

    Kenza Lahkim Bennani-Belhaj

    2010-01-01

    Full Text Available Bloom's syndrome (BS displays one of the strongest known correlations between chromosomal instability and a high risk of cancer at an early age. BS cells combine a reduced average fork velocity with constitutive endogenous replication stress. However, the response of BS cells to replication stress induced by hydroxyurea (HU, which strongly slows the progression of replication forks, remains unclear due to publication of conflicting results. Using two different cellular models of BS, we showed that BLM deficiency is not associated with sensitivity to HU, in terms of clonogenic survival, DSB generation, and SCE induction. We suggest that surviving BLM-deficient cells are selected on the basis of their ability to deal with an endogenous replication stress induced by replication fork slowing, resulting in insensitivity to HU-induced replication stress.

  15. Simple genomes, complex interactions: Epistasis in RNA virus

    Science.gov (United States)

    Elena, Santiago F.; Solé, Ricard V.; Sardanyés, Josep

    2010-06-01

    Owed to their reduced size and low number of proteins encoded, RNA viruses and other subviral pathogens are often considered as being genetically too simple. However, this structural simplicity also creates the necessity for viral RNA sequences to encode for more than one protein and for proteins to carry out multiple functions, all together resulting in complex patterns of genetic interactions. In this work we will first review the experimental studies revealing that the architecture of viral genomes is dominated by antagonistic interactions among loci. Second, we will also review mathematical models and provide a description of computational tools for the study of RNA virus dynamics and evolution. As an application of these tools, we will finish this review article by analyzing a stochastic bit-string model of in silico virus replication. This model analyzes the interplay between epistasis and the mode of replication on determining the population load of deleterious mutations. The model suggests that, for a given mutation rate, the deleterious mutational load is always larger when epistasis is predominantly antagonistic than when synergism is the rule. However, the magnitude of this effect is larger if replication occurs geometrically than if it proceeds linearly.

  16. N-terminally truncated POM121C inhibits HIV-1 replication.

    Directory of Open Access Journals (Sweden)

    Hideki Saito

    Full Text Available Recent studies have identified host cell factors that regulate early stages of HIV-1 infection including viral cDNA synthesis and orientation of the HIV-1 capsid (CA core toward the nuclear envelope, but it remains unclear how viral DNA is imported through the nuclear pore and guided to the host chromosomal DNA. Here, we demonstrate that N-terminally truncated POM121C, a component of the nuclear pore complex, blocks HIV-1 infection. This truncated protein is predominantly localized in the cytoplasm, does not bind to CA, does not affect viral cDNA synthesis, reduces the formation of 2-LTR and diminished the amount of integrated proviral DNA. Studies with an HIV-1-murine leukemia virus (MLV chimeric virus carrying the MLV-derived Gag revealed that Gag is a determinant of this inhibition. Intriguingly, mutational studies have revealed that the blockade by N-terminally-truncated POM121C is closely linked to its binding to importin-β/karyopherin subunit beta 1 (KPNB1. These results indicate that N-terminally-truncated POM121C inhibits HIV-1 infection after completion of reverse transcription and before integration, and suggest an important role for KPNB1 in HIV-1 replication.

  17. A reversible histone H3 acetylation cooperates with mismatch repair and replicative polymerases in maintaining genome stability.

    Directory of Open Access Journals (Sweden)

    Lyudmila Y Kadyrova

    2013-10-01

    Full Text Available Mutations are a major driving force of evolution and genetic disease. In eukaryotes, mutations are produced in the chromatin environment, but the impact of chromatin on mutagenesis is poorly understood. Previous studies have determined that in yeast Saccharomyces cerevisiae, Rtt109-dependent acetylation of histone H3 on K56 is an abundant modification that is introduced in chromatin in S phase and removed by Hst3 and Hst4 in G2/M. We show here that the chromatin deacetylation on histone H3 K56 by Hst3 and Hst4 is required for the suppression of spontaneous gross chromosomal rearrangements, base substitutions, 1-bp insertions/deletions, and complex mutations. The rate of base substitutions in hst3Δ hst4Δ is similar to that in isogenic mismatch repair-deficient msh2Δ mutant. We also provide evidence that H3 K56 acetylation by Rtt109 is important for safeguarding DNA from small insertions/deletions and complex mutations. Furthermore, we reveal that both the deacetylation and acetylation on histone H3 K56 are involved in mutation avoidance mechanisms that cooperate with mismatch repair and the proofreading activities of replicative DNA polymerases in suppressing spontaneous mutagenesis. Our results suggest that cyclic acetylation and deacetylation of chromatin contribute to replication fidelity and play important roles in the protection of nuclear DNA from diverse spontaneous mutations.

  18. Phosphorylation of Large T Antigen Regulates Merkel Cell Polyomavirus Replication

    International Nuclear Information System (INIS)

    Diaz, Jason; Wang, Xin; Tsang, Sabrina H.; Jiao, Jing; You, Jianxin

    2014-01-01

    Merkel Cell Polyomavirus (MCPyV) was recently discovered as a novel human polyomavirus that is associated with ~80% of Merkel Cell Carcinomas. The Large Tumor antigen (LT) is an early viral protein which has a variety of functions, including manipulation of the cell cycle and initiating viral DNA replication. Phosphorylation plays a critical regulatory role for polyomavirus LT proteins, but no investigation of MCPyV LT phosphorylation has been performed to date. In this report mass spectrometry analysis reveals three unique phosphorylation sites: T271, T297 and T299. In vivo replication assays confirm that phosphorylation of T271 does not play a role in viral replication, while modification at T297 and T299 have dramatic and opposing effects on LT’s ability to initiate replication from the viral origin. We test these mutants for their ability to bind, unwind, and act as a functional helicase at the viral origin. These studies provide a framework for understanding how phosphorylation of LT may dynamically regulate viral replication. Although the natural host cell of MCPyV has not yet been established, this work provides a foundation for understanding how LT activity is regulated and provides tools for better exploring this regulation in both natural host cells and Merkel cells

  19. Phosphorylation of Large T Antigen Regulates Merkel Cell Polyomavirus Replication

    Energy Technology Data Exchange (ETDEWEB)

    Diaz, Jason; Wang, Xin; Tsang, Sabrina H. [Department of Microbiology, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104 (United States); Jiao, Jing [Department of Pathology and Laboratory Medicine, Children’s Hospital of Philadelphia, Philadelphia, PA 19104 (United States); You, Jianxin, E-mail: jianyou@mail.med.upenn.edu [Department of Microbiology, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104 (United States)

    2014-07-08

    Merkel Cell Polyomavirus (MCPyV) was recently discovered as a novel human polyomavirus that is associated with ~80% of Merkel Cell Carcinomas. The Large Tumor antigen (LT) is an early viral protein which has a variety of functions, including manipulation of the cell cycle and initiating viral DNA replication. Phosphorylation plays a critical regulatory role for polyomavirus LT proteins, but no investigation of MCPyV LT phosphorylation has been performed to date. In this report mass spectrometry analysis reveals three unique phosphorylation sites: T271, T297 and T299. In vivo replication assays confirm that phosphorylation of T271 does not play a role in viral replication, while modification at T297 and T299 have dramatic and opposing effects on LT’s ability to initiate replication from the viral origin. We test these mutants for their ability to bind, unwind, and act as a functional helicase at the viral origin. These studies provide a framework for understanding how phosphorylation of LT may dynamically regulate viral replication. Although the natural host cell of MCPyV has not yet been established, this work provides a foundation for understanding how LT activity is regulated and provides tools for better exploring this regulation in both natural host cells and Merkel cells.

  20. Replicating animal mitochondrial DNA

    Directory of Open Access Journals (Sweden)

    Emily A. McKinney

    2013-01-01

    Full Text Available The field of mitochondrial DNA (mtDNA replication has been experiencing incredible progress in recent years, and yet little is certain about the mechanism(s used by animal cells to replicate this plasmid-like genome. The long-standing strand-displacement model of mammalian mtDNA replication (for which single-stranded DNA intermediates are a hallmark has been intensively challenged by a new set of data, which suggests that replication proceeds via coupled leading-and lagging-strand synthesis (resembling bacterial genome replication and/or via long stretches of RNA intermediates laid on the mtDNA lagging-strand (the so called RITOLS. The set of proteins required for mtDNA replication is small and includes the catalytic and accessory subunits of DNA polymerase y, the mtDNA helicase Twinkle, the mitochondrial single-stranded DNA-binding protein, and the mitochondrial RNA polymerase (which most likely functions as the mtDNA primase. Mutations in the genes coding for the first three proteins are associated with human diseases and premature aging, justifying the research interest in the genetic, biochemical and structural properties of the mtDNA replication machinery. Here we summarize these properties and discuss the current models of mtDNA replication in animal cells.

  1. Recruitment of Mcm10 to Sites of Replication Initiation Requires Direct Binding to the Minichromosome Maintenance (MCM) Complex*

    Science.gov (United States)

    Douglas, Max E.

    2016-01-01

    Mcm10 is required for the initiation of eukaryotic DNA replication and contributes in some unknown way to the activation of the Cdc45-MCM-GINS (CMG) helicase. How Mcm10 is localized to sites of replication initiation is unclear, as current models indicate that direct binding to minichromosome maintenance (MCM) plays a role, but the details and functional importance of this interaction have not been determined. Here, we show that purified Mcm10 can bind both DNA-bound double hexamers and soluble single hexamers of MCM. The binding of Mcm10 to MCM requires the Mcm10 C terminus. Moreover, the binding site for Mcm10 on MCM includes the Mcm2 and Mcm6 subunits and overlaps that for the loading factor Cdt1. Whether Mcm10 recruitment to replication origins depends on CMG helicase assembly has been unclear. We show that Mcm10 recruitment occurs via two modes: low affinity recruitment in the absence of CMG assembly (“G1-like”) and high affinity recruitment when CMG assembly takes place (“S-phase-like”). Mcm10 that cannot bind directly to MCM is defective in both modes of recruitment and is unable to support DNA replication. These findings indicate that Mcm10 is localized to replication initiation sites by directly binding MCM through the Mcm10 C terminus. PMID:26719337

  2. Recruitment of Mcm10 to Sites of Replication Initiation Requires Direct Binding to the Minichromosome Maintenance (MCM) Complex.

    Science.gov (United States)

    Douglas, Max E; Diffley, John F X

    2016-03-11

    Mcm10 is required for the initiation of eukaryotic DNA replication and contributes in some unknown way to the activation of the Cdc45-MCM-GINS (CMG) helicase. How Mcm10 is localized to sites of replication initiation is unclear, as current models indicate that direct binding to minichromosome maintenance (MCM) plays a role, but the details and functional importance of this interaction have not been determined. Here, we show that purified Mcm10 can bind both DNA-bound double hexamers and soluble single hexamers of MCM. The binding of Mcm10 to MCM requires the Mcm10 C terminus. Moreover, the binding site for Mcm10 on MCM includes the Mcm2 and Mcm6 subunits and overlaps that for the loading factor Cdt1. Whether Mcm10 recruitment to replication origins depends on CMG helicase assembly has been unclear. We show that Mcm10 recruitment occurs via two modes: low affinity recruitment in the absence of CMG assembly ("G1-like") and high affinity recruitment when CMG assembly takes place ("S-phase-like"). Mcm10 that cannot bind directly to MCM is defective in both modes of recruitment and is unable to support DNA replication. These findings indicate that Mcm10 is localized to replication initiation sites by directly binding MCM through the Mcm10 C terminus. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  3. ATAD2 is an epigenetic reader of newly synthesized histone marks during DNA replication.

    Science.gov (United States)

    Koo, Seong Joo; Fernández-Montalván, Amaury E; Badock, Volker; Ott, Christopher J; Holton, Simon J; von Ahsen, Oliver; Toedling, Joern; Vittori, Sarah; Bradner, James E; Gorjánácz, Mátyás

    2016-10-25

    ATAD2 (ATPase family AAA domain-containing protein 2) is a chromatin regulator harboring an AAA+ ATPase domain and a bromodomain, previously proposed to function as an oncogenic transcription co-factor. Here we suggest that ATAD2 is also required for DNA replication. ATAD2 is co-expressed with genes involved in DNA replication in various cancer types and predominantly expressed in S phase cells where it localized on nascent chromatin (replication sites). Our extensive biochemical and cellular analyses revealed that ATAD2 is recruited to replication sites through a direct interaction with di-acetylated histone H4 at K5 and K12, indicative of newly synthesized histones during replication-coupled chromatin reassembly. Similar to ATAD2-depletion, ectopic expression of ATAD2 mutants that are deficient in binding to these di-acetylation marks resulted in reduced DNA replication and impaired loading of PCNA onto chromatin, suggesting relevance of ATAD2 in DNA replication. Taken together, our data show a novel function of ATAD2 in cancer and for the first time identify a reader of newly synthesized histone di-acetylation-marks during replication.

  4. Structural diversity and dynamics of genomic replication origins in Schizosaccharomyces pombe

    Science.gov (United States)

    Cotobal, Cristina; Segurado, Mónica; Antequera, Francisco

    2010-01-01

    DNA replication origins (ORI) in Schizosaccharomyces pombe colocalize with adenine and thymine (A+T)-rich regions, and earlier analyses have established a size from 0.5 to over 3 kb for a DNA fragment to drive replication in plasmid assays. We have asked what are the requirements for ORI function in the chromosomal context. By designing artificial ORIs, we have found that A+T-rich fragments as short as 100 bp without homology to S. pombe DNA are able to initiate replication in the genome. On the other hand, functional dissection of endogenous ORIs has revealed that some of them span a few kilobases and include several modules that may be as short as 25–30 contiguous A+Ts capable of initiating replication from ectopic chromosome positions. The search for elements with these characteristics across the genome has uncovered an earlier unnoticed class of low-efficiency ORIs that fire late during S phase. These results indicate that ORI specification and dynamics varies widely in S. pombe, ranging from very short elements to large regions reminiscent of replication initiation zones in mammals. PMID:20094030

  5. Different nucleosomal architectures at early and late replicating origins in Saccharomyces cerevisiae.

    Science.gov (United States)

    Soriano, Ignacio; Morafraile, Esther C; Vázquez, Enrique; Antequera, Francisco; Segurado, Mónica

    2014-09-13

    Eukaryotic genomes are replicated during S phase according to a temporal program. Several determinants control the timing of origin firing, including the chromatin environment and epigenetic modifications. However, how chromatin structure influences the timing of the activation of specific origins is still poorly understood. By performing high-resolution analysis of genome-wide nucleosome positioning we have identified different chromatin architectures at early and late replication origins. These different patterns are already established in G1 and are tightly correlated with the organization of adjacent transcription units. Moreover, specific early and late nucleosomal patterns are fixed robustly, even in rpd3 mutants in which histone acetylation and origin timing have been significantly altered. Nevertheless, higher histone acetylation levels correlate with the local modulation of chromatin structure, leading to increased origin accessibility. In addition, we conducted parallel analyses of replication and nucleosome dynamics that revealed that chromatin structure at origins is modulated during origin activation. Our results show that early and late replication origins present distinctive nucleosomal configurations, which are preferentially associated to different genomic regions. Our data also reveal that origin structure is dynamic and can be locally modulated by histone deacetylation, as well as by origin activation. These data offer novel insight into the contribution of chromatin structure to origin selection and firing in budding yeast.

  6. Hydra meiosis reveals unexpected conservation of structural synaptonemal complex proteins across metazoans

    OpenAIRE

    Fraune, Johanna; Alsheimer, Manfred; Volff, Jean-Nicolas; Busch, Karoline; Fraune, Sebastian; Bosch, Thomas C. G.; Benavente, Ricardo

    2012-01-01

    The synaptonemal complex (SC) is a key structure of meiosis, mediating the stable pairing (synapsis) of homologous chromosomes during prophase I. Its remarkable tripartite structure is evolutionarily well conserved and can be found in almost all sexually reproducing organisms. However, comparison of the different SC protein components in the common meiosis model organisms Saccharomyces cerevisiae, Arabidopsis thaliana, Caenorhabditis elegans, Drosophila melanogaster, and Mus musculus revealed...

  7. Human ribonuclease H1 resolves R-loops and thereby enables progression of the DNA replication fork.

    Science.gov (United States)

    Parajuli, Shankar; Teasley, Daniel C; Murali, Bhavna; Jackson, Jessica; Vindigni, Alessandro; Stewart, Sheila A

    2017-09-15

    Faithful DNA replication is essential for genome stability. To ensure accurate replication, numerous complex and redundant replication and repair mechanisms function in tandem with the core replication proteins to ensure DNA replication continues even when replication challenges are present that could impede progression of the replication fork. A unique topological challenge to the replication machinery is posed by RNA-DNA hybrids, commonly referred to as R-loops. Although R-loops play important roles in gene expression and recombination at immunoglobulin sites, their persistence is thought to interfere with DNA replication by slowing or impeding replication fork progression. Therefore, it is of interest to identify DNA-associated enzymes that help resolve replication-impeding R-loops. Here, using DNA fiber analysis, we demonstrate that human ribonuclease H1 (RNH1) plays an important role in replication fork movement in the mammalian nucleus by resolving R-loops. We found that RNH1 depletion results in accumulation of RNA-DNA hybrids, slowing of replication forks, and increased DNA damage. Our data uncovered a role for RNH1 in global DNA replication in the mammalian nucleus. Because accumulation of RNA-DNA hybrids is linked to various human cancers and neurodegenerative disorders, our study raises the possibility that replication fork progression might be impeded, adding to increased genomic instability and contributing to disease. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  8. A study of an adaptive replication framework for orchestrated composite web services.

    Science.gov (United States)

    Mohamed, Marwa F; Elyamany, Hany F; Nassar, Hamed M

    2013-01-01

    Replication is considered one of the most important techniques to improve the Quality of Services (QoS) of published Web Services. It has achieved impressive success in managing resource sharing and usage in order to moderate the energy consumed in IT environments. For a robust and successful replication process, attention should be paid to suitable time as well as the constraints and capabilities in which the process runs. The replication process is time-consuming since outsourcing some new replicas into other hosts is lengthy. Furthermore, nowadays, most of the business processes that might be implemented over the Web are composed of multiple Web services working together in two main styles: Orchestration and Choreography. Accomplishing a replication over such business processes is another challenge due to the complexity and flexibility involved. In this paper, we present an adaptive replication framework for regular and orchestrated composite Web services. The suggested framework includes a number of components for detecting unexpected and unhappy events that might occur when consuming the original published web services including failure or overloading. It also includes a specific replication controller to manage the replication process and select the best host that would encapsulate a new replica. In addition, it includes a component for predicting the incoming load in order to decrease the time needed for outsourcing new replicas, enhancing the performance greatly. A simulation environment has been created to measure the performance of the suggested framework. The results indicate that adaptive replication with prediction scenario is the best option for enhancing the performance of the replication process in an online business environment.

  9. Genetic variations in the DNA replication origins of human papillomavirus family correlate with their oncogenic potential.

    Science.gov (United States)

    Yilmaz, Gulden; Biswas-Fiss, Esther E; Biswas, Subhasis B

    2018-04-01

    Human papillomaviruses (HPVs) encompass a large family of viruses that range from benign to highly carcinogenic. The crucial differences between benign and carcinogenic types of HPV remain unknown, except that the two HPV types differ in the frequency of DNA replication. We have systematically analyzed the mechanism of HPV DNA replication initiation in low-risk and high-risk HPVs. Our results demonstrate that HPV-encoded E2 initiator protein and its four binding sites in the replication origin play pivotal roles in determining the destiny of the HPV-infected cell. We have identified strain-specific single nucleotide variations in E2 binding sites found only in the high-risk HPVs. We have demonstrated that these variations result in attenuated formation of the E2-DNA complex. E2 binding to these sites is linked to the activation of the DNA replication origin as well as initiation of DNA replication. Both electrophoretic mobility shift assay and atomic force microscopy studies demonstrated that binding of E2 from either low- or high-risk HPVs with variant binding sequences lacked multimeric E2-DNA complex formation in vitro. These results provided a molecular basis of differential DNA replication in the two types of HPVs and pointed to a correlation with the development of cancer. Copyright © 2017. Published by Elsevier B.V.

  10. Chromatin Controls DNA Replication Origin Selection, Lagging-Strand Synthesis, and Replication Fork Rates.

    Science.gov (United States)

    Kurat, Christoph F; Yeeles, Joseph T P; Patel, Harshil; Early, Anne; Diffley, John F X

    2017-01-05

    The integrity of eukaryotic genomes requires rapid and regulated chromatin replication. How this is accomplished is still poorly understood. Using purified yeast replication proteins and fully chromatinized templates, we have reconstituted this process in vitro. We show that chromatin enforces DNA replication origin specificity by preventing non-specific MCM helicase loading. Helicase activation occurs efficiently in the context of chromatin, but subsequent replisome progression requires the histone chaperone FACT (facilitates chromatin transcription). The FACT-associated Nhp6 protein, the nucleosome remodelers INO80 or ISW1A, and the lysine acetyltransferases Gcn5 and Esa1 each contribute separately to maximum DNA synthesis rates. Chromatin promotes the regular priming of lagging-strand DNA synthesis by facilitating DNA polymerase α function at replication forks. Finally, nucleosomes disrupted during replication are efficiently re-assembled into regular arrays on nascent DNA. Our work defines the minimum requirements for chromatin replication in vitro and shows how multiple chromatin factors might modulate replication fork rates in vivo. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  11. Modeling inhomogeneous DNA replication kinetics.

    Directory of Open Access Journals (Sweden)

    Michel G Gauthier

    Full Text Available In eukaryotic organisms, DNA replication is initiated at a series of chromosomal locations called origins, where replication forks are assembled proceeding bidirectionally to replicate the genome. The distribution and firing rate of these origins, in conjunction with the velocity at which forks progress, dictate the program of the replication process. Previous attempts at modeling DNA replication in eukaryotes have focused on cases where the firing rate and the velocity of replication forks are homogeneous, or uniform, across the genome. However, it is now known that there are large variations in origin activity along the genome and variations in fork velocities can also take place. Here, we generalize previous approaches to modeling replication, to allow for arbitrary spatial variation of initiation rates and fork velocities. We derive rate equations for left- and right-moving forks and for replication probability over time that can be solved numerically to obtain the mean-field replication program. This method accurately reproduces the results of DNA replication simulation. We also successfully adapted our approach to the inverse problem of fitting measurements of DNA replication performed on single DNA molecules. Since such measurements are performed on specified portion of the genome, the examined DNA molecules may be replicated by forks that originate either within the studied molecule or outside of it. This problem was solved by using an effective flux of incoming replication forks at the model boundaries to represent the origin activity outside the studied region. Using this approach, we show that reliable inferences can be made about the replication of specific portions of the genome even if the amount of data that can be obtained from single-molecule experiments is generally limited.

  12. Mechanisms of DNA replication termination.

    Science.gov (United States)

    Dewar, James M; Walter, Johannes C

    2017-08-01

    Genome duplication is carried out by pairs of replication forks that assemble at origins of replication and then move in opposite directions. DNA replication ends when converging replication forks meet. During this process, which is known as replication termination, DNA synthesis is completed, the replication machinery is disassembled and daughter molecules are resolved. In this Review, we outline the steps that are likely to be common to replication termination in most organisms, namely, fork convergence, synthesis completion, replisome disassembly and decatenation. We briefly review the mechanism of termination in the bacterium Escherichia coli and in simian virus 40 (SV40) and also focus on recent advances in eukaryotic replication termination. In particular, we discuss the recently discovered E3 ubiquitin ligases that control replisome disassembly in yeast and higher eukaryotes, and how their activity is regulated to avoid genome instability.

  13. TGF-β Suppression of HBV RNA through AID-Dependent Recruitment of an RNA Exosome Complex

    Science.gov (United States)

    Kitamura, Kouichi; Wang, Zhe; Chowdhury, Sajeda; Monjurul, Ahasan Md; Wakae, Kousho; Koura, Miki; Shimadu, Miyuki; Kinoshita, Kazuo; Muramatsu, Masamichi

    2015-01-01

    Transforming growth factor (TGF)-β inhibits hepatitis B virus (HBV) replication although the intracellular effectors involved are not determined. Here, we report that reduction of HBV transcripts by TGF-β is dependent on AID expression, which significantly decreases both HBV transcripts and viral DNA, resulting in inhibition of viral replication. Immunoprecipitation reveals that AID physically associates with viral P protein that binds to specific virus RNA sequence called epsilon. AID also binds to an RNA degradation complex (RNA exosome proteins), indicating that AID, RNA exosome, and P protein form an RNP complex. Suppression of HBV transcripts by TGF-β was abrogated by depletion of either AID or RNA exosome components, suggesting that AID and the RNA exosome involve in TGF-β mediated suppression of HBV RNA. Moreover, AID-mediated HBV reduction does not occur when P protein is disrupted or when viral transcription is inhibited. These results suggest that induced expression of AID by TGF-β causes recruitment of the RNA exosome to viral RNP complex and the RNA exosome degrades HBV RNA in a transcription-coupled manner. PMID:25836330

  14. Y-box-binding protein 1 interacts with hepatitis C virus NS3/4A and influences the equilibrium between viral RNA replication and infectious particle production.

    Science.gov (United States)

    Chatel-Chaix, Laurent; Melançon, Pierre; Racine, Marie-Ève; Baril, Martin; Lamarre, Daniel

    2011-11-01

    The hepatitis C virus (HCV) NS3/4A protein has several essential roles in the virus life cycle, most probably through dynamic interactions with host factors. To discover cellular cofactors that are co-opted by HCV for its replication, we elucidated the NS3/4A interactome using mass spectrometry and identified Y-box-binding protein 1 (YB-1) as an interacting partner of NS3/4A protein and HCV genomic RNA. Importantly, silencing YB-1 expression decreased viral RNA replication and severely impaired the propagation of the infectious HCV molecular clone JFH-1. Immunofluorescence studies further revealed a drastic HCV-dependent redistribution of YB-1 to the surface of the lipid droplets, an important organelle for HCV assembly. Core and NS3 protein-dependent polyprotein maturation were shown to be required for YB-1 relocalization. Unexpectedly, YB-1 knockdown cells showed the increased production of viral infectious particles while HCV RNA replication was impaired. Our data support that HCV hijacks YB-1-containing ribonucleoparticles and that YB-1-NS3/4A-HCV RNA complexes regulate the equilibrium between HCV RNA replication and viral particle production.

  15. A conserved MCM single-stranded DNA binding element is essential for replication initiation.

    Science.gov (United States)

    Froelich, Clifford A; Kang, Sukhyun; Epling, Leslie B; Bell, Stephen P; Enemark, Eric J

    2014-04-01

    The ring-shaped MCM helicase is essential to all phases of DNA replication. The complex loads at replication origins as an inactive double-hexamer encircling duplex DNA. Helicase activation converts this species to two active single hexamers that encircle single-stranded DNA (ssDNA). The molecular details of MCM DNA interactions during these events are unknown. We determined the crystal structure of the Pyrococcus furiosus MCM N-terminal domain hexamer bound to ssDNA and define a conserved MCM-ssDNA binding motif (MSSB). Intriguingly, ssDNA binds the MCM ring interior perpendicular to the central channel with defined polarity. In eukaryotes, the MSSB is conserved in several Mcm2-7 subunits, and MSSB mutant combinations in S. cerevisiae Mcm2-7 are not viable. Mutant Mcm2-7 complexes assemble and are recruited to replication origins, but are defective in helicase loading and activation. Our findings identify an important MCM-ssDNA interaction and suggest it functions during helicase activation to select the strand for translocation. DOI: http://dx.doi.org/10.7554/eLife.01993.001.

  16. Shared active site architecture between archaeal PolD and multi-subunit RNA polymerases revealed by X-ray crystallography.

    Science.gov (United States)

    Sauguet, Ludovic; Raia, Pierre; Henneke, Ghislaine; Delarue, Marc

    2016-08-22

    Archaeal replicative DNA polymerase D (PolD) constitute an atypical class of DNA polymerases made of a proofreading exonuclease subunit (DP1) and a larger polymerase catalytic subunit (DP2), both with unknown structures. We have determined the crystal structures of Pyrococcus abyssi DP1 and DP2 at 2.5 and 2.2 Å resolution, respectively, revealing a catalytic core strikingly different from all other known DNA polymerases (DNAPs). Rather, the PolD DP2 catalytic core has the same 'double-psi β-barrel' architecture seen in the RNA polymerase (RNAP) superfamily, which includes multi-subunit transcriptases of all domains of life, homodimeric RNA-silencing pathway RNAPs and atypical viral RNAPs. This finding bridges together, in non-viral world, DNA transcription and DNA replication within the same protein superfamily. This study documents further the complex evolutionary history of the DNA replication apparatus in different domains of life and proposes a classification of all extant DNAPs.

  17. Mammalian RAD52 Functions in Break-Induced Replication Repair of Collapsed DNA Replication Forks

    DEFF Research Database (Denmark)

    Sotiriou, Sotirios K; Kamileri, Irene; Lugli, Natalia

    2016-01-01

    Human cancers are characterized by the presence of oncogene-induced DNA replication stress (DRS), making them dependent on repair pathways such as break-induced replication (BIR) for damaged DNA replication forks. To better understand BIR, we performed a targeted siRNA screen for genes whose...... RAD52 facilitates repair of collapsed DNA replication forks in cancer cells....

  18. Molecular mechanism of DNA replication-coupled inactivation of the initiator protein in Escherichia coli: interaction of DnaA with the sliding clamp-loaded DNA and the sliding clamp-Hda complex.

    Science.gov (United States)

    Su'etsugu, Masayuki; Takata, Makoto; Kubota, Toshio; Matsuda, Yusaku; Katayama, Tsutomu

    2004-06-01

    In Escherichia coli, the ATP-DnaA protein initiates chromosomal replication. After the DNA polymerase III holoenzyme is loaded on to DNA, DnaA-bound ATP is hydrolysed in a manner depending on Hda protein and the DNA-loaded form of the DNA polymerase III sliding clamp subunit, which yields ADP-DnaA, an inactivated form for initiation. This regulatory DnaA-inactivation represses extra initiation events. In this study, in vitro replication intermediates and structured DNA mimicking replicational intermediates were first used to identify structural prerequisites in the process of DnaA-ATP hydrolysis. Unlike duplex DNA loaded with sliding clamps, primer RNA-DNA heteroduplexes loaded with clamps were not associated with DnaA-ATP hydrolysis, and duplex DNA provided in trans did not rescue this defect. At least 40-bp duplex DNA is competent for the DnaA-ATP hydrolysis when a single clamp was loaded. The DnaA-ATP hydrolysis was inhibited when ATP-DnaA was tightly bound to a DnaA box-bearing oligonucleotide. These results imply that the DnaA-ATP hydrolysis involves the direct interaction of ATP-DnaA with duplex DNA flanking the sliding clamp. Furthermore, Hda protein formed a stable complex with the sliding clamp. Based on these, we suggest a mechanical basis in the DnaA-inactivation that ATP-DnaA interacts with the Hda-clamp complex with the aid of DNA binding. Copyright Blackwell Publishing Limited

  19. Coordinated leading and lagging strand DNA synthesis by using the herpes simplex virus 1 replication complex and minicircle DNA templates.

    Science.gov (United States)

    Stengel, Gudrun; Kuchta, Robert D

    2011-01-01

    The origin-specific replication of the herpes simplex virus 1 genome requires seven proteins: the helicase-primase (UL5-UL8-UL52), the DNA polymerase (UL30-UL42), the single-strand DNA binding protein (ICP8), and the origin-binding protein (UL9). We reconstituted these proteins, excluding UL9, on synthetic minicircular DNA templates and monitored leading and lagging strand DNA synthesis using the strand-specific incorporation of dTMP and dAMP. Critical features of the assays that led to efficient leading and lagging stand synthesis included high helicase-primase concentrations and a lagging strand template whose sequence resembled that of the viral DNA. Depending on the nature of the minicircle template, the replication complex synthesized leading and lagging strand products at molar ratios varying between 1:1 and 3:1. Lagging strand products (∼0.2 to 0.6 kb) were significantly shorter than leading strand products (∼2 to 10 kb), and conditions that stimulated primer synthesis led to shorter lagging strand products. ICP8 was not essential; however, its presence stimulated DNA synthesis and increased the length of both leading and lagging strand products. Curiously, human DNA polymerase α (p70-p180 or p49-p58-p70-p180), which improves the utilization of RNA primers synthesized by herpesvirus primase on linear DNA templates, had no effect on the replication of the minicircles. The lack of stimulation by polymerase α suggests the existence of a macromolecular assembly that enhances the utilization of RNA primers and may functionally couple leading and lagging strand synthesis. Evidence for functional coupling is further provided by our observations that (i) leading and lagging strand synthesis produce equal amounts of DNA, (ii) leading strand synthesis proceeds faster under conditions that disable primer synthesis on the lagging strand, and (iii) conditions that accelerate helicase-catalyzed DNA unwinding stimulate decoupled leading strand synthesis but not

  20. Registered Replication Report: Strack, Martin, & Stepper (1988).

    Science.gov (United States)

    Acosta, Alberto; Adams, Reginald B; Albohn, Daniel N; Allard, Eric S; Beek, Titia; Benning, Stephen D; Blouin- Hudon, Eve-Marie; Bulnes, Luis Carlo; Caldwell, Tracy L; Calin-Jageman, Robert J; Capaldi, Colin A; Carfagno, Nicholas S; Chasten, Kelsie T; Cleeremans, Axel; Connell, Louise; DeCicco, Jennifer M.; Dijkhoff, Laura; Dijkstra, Katinka; Fischer, Agneta H; Foroni, Francesco; Gronau, Quentin F; Hess, Ursula; Holmes, Kevin J; Jones, Jacob L H; Klein, Olivier; Koch, Christopher; Korb, Sebastian; Lewinski, Peter; Liao, Julia D; Lund, Sophie; Lupiáñez, Juan; Lynott, Dermot; Nance, Christin N; Oosterwijk, Suzanne; Özdog˘ru, Asil Ali; Pacheco-Unguetti, Antonia Pilar; Pearson, Bethany; Powis, Christina; Riding, Sarah; Roberts, Tomi-Ann; Rumiati, Raffaella I; Senden, Morgane; Shea-Shumsky, Noah B; Sobocko, Karin; Soto, Jose A; Steiner, Troy G; Talarico, Jennifer M; vanAllen, Zack M; Wagenmakers, E-J; Vandekerckhove, Marie; Wainwright, Bethany; Wayand, Joseph F; Zeelenberg, Rene; Zetzer, Emily E; Zwaan, Rolf A

    2016-11-01

    According to the facial feedback hypothesis, people's affective responses can be influenced by their own facial expression (e.g., smiling, pouting), even when their expression did not result from their emotional experiences. For example, Strack, Martin, and Stepper (1988) instructed participants to rate the funniness of cartoons using a pen that they held in their mouth. In line with the facial feedback hypothesis, when participants held the pen with their teeth (inducing a "smile"), they rated the cartoons as funnier than when they held the pen with their lips (inducing a "pout"). This seminal study of the facial feedback hypothesis has not been replicated directly. This Registered Replication Report describes the results of 17 independent direct replications of Study 1 from Strack et al. (1988), all of which followed the same vetted protocol. A meta-analysis of these studies examined the difference in funniness ratings between the "smile" and "pout" conditions. The original Strack et al. (1988) study reported a rating difference of 0.82 units on a 10-point Likert scale. Our meta-analysis revealed a rating difference of 0.03 units with a 95% confidence interval ranging from -0.11 to 0.16. © The Author(s) 2016.

  1. Crystal structures of the CPAP/STIL complex reveal its role in centriole assembly and human microcephaly

    Science.gov (United States)

    Cottee, Matthew A; Muschalik, Nadine; Wong, Yao Liang; Johnson, Christopher M; Johnson, Steven; Andreeva, Antonina; Oegema, Karen; Lea, Susan M; Raff, Jordan W; van Breugel, Mark

    2013-01-01

    Centrioles organise centrosomes and template cilia and flagella. Several centriole and centrosome proteins have been linked to microcephaly (MCPH), a neuro-developmental disease associated with small brain size. CPAP (MCPH6) and STIL (MCPH7) are required for centriole assembly, but it is unclear how mutations in them lead to microcephaly. We show that the TCP domain of CPAP constitutes a novel proline recognition domain that forms a 1:1 complex with a short, highly conserved target motif in STIL. Crystal structures of this complex reveal an unusual, all-β structure adopted by the TCP domain and explain how a microcephaly mutation in CPAP compromises complex formation. Through point mutations, we demonstrate that complex formation is essential for centriole duplication in vivo. Our studies provide the first structural insight into how the malfunction of centriole proteins results in human disease and also reveal that the CPAP–STIL interaction constitutes a conserved key step in centriole biogenesis. DOI: http://dx.doi.org/10.7554/eLife.01071.001 PMID:24052813

  2. Replicating a self-affirmation intervention to address gender differences: Successes and challenges

    Science.gov (United States)

    Kost-Smith, Lauren E.; Pollock, Steven J.; Finkelstein, Noah D.; Cohen, Geoffrey L.; Ito, Tiffany A.; Miyake, Akira

    2012-02-01

    We previously reported on the success of a psychological intervention implemented to reduce gender differences in achievement in an introductory college physics course. In this prior study, we found that the gender gap on exams and the FMCE among students who completed two 15-minute self-affirmation writing exercises was significantly reduced compared to the gender gap among students who completed neutral writing exercises. In a follow-up study we replicated the self-affirmation intervention in a later semester of the same course, with the same instructor. In this paper, we report the details and preliminary results of the replication study, where we find similar patterns along exams and course grades, but do not observe these patterns along the FMCE. We begin to investigate the critical features of replicating educational interventions, finding that replicating educational interventions is challenging, complex, and involves potentially subtle factors, some of which we explore and others that require further research.

  3. REPLICATION TOOL AND METHOD OF PROVIDING A REPLICATION TOOL

    DEFF Research Database (Denmark)

    2016-01-01

    The invention relates to a replication tool (1, 1a, 1b) for producing a part (4) with a microscale textured replica surface (5a, 5b, 5c, 5d). The replication tool (1, 1a, 1b) comprises a tool surface (2a, 2b) defining a general shape of the item. The tool surface (2a, 2b) comprises a microscale...... energy directors on flange portions thereof uses the replication tool (1, 1a, 1b) to form an item (4) with a general shape as defined by the tool surface (2a, 2b). The formed item (4) comprises a microscale textured replica surface (5a, 5b, 5c, 5d) with a lateral arrangement of polydisperse microscale...

  4. A unique binding mode enables MCM2 to chaperone histones H3-H4 at replication forks

    DEFF Research Database (Denmark)

    Huang, Hongda; Strømme, Caroline B; Saredi, Giulia

    2015-01-01

    During DNA replication, chromatin is reassembled by recycling of modified old histones and deposition of new ones. How histone dynamics integrates with DNA replication to maintain genome and epigenome information remains unclear. Here, we reveal how human MCM2, part of the replicative helicase......, chaperones histones H3-H4. Our first structure shows an H3-H4 tetramer bound by two MCM2 histone-binding domains (HBDs), which hijack interaction sites used by nucleosomal DNA. Our second structure reveals MCM2 and ASF1 cochaperoning an H3-H4 dimer. Mutational analyses show that the MCM2 HBD is required...... for MCM2-7 histone-chaperone function and normal cell proliferation. Further, we show that MCM2 can chaperone both new and old canonical histones H3-H4 as well as H3.3 and CENPA variants. The unique histone-binding mode of MCM2 thus endows the replicative helicase with ideal properties for recycling...

  5. Recommendations for Replication Research in Special Education: A Framework of Systematic, Conceptual Replications

    Science.gov (United States)

    Coyne, Michael D.; Cook, Bryan G.; Therrien, William J.

    2016-01-01

    Special education researchers conduct studies that can be considered replications. However, they do not often refer to them as replication studies. The purpose of this article is to consider the potential benefits of conceptualizing special education intervention research within a framework of systematic, conceptual replication. Specifically, we…

  6. Regulatory cross-talk links Vibrio cholerae chromosome II replication and segregation.

    Directory of Open Access Journals (Sweden)

    Yoshiharu Yamaichi

    2011-07-01

    Full Text Available There is little knowledge of factors and mechanisms for coordinating bacterial chromosome replication and segregation. Previous studies have revealed that genes (and their products that surround the origin of replication (oriCII of Vibrio cholerae chromosome II (chrII are critical for controlling the replication and segregation of this chromosome. rctB, which flanks one side of oriCII, encodes a protein that initiates chrII replication; rctA, which flanks the other side of oriCII, inhibits rctB activity. The chrII parAB2 operon, which is essential for chrII partitioning, is located immediately downstream of rctA. Here, we explored how rctA exerts negative control over chrII replication. Our observations suggest that RctB has at least two DNA binding domains--one for binding to oriCII and initiating replication and the other for binding to rctA and thereby inhibiting RctB's ability to initiate replication. Notably, the inhibitory effect of rctA could be alleviated by binding of ParB2 to a centromere-like parS site within rctA. Furthermore, by binding to rctA, ParB2 and RctB inversely regulate expression of the parAB2 genes. Together, our findings suggest that fluctuations in binding of the partitioning protein ParB2 and the chrII initiator RctB to rctA underlie a regulatory network controlling both oriCII firing and the production of the essential chrII partitioning proteins. Thus, by binding both RctB and ParB2, rctA serves as a nexus for regulatory cross-talk coordinating chrII replication and segregation.

  7. DNA replication and cancer

    DEFF Research Database (Denmark)

    Boyer, Anne-Sophie; Walter, David; Sørensen, Claus Storgaard

    2016-01-01

    A dividing cell has to duplicate its DNA precisely once during the cell cycle to preserve genome integrity avoiding the accumulation of genetic aberrations that promote diseases such as cancer. A large number of endogenous impacts can challenge DNA replication and cells harbor a battery of pathways...... causing DNA replication stress and genome instability. Further, we describe cellular and systemic responses to these insults with a focus on DNA replication restart pathways. Finally, we discuss the therapeutic potential of exploiting intrinsic replicative stress in cancer cells for targeted therapy....

  8. Effects of Interferon-α/β on HBV Replication Determined by Viral Load

    Science.gov (United States)

    Tian, Yongjun; Chen, Wen-ling; Ou, Jing-hsiung James

    2011-01-01

    Interferons α and β (IFN-α/β) are type I interferons produced by the host to control microbial infections. However, the use of IFN-α to treat hepatitis B virus (HBV) patients generated sustained response to only a minority of patients. By using HBV transgenic mice as a model and by using hydrodynamic injection to introduce HBV DNA into the mouse liver, we studied the effect of IFN-α/β on HBV in vivo. Interestingly, our results indicated that IFN-α/β could have opposite effects on HBV: they suppressed HBV replication when viral load was high and enhanced HBV replication when viral load was low. IFN-α/β apparently suppressed HBV replication via transcriptional and post-transcriptional regulations. In contrast, IFN-α/β enhanced viral replication by inducing the transcription factor HNF3γ and activating STAT3, which together stimulated HBV gene expression and replication. Further studies revealed an important role of IFN-α/β in stimulating viral growth and prolonging viremia when viral load is low. This use of an innate immune response to enhance its replication and persistence may represent a novel strategy that HBV uses to enhance its growth and spread in the early stage of viral infection when the viral level is low. PMID:21829354

  9. Late-replicating X-chromosome: replication patterns in mammalian females

    Directory of Open Access Journals (Sweden)

    Tunin Karen

    2002-01-01

    Full Text Available The GTG-banding and 5-BrdU incorporation patterns of the late-replicating X-chromosome were studied in female dogs and cattle, and compared to human female patterns. The replication patterns of the short arm of the X-chromosomes did not show any difference between human, dog and cattle females. As to the long arm, some bands showed differences among the three studied species regarding the replication kinetics pattern. These differences were observed in a restricted region of the X-chromosome, delimited by Xq11 -> q25 in humans, by Xq1 -> q8 in dogs, and by Xq12 -> q32 in cattle. In an attempt to find out if these differences in the replication kinetics could be a reflection of differences in the localization of genes in that region of the X-chromosome, we used the probe for the human androgen receptor gene (AR localized at Xq12, which is in the region where we observed differences among the three studied species. We did not, however, observe hybridization signals. Our study goes on, using other human probes for genes located in the region Xq11 -> Xq25.

  10. A Replication by Any Other Name: A Systematic Review of Replicative Intervention Studies

    Science.gov (United States)

    Cook, Bryan G.; Collins, Lauren W.; Cook, Sara C.; Cook, Lysandra

    2016-01-01

    Replication research is essential to scientific knowledge. Reviews of replication studies often electronically search for "replicat*" as a textword, which does not identify studies that replicate previous research but do not self-identify as such. We examined whether the 83 intervention studies published in six non-categorical research…

  11. Registered Replication Report

    DEFF Research Database (Denmark)

    Bouwmeester, S.; Verkoeijen, P. P.J.L.; Aczel, B.

    2017-01-01

    and colleagues. The results of studies using time pressure have been mixed, with some replication attempts observing similar patterns (e.g., Rand et al., 2014) and others observing null effects (e.g., Tinghög et al., 2013; Verkoeijen & Bouwmeester, 2014). This Registered Replication Report (RRR) assessed...... the size and variability of the effect of time pressure on cooperative decisions by combining 21 separate, preregistered replications of the critical conditions from Study 7 of the original article (Rand et al., 2012). The primary planned analysis used data from all participants who were randomly assigned...

  12. Inhibition of influenza virus replication by targeting broad host cell pathways.

    Directory of Open Access Journals (Sweden)

    Isabelle Marois

    Full Text Available Antivirals that are currently used to treat influenza virus infections target components of the virus which can mutate rapidly. Consequently, there has been an increase in the number of resistant strains to one or many antivirals in recent years. Here we compared the antiviral effects of lysosomotropic alkalinizing agents (LAAs and calcium modulators (CMs, which interfere with crucial events in the influenza virus replication cycle, against avian, swine, and human viruses of different subtypes in MDCK cells. We observed that treatment with LAAs, CMs, or a combination of both, significantly inhibited viral replication. Moreover, the drugs were effective even when they were administered 8 h after infection. Finally, analysis of the expression of viral acidic polymerase (PA revealed that both drugs classes interfered with early events in the viral replication cycle. This study demonstrates that targeting broad host cellular pathways can be an efficient strategy to inhibit influenza replication. Furthermore, it provides an interesting avenue for drug development where resistance by the virus might be reduced since the virus is not targeted directly.

  13. Replication and virus-induced transcriptome of HAdV-5 in normal host cells versus cancer cells--differences of relevance for adenoviral oncolysis.

    Directory of Open Access Journals (Sweden)

    Dominik E Dorer

    Full Text Available Adenoviruses (Ads, especially HAdV-5, have been genetically equipped with tumor-restricted replication potential to enable applications in oncolytic cancer therapy. Such oncolytic adenoviruses have been well tolerated in cancer patients, but their anti-tumor efficacy needs to be enhanced. In this regard, it should be considered that cancer cells, dependent on their tissue of origin, can differ substantially from the normal host cells to which Ads are adapted by complex virus-host interactions. Consequently, viral replication efficiency, a key determinant of oncolytic activity, might be suboptimal in cancer cells. Therefore, we have analyzed both the replication kinetics of HAdV-5 and the virus-induced transcriptome in human bronchial epithelial cells (HBEC in comparison to cancer cells. This is the first report on genome-wide expression profiling of Ads in their native host cells. We found that E1A expression and onset of viral genome replication are most rapid in HBEC and considerably delayed in melanoma cells. In squamous cell lung carcinoma cells, we observed intermediate HAdV-5 replication kinetics. Infectious particle production, viral spread and lytic activity of HAdV-5 were attenuated in melanoma cells versus HBEC. Expression profiling at the onset of viral genome replication revealed that HAdV-5 induced the strongest changes in the cellular transcriptome in HBEC, followed by lung cancer and melanoma cells. We identified prominent regulation of genes involved in cell cycle and DNA metabolism, replication and packaging in HBEC, which is in accord with the necessity to induce S phase for viral replication. Strikingly, in melanoma cells HAdV-5 triggered opposing regulation of said genes and, in contrast to lung cancer cells, no weak S phase induction was detected when using the E2F promoter as reporter. Our results provide a rationale for improving oncolytic adenoviruses either by adaptation of viral infection to target tumor cells or by

  14. Multiscale analysis of replication technique efficiency for 3D roughness characterization of manufactured surfaces

    Science.gov (United States)

    Jolivet, S.; Mezghani, S.; El Mansori, M.

    2016-09-01

    The replication of topography has been generally restricted to optimizing material processing technologies in terms of statistical and single-scale features such as roughness. By contrast, manufactured surface topography is highly complex, irregular, and multiscale. In this work, we have demonstrated the use of multiscale analysis on replicates of surface finish to assess the precise control of the finished replica. Five commercial resins used for surface replication were compared. The topography of five standard surfaces representative of common finishing processes were acquired both directly and by a replication technique. Then, they were characterized using the ISO 25178 standard and multiscale decomposition based on a continuous wavelet transform, to compare the roughness transfer quality at different scales. Additionally, atomic force microscope force modulation mode was used in order to compare the resins’ stiffness properties. The results showed that less stiff resins are able to replicate the surface finish along a larger wavelength band. The method was then tested for non-destructive quality control of automotive gear tooth surfaces.

  15. International Expansion through Flexible Replication

    DEFF Research Database (Denmark)

    Jonsson, Anna; Foss, Nicolai Juul

    2011-01-01

    Business organizations may expand internationally by replicating a part of their value chain, such as a sales and marketing format, in other countries. However, little is known regarding how such “international replicators” build a format for replication, or how they can adjust it in order to ada......, etc.) are replicated in a uniform manner across stores, and change only very slowly (if at all) in response to learning (“flexible replication”). We conclude by discussing the factors that influence the approach to replication adopted by an international replicator.......Business organizations may expand internationally by replicating a part of their value chain, such as a sales and marketing format, in other countries. However, little is known regarding how such “international replicators” build a format for replication, or how they can adjust it in order to adapt...

  16. A Crystallographic Study of the Role of Sequence Context in Thymine Glycol Bypass by a Replicative DNA Polymerase Serendipitously Sheds Light on the Exonuclease Complex

    Energy Technology Data Exchange (ETDEWEB)

    Aller, Pierre; Duclos, Stéphanie; Wallace, Susan S.; Doublié, Sylvie (Vermont)

    2012-06-27

    Thymine glycol (Tg) is the most common oxidation product of thymine and is known to be a strong block to replicative DNA polymerases. A previously solved structure of the bacteriophage RB69 DNA polymerase (RB69 gp43) in complex with Tg in the sequence context 5'-G-Tg-G shed light on how Tg blocks primer elongation: The protruding methyl group of the oxidized thymine displaces the adjacent 5'-G, which can no longer serve as a template for primer elongation [Aller, P., Rould, M. A., Hogg, M, Wallace, S. S. and Doublie S. (2007). A structural rationale for stalling of a replicative DNA polymerase at the most common oxidative thymine lesion, thymine glycol. Proc. Natl. Acad. Sci. USA, 104, 814-818.]. Several studies showed that in the sequence context 5'-C-Tg-purine, Tg is more likely to be bypassed by Klenow fragment, an A-family DNA polymerase. We set out to investigate the role of sequence context in Tg bypass in a B-family polymerase and to solve the crystal structures of the bacteriophage RB69 DNA polymerase in complex with Tg-containing DNA in the three remaining sequence contexts: 5'-A-Tg-G, 5'-T-Tg-G, and 5'-C-Tg-G. A combination of several factors - including the associated exonuclease activity, the nature of the 3' and 5' bases surrounding Tg, and the cis-trans interconversion of Tg - influences Tg bypass. We also visualized for the first time the structure of a well-ordered exonuclease complex, allowing us to identify and confirm the role of key residues (Phe123, Met256, and Tyr257) in strand separation and in the stabilization of the primer strand in the exonuclease site.

  17. Lymphatic endothelial cells are a replicative niche for Mycobacterium tuberculosis

    Science.gov (United States)

    Lerner, Thomas R.; de Souza Carvalho-Wodarz, Cristiane; Repnik, Urska; Russell, Matthew R.G.; Borel, Sophie; Diedrich, Collin R.; Rohde, Manfred; Wainwright, Helen; Collinson, Lucy M.; Wilkinson, Robert J.; Griffiths, Gareth; Gutierrez, Maximiliano G.

    2016-01-01

    In extrapulmonary tuberculosis, the most common site of infection is within the lymphatic system, and there is growing recognition that lymphatic endothelial cells (LECs) are involved in immune function. Here, we identified LECs, which line the lymphatic vessels, as a niche for Mycobacterium tuberculosis in the lymph nodes of patients with tuberculosis. In cultured primary human LECs (hLECs), we determined that M. tuberculosis replicates both in the cytosol and within autophagosomes, but the bacteria failed to replicate when the virulence locus RD1 was deleted. Activation by IFN-γ induced a cell-autonomous response in hLECs via autophagy and NO production that restricted M. tuberculosis growth. Thus, depending on the activation status of LECs, autophagy can both promote and restrict replication. Together, these findings reveal a previously unrecognized role for hLECs and autophagy in tuberculosis pathogenesis and suggest that hLECs are a potential niche for M. tuberculosis that allows establishment of persistent infection in lymph nodes. PMID:26901813

  18. Hili Inhibits HIV Replication in Activated T Cells.

    Science.gov (United States)

    Peterlin, B Matija; Liu, Pingyang; Wang, Xiaoyun; Cary, Daniele; Shao, Wei; Leoz, Marie; Hong, Tian; Pan, Tao; Fujinaga, Koh

    2017-06-01

    P-element-induced wimpy-like (Piwil) proteins restrict the replication of mobile genetic elements in the germ line. They are also expressed in many transformed cell lines. In this study, we discovered that the human Piwil 2 (Hili) protein can also inhibit HIV replication, especially in activated CD4 + T cells that are the preferred target cells for this virus in the infected host. Although resting cells did not express Hili, its expression was rapidly induced following T cell activation. In these cells and transformed cell lines, depletion of Hili increased levels of viral proteins and new viral particles. Further studies revealed that Hili binds to tRNA. Some of the tRNAs represent rare tRNA species, whose codons are overrepresented in the viral genome. Targeting tRNA Arg (UCU) with an antisense oligonucleotide replicated effects of Hili and also inhibited HIV replication. Finally, Hili also inhibited the retrotransposition of the endogenous intracysternal A particle (IAP) by a similar mechanism. Thus, Hili joins a list of host proteins that inhibit the replication of HIV and other mobile genetic elements. IMPORTANCE Piwil proteins inhibit the movement of mobile genetic elements in the germ line. In their absence, sperm does not form and male mice are sterile. This inhibition is thought to occur via small Piwi-interacting RNAs (piRNAs). However, in some species and in human somatic cells, Piwil proteins bind primarily to tRNA. In this report, we demonstrate that human Piwil proteins, especially Hili, not only bind to select tRNA species, including rare tRNAs, but also inhibit HIV replication. Importantly, T cell activation induces the expression of Hili in CD4 + T cells. Since Hili also inhibited the movement of an endogenous retrovirus (IAP), our finding shed new light on this intracellular resistance to exogenous and endogenous retroviruses as well as other mobile genetic elements. Copyright © 2017 American Society for Microbiology.

  19. Multilayer Stochastic Block Models Reveal the Multilayer Structure of Complex Networks

    Directory of Open Access Journals (Sweden)

    Toni Vallès-Català

    2016-03-01

    Full Text Available In complex systems, the network of interactions we observe between systems components is the aggregate of the interactions that occur through different mechanisms or layers. Recent studies reveal that the existence of multiple interaction layers can have a dramatic impact in the dynamical processes occurring on these systems. However, these studies assume that the interactions between systems components in each one of the layers are known, while typically for real-world systems we do not have that information. Here, we address the issue of uncovering the different interaction layers from aggregate data by introducing multilayer stochastic block models (SBMs, a generalization of single-layer SBMs that considers different mechanisms of layer aggregation. First, we find the complete probabilistic solution to the problem of finding the optimal multilayer SBM for a given aggregate-observed network. Because this solution is computationally intractable, we propose an approximation that enables us to verify that multilayer SBMs are more predictive of network structure in real-world complex systems.

  20. The Proteome of the Isolated Chlamydia trachomatis Containing Vacuole Reveals a Complex Trafficking Platform Enriched for Retromer Components.

    Directory of Open Access Journals (Sweden)

    Lukas Aeberhard

    2015-06-01

    Full Text Available Chlamydia trachomatis is an important human pathogen that replicates inside the infected host cell in a unique vacuole, the inclusion. The formation of this intracellular bacterial niche is essential for productive Chlamydia infections. Despite its importance for Chlamydia biology, a holistic view on the protein composition of the inclusion, including its membrane, is currently missing. Here we describe the host cell-derived proteome of isolated C. trachomatis inclusions by quantitative proteomics. Computational analysis indicated that the inclusion is a complex intracellular trafficking platform that interacts with host cells' antero- and retrograde trafficking pathways. Furthermore, the inclusion is highly enriched for sorting nexins of the SNX-BAR retromer, a complex essential for retrograde trafficking. Functional studies showed that in particular, SNX5 controls the C. trachomatis infection and that retrograde trafficking is essential for infectious progeny formation. In summary, these findings suggest that C. trachomatis hijacks retrograde pathways for effective infection.

  1. Identification of rep-associated factors in herpes simplex virus type 1-induced adeno-associated virus type 2 replication compartments.

    Science.gov (United States)

    Nicolas, Armel; Alazard-Dany, Nathalie; Biollay, Coline; Arata, Loredana; Jolinon, Nelly; Kuhn, Lauriane; Ferro, Myriam; Weller, Sandra K; Epstein, Alberto L; Salvetti, Anna; Greco, Anna

    2010-09-01

    Adeno-associated virus (AAV) is a human parvovirus that replicates only in cells coinfected with a helper virus, such as adenovirus or herpes simplex virus type 1 (HSV-1). We previously showed that nine HSV-1 factors are able to support AAV rep gene expression and genome replication. To elucidate the strategy of AAV replication in the presence of HSV-1, we undertook a proteomic analysis of cellular and HSV-1 factors associated with Rep proteins and thus potentially recruited within AAV replication compartments (AAV RCs). This study resulted in the identification of approximately 60 cellular proteins, among which factors involved in DNA and RNA metabolism represented the largest functional categories. Validation analyses indicated that the cellular DNA replication enzymes RPA, RFC, and PCNA were recruited within HSV-1-induced AAV RCs. Polymerase delta was not identified but subsequently was shown to colocalize with Rep within AAV RCs even in the presence of the HSV-1 polymerase complex. In addition, we found that AAV replication is associated with the recruitment of components of the Mre11/Rad50/Nbs1 complex, Ku70 and -86, and the mismatch repair proteins MSH2, -3, and -6. Finally, several HSV-1 factors were also found to be associated with Rep, including UL12. We demonstrated for the first time that this protein plays a role during AAV replication by enhancing the resolution of AAV replicative forms and AAV particle production. Altogether, these analyses provide the basis to understand how AAV adapts its replication strategy to the nuclear environment induced by the helper virus.

  2. Investigating the role of viral integral membrane proteins in promoting the assembly of nepovirus and comovirus replication factories

    Directory of Open Access Journals (Sweden)

    Helene eSanfacon

    2013-01-01

    Full Text Available Formation of plant virus membrane-associated replication factories requires the association of viral replication proteins and viral RNA with intracellular membranes, the recruitment of host factors and the modification of membranes to form novel structures that house the replication complex. Many viruses encode integral membrane proteins that act as anchors for the replication complex. These hydrophobic proteins contain trans-membrane domains and/or amphipathic helices that associate with the membrane and modify its structure. The comovirus Co-Pro and NTP-binding (NTB, putative helicase proteins and the cognate nepovirus X2 and NTB proteins are among the best characterized plant virus integral membrane replication proteins and are functionally related to the picornavirus 2B, 2C and 3A membrane proteins. The identification of membrane-association domains and analysis of the membrane topology of these proteins is discussed. The evidence suggesting that these proteins have the ability to induce membrane proliferation, alter the structure and integrity of intracellular membranes and modulate the induction of symptoms in infected plants is also reviewed. Finally, areas of research that need further investigation are highlighted.

  3. Replication quality assessment and uncertainty evaluation of a polymer precision injection moulded component

    DEFF Research Database (Denmark)

    Baruffi, Federico; Calaon, Matteo; Tosello, Guido

    2017-01-01

    Precision injection moulding holds a central role in manufacturing as only replication process currently capable of accurately producing complex shaped polymer parts integrating micrometric features on a mass scale production. In this scenario, a study on the replication quality of a polymer...... injection moulded precision component for telecommunication applications is presented. The effects of the process parameters on the component dimensional variation have been investigated using a statistical approach. Replication fidelity of produced parts has been assessed using a focus variation microscope...... with sub-micrometric resolution. Measurement uncertainty has then been evaluated, according to the GUM considering contributions from different process settings combinations and mould geometries. The analysis showed that the injection moulding manufacturing process and the utilized measurement chain...

  4. Dynamic assembly of Hda and the sliding clamp in the regulation of replication licensing

    OpenAIRE

    Kim, Jin S.; Nanfara, Michael T.; Chodavarapu, Sundari; Jin, Kyeong S.; Babu, Vignesh?M.?P.; Ghazy, Mohamed A.; Chung, Scisung; Kaguni, Jon M.; Sutton, Mark D.; Cho, Yunje

    2017-01-01

    Abstract Regulatory inactivation of DnaA (RIDA) is one of the major regulatory mechanisms of prokaryotic replication licensing. In RIDA, the Hda?sliding clamp complex loaded onto DNA directly interacts with adenosine triphosphate (ATP)-bound DnaA and stimulates the hydrolysis of ATP to inactivate DnaA. A prediction is that the activity of Hda is tightly controlled to ensure that replication initiation occurs only once per cell cycle. Here, we determined the crystal structure of the Hda?? clam...

  5. Who Needs Replication?

    Science.gov (United States)

    Porte, Graeme

    2013-01-01

    In this paper, the editor of a recent Cambridge University Press book on research methods discusses replicating previous key studies to throw more light on their reliability and generalizability. Replication research is presented as an accepted method of validating previous research by providing comparability between the original and replicated…

  6. What Should Researchers Expect When They Replicate Studies? A Statistical View of Replicability in Psychological Science.

    Science.gov (United States)

    Patil, Prasad; Peng, Roger D; Leek, Jeffrey T

    2016-07-01

    A recent study of the replicability of key psychological findings is a major contribution toward understanding the human side of the scientific process. Despite the careful and nuanced analysis reported, the simple narrative disseminated by the mass, social, and scientific media was that in only 36% of the studies were the original results replicated. In the current study, however, we showed that 77% of the replication effect sizes reported were within a 95% prediction interval calculated using the original effect size. Our analysis suggests two critical issues in understanding replication of psychological studies. First, researchers' intuitive expectations for what a replication should show do not always match with statistical estimates of replication. Second, when the results of original studies are very imprecise, they create wide prediction intervals-and a broad range of replication effects that are consistent with the original estimates. This may lead to effects that replicate successfully, in that replication results are consistent with statistical expectations, but do not provide much information about the size (or existence) of the true effect. In this light, the results of the Reproducibility Project: Psychology can be viewed as statistically consistent with what one might expect when performing a large-scale replication experiment. © The Author(s) 2016.

  7. Ameliorating replicative senescence of human bone marrow stromal cells by PSMB5 overexpression

    International Nuclear Information System (INIS)

    Lu, Li; Song, Hui-Fang; Wei, Jiao-Long; Liu, Xue-Qin; Song, Wen-Hui; Yan, Ba-Yi; Yang, Gui-Jiao; Li, Ang; Yang, Wu-Lin

    2014-01-01

    Highlights: • PSMB5 overexpression restores the differentiation potential of aged hBMSCs. • PSMB5 overexpression enhances the proteasomal activity of late-stage hBMSCs. • PSMB5 overexpression inhibits replicative senescence and improved cell viability. • PSMB5 overexpression promotes cell growth by upregulating the Cyclin D1/CDK4 complex. - Abstract: Multipotent human bone marrow stromal cells (hBMSCs) potentially serve as a source for cell-based therapy in regenerative medicine. However, in vitro expansion was inescapably accompanied with cell senescence, characterized by inhibited proliferation and compromised pluripotency. We have previously demonstrated that this aging process is closely associated with reduced 20S proteasomal activity, with down-regulation of rate-limiting catalytic β-subunits particularly evident. In the present study, we confirmed that proteasomal activity directly contributes to senescence of hBMSCs, which could be reversed by overexpression of the β5-subunit (PSMB5). Knocking down PSMB5 led to decreased proteasomal activity concurrent with reduced cell proliferation in early-stage hBMSCs, which is similar to the senescent phenotype observed in late-stage cells. In contrast, overexpressing PSMB5 in late-stage cells efficiently restored the normal activity of 20S proteasomes and promoted cell growth, possibly via upregulating the Cyclin D1/CDK4 complex. Additionally, PSMB5 could enhance cell resistance to oxidative stress, as evidenced by the increased cell survival upon exposing senescent hBMSCs to hydrogen peroxide. Furthermore, PSMB5 overexpression retained the pluripotency of late-stage hBMSCs by facilitating their neural differentiation both in vitro and in vivo. Collectively, our work reveals a critical role of PSMB5 in 20S proteasome-mediated protection against replicative senescence, pointing to a possible strategy for maintaining the integrity of culture-expanded hBMSCs by manipulating the expression of PSMB5

  8. Ameliorating replicative senescence of human bone marrow stromal cells by PSMB5 overexpression

    Energy Technology Data Exchange (ETDEWEB)

    Lu, Li, E-mail: luli7300@126.com [Department of Anatomy, Shanxi Medical University, Taiyuan 030001 (China); Song, Hui-Fang; Wei, Jiao-Long; Liu, Xue-Qin [Department of Anatomy, Shanxi Medical University, Taiyuan 030001 (China); Song, Wen-Hui [Department of Orthopaedics, The Second Affiliated Hospital of Shanxi Medical University, Taiyuan 030001 (China); Yan, Ba-Yi; Yang, Gui-Jiao [Department of Anatomy, Shanxi Medical University, Taiyuan 030001 (China); Li, Ang [Department of Medicine, University of Hong Kong Faculty of Medicine, Hong Kong (Hong Kong); Department of Anatomy, University of Hong Kong Faculty of Medicine, Hong Kong (Hong Kong); Yang, Wu-Lin, E-mail: wulinyoung@163.com [School of Biotechnology and Food Engineering, Hefei University of Technology, Hefei 230009 (China); Laboratory of Metabolic Medicine, Singapore Bioimaging Consortium (SBIC), Agency for Science, Technology and Research - A*STAR (Singapore)

    2014-01-24

    Highlights: • PSMB5 overexpression restores the differentiation potential of aged hBMSCs. • PSMB5 overexpression enhances the proteasomal activity of late-stage hBMSCs. • PSMB5 overexpression inhibits replicative senescence and improved cell viability. • PSMB5 overexpression promotes cell growth by upregulating the Cyclin D1/CDK4 complex. - Abstract: Multipotent human bone marrow stromal cells (hBMSCs) potentially serve as a source for cell-based therapy in regenerative medicine. However, in vitro expansion was inescapably accompanied with cell senescence, characterized by inhibited proliferation and compromised pluripotency. We have previously demonstrated that this aging process is closely associated with reduced 20S proteasomal activity, with down-regulation of rate-limiting catalytic β-subunits particularly evident. In the present study, we confirmed that proteasomal activity directly contributes to senescence of hBMSCs, which could be reversed by overexpression of the β5-subunit (PSMB5). Knocking down PSMB5 led to decreased proteasomal activity concurrent with reduced cell proliferation in early-stage hBMSCs, which is similar to the senescent phenotype observed in late-stage cells. In contrast, overexpressing PSMB5 in late-stage cells efficiently restored the normal activity of 20S proteasomes and promoted cell growth, possibly via upregulating the Cyclin D1/CDK4 complex. Additionally, PSMB5 could enhance cell resistance to oxidative stress, as evidenced by the increased cell survival upon exposing senescent hBMSCs to hydrogen peroxide. Furthermore, PSMB5 overexpression retained the pluripotency of late-stage hBMSCs by facilitating their neural differentiation both in vitro and in vivo. Collectively, our work reveals a critical role of PSMB5 in 20S proteasome-mediated protection against replicative senescence, pointing to a possible strategy for maintaining the integrity of culture-expanded hBMSCs by manipulating the expression of PSMB5.

  9. Hepatitis A Virus Genome Organization and Replication Strategy.

    Science.gov (United States)

    McKnight, Kevin L; Lemon, Stanley M

    2018-04-02

    Hepatitis A virus (HAV) is a positive-strand RNA virus classified in the genus Hepatovirus of the family Picornaviridae It is an ancient virus with a long evolutionary history and multiple features of its capsid structure, genome organization, and replication cycle that distinguish it from other mammalian picornaviruses. HAV proteins are produced by cap-independent translation of a single, long open reading frame under direction of an inefficient, upstream internal ribosome entry site (IRES). Genome replication occurs slowly and is noncytopathic, with transcription likely primed by a uridylated protein primer as in other picornaviruses. Newly produced quasi-enveloped virions (eHAV) are released from cells in a nonlytic fashion in a unique process mediated by interactions of capsid proteins with components of the host cell endosomal sorting complexes required for transport (ESCRT) system. Copyright © 2018 Cold Spring Harbor Laboratory Press; all rights reserved.

  10. Planktonic replication is essential for biofilm formation by Legionella pneumophila in a complex medium under static and dynamic flow conditions

    DEFF Research Database (Denmark)

    Mampel, J.; Spirig, T.; Weber, S.S.

    2006-01-01

    Legionella pneumophila persists for a long time in aquatic habitats, where the bacteria associate with biofilms and replicate within protozoan predators. While L. pneumophila serves as a paradigm for intracellular growth within protozoa, it is less clear whether the bacteria form or replicate...

  11. How MCM loading and spreading specify eukaryotic DNA replication initiation sites [version 1; referees: 4 approved

    Directory of Open Access Journals (Sweden)

    Olivier Hyrien

    2016-08-01

    Full Text Available DNA replication origins strikingly differ between eukaryotic species and cell types. Origins are localized and can be highly efficient in budding yeast, are randomly located in early fly and frog embryos, which do not transcribe their genomes, and are clustered in broad (10-100 kb non-transcribed zones, frequently abutting transcribed genes, in mammalian cells. Nonetheless, in all cases, origins are established during the G1-phase of the cell cycle by the loading of double hexamers of the Mcm 2-7 proteins (MCM DHs, the core of the replicative helicase. MCM DH activation in S-phase leads to origin unwinding, polymerase recruitment, and initiation of bidirectional DNA synthesis. Although MCM DHs are initially loaded at sites defined by the binding of the origin recognition complex (ORC, they ultimately bind chromatin in much greater numbers than ORC and only a fraction are activated in any one S-phase. Data suggest that the multiplicity and functional redundancy of MCM DHs provide robustness to the replication process and affect replication time and that MCM DHs can slide along the DNA and spread over large distances around the ORC. Recent studies further show that MCM DHs are displaced along the DNA by collision with transcription complexes but remain functional for initiation after displacement. Therefore, eukaryotic DNA replication relies on intrinsically mobile and flexible origins, a strategy fundamentally different from bacteria but conserved from yeast to human. These properties of MCM DHs likely contribute to the establishment of broad, intergenic replication initiation zones in higher eukaryotes.

  12. 454 sequencing reveals extreme complexity of the class II Major Histocompatibility Complex in the collared flycatcher

    Directory of Open Access Journals (Sweden)

    Gustafsson Lars

    2010-12-01

    Full Text Available Abstract Background Because of their functional significance, the Major Histocompatibility Complex (MHC class I and II genes have been the subject of continuous interest in the fields of ecology, evolution and conservation. In some vertebrate groups MHC consists of multiple loci with similar alleles; therefore, the multiple loci must be genotyped simultaneously. In such complex systems, understanding of the evolutionary patterns and their causes has been limited due to challenges posed by genotyping. Results Here we used 454 amplicon sequencing to characterize MHC class IIB exon 2 variation in the collared flycatcher, an important organism in evolutionary and immuno-ecological studies. On the basis of over 152,000 sequencing reads we identified 194 putative alleles in 237 individuals. We found an extreme complexity of the MHC class IIB in the collared flycatchers, with our estimates pointing to the presence of at least nine expressed loci and a large, though difficult to estimate precisely, number of pseudogene loci. Many similar alleles occurred in the pseudogenes indicating either a series of recent duplications or extensive concerted evolution. The expressed alleles showed unambiguous signals of historical selection and the occurrence of apparent interlocus exchange of alleles. Placing the collared flycatcher's MHC sequences in the context of passerine diversity revealed transspecific MHC class II evolution within the Muscicapidae family. Conclusions 454 amplicon sequencing is an effective tool for advancing our understanding of the MHC class II structure and evolutionary patterns in Passeriformes. We found a highly dynamic pattern of evolution of MHC class IIB genes with strong signals of selection and pronounced sequence divergence in expressed genes, in contrast to the apparent sequence homogenization in pseudogenes. We show that next generation sequencing offers a universal, affordable method for the characterization and, in perspective

  13. DNA Replication Profiling Using Deep Sequencing.

    Science.gov (United States)

    Saayman, Xanita; Ramos-Pérez, Cristina; Brown, Grant W

    2018-01-01

    Profiling of DNA replication during progression through S phase allows a quantitative snap-shot of replication origin usage and DNA replication fork progression. We present a method for using deep sequencing data to profile DNA replication in S. cerevisiae.

  14. Multiple determinants controlling activation of yeast replication origins late in S phase.

    Science.gov (United States)

    Friedman, K L; Diller, J D; Ferguson, B M; Nyland, S V; Brewer, B J; Fangman, W L

    1996-07-01

    Analysis of a 131-kb segment of the left arm of yeast chromosome XIV beginning 157 kb from the telomere reveals four highly active origins of replication that initiate replication late in S phase. Previous work has shown that telomeres act as determinants for late origin activation. However, at least two of the chromosome XIV origins maintain their late activation time when located on large circular plasmids, indicating that late replication is independent of telomeres. Analysis of the replication time of plasmid derivatives containing varying amounts of chromosome XIV DNA show that a minimum of three chromosomal elements, distinct from each tested origin, contribute to late activation time. These late determinants are functionally equivalent, because duplication of one set of contributing sequences can compensate for the removal of another set. Furthermore, insertion of an origin that is normally early activated into this domain results in a shift to late activation, suggesting that the chromosome XIV origins are not unique in their ability to respond to the late determinants.

  15. Long-Term Live Cell Imaging Reveals New Roles For Salmonella Effector Proteins SseG and SteA

    Science.gov (United States)

    McQuate, Sarah E.; Young, Alexandra M.; Silva-Herzog, Eugenia; Bunker, Eric; Hernandez, Mateo; de Chaumont, Fabrice; Liu, Xuedong; Detweiler, Corrella S.; Palmer, Amy E.

    2016-01-01

    Summary Salmonella Typhimurium is an intracellular bacterial pathogen that infects both epithelial cells and macrophages. Salmonella effector proteins, which are translocated into the host cell and manipulate host cell components, control the ability to replicate and/or survive in host cells. Due to the complexity and heterogeneity of Salmonella infections, there is growing recognition of the need for single cell and live-cell imaging approaches to identify and characterize the diversity of cellular phenotypes and how they evolve over time. Here we establish a pipeline for long-term (16 hours) live-cell imaging of infected cells and subsequent image analysis methods. We apply this pipeline to track bacterial replication within the Salmonella-containing vacuole in epithelial cells, quantify vacuolar replication versus survival in macrophages, and investigate the role of individual effector proteins in mediating these parameters. This approach revealed that dispersed bacteria can coalesce at later stages of infection, that the effector protein SseG influences the propensity for cytosolic hyperreplication in epithelial cells, and that while SteA only has a subtle effect on vacuolar replication in epithelial cells, it has a profound impact on infection parameters in immunocompetent macrophages, suggesting differential roles for effector proteins in different infection models. PMID:27376507

  16. R-loops and initiation of DNA replication in human cells: a missing link?

    Directory of Open Access Journals (Sweden)

    Rodrigo eLombraña

    2015-04-01

    Full Text Available The unanticipated widespread occurrence of stable hybrid DNA/RNA structures (R-loops in human cells and the increasing evidence of their involvement in several human malignancies have invigorated the research on R-loop biology in recent years. Here we propose that physiological R-loop formation at CpG island promoters can contribute to DNA replication origin specification at these regions, the most efficient replication initiation sites in mammalian cells. Quite likely, this occurs by the strand-displacement reaction activating the formation of G-quadruplex structures that target the Origin Recognition Complex (ORC in the single-stranded conformation. In agreement with this, we found that R-loops co-localize with the ORC within the same CpG island region in a significant fraction of these efficient replication origins, precisely at the position displaying the highest density of G4 motifs. This scenario builds on the connection between transcription and replication in human cells and suggests that R-loop dysregulation at CpG island promoter-origins might contribute to the phenotype of DNA replication abnormalities and loss of genome integrity detected in cancer cells.

  17. In vitro-reconstituted nucleoids can block mitochondrial DNA replication and transcription.

    Science.gov (United States)

    Farge, Géraldine; Mehmedovic, Majda; Baclayon, Marian; van den Wildenberg, Siet M J L; Roos, Wouter H; Gustafsson, Claes M; Wuite, Gijs J L; Falkenberg, Maria

    2014-07-10

    The mechanisms regulating the number of active copies of mtDNA are still unclear. A mammalian cell typically contains 1,000-10,000 copies of mtDNA, which are packaged into nucleoprotein complexes termed nucleoids. The main protein component of these structures is mitochondrial transcription factor A (TFAM). Here, we reconstitute nucleoid-like particles in vitro and demonstrate that small changes in TFAM levels dramatically impact the fraction of DNA molecules available for transcription and DNA replication. Compaction by TFAM is highly cooperative, and at physiological ratios of TFAM to DNA, there are large variations in compaction, from fully compacted nucleoids to naked DNA. In compacted nucleoids, TFAM forms stable protein filaments on DNA that block melting and prevent progression of the replication and transcription machineries. Based on our observations, we suggest that small variations in the TFAM-to-mtDNA ratio may be used to regulate mitochondrial gene transcription and DNA replication. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

  18. RPA-Binding Protein ETAA1 Is an ATR Activator Involved in DNA Replication Stress Response.

    Science.gov (United States)

    Lee, Yuan-Cho; Zhou, Qing; Chen, Junjie; Yuan, Jingsong

    2016-12-19

    ETAA1 (Ewing tumor-associated antigen 1), also known as ETAA16, was identified as a tumor-specific antigen in the Ewing family of tumors. However, the biological function of this protein remains unknown. Here, we report the identification of ETAA1 as a DNA replication stress response protein. ETAA1 specifically interacts with RPA (Replication protein A) via two conserved RPA-binding domains and is therefore recruited to stalled replication forks. Interestingly, further analysis of ETAA1 function revealed that ETAA1 participates in the activation of ATR signaling pathway via a conserved ATR-activating domain (AAD) located near its N terminus. Importantly, we demonstrate that both RPA binding and ATR activation are required for ETAA1 function at stalled replication forks to maintain genome stability. Therefore, our data suggest that ETAA1 is a new ATR activator involved in replication checkpoint control. Copyright © 2016 Elsevier Ltd. All rights reserved.

  19. Evidence that DNA polymerase δ contributes to initiating leading strand DNA replication in Saccharomyces cerevisiae.

    Science.gov (United States)

    Garbacz, Marta A; Lujan, Scott A; Burkholder, Adam B; Cox, Phillip B; Wu, Qiuqin; Zhou, Zhi-Xiong; Haber, James E; Kunkel, Thomas A

    2018-02-27

    To investigate nuclear DNA replication enzymology in vivo, we have studied Saccharomyces cerevisiae strains containing a pol2-16 mutation that inactivates the catalytic activities of DNA polymerase ε (Pol ε). Although pol2-16 mutants survive, they present very tiny spore colonies, increased doubling time, larger than normal cells, aberrant nuclei, and rapid acquisition of suppressor mutations. These phenotypes reveal a severe growth defect that is distinct from that of strains that lack only Pol ε proofreading (pol2-4), consistent with the idea that Pol ε is the major leading-strand polymerase used for unstressed DNA replication. Ribonucleotides are incorporated into the pol2-16 genome in patterns consistent with leading-strand replication by Pol δ when Pol ε is absent. More importantly, ribonucleotide distributions at replication origins suggest that in strains encoding all three replicases, Pol δ contributes to initiation of leading-strand replication. We describe two possible models.

  20. Multiple conformational states of DnaA protein regulate its interaction with DnaA boxes in the initiation of DNA replication.

    Science.gov (United States)

    Patel, Meera J; Bhatia, Lavesh; Yilmaz, Gulden; Biswas-Fiss, Esther E; Biswas, Subhasis B

    2017-09-01

    DnaA protein is the initiator of genomic DNA replication in prokaryotes. It binds to specific DNA sequences in the origin of DNA replication and unwinds small AT-rich sequences downstream for the assembly of the replisome. The mechanism of activation of DnaA that enables it to bind and organize the origin DNA and leads to replication initiation remains unclear. In this study, we have developed double-labeled fluorescent DnaA probes to analyze conformational states of DnaA protein upon binding DNA, nucleotide, and Soj sporulation protein using Fluorescence Resonance Energy Transfer (FRET). Our studies demonstrate that DnaA protein undergoes large conformational changes upon binding to substrates and there are multiple distinct conformational states that enable it to initiate DNA replication. DnaA protein adopted a relaxed conformation by expanding ~15Å upon binding ATP and DNA to form the ATP·DnaA·DNA complex. Hydrolysis of bound ATP to ADP led to a contraction of DnaA within the complex. The relaxed conformation of DnaA is likely required for the formation of the multi-protein ATP·DnaA·DNA complex. In the initiation of sporulation, Soj binding to DnaA prevented relaxation of its conformation. Soj·ADP appeared to block the activation of DnaA, suggesting a mechanism for Soj·ADP in switching initiation of DNA replication to sporulation. Our studies demonstrate that multiple conformational states of DnaA protein regulate its binding to DNA in the initiation of DNA replication. Copyright © 2017 Elsevier B.V. All rights reserved.

  1. Viral precursor protein P3 and its processed products perform discrete and essential functions in the poliovirus RNA replication complex

    Science.gov (United States)

    The differential use of protein precursors and their products is a key strategy used during poliovirus replication. To characterize the role of protein precursors during replication, we examined the complementation profiles of mutants that inhibited 3D polymerase or 3C-RNA binding activity. We showe...

  2. Potential roles of DNA methylation in the initiation and establishment of replicative senescence revealed by array-based methylome and transcriptome analyses.

    Directory of Open Access Journals (Sweden)

    Mizuho Sakaki

    Full Text Available Cellular senescence is classified into two groups: replicative and premature senescence. Gene expression and epigenetic changes are reported to differ between these two groups and cell types. Normal human diploid fibroblast TIG-3 cells have often been used in cellular senescence research; however, their epigenetic profiles are still not fully understood. To elucidate how cellular senescence is epigenetically regulated in TIG-3 cells, we analyzed the gene expression and DNA methylation profiles of three types of senescent cells, namely, replicatively senescent, ras-induced senescent (RIS, and non-permissive temperature-induced senescent SVts8 cells, using gene expression and DNA methylation microarrays. The expression of genes involved in the cell cycle and immune response was commonly either down- or up-regulated in the three types of senescent cells, respectively. The altered DNA methylation patterns were observed in replicatively senescent cells, but not in prematurely senescent cells. Interestingly, hypomethylated CpG sites detected on non-CpG island regions ("open sea" were enriched in immune response-related genes that had non-CpG island promoters. The integrated analysis of gene expression and methylation in replicatively senescent cells demonstrated that differentially expressed 867 genes, including cell cycle- and immune response-related genes, were associated with DNA methylation changes in CpG sites close to the transcription start sites (TSSs. Furthermore, several miRNAs regulated in part through DNA methylation were found to affect the expression of their targeted genes. Taken together, these results indicate that the epigenetic changes of DNA methylation regulate the expression of a certain portion of genes and partly contribute to the introduction and establishment of replicative senescence.

  3. Induction of UV-resistant DNA replication in Escherichia coli: Induced stable DNA replication as an SOS function

    International Nuclear Information System (INIS)

    Kogoma, T.; Torrey, T.A.; Connaughton, M.J.

    1979-01-01

    The striking similarity between the treatments that induce SOS functions and those that result in stable DNA replication (continuous DNA replication in the absence of protein synthesis) prompted us to examine the possibility of stable DNA replication being a recA + lexA + -dependent SOS function. In addition to the treatments previously reported, ultraviolet (UV) irradiation or treatment with mitomycin C was also found to induce stable DNA replication. The thermal treatment of tif-1 strains did not result in detectable levels of stable DNA replication, but nalidixic acid readily induced the activity in these strains. The induction of stable DNA replication with nalidixic acid was severely suppressed in tif-1 lex A mutant strains. The inhibitory activity of lexA3 was negated by the presence of the spr-5l mutation, an intragenic suppressor of lexA3. Induced stable DNA replication was found to be considerably more resistant to UV irradiation than normal replication both in a uvr A6 strain and a uvr + strain. The UV-resistant replication occurred mostly in the semiconservative manner. The possible roles of stable DNA replication in repair of damaged DNA are discussed. (orig.)

  4. RIPK1 and PGAM5 Control Leishmania Replication through Distinct Mechanisms.

    Science.gov (United States)

    Farias Luz, Nivea; Balaji, Sakthi; Okuda, Kendi; Barreto, Aline Silva; Bertin, John; Gough, Peter J; Gazzinelli, Ricardo; Almeida, Roque P; Bozza, Marcelo T; Borges, Valeria M; Chan, Francis Ka-Ming

    2016-06-15

    Leishmaniasis is an important parasitic disease found in the tropics and subtropics. Cutaneous and visceral leishmaniasis affect an estimated 1.5 million people worldwide. Despite its human health relevance, relatively little is known about the cell death pathways that control Leishmania replication in the host. Necroptosis is a recently identified form of cell death with potent antiviral effects. Receptor interacting protein kinase 1 (RIPK1) is a critical kinase that mediates necroptosis downstream of death receptors and TLRs. Heme, a product of hemoglobin catabolism during certain intracellular pathogen infections, is also a potent inducer of macrophage necroptosis. We found that human visceral leishmaniasis patients exhibit elevated serum levels of heme. Therefore, we examined the impact of heme and necroptosis on Leishmania replication. Indeed, heme potently inhibited Leishmania replication in bone marrow-derived macrophages. Moreover, we found that inhibition of RIPK1 kinase activity also enhanced parasite replication in the absence of heme. We further found that the mitochondrial phosphatase phosphoglycerate mutase family member 5 (PGAM5), a putative downstream effector of RIPK1, was also required for inhibition of Leishmania replication. In mouse infection, both PGAM5 and RIPK1 kinase activity are required for IL-1β expression in response to Leishmania However, PGAM5, but not RIPK1 kinase activity, was directly responsible for Leishmania-induced IL-1β secretion and NO production in bone marrow-derived macrophages. Collectively, these results revealed that RIPK1 and PGAM5 function independently to exert optimal control of Leishmania replication in the host. Copyright © 2016 by The American Association of Immunologists, Inc.

  5. Mechanism for Coordinated RNA Packaging and Genome Replication by Rotavirus Polymerase VP1

    Energy Technology Data Exchange (ETDEWEB)

    Lu, Xiaohui; McDonald, Sarah M.; Tortorici, M. Alejandra; Tao, Yizhi Jane; Vasquez-Del Carpio, Rodrigo; Nibert, Max L.; Patton, John T.; Harrison, Stephen C. (Harvard-Med); (NIH); (CH-Boston)

    2009-04-08

    Rotavirus RNA-dependent RNA polymerase VP1 catalyzes RNA synthesis within a subviral particle. This activity depends on core shell protein VP2. A conserved sequence at the 3' end of plus-strand RNA templates is important for polymerase association and genome replication. We have determined the structure of VP1 at 2.9 {angstrom} resolution, as apoenzyme and in complex with RNA. The cage-like enzyme is similar to reovirus {lambda}3, with four tunnels leading to or from a central, catalytic cavity. A distinguishing characteristic of VP1 is specific recognition, by conserved features of the template-entry channel, of four bases, UGUG, in the conserved 3' sequence. Well-defined interactions with these bases position the RNA so that its 3' end overshoots the initiating register, producing a stable but catalytically inactive complex. We propose that specific 3' end recognition selects rotavirus RNA for packaging and that VP2 activates the autoinhibited VP1/RNA complex to coordinate packaging and genome replication.

  6. Rhinovirus uses a phosphatidylinositol 4-phosphate/cholesterol counter-current for the formation of replication compartments at the ER-Golgi interface

    NARCIS (Netherlands)

    Roulin, Pascal S; Lötzerich, Mark; Torta, Federico; Tanner, Lukas B; van Kuppeveld, Frank J M|info:eu-repo/dai/nl/156614723; Wenk, Markus R; Greber, Urs F

    2014-01-01

    Similar to other positive-strand RNA viruses, rhinovirus, the causative agent of the common cold, replicates on a web of cytoplasmic membranes, orchestrated by host proteins and lipids. The host pathways that facilitate the formation and function of the replication membranes and complexes are poorly

  7. Chromatin replication and epigenome maintenance

    DEFF Research Database (Denmark)

    Alabert, Constance; Groth, Anja

    2012-01-01

    Stability and function of eukaryotic genomes are closely linked to chromatin structure and organization. During cell division the entire genome must be accurately replicated and the chromatin landscape reproduced on new DNA. Chromatin and nuclear structure influence where and when DNA replication...... initiates, whereas the replication process itself disrupts chromatin and challenges established patterns of genome regulation. Specialized replication-coupled mechanisms assemble new DNA into chromatin, but epigenome maintenance is a continuous process taking place throughout the cell cycle. If DNA...

  8. Looking Is Not Seeing and Listening Is Not Hearing: A Replication Study With Accelerated BSN Students.

    Science.gov (United States)

    Honan, Linda; Shealy, Sarah; Fennie, Kristopher; Duffy, Thomas C; Friedlaender, Linda; Del Vecchio, Megan

    2016-01-01

    Development of perceptual aptitude is a critical yet complex skill that requires the effective organization and interpretation of data using visual and auditory clinical observation. Educators face challenges in creating pedagogy that consistently demonstrates reliability and validity in fostering clinical skills. We have dependably used the arts as a means to improve students' auditory and visual skills, and this article will describe replication of our work with accelerated nursing students in a bachelor's program in their last semester of nursing school (n=23). Our results reveal that auscultative and observational abilities of soon-to-be registered nurses are in need of improvement. The use of art in a museum improves observational and assessment abilities, and music training increases auscultative interpretive skills significantly. Copyright © 2016 Elsevier Inc. All rights reserved.

  9. 36 CFR 910.64 - Replication.

    Science.gov (United States)

    2010-07-01

    ... 36 Parks, Forests, and Public Property 3 2010-07-01 2010-07-01 false Replication. 910.64 Section 910.64 Parks, Forests, and Public Property PENNSYLVANIA AVENUE DEVELOPMENT CORPORATION GENERAL... DEVELOPMENT AREA Glossary of Terms § 910.64 Replication. Replication means the process of using modern methods...

  10. The difference between LSMC and replicating portfolio in insurance liability modeling.

    Science.gov (United States)

    Pelsser, Antoon; Schweizer, Janina

    2016-01-01

    Solvency II requires insurers to calculate the 1-year value at risk of their balance sheet. This involves the valuation of the balance sheet in 1 year's time. As for insurance liabilities, closed-form solutions to their value are generally not available, insurers turn to estimation procedures. While pure Monte Carlo simulation set-ups are theoretically sound, they are often infeasible in practice. Therefore, approximation methods are exploited. Among these, least squares Monte Carlo (LSMC) and portfolio replication are prominent and widely applied in practice. In this paper, we show that, while both are variants of regression-based Monte Carlo methods, they differ in one significant aspect. While the replicating portfolio approach only contains an approximation error, which converges to zero in the limit, in LSMC a projection error is additionally present, which cannot be eliminated. It is revealed that the replicating portfolio technique enjoys numerous advantages and is therefore an attractive model choice.

  11. Replication stress-induced chromosome breakage is correlated with replication fork progression and is preceded by single-stranded DNA formation.

    Science.gov (United States)

    Feng, Wenyi; Di Rienzi, Sara C; Raghuraman, M K; Brewer, Bonita J

    2011-10-01

    Chromosome breakage as a result of replication stress has been hypothesized to be the direct consequence of defective replication fork progression, or "collapsed" replication forks. However, direct and genome-wide evidence that collapsed replication forks give rise to chromosome breakage is still lacking. Previously we showed that a yeast replication checkpoint mutant mec1-1, after transient exposure to replication impediment imposed by hydroxyurea (HU), failed to complete DNA replication, accumulated single-stranded DNA (ssDNA) at the replication forks, and fragmented its chromosomes. In this study, by following replication fork progression genome-wide via ssDNA detection and by direct mapping of chromosome breakage after HU exposure, we have tested the hypothesis that the chromosome breakage in mec1 cells occurs at collapsed replication forks. We demonstrate that sites of chromosome breakage indeed correlate with replication fork locations. Moreover, ssDNA can be detected prior to chromosome breakage, suggesting that ssDNA accumulation is the common precursor to double strand breaks at collapsed replication forks.

  12. A serine palmitoyltransferase inhibitor blocks hepatitis C virus replication in human hepatocytes.

    Science.gov (United States)

    Katsume, Asao; Tokunaga, Yuko; Hirata, Yuichi; Munakata, Tsubasa; Saito, Makoto; Hayashi, Hitohisa; Okamoto, Koichi; Ohmori, Yusuke; Kusanagi, Isamu; Fujiwara, Shinya; Tsukuda, Takuo; Aoki, Yuko; Klumpp, Klaus; Tsukiyama-Kohara, Kyoko; El-Gohary, Ahmed; Sudoh, Masayuki; Kohara, Michinori

    2013-10-01

    Host cell lipid rafts form a scaffold required for replication of hepatitis C virus (HCV). Serine palmitoyltransferases (SPTs) produce sphingolipids, which are essential components of the lipid rafts that associate with HCV nonstructural proteins. Prevention of the de novo synthesis of sphingolipids by an SPT inhibitor disrupts the HCV replication complex and thereby inhibits HCV replication. We investigated the ability of the SPT inhibitor NA808 to prevent HCV replication in cells and mice. We tested the ability of NA808 to inhibit SPT's enzymatic activity in FLR3-1 replicon cells. We used a replicon system to select for HCV variants that became resistant to NA808 at concentrations 4- to 6-fold the 50% inhibitory concentration, after 14 rounds of cell passage. We assessed the ability of NA808 or telaprevir to inhibit replication of HCV genotypes 1a, 1b, 2a, 3a, and 4a in mice with humanized livers (transplanted with human hepatocytes). NA808 was injected intravenously, with or without pegylated interferon alfa-2a and HCV polymerase and/or protease inhibitors. NA808 prevented HCV replication via noncompetitive inhibition of SPT; no resistance mutations developed. NA808 prevented replication of all HCV genotypes tested in mice with humanized livers. Intravenous NA808 significantly reduced viral load in the mice and had synergistic effects with pegylated interferon alfa-2a and HCV polymerase and protease inhibitors. The SPT inhibitor NA808 prevents replication of HCV genotypes 1a, 1b, 2a, 3a, and 4a in cultured hepatocytes and in mice with humanized livers. It might be developed for treatment of HCV infection or used in combination with pegylated interferon alfa-2a or HCV polymerase or protease inhibitors. Copyright © 2013 AGA Institute. Published by Elsevier Inc. All rights reserved.

  13. Complex Virus-Host Interactions Involved in the Regulation of Classical Swine Fever Virus Replication: A Minireview.

    Science.gov (United States)

    Li, Su; Wang, Jinghan; Yang, Qian; Naveed Anwar, Muhammad; Yu, Shaoxiong; Qiu, Hua-Ji

    2017-07-05

    Classical swine fever (CSF), caused by classical swine fever virus (CSFV), is one of the most devastating epizootic diseases of pigs in many countries. Viruses are small intracellular parasites and thus rely on the cellular factors for replication. Fundamental aspects of CSFV-host interactions have been well described, such as factors contributing to viral attachment, modulation of genomic replication and translation, antagonism of innate immunity, and inhibition of cell apoptosis. However, those host factors that participate in the viral entry, assembly, and release largely remain to be elucidated. In this review, we summarize recent progress in the virus-host interactions involved in the life cycle of CSFV and analyze the potential mechanisms of viral entry, assembly, and release. We conclude with future perspectives and highlight areas that require further understanding.

  14. Beyond Contagion: Reality Mining Reveals Complex Patterns of Social Influence.

    Directory of Open Access Journals (Sweden)

    Aamena Alshamsi

    Full Text Available Contagion, a concept from epidemiology, has long been used to characterize social influence on people's behavior and affective (emotional states. While it has revealed many useful insights, it is not clear whether the contagion metaphor is sufficient to fully characterize the complex dynamics of psychological states in a social context. Using wearable sensors that capture daily face-to-face interaction, combined with three daily experience sampling surveys, we collected the most comprehensive data set of personality and emotion dynamics of an entire community of work. From this high-resolution data about actual (rather than self-reported face-to-face interaction, a complex picture emerges where contagion (that can be seen as adaptation of behavioral responses to the behavior of other people cannot fully capture the dynamics of transitory states. We found that social influence has two opposing effects on states: adaptation effects that go beyond mere contagion, and complementarity effects whereby individuals' behaviors tend to complement the behaviors of others. Surprisingly, these effects can exhibit completely different directions depending on the stable personality or emotional dispositions (stable traits of target individuals. Our findings provide a foundation for richer models of social dynamics, and have implications on organizational engineering and workplace well-being.

  15. Beyond Contagion: Reality Mining Reveals Complex Patterns of Social Influence.

    Science.gov (United States)

    Alshamsi, Aamena; Pianesi, Fabio; Lepri, Bruno; Pentland, Alex; Rahwan, Iyad

    2015-01-01

    Contagion, a concept from epidemiology, has long been used to characterize social influence on people's behavior and affective (emotional) states. While it has revealed many useful insights, it is not clear whether the contagion metaphor is sufficient to fully characterize the complex dynamics of psychological states in a social context. Using wearable sensors that capture daily face-to-face interaction, combined with three daily experience sampling surveys, we collected the most comprehensive data set of personality and emotion dynamics of an entire community of work. From this high-resolution data about actual (rather than self-reported) face-to-face interaction, a complex picture emerges where contagion (that can be seen as adaptation of behavioral responses to the behavior of other people) cannot fully capture the dynamics of transitory states. We found that social influence has two opposing effects on states: adaptation effects that go beyond mere contagion, and complementarity effects whereby individuals' behaviors tend to complement the behaviors of others. Surprisingly, these effects can exhibit completely different directions depending on the stable personality or emotional dispositions (stable traits) of target individuals. Our findings provide a foundation for richer models of social dynamics, and have implications on organizational engineering and workplace well-being.

  16. Replication fork stability confers chemoresistance in BRCA-deficient cells

    DEFF Research Database (Denmark)

    Chaudhuri, Arnab Ray; Callen, Elsa; Ding, Xia

    2016-01-01

    /4 complex protein, PTIP, protects Brca1/2-deficient cells from DNA damage and rescues the lethality of Brca2-deficient embryonic stem cells. However, PTIP deficiency does not restore homologous recombination activity at double-strand breaks. Instead, its absence inhibits the recruitment of the MRE11......Cells deficient in the Brca1 and Brca2 genes have reduced capacity to repair DNA double-strand breaks by homologous recombination and consequently are hypersensitive to DNA-damaging agents, including cisplatin and poly(ADP-ribose) polymerase (PARP) inhibitors. Here we show that loss of the MLL3...... nuclease to stalled replication forks, which in turn protects nascent DNA strands from extensive degradation. More generally, acquisition of PARP inhibitors and cisplatin resistance is associated with replication fork protection in Brca2-deficient tumour cells that do not develop Brca2 reversion mutations...

  17. ATR-Chk1-APC/C-dependent stabilization of Cdc7-ASK (Dbf4) kinase is required for DNA lesion bypass under replication stress

    DEFF Research Database (Denmark)

    Yamada, M.; Watanabe, K.; Mistrik, M.

    2013-01-01

    replication. Stalled DNA replication evoked stabilization of the Cdc7-ASK (Dbf4) complex in a manner dependent on ATR-Chk1-mediated checkpoint signaling and its interplay with the anaphase-promoting complex/cyclosomeCdh1 (APC/C) ubiquitin ligase. Mechanistically, Chk1 kinase inactivates APC/C through...... degradation of Cdh1 upon replication block, thereby stabilizing APC/C substrates, including Cdc7-ASK (Dbf4). Furthermore, motif C of ASK (Dbf4) interacts with the N-terminal region of RAD18 ubiquitin ligase, and this interaction is required for chromatin binding of RAD18. Impaired interaction of ASK (Dbf4...

  18. Genome-scale cluster analysis of replicated microarrays using shrinkage correlation coefficient.

    Science.gov (United States)

    Yao, Jianchao; Chang, Chunqi; Salmi, Mari L; Hung, Yeung Sam; Loraine, Ann; Roux, Stanley J

    2008-06-18

    Currently, clustering with some form of correlation coefficient as the gene similarity metric has become a popular method for profiling genomic data. The Pearson correlation coefficient and the standard deviation (SD)-weighted correlation coefficient are the two most widely-used correlations as the similarity metrics in clustering microarray data. However, these two correlations are not optimal for analyzing replicated microarray data generated by most laboratories. An effective correlation coefficient is needed to provide statistically sufficient analysis of replicated microarray data. In this study, we describe a novel correlation coefficient, shrinkage correlation coefficient (SCC), that fully exploits the similarity between the replicated microarray experimental samples. The methodology considers both the number of replicates and the variance within each experimental group in clustering expression data, and provides a robust statistical estimation of the error of replicated microarray data. The value of SCC is revealed by its comparison with two other correlation coefficients that are currently the most widely-used (Pearson correlation coefficient and SD-weighted correlation coefficient) using statistical measures on both synthetic expression data as well as real gene expression data from Saccharomyces cerevisiae. Two leading clustering methods, hierarchical and k-means clustering were applied for the comparison. The comparison indicated that using SCC achieves better clustering performance. Applying SCC-based hierarchical clustering to the replicated microarray data obtained from germinating spores of the fern Ceratopteris richardii, we discovered two clusters of genes with shared expression patterns during spore germination. Functional analysis suggested that some of the genetic mechanisms that control germination in such diverse plant lineages as mosses and angiosperms are also conserved among ferns. This study shows that SCC is an alternative to the Pearson

  19. Replication Research and Special Education

    Science.gov (United States)

    Travers, Jason C.; Cook, Bryan G.; Therrien, William J.; Coyne, Michael D.

    2016-01-01

    Replicating previously reported empirical research is a necessary aspect of an evidence-based field of special education, but little formal investigation into the prevalence of replication research in the special education research literature has been conducted. Various factors may explain the lack of attention to replication of special education…

  20. Origin Licensing Requires ATP Binding and Hydrolysis by the MCM Replicative Helicase

    Science.gov (United States)

    Coster, Gideon; Frigola, Jordi; Beuron, Fabienne; Morris, Edward P.; Diffley, John F.X.

    2014-01-01

    Summary Loading of the six related Minichromosome Maintenance (MCM) proteins as head-to-head double hexamers during DNA replication origin licensing is crucial for ensuring once-per-cell-cycle DNA replication in eukaryotic cells. Assembly of these prereplicative complexes (pre-RCs) requires the Origin Recognition Complex (ORC), Cdc6, and Cdt1. ORC, Cdc6, and MCM are members of the AAA+ family of ATPases, and pre-RC assembly requires ATP hydrolysis. Here we show that ORC and Cdc6 mutants defective in ATP hydrolysis are competent for origin licensing. However, ATP hydrolysis by Cdc6 is required to release nonproductive licensing intermediates. We show that ATP binding stabilizes the wild-type MCM hexamer. Moreover, by analyzing MCM containing mutant subunits, we show that ATP binding and hydrolysis by MCM are required for Cdt1 release and double hexamer formation. This work alters our view of how ATP is used by licensing factors to assemble pre-RCs. PMID:25087873

  1. The molecular biology of Bluetongue virus replication.

    Science.gov (United States)

    Patel, Avnish; Roy, Polly

    2014-03-01

    The members of Orbivirus genus within the Reoviridae family are arthropod-borne viruses which are responsible for high morbidity and mortality in ruminants. Bluetongue virus (BTV) which causes disease in livestock (sheep, goat, cattle) has been in the forefront of molecular studies for the last three decades and now represents the best understood orbivirus at a molecular and structural level. The complex nature of the virion structure has been well characterised at high resolution along with the definition of the virus encoded enzymes required for RNA replication; the ordered assembly of the capsid shell as well as the protein and genome sequestration required for it; and the role of host proteins in virus entry and virus release. More recent developments of Reverse Genetics and Cell-Free Assembly systems have allowed integration of the accumulated structural and molecular knowledge to be tested at meticulous level, yielding higher insight into basic molecular virology, from which the rational design of safe efficacious vaccines has been possible. This article is centred on the molecular dissection of BTV with a view to understanding the role of each protein in the virus replication cycle. These areas are important in themselves for BTV replication but they also indicate the pathways that related viruses, which includes viruses that are pathogenic to man and animals, might also use providing an informed starting point for intervention or prevention. Copyright © 2014 Elsevier B.V. All rights reserved.

  2. Levels of the E2 interacting protein TopBP1 modulate papillomavirus maintenance stage replication

    International Nuclear Information System (INIS)

    Kanginakudru, Sriramana; DeSmet, Marsha; Thomas, Yanique; Morgan, Iain M.; Androphy, Elliot J.

    2015-01-01

    The evolutionarily conserved DNA topoisomerase II beta-binding protein 1 (TopBP1) functions in DNA replication, DNA damage response, and cell survival. We analyzed the role of TopBP1 in human and bovine papillomavirus genome replication. Consistent with prior reports, TopBP1 co-localized in discrete nuclear foci and was in complex with papillomavirus E2 protein. Similar to E2, TopBP1 is recruited to the region of the viral origin of replication during G1/S and early S phase. TopBP1 knockdown increased, while over-expression decreased transient virus replication, without affecting cell cycle. Similarly, using cell lines harboring HPV-16 or HPV-31 genome, TopBP1 knockdown increased while over-expression reduced viral copy number relative to genomic DNA. We propose a model in which TopBP1 serves dual roles in viral replication: it is essential for initiation of replication yet it restricts viral copy number. - Highlights: • Protein interaction study confirmed In-situ interaction between TopBP1 and E2. • TopBP1 present at papillomavirus ori in G1/S and early S phase of cell cycle. • TopBP1 knockdown increased, over-expression reduced virus replication. • TopBP1 protein level change did not influence cell survival or cell cycle. • TopBP1 displaced from papillomavirus ori after initiation of replication

  3. Levels of the E2 interacting protein TopBP1 modulate papillomavirus maintenance stage replication

    Energy Technology Data Exchange (ETDEWEB)

    Kanginakudru, Sriramana, E-mail: skangina@iu.edu [Department of Dermatology, Indiana University School of Medicine, Indianapolis, IN (United States); DeSmet, Marsha, E-mail: mdesmet@iupui.edu [Department of Dermatology, Indiana University School of Medicine, Indianapolis, IN (United States); Thomas, Yanique, E-mail: ysthomas@umail.iu.edu [Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, IN (United States); Morgan, Iain M., E-mail: immorgan@vcu.edu [VCU Philips Institute for Oral Health Research, Virginia Commonwealth University, Richmond, Virginia (United States); Androphy, Elliot J., E-mail: eandro@iu.edu [Department of Dermatology, Indiana University School of Medicine, Indianapolis, IN (United States); Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, IN (United States)

    2015-04-15

    The evolutionarily conserved DNA topoisomerase II beta-binding protein 1 (TopBP1) functions in DNA replication, DNA damage response, and cell survival. We analyzed the role of TopBP1 in human and bovine papillomavirus genome replication. Consistent with prior reports, TopBP1 co-localized in discrete nuclear foci and was in complex with papillomavirus E2 protein. Similar to E2, TopBP1 is recruited to the region of the viral origin of replication during G1/S and early S phase. TopBP1 knockdown increased, while over-expression decreased transient virus replication, without affecting cell cycle. Similarly, using cell lines harboring HPV-16 or HPV-31 genome, TopBP1 knockdown increased while over-expression reduced viral copy number relative to genomic DNA. We propose a model in which TopBP1 serves dual roles in viral replication: it is essential for initiation of replication yet it restricts viral copy number. - Highlights: • Protein interaction study confirmed In-situ interaction between TopBP1 and E2. • TopBP1 present at papillomavirus ori in G1/S and early S phase of cell cycle. • TopBP1 knockdown increased, over-expression reduced virus replication. • TopBP1 protein level change did not influence cell survival or cell cycle. • TopBP1 displaced from papillomavirus ori after initiation of replication.

  4. Eukaryotic DNA Replication Fork.

    Science.gov (United States)

    Burgers, Peter M J; Kunkel, Thomas A

    2017-06-20

    This review focuses on the biogenesis and composition of the eukaryotic DNA replication fork, with an emphasis on the enzymes that synthesize DNA and repair discontinuities on the lagging strand of the replication fork. Physical and genetic methodologies aimed at understanding these processes are discussed. The preponderance of evidence supports a model in which DNA polymerase ε (Pol ε) carries out the bulk of leading strand DNA synthesis at an undisturbed replication fork. DNA polymerases α and δ carry out the initiation of Okazaki fragment synthesis and its elongation and maturation, respectively. This review also discusses alternative proposals, including cellular processes during which alternative forks may be utilized, and new biochemical studies with purified proteins that are aimed at reconstituting leading and lagging strand DNA synthesis separately and as an integrated replication fork.

  5. Hydra meiosis reveals unexpected conservation of structural synaptonemal complex proteins across metazoans.

    Science.gov (United States)

    Fraune, Johanna; Alsheimer, Manfred; Volff, Jean-Nicolas; Busch, Karoline; Fraune, Sebastian; Bosch, Thomas C G; Benavente, Ricardo

    2012-10-09

    The synaptonemal complex (SC) is a key structure of meiosis, mediating the stable pairing (synapsis) of homologous chromosomes during prophase I. Its remarkable tripartite structure is evolutionarily well conserved and can be found in almost all sexually reproducing organisms. However, comparison of the different SC protein components in the common meiosis model organisms Saccharomyces cerevisiae, Arabidopsis thaliana, Caenorhabditis elegans, Drosophila melanogaster, and Mus musculus revealed no sequence homology. This discrepancy challenged the hypothesis that the SC arose only once in evolution. To pursue this matter we focused on the evolution of SYCP1 and SYCP3, the two major structural SC proteins of mammals. Remarkably, our comparative bioinformatic and expression studies revealed that SYCP1 and SYCP3 are also components of the SC in the basal metazoan Hydra. In contrast to previous assumptions, we therefore conclude that SYCP1 and SYCP3 form monophyletic groups of orthologous proteins across metazoans.

  6. Identification of Persistent RNA-DNA Hybrid Structures within the Origin of Replication of Human Cytomegalovirus

    OpenAIRE

    Prichard, Mark N.; Jairath, Sanju; Penfold, Mark E. T.; Jeor, Stephen St.; Bohlman, Marlene C.; Pari, Gregory S.

    1998-01-01

    Human cytomegalovirus (HCMV) lytic-phase DNA replication initiates at the cis-acting origin of replication, oriLyt. oriLyt is a structurally complex region containing repeat elements and transcription factor binding sites. We identified two site-specific alkali-labile regions within oriLyt which flank an alkali-resistant DNA segment. These alkali-sensitive regions were the result of the degradation of two RNA species embedded within oriLyt and covalently linked to viral DNA. The virus-associa...

  7. Slow Replication Fork Velocity of Homologous Recombination-Defective Cells Results from Endogenous Oxidative Stress

    Science.gov (United States)

    Magdalou, Indiana; Machon, Christelle; Dardillac, Elodie; Técher, Hervé; Guitton, Jérôme; Debatisse, Michelle; Lopez, Bernard S.

    2016-01-01

    Replications forks are routinely hindered by different endogenous stresses. Because homologous recombination plays a pivotal role in the reactivation of arrested replication forks, defects in homologous recombination reveal the initial endogenous stress(es). Homologous recombination-defective cells consistently exhibit a spontaneously reduced replication speed, leading to mitotic extra centrosomes. Here, we identify oxidative stress as a major endogenous source of replication speed deceleration in homologous recombination-defective cells. The treatment of homologous recombination-defective cells with the antioxidant N-acetyl-cysteine or the maintenance of the cells at low O2 levels (3%) rescues both the replication fork speed, as monitored by single-molecule analysis (molecular combing), and the associated mitotic extra centrosome frequency. Reciprocally, the exposure of wild-type cells to H2O2 reduces the replication fork speed and generates mitotic extra centrosomes. Supplying deoxynucleotide precursors to H2O2-exposed cells rescued the replication speed. Remarkably, treatment with N-acetyl-cysteine strongly expanded the nucleotide pool, accounting for the replication speed rescue. Remarkably, homologous recombination-defective cells exhibit a high level of endogenous reactive oxygen species. Consistently, homologous recombination-defective cells accumulate spontaneous γH2AX or XRCC1 foci that are abolished by treatment with N-acetyl-cysteine or maintenance at 3% O2. Finally, oxidative stress stimulated homologous recombination, which is suppressed by supplying deoxynucleotide precursors. Therefore, the cellular redox status strongly impacts genome duplication and transmission. Oxidative stress should generate replication stress through different mechanisms, including DNA damage and nucleotide pool imbalance. These data highlight the intricacy of endogenous replication and oxidative stresses, which are both evoked during tumorigenesis and senescence initiation

  8. Slow Replication Fork Velocity of Homologous Recombination-Defective Cells Results from Endogenous Oxidative Stress.

    Directory of Open Access Journals (Sweden)

    Therese Wilhelm

    2016-05-01

    Full Text Available Replications forks are routinely hindered by different endogenous stresses. Because homologous recombination plays a pivotal role in the reactivation of arrested replication forks, defects in homologous recombination reveal the initial endogenous stress(es. Homologous recombination-defective cells consistently exhibit a spontaneously reduced replication speed, leading to mitotic extra centrosomes. Here, we identify oxidative stress as a major endogenous source of replication speed deceleration in homologous recombination-defective cells. The treatment of homologous recombination-defective cells with the antioxidant N-acetyl-cysteine or the maintenance of the cells at low O2 levels (3% rescues both the replication fork speed, as monitored by single-molecule analysis (molecular combing, and the associated mitotic extra centrosome frequency. Reciprocally, the exposure of wild-type cells to H2O2 reduces the replication fork speed and generates mitotic extra centrosomes. Supplying deoxynucleotide precursors to H2O2-exposed cells rescued the replication speed. Remarkably, treatment with N-acetyl-cysteine strongly expanded the nucleotide pool, accounting for the replication speed rescue. Remarkably, homologous recombination-defective cells exhibit a high level of endogenous reactive oxygen species. Consistently, homologous recombination-defective cells accumulate spontaneous γH2AX or XRCC1 foci that are abolished by treatment with N-acetyl-cysteine or maintenance at 3% O2. Finally, oxidative stress stimulated homologous recombination, which is suppressed by supplying deoxynucleotide precursors. Therefore, the cellular redox status strongly impacts genome duplication and transmission. Oxidative stress should generate replication stress through different mechanisms, including DNA damage and nucleotide pool imbalance. These data highlight the intricacy of endogenous replication and oxidative stresses, which are both evoked during tumorigenesis and

  9. Slow Replication Fork Velocity of Homologous Recombination-Defective Cells Results from Endogenous Oxidative Stress.

    Science.gov (United States)

    Wilhelm, Therese; Ragu, Sandrine; Magdalou, Indiana; Machon, Christelle; Dardillac, Elodie; Técher, Hervé; Guitton, Jérôme; Debatisse, Michelle; Lopez, Bernard S

    2016-05-01

    Replications forks are routinely hindered by different endogenous stresses. Because homologous recombination plays a pivotal role in the reactivation of arrested replication forks, defects in homologous recombination reveal the initial endogenous stress(es). Homologous recombination-defective cells consistently exhibit a spontaneously reduced replication speed, leading to mitotic extra centrosomes. Here, we identify oxidative stress as a major endogenous source of replication speed deceleration in homologous recombination-defective cells. The treatment of homologous recombination-defective cells with the antioxidant N-acetyl-cysteine or the maintenance of the cells at low O2 levels (3%) rescues both the replication fork speed, as monitored by single-molecule analysis (molecular combing), and the associated mitotic extra centrosome frequency. Reciprocally, the exposure of wild-type cells to H2O2 reduces the replication fork speed and generates mitotic extra centrosomes. Supplying deoxynucleotide precursors to H2O2-exposed cells rescued the replication speed. Remarkably, treatment with N-acetyl-cysteine strongly expanded the nucleotide pool, accounting for the replication speed rescue. Remarkably, homologous recombination-defective cells exhibit a high level of endogenous reactive oxygen species. Consistently, homologous recombination-defective cells accumulate spontaneous γH2AX or XRCC1 foci that are abolished by treatment with N-acetyl-cysteine or maintenance at 3% O2. Finally, oxidative stress stimulated homologous recombination, which is suppressed by supplying deoxynucleotide precursors. Therefore, the cellular redox status strongly impacts genome duplication and transmission. Oxidative stress should generate replication stress through different mechanisms, including DNA damage and nucleotide pool imbalance. These data highlight the intricacy of endogenous replication and oxidative stresses, which are both evoked during tumorigenesis and senescence initiation

  10. Interaction of the retinoblastoma protein with Orc1 and its recruitment to human origins of DNA replication.

    Directory of Open Access Journals (Sweden)

    Ramiro Mendoza-Maldonado

    Full Text Available BACKGROUND: The retinoblastoma protein (Rb is a crucial regulator of cell cycle progression by binding with E2F transcription factor and repressing the expression of a variety of genes required for the G1-S phase transition. METHODOLOGY/PRINCIPAL FINDINGS: Here we show that Rb and E2F1 directly participate in the control of initiation of DNA replication in human HeLa, U2OS and T98G cells by specifically binding to origins of DNA replication in a cell cycle regulated manner. We show that, both in vitro and inside the cells, the largest subunit of the origin recognition complex (Orc1 specifically binds hypo-phosphorylated Rb and that this interaction is competitive with the binding of Rb to E2F1. The displacement of Rb-bound Orc1 by E2F1 at origins of DNA replication marks the progression of the G1 phase of the cell cycle toward the G1-S border. CONCLUSIONS/SIGNIFICANCE: The participation of Rb and E2F1 in the formation of the multiprotein complex that binds origins of DNA replication in mammalian cells appears to represent an effective mechanism to couple the expression of genes required for cell cycle progression to the activation of DNA replication.

  11. Physical Interaction between Replication Protein A (RPA) and MRN: Involvement of RPA2 Phosphorylation and the N-terminus of RPA1

    OpenAIRE

    Oakley, Greg; Tillison, Kristin; Opiyo, Stephen; Glanzer, Jason; Horn, Jeffrey M.; Patrick, Steve M.

    2009-01-01

    Replication protein A (RPA) is a heterotrimeric protein consisting of RPA1, RPA2 and RPA3 subunits that binds to ssDNA with high affinity. The response to replication stress requires the recruitment of RPA and the MRE11/RAD50/NBS1 (MRN) complex. RPA bound to ssDNA stabilizes stalled replication forks by recruiting checkpoint proteins involved in fork stabilization. MRN can bind DNA structures encountered at stalled or collapsed replication forks, such as ssDNA-dsDNA junctions or breaks and pr...

  12. Flock House virus subgenomic RNA3 is replicated and its replication correlates with transactivation of RNA2

    International Nuclear Information System (INIS)

    Eckerle, Lance D.; Albarino, Cesar G.; Ball, L. Andrew.

    2003-01-01

    The nodavirus Flock House virus has a bipartite genome composed of RNAs 1 and 2, which encode the catalytic component of the RNA-dependent RNA polymerase (RdRp) and the capsid protein precursor, respectively. In addition to catalyzing replication of the viral genome, the RdRp also transcribes from RNA1 a subgenomic RNA3, which is both required for and suppressed by RNA2 replication. Here, we show that in the absence of RNA1 replication, FHV RdRp replicated positive-sense RNA3 transcripts fully and copied negative-sense RNA3 transcripts into positive strands. The two nonstructural proteins encoded by RNA3 were dispensable for replication, but sequences in the 3'-terminal 58 nucleotides were required. RNA3 variants that failed to replicate also failed to transactivate RNA2. These results imply that RNA3 is naturally produced both by transcription from RNA1 and by subsequent RNA1-independent replication and that RNA3 replication may be necessary for transactivation of RNA2

  13. Geometric Mechanics Reveals Optimal Complex Terrestrial Undulation Patterns

    Science.gov (United States)

    Gong, Chaohui; Astley, Henry; Schiebel, Perrin; Dai, Jin; Travers, Matthew; Goldman, Daniel; Choset, Howie; CMU Team; GT Team

    Geometric mechanics offers useful tools for intuitively analyzing biological and robotic locomotion. However, utility of these tools were previously restricted to systems that have only two internal degrees of freedom and in uniform media. We show kinematics of complex locomotors that make intermittent contacts with substrates can be approximated as a linear combination of two shape bases, and can be represented using two variables. Therefore, the tools of geometric mechanics can be used to analyze motions of locomotors with many degrees of freedom. To demonstrate the proposed technique, we present studies on two different types of snake gaits which utilize combinations of waves in the horizontal and vertical planes: sidewinding (in the sidewinder rattlesnake C. cerastes) and lateral undulation (in the desert specialist snake C. occipitalis). C. cerastes moves by generating posteriorly traveling body waves in the horizontal and vertical directions, with a relative phase offset equal to +/-π/2 while C. occipitalismaintains a π/2 offset of a frequency doubled vertical wave. Geometric analysis reveals these coordination patterns enable optimal movement in the two different styles of undulatory terrestrial locomotion. More broadly, these examples demonstrate the utility of geometric mechanics in analyzing realistic biological and robotic locomotion.

  14. Comparative analysis of seven viral nuclear export signals (NESs reveals the crucial role of nuclear export mediated by the third NES consensus sequence of nucleoprotein (NP in influenza A virus replication.

    Directory of Open Access Journals (Sweden)

    Nopporn Chutiwitoonchai

    Full Text Available The assembly of influenza virus progeny virions requires machinery that exports viral genomic ribonucleoproteins from the cell nucleus. Currently, seven nuclear export signal (NES consensus sequences have been identified in different viral proteins, including NS1, NS2, M1, and NP. The present study examined the roles of viral NES consensus sequences and their significance in terms of viral replication and nuclear export. Mutation of the NP-NES3 consensus sequence resulted in a failure to rescue viruses using a reverse genetics approach, whereas mutation of the NS2-NES1 and NS2-NES2 sequences led to a strong reduction in viral replication kinetics compared with the wild-type sequence. While the viral replication kinetics for other NES mutant viruses were also lower than those of the wild-type, the difference was not so marked. Immunofluorescence analysis after transient expression of NP-NES3, NS2-NES1, or NS2-NES2 proteins in host cells showed that they accumulated in the cell nucleus. These results suggest that the NP-NES3 consensus sequence is mostly required for viral replication. Therefore, each of the hydrophobic (Φ residues within this NES consensus sequence (Φ1, Φ2, Φ3, or Φ4 was mutated, and its viral replication and nuclear export function were analyzed. No viruses harboring NP-NES3 Φ2 or Φ3 mutants could be rescued. Consistent with this, the NP-NES3 Φ2 and Φ3 mutants showed reduced binding affinity with CRM1 in a pull-down assay, and both accumulated in the cell nucleus. Indeed, a nuclear export assay revealed that these mutant proteins showed lower nuclear export activity than the wild-type protein. Moreover, the Φ2 and Φ3 residues (along with other Φ residues within the NP-NES3 consensus were highly conserved among different influenza A viruses, including human, avian, and swine. Taken together, these results suggest that the Φ2 and Φ3 residues within the NP-NES3 protein are important for its nuclear export function

  15. Education: DNA replication using microscale natural convection.

    Science.gov (United States)

    Priye, Aashish; Hassan, Yassin A; Ugaz, Victor M

    2012-12-07

    There is a need for innovative educational experiences that unify and reinforce fundamental principles at the interface between the physical, chemical, and life sciences. These experiences empower and excite students by helping them recognize how interdisciplinary knowledge can be applied to develop new products and technologies that benefit society. Microfluidics offers an incredibly versatile tool to address this need. Here we describe our efforts to create innovative hands-on activities that introduce chemical engineering students to molecular biology by challenging them to harness microscale natural convection phenomena to perform DNA replication via the polymerase chain reaction (PCR). Experimentally, we have constructed convective PCR stations incorporating a simple design for loading and mounting cylindrical microfluidic reactors between independently controlled thermal plates. A portable motion analysis microscope enables flow patterns inside the convective reactors to be directly visualized using fluorescent bead tracers. We have also developed a hands-on computational fluid dynamics (CFD) exercise based on modeling microscale thermal convection to identify optimal geometries for DNA replication. A cognitive assessment reveals that these activities strongly impact student learning in a positive way.

  16. Interaction of RECQ4 and MCM10 is important for efficient DNA replication origin firing in human cells

    DEFF Research Database (Denmark)

    Kliszczak, Maciej; Sedlackova, Hana; Pitchai, Ganesha P

    2015-01-01

    DNA replication is a highly coordinated process that is initiated at multiple replication origins in eukaryotes. These origins are bound by the origin recognition complex (ORC), which subsequently recruits the Mcm2-7 replicative helicase in a Cdt1/Cdc6-dependent manner. In budding yeast, two...... essential replication factors, Sld2 and Mcm10, are then important for the activation of replication origins. In humans, the putative Sld2 homolog, RECQ4, interacts with MCM10. Here, we have identified two mutants of human RECQ4 that are deficient in binding to MCM10. We show that these RECQ4 variants...... are able to complement the lethality of an avian cell RECQ4 deletion mutant, indicating that the essential function of RECQ4 in vertebrates is unlikely to require binding to MCM10. Nevertheless, we show that the RECQ4-MCM10 interaction is important for efficient replication origin firing....

  17. DNA replication origin function is promoted by H3K4 di-methylation in Saccharomyces cerevisiae.

    Science.gov (United States)

    Rizzardi, Lindsay F; Dorn, Elizabeth S; Strahl, Brian D; Cook, Jeanette Gowen

    2012-10-01

    DNA replication is a highly regulated process that is initiated from replication origins, but the elements of chromatin structure that contribute to origin activity have not been fully elucidated. To identify histone post-translational modifications important for DNA replication, we initiated a genetic screen to identify interactions between genes encoding chromatin-modifying enzymes and those encoding proteins required for origin function in the budding yeast Saccharomyces cerevisiae. We found that enzymes required for histone H3K4 methylation, both the histone methyltransferase Set1 and the E3 ubiquitin ligase Bre1, are required for robust growth of several hypomorphic replication mutants, including cdc6-1. Consistent with a role for these enzymes in DNA replication, we found that both Set1 and Bre1 are required for efficient minichromosome maintenance. These phenotypes are recapitulated in yeast strains bearing mutations in the histone substrates (H3K4 and H2BK123). Set1 functions as part of the COMPASS complex to mono-, di-, and tri-methylate H3K4. By analyzing strains lacking specific COMPASS complex members or containing H2B mutations that differentially affect H3K4 methylation states, we determined that these replication defects were due to loss of H3K4 di-methylation. Furthermore, histone H3K4 di-methylation is enriched at chromosomal origins. These data suggest that H3K4 di-methylation is necessary and sufficient for normal origin function. We propose that histone H3K4 di-methylation functions in concert with other histone post-translational modifications to support robust genome duplication.

  18. NB protein does not affect influenza B virus replication in vitro and is not required for replication in or transmission between ferrets

    Science.gov (United States)

    Elderfield, Ruth A.; Koutsakos, Marios; Frise, Rebecca; Bradley, Konrad; Ashcroft, Jonathan; Miah, Shanhjahan; Lackenby, Angie

    2016-01-01

    The influenza B virus encodes a unique protein, NB, a membrane protein whose function in the replication cycle is not, as yet, understood. We engineered a recombinant influenza B virus lacking NB expression, with no concomitant difference in expression or activity of viral neuraminidase (NA) protein, an important caveat since NA is encoded on the same segment and initiated from a start codon just 4 nt downstream of NB. Replication of the virus lacking NB was not different to wild-type virus with full-length NB in clonal immortalized or complex primary cell cultures. In the mouse model, virus lacking NB induced slightly lower IFN-α levels in infected lungs, but this did not affect virus titres or weight loss. In ferrets infected with a mixture of viruses that did or did not express NB, there was no fitness advantage for the virus that retained NB. Moreover, virus lacking NB protein was transmitted following respiratory droplet exposure of sentinel animals. These data suggest no role for NB in supporting replication or transmission in vivo in this animal model. The role of NB and the nature of selection to retain it in all natural influenza B viruses remain unclear. PMID:26703440

  19. DNA Replication Control During Drosophila Development: Insights into the Onset of S Phase, Replication Initiation, and Fork Progression

    Science.gov (United States)

    Hua, Brian L.; Orr-Weaver, Terry L.

    2017-01-01

    Proper control of DNA replication is critical to ensure genomic integrity during cell proliferation. In addition, differential regulation of the DNA replication program during development can change gene copy number to influence cell size and gene expression. Drosophila melanogaster serves as a powerful organism to study the developmental control of DNA replication in various cell cycle contexts in a variety of differentiated cell and tissue types. Additionally, Drosophila has provided several developmentally regulated replication models to dissect the molecular mechanisms that underlie replication-based copy number changes in the genome, which include differential underreplication and gene amplification. Here, we review key findings and our current understanding of the developmental control of DNA replication in the contexts of the archetypal replication program as well as of underreplication and differential gene amplification. We focus on the use of these latter two replication systems to delineate many of the molecular mechanisms that underlie the developmental control of replication initiation and fork elongation. PMID:28874453

  20. Role of the Escherichia coli grpE heat shock protein in the initiation of bacteriophage lambda DNA replication.

    Science.gov (United States)

    Osipiuk, J; Zylicz, M

    1991-01-01

    Initiation of replication of lambda DNA requires assembly of the proper nucleoprotein complex consisting of the lambda origin of replication-lambda O-lambda P-dnaB proteins. The dnaJ, dnaK and grpE heat shock proteins destabilize the lambda P-dnaB interaction in this complex permitting dnaB helicase to unwind lambda DNA near ori lambda sequence. First step of this disassembling reaction is the binding of dnaK protein to lambda P protein. In this report we examined the influence of dnaJ and grpE proteins on stability of the lambda P-dnaK complex. Our results show that grpE alone dissociates this complex, but both grpE and dnaJ together do not. These results suggest that, in the presence of grpE protein, dnaK protein has a higher affinity for lambda P protein complexed with dnaJ protein than in the situation where grpE protein is not used.

  1. Mechanistic Studies with DNA Polymerases Reveal Complex Outcomes following Bypass of DNA Damage

    Directory of Open Access Journals (Sweden)

    Robert L. Eoff

    2010-01-01

    Full Text Available DNA is a chemically reactive molecule that is subject to many different covalent modifications from sources that are both endogenous and exogenous in origin. The inherent instability of DNA is a major obstacle to genomic maintenance and contributes in varying degrees to cellular dysfunction and disease in multi-cellular organisms. Investigations into the chemical and biological aspects of DNA damage have identified multi-tiered and overlapping cellular systems that have evolved as a means of stabilizing the genome. One of these pathways supports DNA replication events by in a sense adopting the mantra that one must “make the best of a bad situation” and tolerating covalent modification to DNA through less accurate copying of the damaged region. Part of this so-called DNA damage tolerance pathway involves the recruitment of specialized DNA polymerases to sites of stalled or collapsed replication forks. These enzymes have unique structural and functional attributes that often allow bypass of adducted template DNA and successful completion of genomic replication. What follows is a selective description of the salient structural features and bypass properties of specialized DNA polymerases with an emphasis on Y-family members.

  2. The logic of DNA replication in double-stranded DNA viruses: insights from global analysis of viral genomes.

    Science.gov (United States)

    Kazlauskas, Darius; Krupovic, Mart; Venclovas, Česlovas

    2016-06-02

    Genomic DNA replication is a complex process that involves multiple proteins. Cellular DNA replication systems are broadly classified into only two types, bacterial and archaeo-eukaryotic. In contrast, double-stranded (ds) DNA viruses feature a much broader diversity of DNA replication machineries. Viruses differ greatly in both completeness and composition of their sets of DNA replication proteins. In this study, we explored whether there are common patterns underlying this extreme diversity. We identified and analyzed all major functional groups of DNA replication proteins in all available proteomes of dsDNA viruses. Our results show that some proteins are common to viruses infecting all domains of life and likely represent components of the ancestral core set. These include B-family polymerases, SF3 helicases, archaeo-eukaryotic primases, clamps and clamp loaders of the archaeo-eukaryotic type, RNase H and ATP-dependent DNA ligases. We also discovered a clear correlation between genome size and self-sufficiency of viral DNA replication, the unanticipated dominance of replicative helicases and pervasive functional associations among certain groups of DNA replication proteins. Altogether, our results provide a comprehensive view on the diversity and evolution of replication systems in the DNA virome and uncover fundamental principles underlying the orchestration of viral DNA replication. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

  3. Molecular architecture of the recombinant human MCM2-7 helicase in complex with nucleotides and DNA

    DEFF Research Database (Denmark)

    Boskovic, Jasminka; Bragado-Nilsson, Elisabeth; Saligram Prabhakar, Bhargav

    2016-01-01

    DNA replication is a key biological process that involves different protein complexes whose assembly is rigorously regulated in a successive order. One of these complexes is a replicative hexameric helicase, the MCM complex, which is essential for the initiation and elongation phases of replicati...

  4. Suppression of Rac1 Signaling by Influenza A Virus NS1 Facilitates Viral Replication

    Science.gov (United States)

    Jiang, Wei; Sheng, Chunjie; Gu, Xiuling; Liu, Dong; Yao, Chen; Gao, Shijuan; Chen, Shuai; Huang, Yinghui; Huang, Wenlin; Fang, Min

    2016-01-01

    Influenza A virus (IAV) is a major human pathogen with the potential to become pandemic. IAV contains only eight RNA segments; thus, the virus must fully exploit the host cellular machinery to facilitate its own replication. In an effort to comprehensively characterize the host machinery taken over by IAV in mammalian cells, we generated stable A549 cell lines with over-expression of the viral non-structural protein (NS1) to investigate the potential host factors that might be modulated by the NS1 protein. We found that the viral NS1 protein directly interacted with cellular Rac1 and facilitated viral replication. Further research revealed that NS1 down-regulated Rac1 activity via post-translational modifications. Therefore, our results demonstrated that IAV blocked Rac1-mediated host cell signal transduction through the NS1 protein to facilitate its own replication. Our findings provide a novel insight into the mechanism of IAV replication and indicate new avenues for the development of potential therapeutic targets. PMID:27869202

  5. Primer retention owing to the absence of RNase H1 is catastrophic for mitochondrial DNA replication.

    Science.gov (United States)

    Holmes, J Bradley; Akman, Gokhan; Wood, Stuart R; Sakhuja, Kiran; Cerritelli, Susana M; Moss, Chloe; Bowmaker, Mark R; Jacobs, Howard T; Crouch, Robert J; Holt, Ian J

    2015-07-28

    Encoding ribonuclease H1 (RNase H1) degrades RNA hybridized to DNA, and its function is essential for mitochondrial DNA maintenance in the developing mouse. Here we define the role of RNase H1 in mitochondrial DNA replication. Analysis of replicating mitochondrial DNA in embryonic fibroblasts lacking RNase H1 reveals retention of three primers in the major noncoding region (NCR) and one at the prominent lagging-strand initiation site termed Ori-L. Primer retention does not lead immediately to depletion, as the persistent RNA is fully incorporated in mitochondrial DNA. However, the retained primers present an obstacle to the mitochondrial DNA polymerase γ in subsequent rounds of replication and lead to the catastrophic generation of a double-strand break at the origin when the resulting gapped molecules are copied. Hence, the essential role of RNase H1 in mitochondrial DNA replication is the removal of primers at the origin of replication.

  6. Genetic characterization of an adapted pandemic 2009 H1N1 influenza virus that reveals improved replication rates in human lung epithelial cells

    International Nuclear Information System (INIS)

    Wörmann, Xenia; Lesch, Markus; Welke, Robert-William; Okonechnikov, Konstantin; Abdurishid, Mirshat; Sieben, Christian; Geissner, Andreas; Brinkmann, Volker; Kastner, Markus; Karner, Andreas; Zhu, Rong; Hinterdorfer, Peter; Anish, Chakkumkal; Seeberger, Peter H.; Herrmann, Andreas

    2016-01-01

    The 2009 influenza pandemic originated from a swine-origin H1N1 virus, which, although less pathogenic than anticipated, may acquire additional virulence-associated mutations in the future. To estimate the potential risk, we sequentially passaged the isolate A/Hamburg/04/2009 in A549 human lung epithelial cells. After passage 6, we observed a 100-fold increased replication rate. High-throughput sequencing of viral gene segments identified five dominant mutations, whose contribution to the enhanced growth was analyzed by reverse genetics. The increased replication rate was pinpointed to two mutations within the hemagglutinin (HA) gene segment (HA_1 D130E, HA_2 I91L), near the receptor binding site and the stem domain. The adapted virus also replicated more efficiently in mice in vivo. Enhanced replication rate correlated with increased fusion pH of the HA protein and a decrease in receptor affinity. Our data might be relevant for surveillance of pre-pandemic strains and development of high titer cell culture strains for vaccine production. - Highlights: • We observed a spontaneous mutation of a 2009-pandemic H1N1 influenza virus in vitro. • The adaptation led to a 100-fold rise in replication rate in human A549 cells. • Adaptation was caused by two mutations in the HA gene segment. • Adaptation correlates with increased fusion pH and decreased receptor affinity.

  7. Genetic characterization of an adapted pandemic 2009 H1N1 influenza virus that reveals improved replication rates in human lung epithelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Wörmann, Xenia [Department of Molecular Biology, Max Planck Institute for Infection Biology, Berlin (Germany); Lesch, Markus [Department of Molecular Biology, Max Planck Institute for Infection Biology, Berlin (Germany); Steinbeis Innovation gGmbH, Center for Systems Biomedicine, Falkensee (Germany); Welke, Robert-William [Department of Biology, Molecular Biophysics, IRI Life Sciences, Humboldt-Universität zu Berlin (Germany); Okonechnikov, Konstantin; Abdurishid, Mirshat [Department of Molecular Biology, Max Planck Institute for Infection Biology, Berlin (Germany); Sieben, Christian [Department of Biology, Molecular Biophysics, IRI Life Sciences, Humboldt-Universität zu Berlin (Germany); Geissner, Andreas [Department for Biomolecular Systems, Max Planck Institute for Colloids and Interfaces, Potsdam (Germany); Institute of Chemistry and Biochemistry, Free University, Berlin (Germany); Brinkmann, Volker [Department of Molecular Biology, Max Planck Institute for Infection Biology, Berlin (Germany); Kastner, Markus [Institute for Biophysics, Johannes Kepler University, Linz (Austria); Karner, Andreas [Center for Advanced Bioanalysis GmbH (CBL), Linz (Austria); Zhu, Rong; Hinterdorfer, Peter [Institute for Biophysics, Johannes Kepler University, Linz (Austria); Anish, Chakkumkal [Department for Biomolecular Systems, Max Planck Institute for Colloids and Interfaces, Potsdam (Germany); Seeberger, Peter H. [Department for Biomolecular Systems, Max Planck Institute for Colloids and Interfaces, Potsdam (Germany); Institute of Chemistry and Biochemistry, Free University, Berlin (Germany); Herrmann, Andreas [Department of Biology, Molecular Biophysics, IRI Life Sciences, Humboldt-Universität zu Berlin (Germany); and others

    2016-05-15

    The 2009 influenza pandemic originated from a swine-origin H1N1 virus, which, although less pathogenic than anticipated, may acquire additional virulence-associated mutations in the future. To estimate the potential risk, we sequentially passaged the isolate A/Hamburg/04/2009 in A549 human lung epithelial cells. After passage 6, we observed a 100-fold increased replication rate. High-throughput sequencing of viral gene segments identified five dominant mutations, whose contribution to the enhanced growth was analyzed by reverse genetics. The increased replication rate was pinpointed to two mutations within the hemagglutinin (HA) gene segment (HA{sub 1} D130E, HA{sub 2} I91L), near the receptor binding site and the stem domain. The adapted virus also replicated more efficiently in mice in vivo. Enhanced replication rate correlated with increased fusion pH of the HA protein and a decrease in receptor affinity. Our data might be relevant for surveillance of pre-pandemic strains and development of high titer cell culture strains for vaccine production. - Highlights: • We observed a spontaneous mutation of a 2009-pandemic H1N1 influenza virus in vitro. • The adaptation led to a 100-fold rise in replication rate in human A549 cells. • Adaptation was caused by two mutations in the HA gene segment. • Adaptation correlates with increased fusion pH and decreased receptor affinity.

  8. A Novel, Broad-Spectrum Inhibitor of Enterovirus Replication That Targets Host Cell Factor Phosphatidylinositol 4-Kinase IIIβ

    Science.gov (United States)

    van der Schaar, Hilde M.; Leyssen, Pieter; Thibaut, Hendrik J.; de Palma, Armando; van der Linden, Lonneke; Lanke, Kjerstin H. W.; Lacroix, Céline; Verbeken, Erik; Conrath, Katja; MacLeod, Angus M.; Mitchell, Dale R.; Palmer, Nicholas J.; van de Poël, Hervé; Andrews, Martin

    2013-01-01

    Despite their high clinical and socioeconomic impacts, there is currently no approved antiviral therapy for the prophylaxis or treatment of enterovirus infections. Here we report on a novel inhibitor of enterovirus replication, compound 1, 2-fluoro-4-(2-methyl-8-(3-(methylsulfonyl)benzylamino)imidazo[1,2-a]pyrazin-3-yl)phenol. This compound exhibited a broad spectrum of antiviral activity, as it inhibited all tested species of enteroviruses and rhinoviruses, with 50% effective concentrations ranging between 4 and 71 nM. After a lengthy resistance selection process, coxsackievirus mutants resistant to compound 1 were isolated that carried substitutions in their 3A protein. Remarkably, the same substitutions were recently shown to provide resistance to inhibitors of phosphatidylinositol 4-kinase IIIβ (PI4KIIIβ), a lipid kinase that is essential for enterovirus replication, suggesting that compound 1 may also target this host factor. Accordingly, compound 1 directly inhibited PI4KIIIβ in an in vitro kinase activity assay. Furthermore, the compound strongly reduced the PI 4-phosphate levels of the Golgi complex in cells. Rescue of coxsackievirus replication in the presence of compound 1 by a mutant PI4KIIIβ carrying a substitution in its ATP-binding pocket revealed that the compound directly binds the kinase at this site. Finally, we determined that an analogue of compound 1, 3-(3-fluoro-4-methoxyphenyl)-2-methyl-N-(pyridin-4-ylmethyl)imidazo[1,2-a]pyrazin-8-amine, is well tolerated in mice and has a dose-dependent protective activity in a coxsackievirus serotype B4-induced pancreatitis model. PMID:23896472

  9. Substrate recognition by complement convertases revealed in the C5-cobra venom factor complex

    DEFF Research Database (Denmark)

    Laursen, Nick Stub; Andersen, Kasper Røjkjær; Braren, Ingke

    2011-01-01

    with a protease subunit (Bb or C2a). We determined the crystal structures of the C3b homologue cobra venom factor (CVF) in complex with C5, and in complex with C5 and the inhibitor SSL7 at 4.3 Å resolution. The structures reveal a parallel two-point attachment between C5 and CVF, where the presence of SSL7 only...... slightly affects the C5-CVF interface, explaining the IgA dependence for SSL7-mediated inhibition of C5 cleavage. CVF functions as a relatively rigid binding scaffold inducing a conformational change in C5, which positions its cleavage site in proximity to the serine protease Bb. A general model...

  10. Replication of micro and nano surface geometries

    DEFF Research Database (Denmark)

    Hansen, Hans Nørgaard; Hocken, R.J.; Tosello, Guido

    2011-01-01

    The paper describes the state-of-the-art in replication of surface texture and topography at micro and nano scale. The description includes replication of surfaces in polymers, metals and glass. Three different main technological areas enabled by surface replication processes are presented......: manufacture of net-shape micro/nano surfaces, tooling (i.e. master making), and surface quality control (metrology, inspection). Replication processes and methods as well as the metrology of surfaces to determine the degree of replication are presented and classified. Examples from various application areas...... are given including replication for surface texture measurements, surface roughness standards, manufacture of micro and nano structured functional surfaces, replicated surfaces for optical applications (e.g. optical gratings), and process chains based on combinations of repeated surface replication steps....

  11. A New Replication Norm for Psychology

    Directory of Open Access Journals (Sweden)

    Etienne P LeBel

    2015-10-01

    Full Text Available In recent years, there has been a growing concern regarding the replicability of findings in psychology, including a mounting number of prominent findings that have failed to replicate via high-powered independent replication attempts. In the face of this replicability “crisis of confidence”, several initiatives have been implemented to increase the reliability of empirical findings. In the current article, I propose a new replication norm that aims to further boost the dependability of findings in psychology. Paralleling the extant social norm that researchers should peer review about three times as many articles that they themselves publish per year, the new replication norm states that researchers should aim to independently replicate important findings in their own research areas in proportion to the number of original studies they themselves publish per year (e.g., a 4:1 original-to-replication studies ratio. I argue this simple approach could significantly advance our science by increasing the reliability and cumulative nature of our empirical knowledge base, accelerating our theoretical understanding of psychological phenomena, instilling a focus on quality rather than quantity, and by facilitating our transformation toward a research culture where executing and reporting independent direct replications is viewed as an ordinary part of the research process. To help promote the new norm, I delineate (1 how each of the major constituencies of the research process (i.e., funders, journals, professional societies, departments, and individual researchers can incentivize replications and promote the new norm and (2 any obstacles each constituency faces in supporting the new norm.

  12. Human CST Facilitates Genome-wide RAD51 Recruitment to GC-Rich Repetitive Sequences in Response to Replication Stress.

    Science.gov (United States)

    Chastain, Megan; Zhou, Qing; Shiva, Olga; Fadri-Moskwik, Maria; Whitmore, Leanne; Jia, Pingping; Dai, Xueyu; Huang, Chenhui; Ye, Ping; Chai, Weihang

    2016-08-02

    The telomeric CTC1/STN1/TEN1 (CST) complex has been implicated in promoting replication recovery under replication stress at genomic regions, yet its precise role is unclear. Here, we report that STN1 is enriched at GC-rich repetitive sequences genome-wide in response to hydroxyurea (HU)-induced replication stress. STN1 deficiency exacerbates the fragility of these sequences under replication stress, resulting in chromosome fragmentation. We find that upon fork stalling, CST proteins form distinct nuclear foci that colocalize with RAD51. Furthermore, replication stress induces physical association of CST with RAD51 in an ATR-dependent manner. Strikingly, CST deficiency diminishes HU-induced RAD51 foci formation and reduces RAD51 recruitment to telomeres and non-telomeric GC-rich fragile sequences. Collectively, our findings establish that CST promotes RAD51 recruitment to GC-rich repetitive sequences in response to replication stress to facilitate replication restart, thereby providing insights into the mechanism underlying genome stability maintenance. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

  13. STAT2 Knockout Syrian Hamsters Support Enhanced Replication and Pathogenicity of Human Adenovirus, Revealing an Important Role of Type I Interferon Response in Viral Control.

    Directory of Open Access Journals (Sweden)

    Karoly Toth

    2015-08-01

    Full Text Available Human adenoviruses have been studied extensively in cell culture and have been a model for studies in molecular, cellular, and medical biology. However, much less is known about adenovirus replication and pathogenesis in vivo in a permissive host because of the lack of an adequate animal model. Presently, the most frequently used permissive immunocompetent animal model for human adenovirus infection is the Syrian hamster. Species C human adenoviruses replicate in these animals and cause pathology that is similar to that seen with humans. Here, we report findings with a new Syrian hamster strain in which the STAT2 gene was functionally knocked out by site-specific gene targeting. Adenovirus-infected STAT2 knockout hamsters demonstrated an accentuated pathology compared to the wild-type control animals, and the virus load in the organs of STAT2 knockout animals was 100- to 1000-fold higher than that in wild-type hamsters. Notably, the adaptive immune response to adenovirus is not adversely affected in STAT2 knockout hamsters, and surviving hamsters cleared the infection by 7 to 10 days post challenge. We show that the Type I interferon pathway is disrupted in these hamsters, revealing the critical role of interferon-stimulated genes in controlling adenovirus infection. This is the first study to report findings with a genetically modified Syrian hamster infected with a virus. Further, this is the first study to show that the Type I interferon pathway plays a role in inhibiting human adenovirus replication in a permissive animal model. Besides providing an insight into adenovirus infection in humans, our results are also interesting from the perspective of the animal model: STAT2 knockout Syrian hamster may also be an important animal model for studying other viral infections, including Ebola-, hanta-, and dengue viruses, where Type I interferon-mediated innate immunity prevents wild type hamsters from being effectively infected to be used as

  14. Chromosome replication, cell growth, division and shape: a personal perspective

    Directory of Open Access Journals (Sweden)

    Arieh eZaritsky

    2015-08-01

    Full Text Available The origins of Molecular Biology and Bacterial Physiology are reviewed, from our personal standpoints, emphasizing the coupling between bacterial growth, chromosome replication and cell division, dimensions and shape. Current knowledge is discussed with historical perspective, summarizing past and present achievements and enlightening ideas for future studies. An interactive simulation program of the Bacterial Cell Division Cycle (BCD, described as The Central Dogma in Bacteriology, is briefly represented. The coupled process of transcription/translation of genes encoding membrane proteins and insertion into the membrane (so-called transertion is invoked as the functional relationship between the only two unique macromolecules in the cell, DNA and peptidoglycan embodying the nucleoid and the sacculus respectively. We envision that nucleoid complexity, defined as the weighted-mean DNA content associated with the replication terminus, is directly related to cell shape through the transertion process. Accordingly, the primary signal for cell division transmitted by DNA dynamics (replication, transcription and segregation to the peptidoglycan biosynthetic machinery is of a physico-chemical nature, eg stress in the plasma membrane, relieving nucleoid occlusion in the cell's center hence enabling the divisome to assemble and function between segregated daughter nucleoids.

  15. The RecJ2 protein in the thermophilic archaeon Thermoplasma acidophilum is a 3'-5' exonuclease that associates with a DNA replication complex.

    Science.gov (United States)

    Ogino, Hiromi; Ishino, Sonoko; Kohda, Daisuke; Ishino, Yoshizumi

    2017-05-12

    RecJ/cell division cycle 45 (Cdc45) proteins are widely conserved in the three domains of life, i.e. in bacteria, Eukarya, and Archaea. Bacterial RecJ is a 5'-3' exonuclease and functions in DNA repair pathways by using its 5'-3' exonuclease activity. Eukaryotic Cdc45 has no identified enzymatic activity but participates in the CMG complex, so named because it is composed of Cdc45, minichromosome maintenance protein complex (MCM) proteins 2-7, and GINS complex proteins (Sld5, Psf11-3). Eukaryotic Cdc45 and bacterial/archaeal RecJ share similar amino acid sequences and are considered functional counterparts. In Archaea, a RecJ homolog in Thermococcus kodakarensis was shown to associate with GINS and accelerate its nuclease activity and was, therefore, designated GAN ( G INS- a ssociated n uclease); however, to date, no archaeal RecJ·MCM·GINS complex has been isolated. The thermophilic archaeon Thermoplasma acidophilum has two RecJ-like proteins, designated TaRecJ1 and TaRecJ2. TaRecJ1 exhibited DNA-specific 5'-3' exonuclease activity, whereas TaRecJ2 had 3'-5' exonuclease activity and preferred RNA over DNA. TaRecJ2, but not TaRecJ1, formed a stable complex with TaGINS in a 2:1 molar ratio. Furthermore, the TaRecJ2·TaGINS complex stimulated activity of TaMCM ( T. acidophilum MCM) helicase in vitro , and the TaRecJ2·TaMCM·TaGINS complex was also observed in vivo However, TaRecJ2 did not interact with TaMCM directly and was not required for the helicase activation in vitro These findings suggest that the function of archaeal RecJ in DNA replication evolved divergently from Cdc45 despite conservation of the CMG-like complex formation between Archaea and Eukarya. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  16. Individual Differences among Employees Management Communication Style and Employee Satisfaction: Replication and Extension.

    Science.gov (United States)

    McCroskey, James C.; And Others

    Portions of three earlier studies relating differences in employees to employee satisfaction and one study relating management communication style (MCS) to employee satisfaction were replicated across four organizational contexts. Major findings supported the generalizability of the results revealed in the earlier research. The role of…

  17. [Replication of Streptomyces plasmids: the DNA nucleotide sequence of plasmid pSB 24.2].

    Science.gov (United States)

    Bolotin, A P; Sorokin, A V; Aleksandrov, N N; Danilenko, V N; Kozlov, Iu I

    1985-11-01

    The nucleotide sequence of DNA in plasmid pSB 24.2, a natural deletion derivative of plasmid pSB 24.1 isolated from S. cyanogenus was studied. The plasmid amounted by its size to 3706 nucleotide pairs. The G-C composition was equal to 73 per cent. The analysis of the DNA structure in plasmid pSB 24.2 revealed the protein-encoding sequence of DNA, the continuity of which was significant for replication of the plasmid containing more than 1300 nucleotide pairs. The analysis also revealed two A-T-rich areas of DNA, the G-C composition of which was less than 55 per cent and a DNA area with a branched pin structure. The results may be of value in investigation of plasmid replication in actinomycetes and experimental cloning of DNA with this plasmid as a vector.

  18. DNA lesions induced by replication stress trigger mitotic aberration and tetraploidy development.

    Directory of Open Access Journals (Sweden)

    Yosuke Ichijima

    Full Text Available During tumorigenesis, cells acquire immortality in association with the development of genomic instability. However, it is still elusive how genomic instability spontaneously generates during the process of tumorigenesis. Here, we show that precancerous DNA lesions induced by oncogene acceleration, which induce situations identical to the initial stages of cancer development, trigger tetraploidy/aneuploidy generation in association with mitotic aberration. Although oncogene acceleration primarily induces DNA replication stress and the resulting lesions in the S phase, these lesions are carried over into the M phase and cause cytokinesis failure and genomic instability. Unlike directly induced DNA double-strand breaks, DNA replication stress-associated lesions are cryptogenic and pass through cell-cycle checkpoints due to limited and ineffective activation of checkpoint factors. Furthermore, since damaged M-phase cells still progress in mitotic steps, these cells result in chromosomal mis-segregation, cytokinesis failure and the resulting tetraploidy generation. Thus, our results reveal a process of genomic instability generation triggered by precancerous DNA replication stress.

  19. The Inherent Asymmetry of DNA Replication.

    Science.gov (United States)

    Snedeker, Jonathan; Wooten, Matthew; Chen, Xin

    2017-10-06

    Semiconservative DNA replication has provided an elegant solution to the fundamental problem of how life is able to proliferate in a way that allows cells, organisms, and populations to survive and replicate many times over. Somewhat lost, however, in our admiration for this mechanism is an appreciation for the asymmetries that occur in the process of DNA replication. As we discuss in this review, these asymmetries arise as a consequence of the structure of the DNA molecule and the enzymatic mechanism of DNA synthesis. Increasing evidence suggests that asymmetries in DNA replication are able to play a central role in the processes of adaptation and evolution by shaping the mutagenic landscape of cells. Additionally, in eukaryotes, recent work has demonstrated that the inherent asymmetries in DNA replication may play an important role in the process of chromatin replication. As chromatin plays an essential role in defining cell identity, asymmetries generated during the process of DNA replication may play critical roles in cell fate decisions related to patterning and development.

  20. Defective replication initiation results in locus specific chromosome breakage and a ribosomal RNA deficiency in yeast.

    Directory of Open Access Journals (Sweden)

    Joseph C Sanchez

    2017-10-01

    Full Text Available A form of dwarfism known as Meier-Gorlin syndrome (MGS is caused by recessive mutations in one of six different genes (ORC1, ORC4, ORC6, CDC6, CDT1, and MCM5. These genes encode components of the pre-replication complex, which assembles at origins of replication prior to S phase. Also, variants in two additional replication initiation genes have joined the list of causative mutations for MGS (Geminin and CDC45. The identity of the causative MGS genetic variants strongly suggests that some aspect of replication is amiss in MGS patients; however, little evidence has been obtained regarding what aspect of chromosome replication is faulty. Since the site of one of the missense mutations in the human ORC4 alleles is conserved between humans and yeast, we sought to determine in what way this single amino acid change affects the process of chromosome replication, by introducing the comparable mutation into yeast (orc4Y232C. We find that yeast cells with the orc4Y232C allele have a prolonged S-phase, due to compromised replication initiation at the ribosomal DNA (rDNA locus located on chromosome XII. The inability to initiate replication at the rDNA locus results in chromosome breakage and a severely reduced rDNA copy number in the survivors, presumably helping to ensure complete replication of chromosome XII. Although reducing rDNA copy number may help ensure complete chromosome replication, orc4Y232C cells struggle to meet the high demand for ribosomal RNA synthesis. This finding provides additional evidence linking two essential cellular pathways-DNA replication and ribosome biogenesis.

  1. Defective replication initiation results in locus specific chromosome breakage and a ribosomal RNA deficiency in yeast.

    Science.gov (United States)

    Sanchez, Joseph C; Kwan, Elizabeth X; Pohl, Thomas J; Amemiya, Haley M; Raghuraman, M K; Brewer, Bonita J

    2017-10-01

    A form of dwarfism known as Meier-Gorlin syndrome (MGS) is caused by recessive mutations in one of six different genes (ORC1, ORC4, ORC6, CDC6, CDT1, and MCM5). These genes encode components of the pre-replication complex, which assembles at origins of replication prior to S phase. Also, variants in two additional replication initiation genes have joined the list of causative mutations for MGS (Geminin and CDC45). The identity of the causative MGS genetic variants strongly suggests that some aspect of replication is amiss in MGS patients; however, little evidence has been obtained regarding what aspect of chromosome replication is faulty. Since the site of one of the missense mutations in the human ORC4 alleles is conserved between humans and yeast, we sought to determine in what way this single amino acid change affects the process of chromosome replication, by introducing the comparable mutation into yeast (orc4Y232C). We find that yeast cells with the orc4Y232C allele have a prolonged S-phase, due to compromised replication initiation at the ribosomal DNA (rDNA) locus located on chromosome XII. The inability to initiate replication at the rDNA locus results in chromosome breakage and a severely reduced rDNA copy number in the survivors, presumably helping to ensure complete replication of chromosome XII. Although reducing rDNA copy number may help ensure complete chromosome replication, orc4Y232C cells struggle to meet the high demand for ribosomal RNA synthesis. This finding provides additional evidence linking two essential cellular pathways-DNA replication and ribosome biogenesis.

  2. The BAH domain of ORC1 links H4K20me2 to DNA replication licensing and Meier-Gorlin syndrome

    Energy Technology Data Exchange (ETDEWEB)

    Kuo, Alex J; Song, Jikui; Cheung, Peggie; Ishibe-Murakami, Satoko; Yamazoe, Sayumi; Chen, James K; Patel, Dinshaw J; Gozani, Or [Stanford; (MSKCC); (Stanford-MED)

    2012-07-11

    The recognition of distinctly modified histones by specialized 'effector' proteins constitutes a key mechanism for transducing molecular events at chromatin to biological outcomes. Effector proteins influence DNA-templated processes, including transcription, DNA recombination and DNA repair; however, no effector functions have yet been identified within the mammalian machinery that regulate DNA replication. Here we show that ORC1 - a component of ORC (origin of replication complex), which mediates pre-DNA replication licensing - contains a bromo adjacent homology (BAH) domain that specifically recognizes histone H4 dimethylated at lysine 20 (H4K20me2). Recognition of H4K20me2 is a property common to BAH domains present within diverse metazoan ORC1 proteins. Structural studies reveal that the specificity of the BAH domain for H4K20me2 is mediated by a dynamic aromatic dimethyl-lysine-binding cage and multiple intermolecular contacts involving the bound peptide. H4K20me2 is enriched at replication origins, and abrogating ORC1 recognition of H4K20me2 in cells impairs ORC1 occupancy at replication origins, ORC chromatin loading and cell-cycle progression. Mutation of the ORC1 BAH domain has been implicated in the aetiology of Meier-Gorlin syndrome (MGS), a form of primordial dwarfism, and ORC1 depletion in zebrafish results in an MGS-like phenotype. We find that wild-type human ORC1, but not ORC1-H4K20me2-binding mutants, rescues the growth retardation of orc1 morphants. Moreover, zebrafish depleted of H4K20me2 have diminished body size, mirroring the phenotype of orc1 morphants. Together, our results identify the BAH domain as a novel methyl-lysine-binding module, thereby establishing the first direct link between histone methylation and the metazoan DNA replication machinery, and defining a pivotal aetiological role for the canonical H4K20me2 mark, via ORC1, in primordial dwarfism.

  3. Replication dynamics of the yeast genome.

    Science.gov (United States)

    Raghuraman, M K; Winzeler, E A; Collingwood, D; Hunt, S; Wodicka, L; Conway, A; Lockhart, D J; Davis, R W; Brewer, B J; Fangman, W L

    2001-10-05

    Oligonucleotide microarrays were used to map the detailed topography of chromosome replication in the budding yeast Saccharomyces cerevisiae. The times of replication of thousands of sites across the genome were determined by hybridizing replicated and unreplicated DNAs, isolated at different times in S phase, to the microarrays. Origin activations take place continuously throughout S phase but with most firings near mid-S phase. Rates of replication fork movement vary greatly from region to region in the genome. The two ends of each of the 16 chromosomes are highly correlated in their times of replication. This microarray approach is readily applicable to other organisms, including humans.

  4. LHCb experience with LFC replication

    CERN Document Server

    Bonifazi, F; Perez, E D; D'Apice, A; dell'Agnello, L; Düllmann, D; Girone, M; Re, G L; Martelli, B; Peco, G; Ricci, P P; Sapunenko, V; Vagnoni, V; Vitlacil, D

    2008-01-01

    Database replication is a key topic in the framework of the LHC Computing Grid to allow processing of data in a distributed environment. In particular, the LHCb computing model relies on the LHC File Catalog, i.e. a database which stores information about files spread across the GRID, their logical names and the physical locations of all the replicas. The LHCb computing model requires the LFC to be replicated at Tier-1s. The LCG 3D project deals with the database replication issue and provides a replication service based on Oracle Streams technology. This paper describes the deployment of the LHC File Catalog replication to the INFN National Center for Telematics and Informatics (CNAF) and to other LHCb Tier-1 sites. We performed stress tests designed to evaluate any delay in the propagation of the streams and the scalability of the system. The tests show the robustness of the replica implementation with performance going much beyond the LHCb requirements.

  5. LHCb experience with LFC replication

    International Nuclear Information System (INIS)

    Bonifazi, F; Carbone, A; D'Apice, A; Dell'Agnello, L; Re, G L; Martelli, B; Ricci, P P; Sapunenko, V; Vitlacil, D; Perez, E D; Duellmann, D; Girone, M; Peco, G; Vagnoni, V

    2008-01-01

    Database replication is a key topic in the framework of the LHC Computing Grid to allow processing of data in a distributed environment. In particular, the LHCb computing model relies on the LHC File Catalog, i.e. a database which stores information about files spread across the GRID, their logical names and the physical locations of all the replicas. The LHCb computing model requires the LFC to be replicated at Tier-1s. The LCG 3D project deals with the database replication issue and provides a replication service based on Oracle Streams technology. This paper describes the deployment of the LHC File Catalog replication to the INFN National Center for Telematics and Informatics (CNAF) and to other LHCb Tier-1 sites. We performed stress tests designed to evaluate any delay in the propagation of the streams and the scalability of the system. The tests show the robustness of the replica implementation with performance going much beyond the LHCb requirements

  6. Mutant p53 perturbs DNA replication checkpoint control through TopBP1 and Treslin.

    Science.gov (United States)

    Liu, Kang; Lin, Fang-Tsyr; Graves, Joshua D; Lee, Yu-Ju; Lin, Weei-Chin

    2017-05-09

    Accumulating evidence supports the gain-of-function of mutant forms of p53 (mutp53s). However, whether mutp53 directly perturbs the DNA replication checkpoint remains unclear. Previously, we have demonstrated that TopBP1 forms a complex with mutp53s and mediates their gain-of-function through NF-Y and p63/p73. Akt phosphorylates TopBP1 and induces its oligomerization, which inhibits its ATR-activating function. Here we show that various contact and conformational mutp53s bypass Akt to induce TopBP1 oligomerization and attenuate ATR checkpoint response during replication stress. The effect on ATR response caused by mutp53 can be exploited in a synthetic lethality strategy, as depletion of another ATR activator, DNA2, in mutp53-R273H-expressing cancer cells renders cells hypersensitive to cisplatin. Expression of mutp53-R273H also makes cancer cells more sensitive to DNA2 depletion or DNA2 inhibitors. In addition to ATR-activating function during replication stress, TopBP1 interacts with Treslin in a Cdk-dependent manner to initiate DNA replication during normal growth. We find that mutp53 also interferes with TopBP1 replication function. Several contact, but not conformational, mutp53s enhance the interaction between TopBP1 and Treslin and promote DNA replication despite the presence of a Cdk2 inhibitor. Together, these data uncover two distinct mechanisms by which mutp53 enhances DNA replication: ( i ) Both contact and conformational mutp53s can bind TopBP1 and attenuate the checkpoint response to replication stress, and ( ii ) during normal growth, contact (but not conformational) mutp53s can override the Cdk2 requirement to promote replication by facilitating the TopBP1/Treslin interaction.

  7. Role of exonucleolytic processing and polymerase-DNA association in bypass of lesions during replication in vitro. Significance for SOS-targeted mutagenesis

    International Nuclear Information System (INIS)

    Shwartz, H.; Shavitt, O.; Livneh, Z.

    1988-01-01

    The role of exonuclease activity in trans-lesion DNA replication with Escherichia coli DNA polymerase III holoenzyme was investigated. RecA protein inhibited the 3'----5' exonuclease activity of the polymerase 2-fold when assayed in the absence of replication and had no effect on turnover of dNTPs into dNMPs. In contrast, single-stranded DNA-binding protein, which had no effect on the exonuclease activity in the absence of replication, showed a pronounced 7-fold suppression of the 3'----5' exonuclease activity during replication. The excision of incorporated dNMP alpha S residues from DNA by the 3'----5' exonuclease activity of DNA polymerase III holoenzyme was inhibited 10-20-fold; still no increase in bypass of pyrimidine photodimers was observed. Thus, in agreement with our previous results in which the exonuclease activity was inhibited at the protein level, inhibition at the DNA level also did not increase bypass of photodimers. Fractionation of the replication mixture after termination of DNA synthesis on a Bio-Gel A-5m column under conditions which favor polymerase-DNA binding yielded a termination complex which could perform turnover of dNTPs into dNMPs. Adding challenge-primed single-stranded DNA to the complex yielded a burst of DNA synthesis which was promoted most likely by DNA polymerase III holoenzyme molecules transferred from the termination complex to the challenge DNA thus demonstrating the instability of the polymerase-DNA association. Addition of a fresh sample of DNA polymerase III holoenzyme to purified termination products, which consist primarily of partially replicated molecules with nascent chains terminated at UV lesions, did not result in any net DNA synthesis as expected

  8. The interactomes of influenza virus NS1 and NS2 proteins identify new host factors and provide insights for ADAR1 playing a supportive role in virus replication.

    Science.gov (United States)

    de Chassey, Benoît; Aublin-Gex, Anne; Ruggieri, Alessia; Meyniel-Schicklin, Laurène; Pradezynski, Fabrine; Davoust, Nathalie; Chantier, Thibault; Tafforeau, Lionel; Mangeot, Philippe-Emmanuel; Ciancia, Claire; Perrin-Cocon, Laure; Bartenschlager, Ralf; André, Patrice; Lotteau, Vincent

    2013-01-01

    Influenza A NS1 and NS2 proteins are encoded by the RNA segment 8 of the viral genome. NS1 is a multifunctional protein and a virulence factor while NS2 is involved in nuclear export of viral ribonucleoprotein complexes. A yeast two-hybrid screening strategy was used to identify host factors supporting NS1 and NS2 functions. More than 560 interactions between 79 cellular proteins and NS1 and NS2 proteins from 9 different influenza virus strains have been identified. These interacting proteins are potentially involved in each step of the infectious process and their contribution to viral replication was tested by RNA interference. Validation of the relevance of these host cell proteins for the viral replication cycle revealed that 7 of the 79 NS1 and/or NS2-interacting proteins positively or negatively controlled virus replication. One of the main factors targeted by NS1 of all virus strains was double-stranded RNA binding domain protein family. In particular, adenosine deaminase acting on RNA 1 (ADAR1) appeared as a pro-viral host factor whose expression is necessary for optimal viral protein synthesis and replication. Surprisingly, ADAR1 also appeared as a pro-viral host factor for dengue virus replication and directly interacted with the viral NS3 protein. ADAR1 editing activity was enhanced by both viruses through dengue virus NS3 and influenza virus NS1 proteins, suggesting a similar virus-host co-evolution.

  9. The interactomes of influenza virus NS1 and NS2 proteins identify new host factors and provide insights for ADAR1 playing a supportive role in virus replication.

    Directory of Open Access Journals (Sweden)

    Benoît de Chassey

    Full Text Available Influenza A NS1 and NS2 proteins are encoded by the RNA segment 8 of the viral genome. NS1 is a multifunctional protein and a virulence factor while NS2 is involved in nuclear export of viral ribonucleoprotein complexes. A yeast two-hybrid screening strategy was used to identify host factors supporting NS1 and NS2 functions. More than 560 interactions between 79 cellular proteins and NS1 and NS2 proteins from 9 different influenza virus strains have been identified. These interacting proteins are potentially involved in each step of the infectious process and their contribution to viral replication was tested by RNA interference. Validation of the relevance of these host cell proteins for the viral replication cycle revealed that 7 of the 79 NS1 and/or NS2-interacting proteins positively or negatively controlled virus replication. One of the main factors targeted by NS1 of all virus strains was double-stranded RNA binding domain protein family. In particular, adenosine deaminase acting on RNA 1 (ADAR1 appeared as a pro-viral host factor whose expression is necessary for optimal viral protein synthesis and replication. Surprisingly, ADAR1 also appeared as a pro-viral host factor for dengue virus replication and directly interacted with the viral NS3 protein. ADAR1 editing activity was enhanced by both viruses through dengue virus NS3 and influenza virus NS1 proteins, suggesting a similar virus-host co-evolution.

  10. High-Resolution Replication Profiles Define the Stochastic Nature of Genome Replication Initiation and Termination

    Directory of Open Access Journals (Sweden)

    Michelle Hawkins

    2013-11-01

    Full Text Available Eukaryotic genome replication is stochastic, and each cell uses a different cohort of replication origins. We demonstrate that interpreting high-resolution Saccharomyces cerevisiae genome replication data with a mathematical model allows quantification of the stochastic nature of genome replication, including the efficiency of each origin and the distribution of termination events. Single-cell measurements support the inferred values for stochastic origin activation time. A strain, in which three origins were inactivated, confirmed that the distribution of termination events is primarily dictated by the stochastic activation time of origins. Cell-to-cell variability in origin activity ensures that termination events are widely distributed across virtually the whole genome. We propose that the heterogeneity in origin usage contributes to genome stability by limiting potentially deleterious events from accumulating at particular loci.

  11. Dynamic assembly of Hda and the sliding clamp in the regulation of replication licensing.

    Science.gov (United States)

    Kim, Jin S; Nanfara, Michael T; Chodavarapu, Sundari; Jin, Kyeong S; Babu, Vignesh M P; Ghazy, Mohamed A; Chung, Scisung; Kaguni, Jon M; Sutton, Mark D; Cho, Yunje

    2017-04-20

    Regulatory inactivation of DnaA (RIDA) is one of the major regulatory mechanisms of prokaryotic replication licensing. In RIDA, the Hda-sliding clamp complex loaded onto DNA directly interacts with adenosine triphosphate (ATP)-bound DnaA and stimulates the hydrolysis of ATP to inactivate DnaA. A prediction is that the activity of Hda is tightly controlled to ensure that replication initiation occurs only once per cell cycle. Here, we determined the crystal structure of the Hda-β clamp complex. This complex contains two pairs of Hda dimers sandwiched between two β clamp rings to form an octamer that is stabilized by three discrete interfaces. Two separate surfaces of Hda make contact with the β clamp, which is essential for Hda function in RIDA. The third interface between Hda monomers occludes the active site arginine finger, blocking its access to DnaA. Taken together, our structural and mutational analyses of the Hda-β clamp complex indicate that the interaction of the β clamp with Hda controls the ability of Hda to interact with DnaA. In the octameric Hda-β clamp complex, the inability of Hda to interact with DnaA is a novel mechanism that may regulate Hda function. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  12. DNA replication and post-replication repair in U.V.-sensitive mouse neuroblastoma cells

    International Nuclear Information System (INIS)

    Lavin, M.F.; McCombe, P.; Kidson, C.

    1976-01-01

    Mouse neuroblastoma cells differentiated when grown in the absence of serum; differentiation was reversed on the addition of serum. Differentiated cells were more sensitive to U.V.-radiation than proliferating cells. Whereas addition of serum to differentiated neuroblastoma cells normally resulted in immediate, synchronous entry into S phase, irradiation just before the addition of serum resulted in a long delay in the onset of DNA replication. During this lag period, incorporated 3 H-thymidine appeared in the light density region of CsCl gradients, reflecting either repair synthesis or abortive replication. Post-replication repair (gap-filling) was found to be present in proliferating cells and at certain times in differentiated cells. It is suggested that the sensitivity of differentiated neuroblastoma cells to U.V.-radiation may have been due to ineffective post-replication repair or to deficiencies in more than one repair mechanism, with reduction in repair capacity beyond a critical threshold. (author)

  13. Replication, refinement & reachability: complexity in dynamic condition-response graphs

    DEFF Research Database (Denmark)

    Debois, Søren; Hildebrandt, Thomas T.; Slaats, Tijs

    2017-01-01

    We explore the complexity of reachability and run-time refinement under safety and liveness constraints in event-based process models. Our study is framed in the DCR? process language, which supports modular specification through a compositional operational semantics. DCR? encompasses the “Dynami...

  14. 3D Spatially Resolved Models of the Intracellular Dynamics of the Hepatitis C Genome Replication Cycle

    KAUST Repository

    Knodel, Markus; Reiter, Sebastian; Targett-Adams, Paul; Grillo, Alfio; Herrmann, Eva; Wittum, Gabriel

    2017-01-01

    virus (HCV) viral RNA (vRNA) occurs within special replication complexes formed from membranes derived from endoplasmatic reticulum (ER). These regions, termed membranous webs, are generated primarily through specific interactions between nonstructural

  15. Sp100 colocalizes with HPV replication foci and restricts the productive stage of the infectious cycle.

    Directory of Open Access Journals (Sweden)

    Wesley H Stepp

    2017-10-01

    Full Text Available We have shown previously that Sp100 (a component of the ND10 nuclear body represses transcription, replication and establishment of incoming human papillomavirus (HPV DNA in the early stages of infection. In this follow up study, we show that Sp100 does not substantially regulate viral infection in the maintenance phase, however at late stages of infection Sp100 interacts with amplifying viral genomes to repress viral processes. We find that Sp100 localizes to HPV16 replication foci generated in primary keratinocytes, to HPV31 replication foci that form in differentiated cells, and to HPV16 replication foci in CIN 1 cervical biopsies. To analyze this further, Sp100 was down regulated by siRNA treatment of differentiating HPV31 containing cells and levels of viral transcription and replication were assessed. This revealed that Sp100 represses viral transcription and replication in differentiated cells. Analysis of Sp100 binding to viral chromatin showed that Sp100 bound across the viral genome, and that binding increased at late stages of infection. Therefore, Sp100 represses the HPV life cycle at both early and late stages of infection.

  16. DATABASE REPLICATION IN HETEROGENOUS PLATFORM

    OpenAIRE

    Hendro Nindito; Evaristus Didik Madyatmadja; Albert Verasius Dian Sano

    2014-01-01

    The application of diverse database technologies in enterprises today is increasingly a common practice. To provide high availability and survavibality of real-time information, a database replication technology that has capability to replicate databases under heterogenous platforms is required. The purpose of this research is to find the technology with such capability. In this research, the data source is stored in MSSQL database server running on Windows. The data will be replicated to MyS...

  17. Endoplasmic Reticulum Stress Induced Synthesis of a Novel Viral Factor Mediates Efficient Replication of Genotype-1 Hepatitis E Virus.

    Directory of Open Access Journals (Sweden)

    Vidya P Nair

    2016-04-01

    Full Text Available Hepatitis E virus (HEV causes acute hepatitis in many parts of the world including Asia, Africa and Latin America. Though self-limiting in normal individuals, it results in ~30% mortality in infected pregnant women. It has also been reported to cause acute and chronic hepatitis in organ transplant patients. Of the seven viral genotypes, genotype-1 virus infects humans and is a major public health concern in South Asian countries. Sporadic cases of genotype-3 and 4 infection in human and animals such as pigs, deer, mongeese have been reported primarily from industrialized countries. Genotype-5, 6 and 7 viruses are known to infect animals such as wild boar and camel, respectively. Genotype-3 and 4 viruses have been successfully propagated in the laboratory in mammalian cell culture. However, genotype-1 virus replicates poorly in mammalian cell culture and no other efficient model exists to study its life cycle. Here, we report that endoplasmic reticulum (ER stress promotes genotype-1 HEV replication by inducing cap-independent, internal initiation mediated translation of a novel viral protein (named ORF4. Importantly, ORF4 expression and stimulatory effect of ER stress inducers on viral replication is specific to genotype-1. ORF4 protein sequence is mostly conserved among genotype-1 HEV isolates and ORF4 specific antibodies were detected in genotype-1 HEV patient serum. ORF4 interacted with multiple viral and host proteins and assembled a protein complex consisting of viral helicase, RNA dependent RNA polymerase (RdRp, X, host eEF1α1 (eukaryotic elongation factor 1 isoform-1 and tubulinβ. In association with eEF1α1, ORF4 stimulated viral RdRp activity. Furthermore, human hepatoma cells that stably express ORF4 or engineered proteasome resistant ORF4 mutant genome permitted enhanced viral replication. These findings reveal a positive role of ER stress in promoting genotype-1 HEV replication and pave the way towards development of an efficient

  18. Exploiting replicative stress to treat cancer

    DEFF Research Database (Denmark)

    Dobbelstein, Matthias; Sørensen, Claus Storgaard

    2015-01-01

    DNA replication in cancer cells is accompanied by stalling and collapse of the replication fork and signalling in response to DNA damage and/or premature mitosis; these processes are collectively known as 'replicative stress'. Progress is being made to increase our understanding of the mechanisms...

  19. Single-molecule spectroscopy reveals photosynthetic LH2 complexes switch between emissive states.

    Science.gov (United States)

    Schlau-Cohen, Gabriela S; Wang, Quan; Southall, June; Cogdell, Richard J; Moerner, W E

    2013-07-02

    Photosynthetic organisms flourish under low light intensities by converting photoenergy to chemical energy with near unity quantum efficiency and under high light intensities by safely dissipating excess photoenergy and deleterious photoproducts. The molecular mechanisms balancing these two functions remain incompletely described. One critical barrier to characterizing the mechanisms responsible for these processes is that they occur within proteins whose excited-state properties vary drastically among individual proteins and even within a single protein over time. In ensemble measurements, these excited-state properties appear only as the average value. To overcome this averaging, we investigate the purple bacterial antenna protein light harvesting complex 2 (LH2) from Rhodopseudomonas acidophila at the single-protein level. We use a room-temperature, single-molecule technique, the anti-Brownian electrokinetic trap, to study LH2 in a solution-phase (nonperturbative) environment. By performing simultaneous measurements of fluorescence intensity, lifetime, and spectra of single LH2 complexes, we identify three distinct states and observe transitions occurring among them on a timescale of seconds. Our results reveal that LH2 complexes undergo photoactivated switching to a quenched state, likely by a conformational change, and thermally revert to the ground state. This is a previously unobserved, reversible quenching pathway, and is one mechanism through which photosynthetic organisms can adapt to changes in light intensities.

  20. Chromatin maturation depends on continued DNA-replication

    International Nuclear Information System (INIS)

    Schlaeger, E.J.; Puelm, W.; Knippers, R.

    1983-01-01

    The structure of [ 3 H]thymidine pulse-labeled chromatin in lymphocytes differs from that of non-replicating chromatin by several operational criteria which are related to the higher nuclease sensitivity of replicating chromatin. These structural features of replicating chromatin rapidly disappear when the [ 3 H]thymidine pulse is followed by a chase in the presence of an excess of non-radioactive thymidine. However, when the rate of DNA replication is reduced, as in cycloheximide-treated lymphocytes, chromatin maturation is retarded. No chromatin maturation is observed when nuclei from pulse-labeled lymphocytes are incubated in vitro in the absence of DNA precursors. In contrast, when these nuclei are incubated under conditions known to be optimal for DNA replication, the structure of replicating chromatin is efficiently converted to that of 'mature', non-replicating chromatin. The authors conclude that the properties of nascent DNA and/or the distance from the replication fork are important factors in chromatin maturation. (Auth.)

  1. The Escherichia coli Tus-Ter replication fork barrier causes site-specific DNA replication perturbation in yeast

    DEFF Research Database (Denmark)

    Larsen, Nicolai B; Sass, Ehud; Suski, Catherine

    2014-01-01

    Replication fork (RF) pausing occurs at both 'programmed' sites and non-physiological barriers (for example, DNA adducts). Programmed RF pausing is required for site-specific DNA replication termination in Escherichia coli, and this process requires the binding of the polar terminator protein, Tus...... as a versatile, site-specific, heterologous DNA replication-perturbing system, with a variety of potential applications....

  2. Functions of mammalian Cdc7 kinase in initiation/monitoring of DNA replication and development

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Jung Min; Yamada, Masayuki; Masai, Hisao

    2003-11-27

    Cdc7 kinase plays an essential role in firing of replication origins by phosphorylating components of the replication complexes. Cdc7 kinase has also been implicated in S phase checkpoint signaling downstream of the ATR and Chk1 kinases. Inactivation of Cdc7 in yeast results in arrest of cell growth with 1C DNA content after completion of the ongoing DNA replication. In contrast, conditional inactivation of Cdc7 in undifferentiated mouse embryonic stem (ES) cells leads to growth arrest with rapid cessation of DNA synthesis, suggesting requirement of Cdc7 functions for continuation of ongoing DNA synthesis. Furthermore, loss of Cdc7 function induces recombinational repair (nuclear Rad51 foci) and G2/M checkpoint responses (inhibition of Cdc2 kinase). Eventually, p53 becomes highly activated and the cells undergo massive p53-dependent apoptosis. Thus, defective origin activation in mammalian cells can generate DNA replication checkpoint signals. Efficient removal of those cells in which replication has been perturbed, through cell death, may be beneficial to maintain the highest level of genetic integrity in totipotent stem cells. Partial, rather than total, loss of Cdc7 kinase expression results in retarded growth at both cellular and whole body levels, with especially profound impairment of germ cell development.

  3. Functions of mammalian Cdc7 kinase in initiation/monitoring of DNA replication and development

    International Nuclear Information System (INIS)

    Kim, Jung Min; Yamada, Masayuki; Masai, Hisao

    2003-01-01

    Cdc7 kinase plays an essential role in firing of replication origins by phosphorylating components of the replication complexes. Cdc7 kinase has also been implicated in S phase checkpoint signaling downstream of the ATR and Chk1 kinases. Inactivation of Cdc7 in yeast results in arrest of cell growth with 1C DNA content after completion of the ongoing DNA replication. In contrast, conditional inactivation of Cdc7 in undifferentiated mouse embryonic stem (ES) cells leads to growth arrest with rapid cessation of DNA synthesis, suggesting requirement of Cdc7 functions for continuation of ongoing DNA synthesis. Furthermore, loss of Cdc7 function induces recombinational repair (nuclear Rad51 foci) and G2/M checkpoint responses (inhibition of Cdc2 kinase). Eventually, p53 becomes highly activated and the cells undergo massive p53-dependent apoptosis. Thus, defective origin activation in mammalian cells can generate DNA replication checkpoint signals. Efficient removal of those cells in which replication has been perturbed, through cell death, may be beneficial to maintain the highest level of genetic integrity in totipotent stem cells. Partial, rather than total, loss of Cdc7 kinase expression results in retarded growth at both cellular and whole body levels, with especially profound impairment of germ cell development

  4. NMR structure of the N-terminal domain of the replication initiator protein DnaA

    Energy Technology Data Exchange (ETDEWEB)

    Wemmer, David E.; Lowery, Thomas J.; Pelton, Jeffrey G.; Chandonia, John-Marc; Kim, Rosalind; Yokota, Hisao; Wemmer, David E.

    2007-08-07

    DnaA is an essential component in the initiation of bacterial chromosomal replication. DnaA binds to a series of 9 base pair repeats leading to oligomerization, recruitment of the DnaBC helicase, and the assembly of the replication fork machinery. The structure of the N-terminal domain (residues 1-100) of DnaA from Mycoplasma genitalium was determined by NMR spectroscopy. The backbone r.m.s.d. for the first 86 residues was 0.6 +/- 0.2 Angstrom based on 742 NOE, 50 hydrogen bond, 46 backbone angle, and 88 residual dipolar coupling restraints. Ultracentrifugation studies revealed that the domain is monomeric in solution. Features on the protein surface include a hydrophobic cleft flanked by several negative residues on one side, and positive residues on the other. A negatively charged ridge is present on the opposite face of the protein. These surfaces may be important sites of interaction with other proteins involved in the replication process. Together, the structure and NMR assignments should facilitate the design of new experiments to probe the protein-protein interactions essential for the initiation of DNA replication.

  5. Enzymatic recognition of DNA replication origins

    International Nuclear Information System (INIS)

    Stayton, M.M.; Bertsch, L.; Biswas, S.

    1983-01-01

    In this paper we discuss the process of recognition of the complementary-strand origin with emphasis on RNA polymerase action in priming M13 DNA replication, the role of primase in G4 DNA replication, and the function of protein n, a priming protein, during primosome assembly. These phage systems do not require several of the bacterial DNA replication enzymes, particularly those involved in the regulation of chromosome copy number of the initiatiion of replication of duplex DNA. 51 references, 13 figures, 1 table

  6. Repurposing Lesogaberan to Promote Human Islet Cell Survival and β-Cell Replication

    Directory of Open Access Journals (Sweden)

    Jide Tian

    2017-01-01

    Full Text Available The activation of β-cell’s A- and B-type gamma-aminobutyric acid receptors (GABAA-Rs and GABAB-Rs can promote their survival and replication, and the activation of α-cell GABAA-Rs promotes their conversion into β-cells. However, GABA and the most clinically applicable GABA-R ligands may be suboptimal for the long-term treatment of diabetes due to their pharmacological properties or potential side-effects on the central nervous system (CNS. Lesogaberan (AZD3355 is a peripherally restricted high-affinity GABAB-R-specific agonist, originally developed for the treatment of gastroesophageal reflux disease (GERD that appears to be safe for human use. This study tested the hypothesis that lesogaberan could be repurposed to promote human islet cell survival and β-cell replication. Treatment with lesogaberan significantly enhanced replication of human islet cells in vitro, which was abrogated by a GABAB-R antagonist. Immunohistochemical analysis of human islets that were grafted into immune-deficient mice revealed that oral treatment with lesogaberan promoted human β-cell replication and islet cell survival in vivo as effectively as GABA (which activates both GABAA-Rs and GABAB-Rs, perhaps because of its more favorable pharmacokinetics. Lesogaberan may be a promising drug candidate for clinical studies of diabetes intervention and islet transplantation.

  7. Escherichia coli DNA polymerase I can disrupt G-quadruplex structures during DNA replication.

    Science.gov (United States)

    Teng, Fang-Yuan; Hou, Xi-Miao; Fan, San-Hong; Rety, Stephane; Dou, Shuo-Xing; Xi, Xu-Guang

    2017-12-01

    Non-canonical four-stranded G-quadruplex (G4) DNA structures can form in G-rich sequences that are widely distributed throughout the genome. The presence of G4 structures can impair DNA replication by hindering the progress of replicative polymerases (Pols), and failure to resolve these structures can lead to genetic instability. In the present study, we combined different approaches to address the question of whether and how Escherichia coli Pol I resolves G4 obstacles during DNA replication and/or repair. We found that E. coli Pol I-catalyzed DNA synthesis could be arrested by G4 structures at low protein concentrations and the degree of inhibition was strongly dependent on the stability of the G4 structures. Interestingly, at high protein concentrations, E. coli Pol I was able to overcome some kinds of G4 obstacles without the involvement of other molecules and could achieve complete replication of G4 DNA. Mechanistic studies suggested that multiple Pol I proteins might be implicated in G4 unfolding, and the disruption of G4 structures requires energy derived from dNTP hydrolysis. The present work not only reveals an unrealized function of E. coli Pol I, but also presents a possible mechanism by which G4 structures can be resolved during DNA replication and/or repair in E. coli. © 2017 Federation of European Biochemical Societies.

  8. Replication Protein A (RPA) Phosphorylation Prevents RPA Association with Replication Centers

    OpenAIRE

    Vassin, Vitaly M.; Wold, Marc S.; Borowiec, James A.

    2004-01-01

    Mammalian replication protein A (RPA) undergoes DNA damage-dependent phosphorylation at numerous sites on the N terminus of the RPA2 subunit. To understand the functional significance of RPA phosphorylation, we expressed RPA2 variants in which the phosphorylation sites were converted to aspartate (RPA2D) or alanine (RPA2A). Although RPA2D was incorporated into RPA heterotrimers and supported simian virus 40 DNA replication in vitro, the RPA2D mutant was selectively unable to associate with re...

  9. Enhanced replication of attenuated HSV-1 in irradiated human glioma xenografts

    International Nuclear Information System (INIS)

    Advani, Sunil J.; Kataoka, Yasushi; Sibley, Greg S.; Song, Paul Y.; Hallahan, Dennis E.; Roizman, Bernard; Weichselbaum, Ralph R.

    1997-01-01

    Purpose: Previously we had shown that combining ionizing radiation (IR) with attenuated replication competent HSV-1 (R3616) significantly increased glioma xenograft eradication compared to IR or virus alone. One hypothesis is that IR induces cell factors that contribute to augment viral replication thereby increasing the efficacy of attenuated HSV-1. The purpose of this study was to examine if IR altered viral replication of attenuated HSV-1 in glioma xenografts Material and Methods: Human U-87MG glioma cells were grown in the hindlimb of athymic mice and grown to >200 mm 3 . Tumors were infected with 2x10 7 plaque forming units (pfu) of R3616 ( γ1 34.5 - ) or R7020 (multimutated, γ1 34.5 + ) on day 0 and irradiated with 20 Gy on day 1 and 25 Gy on day 2. Tumors were harvested 3, 5, 7, and 14 days after viral injection. Tumors were homogenized and sonnicated. Serial dilutions of tumor extract were overlaid on Vero cells to determine the number of pfu. In addition, in-situ hybridization to HSV-1 DNA was performed on tumors harvested at day 7. Results: In-situ hybridization revealed larger numbers of glial cells infected with HSV along with a greater distribution in the irradiated tumors compared to non-irradiated tumors. We next quantified viral particles in infected tumors +/- IR: Conclusion: Herein we demonstrate radiation enhanced viral replication as one of the interactive effects of combining IR and attenuated HSV in treating glioma xenografts and a potential therapeutic motif in the treatment of gliomas. To reduce normal tissue toxicity of HSV in glioma therapy, viruses must be attenuated. However, attenuating the virus compromises its replication and thus its potential efficacy. Our results indicate that IR augments the amount of virus recovered from human glioma xenografts for up to 3 days post IR. The results do not appear to be related to a specific mutation in the herpes genome but rather to herpes viruses in general. Yields of R7020 were greater than R

  10. The Impact of Message Replication on the Performance of Opportunistic Networks for Sensed Data Collection

    Directory of Open Access Journals (Sweden)

    Tekenate E. Amah

    2017-11-01

    Full Text Available Opportunistic networks (OppNets provide a scalable solution for collecting delay‑tolerant data from sensors for their respective gateways. Portable handheld user devices contribute significantly to the scalability of OppNets since their number increases according to user population and they closely follow human movement patterns. Hence, OppNets for sensed data collection are characterised by high node population and degrees of spatial locality inherent to user movement. We study the impact of these characteristics on the performance of existing OppNet message replication techniques. Our findings reveal that the existing replication techniques are not specifically designed to cope with these characteristics. This raises concerns regarding excessive message transmission overhead and throughput degradations due to resource constraints and technological limitations associated with portable handheld user devices. Based on concepts derived from the study, we suggest design guidelines to augment existing message replication techniques. We also follow our design guidelines to propose a message replication technique, namely Locality Aware Replication (LARep. Simulation results show that LARep achieves better network performance under high node population and degrees of spatial locality as compared with existing techniques.

  11. Variance Swap Replication: Discrete or Continuous?

    Directory of Open Access Journals (Sweden)

    Fabien Le Floc’h

    2018-02-01

    Full Text Available The popular replication formula to price variance swaps assumes continuity of traded option strikes. In practice, however, there is only a discrete set of option strikes traded on the market. We present here different discrete replication strategies and explain why the continuous replication price is more relevant.

  12. Group Therapy for Repeated Deliberate Self-Harm in Adolescents: Failure of Replication of a Randomized Trial

    Science.gov (United States)

    Hazell, Philip L.; Martin, Graham; McGill, Katherine; Kay, Tracey; Wood, Alison; Trainor, Gemma; Harrington, Richard

    2009-01-01

    A study revealing the superiority of group therapy to routine care in preventing the recurrence of self-harming behavior among adolescents is unsuccessfully replicated. The study's findings contradicted those of the original study.

  13. Replication of somatic micronuclei in bovine enucleated oocytes

    Directory of Open Access Journals (Sweden)

    Canel Natalia

    2012-11-01

    Full Text Available Abstract Background Microcell-mediated chromosome transfer (MMCT was developed to introduce a low number of chromosomes into a host cell. We have designed a novel technique combining part of MMCT with somatic cell nuclear transfer, which consists of injecting a somatic micronucleus into an enucleated oocyte, and inducing its cellular machinery to replicate such micronucleus. It would allow the isolation and manipulation of a single or a low number of somatic chromosomes. Methods Micronuclei from adult bovine fibroblasts were produced by incubation in 0.05 μg/ml demecolcine for 46 h followed by 2 mg/ml mitomycin for 2 h. Cells were finally treated with 10 μg/ml cytochalasin B for 1 h. In vitro matured bovine oocytes were mechanically enucleated and intracytoplasmatically injected with one somatic micronucleus, which had been previously exposed [Micronucleus- injected (+] or not [Micronucleus- injected (−] to a transgene (50 ng/μl pCX-EGFP during 5 min. Enucleated oocytes [Enucleated (+] and parthenogenetic [Parthenogenetic (+] controls were injected into the cytoplasm with less than 10 pl of PVP containing 50 ng/μl pCX-EGFP. A non-injected parthenogenetic control [Parthenogenetic (−] was also included. Two hours after injection, oocytes and reconstituted embryos were activated by incubation in 5 μM ionomycin for 4 min + 1.9 mM 6-DMAP for 3 h. Cleavage stage and egfp expression were evaluated. DNA replication was confirmed by DAPI staining. On day 2, Micronucleus- injected (−, Parthenogenetic (− and in vitro fertilized (IVF embryos were karyotyped. Differences among treatments were determined by Fisher′s exact test (p≤0.05. Results All the experimental groups underwent the first cell divisions. Interestingly, a low number of Micronucleus-injected embryos showed egfp expression. DAPI staining confirmed replication of micronuclei in most of the evaluated embryos. Karyotype analysis revealed that all Micronucleus-injected embryos had

  14. Replication Stalling and Heteroduplex Formation within CAG/CTG Trinucleotide Repeats by Mismatch Repair

    KAUST Repository

    Viterbo, David; Michoud, Gregoire; Mosbach, Valentine; Dujon, Bernard; Richard, Guy-Franck

    2016-01-01

    Trinucleotide repeat expansions are responsible for at least two dozen neurological disorders. Mechanisms leading to these large expansions of repeated DNA are still poorly understood. It was proposed that transient stalling of the replication fork by the repeat tract might trigger slippage of the newly-synthesized strand over its template, leading to expansions or contractions of the triplet repeat. However, such mechanism was never formally proven. Here we show that replication fork pausing and CAG/CTG trinucleotide repeat instability are not linked, stable and unstable repeats exhibiting the same propensity to stall replication forks when integrated in a yeast natural chromosome. We found that replication fork stalling was dependent on the integrity of the mismatch-repair system, especially the Msh2p-Msh6p complex, suggesting that direct interaction of MMR proteins with secondary structures formed by trinucleotide repeats in vivo, triggers replication fork pauses. We also show by chromatin immunoprecipitation that Msh2p is enriched at trinucleotide repeat tracts, in both stable and unstable orientations, this enrichment being dependent on MSH3 and MSH6. Finally, we show that overexpressing MSH2 favors the formation of heteroduplex regions, leading to an increase in contractions and expansions of CAG/CTG repeat tracts during replication, these heteroduplexes being dependent on both MSH3 and MSH6. These heteroduplex regions were not detected when a mutant msh2-E768A gene in which the ATPase domain was mutated was overexpressed. Our results unravel two new roles for mismatch-repair proteins: stabilization of heteroduplex regions and transient blocking of replication forks passing through such repeats. Both roles may involve direct interactions between MMR proteins and secondary structures formed by trinucleotide repeat tracts, although indirect interactions may not be formally excluded.

  15. Replication Stalling and Heteroduplex Formation within CAG/CTG Trinucleotide Repeats by Mismatch Repair

    KAUST Repository

    Viterbo, David

    2016-03-16

    Trinucleotide repeat expansions are responsible for at least two dozen neurological disorders. Mechanisms leading to these large expansions of repeated DNA are still poorly understood. It was proposed that transient stalling of the replication fork by the repeat tract might trigger slippage of the newly-synthesized strand over its template, leading to expansions or contractions of the triplet repeat. However, such mechanism was never formally proven. Here we show that replication fork pausing and CAG/CTG trinucleotide repeat instability are not linked, stable and unstable repeats exhibiting the same propensity to stall replication forks when integrated in a yeast natural chromosome. We found that replication fork stalling was dependent on the integrity of the mismatch-repair system, especially the Msh2p-Msh6p complex, suggesting that direct interaction of MMR proteins with secondary structures formed by trinucleotide repeats in vivo, triggers replication fork pauses. We also show by chromatin immunoprecipitation that Msh2p is enriched at trinucleotide repeat tracts, in both stable and unstable orientations, this enrichment being dependent on MSH3 and MSH6. Finally, we show that overexpressing MSH2 favors the formation of heteroduplex regions, leading to an increase in contractions and expansions of CAG/CTG repeat tracts during replication, these heteroduplexes being dependent on both MSH3 and MSH6. These heteroduplex regions were not detected when a mutant msh2-E768A gene in which the ATPase domain was mutated was overexpressed. Our results unravel two new roles for mismatch-repair proteins: stabilization of heteroduplex regions and transient blocking of replication forks passing through such repeats. Both roles may involve direct interactions between MMR proteins and secondary structures formed by trinucleotide repeat tracts, although indirect interactions may not be formally excluded.

  16. Activation of human herpesvirus replication by apoptosis.

    Science.gov (United States)

    Prasad, Alka; Remick, Jill; Zeichner, Steven L

    2013-10-01

    A central feature of herpesvirus biology is the ability of herpesviruses to remain latent within host cells. Classically, exposure to inducing agents, like activating cytokines or phorbol esters that stimulate host cell signal transduction events, and epigenetic agents (e.g., butyrate) was thought to end latency. We recently showed that Kaposi's sarcoma-associated herpesvirus (KSHV, or human herpesvirus-8 [HHV-8]) has another, alternative emergency escape replication pathway that is triggered when KSHV's host cell undergoes apoptosis, characterized by the lack of a requirement for the replication and transcription activator (RTA) protein, accelerated late gene kinetics, and production of virus with decreased infectivity. Caspase-3 is necessary and sufficient to initiate the alternative replication program. HSV-1 was also recently shown to initiate replication in response to host cell apoptosis. These observations suggested that an alternative apoptosis-triggered replication program might be a general feature of herpesvirus biology and that apoptosis-initiated herpesvirus replication may have clinical implications, particularly for herpesviruses that almost universally infect humans. To explore whether an alternative apoptosis-initiated replication program is a common feature of herpesvirus biology, we studied cell lines latently infected with Epstein-Barr virus/HHV-4, HHV-6A, HHV-6B, HHV-7, and KSHV. We found that apoptosis triggers replication for each HHV studied, with caspase-3 being necessary and sufficient for HHV replication. An alternative apoptosis-initiated replication program appears to be a common feature of HHV biology. We also found that commonly used cytotoxic chemotherapeutic agents activate HHV replication, which suggests that treatments that promote apoptosis may lead to activation of latent herpesviruses, with potential clinical significance.

  17. EM structure of a helicase-loader complex depicting a 6:2 binding sub-stoichiometry from Geobacillus kaustophilus HTA426

    International Nuclear Information System (INIS)

    Lin, Yen-Chen; Naveen, Vankadari; Hsiao, Chwan-Deng

    2016-01-01

    During DNA replication, bacterial helicase is recruited as a complex in association with loader proteins to unwind the parental duplex. Previous structural studies have reported saturated 6:6 helicase-loader complexes with different conformations. However, structural information on the sub-stoichiometric conformations of these previously-documented helicase-loader complexes remains elusive. Here, with the aid of single particle electron-microscopy (EM) image reconstruction, we present the Geobacillus kaustophilus HTA426 helicase-loader (DnaC-DnaI) complex with a 6:2 binding stoichiometry in the presence of ATPγS. In the 19 Å resolution EM map, the undistorted and unopened helicase ring holds a robust loader density above the C-terminal RecA-like domain. Meanwhile, the path of the central DNA binding channel appears to be obstructed by the reconstructed loader density, implying its potential role as a checkpoint conformation to prevent the loading of immature complex onto DNA. Our data also reveals that the bound nucleotides and the consequently induced conformational changes in the helicase hexamer are essential for active association with loader proteins. These observations provide fundamental insights into the formation of the helicase-loader complex in bacteria that regulates the DNA replication process. - Highlights: • Helicase-loader complex structure with 6:2 sub-stoichiometry is resolved by EM. • Helicase hexamer in 6:2 sub-stoichiometry is constricted and un-opened. • 6:2 binding ratio of helicase-loader complex could act as a DNA loading checkpoint. • Nucleotides stabilize helicase-loader complex at low protein concentrations.

  18. EM structure of a helicase-loader complex depicting a 6:2 binding sub-stoichiometry from Geobacillus kaustophilus HTA426

    Energy Technology Data Exchange (ETDEWEB)

    Lin, Yen-Chen [Institute of Molecular Biology, Academia Sinica, Taipei 115, Taiwan (China); Naveen, Vankadari [Institute of Molecular Biology, Academia Sinica, Taipei 115, Taiwan (China); Molecular Cell Biology, Taiwan International Graduate Program, Institute of Molecular Biology, Academia Sinica, and Graduate Institute of Life Sciences, National Defense Medical Center, Taipei, Taiwan (China); Hsiao, Chwan-Deng, E-mail: hsiao@gate.sinica.edu.tw [Institute of Molecular Biology, Academia Sinica, Taipei 115, Taiwan (China); Molecular Cell Biology, Taiwan International Graduate Program, Institute of Molecular Biology, Academia Sinica, and Graduate Institute of Life Sciences, National Defense Medical Center, Taipei, Taiwan (China)

    2016-04-22

    During DNA replication, bacterial helicase is recruited as a complex in association with loader proteins to unwind the parental duplex. Previous structural studies have reported saturated 6:6 helicase-loader complexes with different conformations. However, structural information on the sub-stoichiometric conformations of these previously-documented helicase-loader complexes remains elusive. Here, with the aid of single particle electron-microscopy (EM) image reconstruction, we present the Geobacillus kaustophilus HTA426 helicase-loader (DnaC-DnaI) complex with a 6:2 binding stoichiometry in the presence of ATPγS. In the 19 Å resolution EM map, the undistorted and unopened helicase ring holds a robust loader density above the C-terminal RecA-like domain. Meanwhile, the path of the central DNA binding channel appears to be obstructed by the reconstructed loader density, implying its potential role as a checkpoint conformation to prevent the loading of immature complex onto DNA. Our data also reveals that the bound nucleotides and the consequently induced conformational changes in the helicase hexamer are essential for active association with loader proteins. These observations provide fundamental insights into the formation of the helicase-loader complex in bacteria that regulates the DNA replication process. - Highlights: • Helicase-loader complex structure with 6:2 sub-stoichiometry is resolved by EM. • Helicase hexamer in 6:2 sub-stoichiometry is constricted and un-opened. • 6:2 binding ratio of helicase-loader complex could act as a DNA loading checkpoint. • Nucleotides stabilize helicase-loader complex at low protein concentrations.

  19. Proteomic amino-termini profiling reveals targeting information for protein import into complex plastids.

    Directory of Open Access Journals (Sweden)

    Pitter F Huesgen

    Full Text Available In organisms with complex plastids acquired by secondary endosymbiosis from a photosynthetic eukaryote, the majority of plastid proteins are nuclear-encoded, translated on cytoplasmic ribosomes, and guided across four membranes by a bipartite targeting sequence. In-depth understanding of this vital import process has been impeded by a lack of information about the transit peptide part of this sequence, which mediates transport across the inner three membranes. We determined the mature N-termini of hundreds of proteins from the model diatom Thalassiosira pseudonana, revealing extensive N-terminal modification by acetylation and proteolytic processing in both cytosol and plastid. We identified 63 mature N-termini of nucleus-encoded plastid proteins, deduced their complete transit peptide sequences, determined a consensus motif for their cleavage by the stromal processing peptidase, and found evidence for subsequent processing by a plastid methionine aminopeptidase. The cleavage motif differs from that of higher plants, but is shared with other eukaryotes with complex plastids.

  20. The host-dependent interaction of alpha-importins with influenza PB2 polymerase subunit is required for virus RNA replication.

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    Patricia Resa-Infante

    Full Text Available The influenza virus polymerase is formed by the PB1, PB2 and PA subunits and is required for virus transcription and replication in the nucleus of infected cells. As PB2 is a relevant host-range determinant we expressed a TAP-tagged PB2 in human cells and isolated intracellular complexes. Alpha-importin was identified as a PB2-associated factor by proteomic analyses. To study the relevance of this interaction for virus replication we mutated the PB2 NLS and analysed the phenotype of mutant subunits, polymerase complexes and RNPs. While mutant PB2 proteins showed reduced nuclear accumulation, they formed polymerase complexes normally when co expressed with PB1 and PA. However, mutant RNPs generated with a viral CAT replicon showed up to hundred-fold reduced CAT accumulation. Rescue of nuclear localisation of mutant PB2 by insertion of an additional SV40 TAg-derived NLS did not revert the mutant phenotype of RNPs. Furthermore, determination of recombinant RNP accumulation in vivo indicated that PB2 NLS mutations drastically reduced virus RNA replication. These results indicate that, above and beyond its role in nuclear accumulation, PB2 interaction with alpha-importins is required for virus RNA replication. To ascertain whether PB2-alpha-importin binding could contribute to the adaptation of H5N1 avian viruses to man, their association in vivo was determined. Human alpha importin isoforms associated efficiently to PB2 protein of an H3N2 human virus but bound to diminished and variable extents to PB2 from H5N1 avian or human strains, suggesting that the function of alpha importin during RNA replication is important for the adaptation of avian viruses to the human host.

  1. Quantitative Multiplex Immunohistochemistry Reveals Myeloid-Inflamed Tumor-Immune Complexity Associated with Poor Prognosis

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    Takahiro Tsujikawa

    2017-04-01

    Full Text Available Here, we describe a multiplexed immunohistochemical platform with computational image processing workflows, including image cytometry, enabling simultaneous evaluation of 12 biomarkers in one formalin-fixed paraffin-embedded tissue section. To validate this platform, we used tissue microarrays containing 38 archival head and neck squamous cell carcinomas and revealed differential immune profiles based on lymphoid and myeloid cell densities, correlating with human papilloma virus status and prognosis. Based on these results, we investigated 24 pancreatic ductal adenocarcinomas from patients who received neoadjuvant GVAX vaccination and revealed that response to therapy correlated with degree of mono-myelocytic cell density and percentages of CD8+ T cells expressing T cell exhaustion markers. These data highlight the utility of in situ immune monitoring for patient stratification and provide digital image processing pipelines to the community for examining immune complexity in precious tissue sections, where phenotype and tissue architecture are preserved to improve biomarker discovery and assessment.

  2. Forging Ahead through Darkness: PCNA, Still the Principal Conductor at the Replication Fork.

    Science.gov (United States)

    Choe, Katherine N; Moldovan, George-Lucian

    2017-02-02

    Proliferating cell nuclear antigen (PCNA) lies at the center of the faithful duplication of eukaryotic genomes. With its distinctive doughnut-shaped molecular structure, PCNA was originally studied for its role in stimulating DNA polymerases. However, we now know that PCNA does much more than promote processive DNA synthesis. Because of the complexity of the events involved, cellular DNA replication poses major threats to genomic integrity. Whatever predicament lies ahead for the replication fork, PCNA is there to orchestrate the events necessary to handle it. Through its many protein interactions and various post-translational modifications, PCNA has far-reaching impacts on a myriad of cellular functions. Copyright © 2017 Elsevier Inc. All rights reserved.

  3. The pathological consequences of impaired genome integrity in humans; disorders of the DNA replication machinery.

    Science.gov (United States)

    O'Driscoll, Mark

    2017-01-01

    Accurate and efficient replication of the human genome occurs in the context of an array of constitutional barriers, including regional topological constraints imposed by chromatin architecture and processes such as transcription, catenation of the helical polymer and spontaneously generated DNA lesions, including base modifications and strand breaks. DNA replication is fundamentally important for tissue development and homeostasis; differentiation programmes are intimately linked with stem cell division. Unsurprisingly, impairments of the DNA replication machinery can have catastrophic consequences for genome stability and cell division. Functional impacts on DNA replication and genome stability have long been known to play roles in malignant transformation through a variety of complex mechanisms, and significant further insights have been gained from studying model organisms in this context. Congenital hypomorphic defects in components of the DNA replication machinery have been and continue to be identified in humans. These disorders present with a wide range of clinical features. Indeed, in some instances, different mutations in the same gene underlie different clinical presentations. Understanding the origin and molecular basis of these features opens a window onto the range of developmental impacts of suboptimal DNA replication and genome instability in humans. Here, I will briefly overview the basic steps involved in DNA replication and the key concepts that have emerged from this area of research, before switching emphasis to the pathological consequences of defects within the DNA replication network; the human disorders. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

  4. The glyceraldehyde-3-phosphate dehydrogenase and the small GTPase Rab 2 are crucial for Brucella replication.

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    Emilie Fugier

    2009-06-01

    Full Text Available The intracellular pathogen Brucella abortus survives and replicates inside host cells within an endoplasmic reticulum (ER-derived replicative organelle named the "Brucella-containing vacuole" (BCV. Here, we developed a subcellular fractionation method to isolate BCVs and characterize for the first time the protein composition of its replicative niche. After identification of BCV membrane proteins by 2 dimensional (2D gel electrophoresis and mass spectrometry, we focused on two eukaryotic proteins: the glyceraldehyde-3-phosphate dehydrogenase (GAPDH and the small GTPase Rab 2 recruited to the vacuolar membrane of Brucella. These proteins were previously described to localize on vesicular and tubular clusters (VTC and to regulate the VTC membrane traffic between the endoplasmic reticulum (ER and the Golgi. Inhibition of either GAPDH or Rab 2 expression by small interfering RNA strongly inhibited B. abortus replication. Consistent with this result, inhibition of other partners of GAPDH and Rab 2, such as COPI and PKC iota, reduced B. abortus replication. Furthermore, blockage of Rab 2 GTPase in a GDP-locked form also inhibited B. abortus replication. Bacteria did not fuse with the ER and instead remained in lysosomal-associated membrane vacuoles. These results reveal an essential role for GAPDH and the small GTPase Rab 2 in B. abortus virulence within host cells.

  5. Evolution of Replication Machines

    Science.gov (United States)

    Yao, Nina Y.; O'Donnell, Mike E.

    2016-01-01

    The machines that decode and regulate genetic information require the translation, transcription and replication pathways essential to all living cells. Thus, it might be expected that all cells share the same basic machinery for these pathways that were inherited from the primordial ancestor cell from which they evolved. A clear example of this is found in the translation machinery that converts RNA sequence to protein. The translation process requires numerous structural and catalytic RNAs and proteins, the central factors of which are homologous in all three domains of life, bacteria, archaea and eukarya. Likewise, the central actor in transcription, RNA polymerase, shows homology among the catalytic subunits in bacteria, archaea and eukarya. In contrast, while some “gears” of the genome replication machinery are homologous in all domains of life, most components of the replication machine appear to be unrelated between bacteria and those of archaea and eukarya. This review will compare and contrast the central proteins of the “replisome” machines that duplicate DNA in bacteria, archaea and eukarya, with an eye to understanding the issues surrounding the evolution of the DNA replication apparatus. PMID:27160337

  6. DNA replication origins—where do we begin?

    Science.gov (United States)

    Prioleau, Marie-Noëlle; MacAlpine, David M.

    2016-01-01

    For more than three decades, investigators have sought to identify the precise locations where DNA replication initiates in mammalian genomes. The development of molecular and biochemical approaches to identify start sites of DNA replication (origins) based on the presence of defining and characteristic replication intermediates at specific loci led to the identification of only a handful of mammalian replication origins. The limited number of identified origins prevented a comprehensive and exhaustive search for conserved genomic features that were capable of specifying origins of DNA replication. More recently, the adaptation of origin-mapping assays to genome-wide approaches has led to the identification of tens of thousands of replication origins throughout mammalian genomes, providing an unprecedented opportunity to identify both genetic and epigenetic features that define and regulate their distribution and utilization. Here we summarize recent advances in our understanding of how primary sequence, chromatin environment, and nuclear architecture contribute to the dynamic selection and activation of replication origins across diverse cell types and developmental stages. PMID:27542827

  7. ATM and KAT5 safeguard replicating chromatin against formaldehyde damage

    Science.gov (United States)

    Ortega-Atienza, Sara; Wong, Victor C.; DeLoughery, Zachary; Luczak, Michal W.; Zhitkovich, Anatoly

    2016-01-01

    Many carcinogens damage both DNA and protein constituents of chromatin, and it is unclear how cells respond to this compound injury. We examined activation of the main DNA damage-responsive kinase ATM and formation of DNA double-strand breaks (DSB) by formaldehyde (FA) that forms histone adducts and replication-blocking DNA-protein crosslinks (DPC). We found that low FA doses caused a strong and rapid activation of ATM signaling in human cells, which was ATR-independent and restricted to S-phase. High FA doses inactivated ATM via its covalent dimerization and formation of larger crosslinks. FA-induced ATM signaling showed higher CHK2 phosphorylation but much lower phospho-KAP1 relative to DSB inducers. Replication blockage by DPC did not produce damaged forks or detectable amounts of DSB during the main wave of ATM activation, which did not require MRE11. Chromatin-monitoring KAT5 (Tip60) acetyltransferase was responsible for acetylation and activation of ATM by FA. KAT5 and ATM were equally important for triggering of intra-S-phase checkpoint and ATM signaling promoted recovery of normal human cells after low-dose FA. Our results revealed a major role of the KAT5-ATM axis in protection of replicating chromatin against damage by the endogenous carcinogen FA. PMID:26420831

  8. Replication and Pedagogy in the History of Psychology II: Fowler & Wells's Phrenology

    Science.gov (United States)

    Trevino, Kelly M.; Konrad, Krista K.

    2008-05-01

    Phrenologists believed that specific brain regions corresponded to certain character traits. In addition, the size of each brain region was believed to determine the strength of the respective trait. Phrenology originated in Austria with Franz Josef Gall and was popularized and commercialized in America at the end of the 19th century by Orson Squire Fowler. In this project, we conducted a replication of Fowler’s phrenology in order to better understand the specificity of the manualized methodology, the extent to which the methodology allowed for positive versus negative analyses, and the implications for the scientific rejection and public acceptance of phrenology. The results of our replication revealed that the subjective judgments and biases of the examiner strongly influence the results of phrenological analyses.

  9. [Mediate evaluation of replicating a Training Program in Nonverbal Communication in Gerontology].

    Science.gov (United States)

    Schimidt, Teresa Cristina Gioia; Duarte, Yeda Aparecida de Oliveira; Silva, Maria Julia Paes da

    2015-04-01

    Replicating the training program in non-verbal communication based on the theoretical framework of interpersonal communication; non-verbal coding, valuing the aging aspects in the perspective of active aging, checking its current relevance through the content assimilation index after 90 days (mediate) of its application. A descriptive and exploratory field study was conducted in three hospitals under direct administration of the state of São Paulo that caters exclusively to Unified Health System (SUS) patients. The training lasted 12 hours divided in three meetings, applied to 102 health professionals. Revealed very satisfactory and satisfactory mediate content assimilation index in 82.9%. The program replication proved to be relevant and updated the setting of hospital services, while remaining efficient for healthcare professionals.

  10. High Resolution Genomic Scans Reveal Genetic Architecture Controlling Alcohol Preference in Bidirectionally Selected Rat Model.

    Directory of Open Access Journals (Sweden)

    Chiao-Ling Lo

    2016-08-01

    Full Text Available Investigations on the influence of nature vs. nurture on Alcoholism (Alcohol Use Disorder in human have yet to provide a clear view on potential genomic etiologies. To address this issue, we sequenced a replicated animal model system bidirectionally-selected for alcohol preference (AP. This model is uniquely suited to map genetic effects with high reproducibility, and resolution. The origin of the rat lines (an 8-way cross resulted in small haplotype blocks (HB with a corresponding high level of resolution. We sequenced DNAs from 40 samples (10 per line of each replicate to determine allele frequencies and HB. We achieved ~46X coverage per line and replicate. Excessive differentiation in the genomic architecture between lines, across replicates, termed signatures of selection (SS, were classified according to gene and region. We identified SS in 930 genes associated with AP. The majority (50% of the SS were confined to single gene regions, the greatest numbers of which were in promoters (284 and intronic regions (169 with the least in exon's (4, suggesting that differences in AP were primarily due to alterations in regulatory regions. We confirmed previously identified genes and found many new genes associated with AP. Of those newly identified genes, several demonstrated neuronal function involved in synaptic memory and reward behavior, e.g. ion channels (Kcnf1, Kcnn3, Scn5a, excitatory receptors (Grin2a, Gria3, Grip1, neurotransmitters (Pomc, and synapses (Snap29. This study not only reveals the polygenic architecture of AP, but also emphasizes the importance of regulatory elements, consistent with other complex traits.

  11. High Resolution Genomic Scans Reveal Genetic Architecture Controlling Alcohol Preference in Bidirectionally Selected Rat Model.

    Science.gov (United States)

    Lo, Chiao-Ling; Lossie, Amy C; Liang, Tiebing; Liu, Yunlong; Xuei, Xiaoling; Lumeng, Lawrence; Zhou, Feng C; Muir, William M

    2016-08-01

    Investigations on the influence of nature vs. nurture on Alcoholism (Alcohol Use Disorder) in human have yet to provide a clear view on potential genomic etiologies. To address this issue, we sequenced a replicated animal model system bidirectionally-selected for alcohol preference (AP). This model is uniquely suited to map genetic effects with high reproducibility, and resolution. The origin of the rat lines (an 8-way cross) resulted in small haplotype blocks (HB) with a corresponding high level of resolution. We sequenced DNAs from 40 samples (10 per line of each replicate) to determine allele frequencies and HB. We achieved ~46X coverage per line and replicate. Excessive differentiation in the genomic architecture between lines, across replicates, termed signatures of selection (SS), were classified according to gene and region. We identified SS in 930 genes associated with AP. The majority (50%) of the SS were confined to single gene regions, the greatest numbers of which were in promoters (284) and intronic regions (169) with the least in exon's (4), suggesting that differences in AP were primarily due to alterations in regulatory regions. We confirmed previously identified genes and found many new genes associated with AP. Of those newly identified genes, several demonstrated neuronal function involved in synaptic memory and reward behavior, e.g. ion channels (Kcnf1, Kcnn3, Scn5a), excitatory receptors (Grin2a, Gria3, Grip1), neurotransmitters (Pomc), and synapses (Snap29). This study not only reveals the polygenic architecture of AP, but also emphasizes the importance of regulatory elements, consistent with other complex traits.

  12. Knockdown of SCF(Skp2 function causes double-parked accumulation in the nucleus and DNA re-replication in Drosophila plasmatocytes.

    Directory of Open Access Journals (Sweden)

    Paul T Kroeger

    Full Text Available In Drosophila, circulating hemocytes are derived from the cephalic mesoderm during the embryonic wave of hematopoiesis. These cells are contributed to the larva and persist through metamorphosis into the adult. To analyze this population of hemocytes, we considered data from a previously published RNAi screen in the hematopoietic niche, which suggested several members of the SCF complex play a role in lymph gland development. eater-Gal4;UAS-GFP flies were crossed to UAS-RNAi lines to knockdown the function of all known SCF complex members in a plasmatocyte-specific fashion, in order to identify which members are novel regulators of plasmatocytes. This specific SCF complex contains five core members: Lin-19-like, SkpA, Skp2, Roc1a and complex activator Nedd8. The complex was identified by its very distinctive large cell phenotype. Furthermore, these large cells stained for anti-P1, a plasmatocyte-specific antibody. It was also noted that the DNA in these cells appeared to be over-replicated. Gamma-tubulin and DAPI staining suggest the cells are undergoing re-replication as they had multiple centrioles and excessive DNA content. Further experimentation determined enlarged cells were BrdU-positive indicating they have progressed through S-phase. To determine how these cells become enlarged and undergo re-replication, cell cycle proteins were analyzed by immunofluorescence. This analysis identified three proteins that had altered subcellular localization in these enlarged cells: Cyclin E, Geminin and Double-parked. Previous research has shown that Double-parked must be degraded to exit S-phase, otherwise the DNA will undergo re-replication. When Double-parked was titrated from the nucleus by an excess of its inhibitor, geminin, the enlarged cells and aberrant protein localization phenotypes were partially rescued. The data in this report suggests that the SCF(Skp2 complex is necessary to ubiquitinate Double-parked during plasmatocyte cell division

  13. Is Implicit Theory of Mind a Real and Robust Phenomenon? Results From a Systematic Replication Study.

    Science.gov (United States)

    Kulke, Louisa; von Duhn, Britta; Schneider, Dana; Rakoczy, Hannes

    2018-06-01

    Recently, theory-of-mind research has been revolutionized by findings from novel implicit tasks suggesting that at least some aspects of false-belief reasoning develop earlier in ontogeny than previously assumed and operate automatically throughout adulthood. Although these findings are the empirical basis for far-reaching theories, systematic replications are still missing. This article reports a preregistered large-scale attempt to replicate four influential anticipatory-looking implicit theory-of-mind tasks using original stimuli and procedures. Results showed that only one of the four paradigms was reliably replicated. A second set of studies revealed, further, that this one paradigm was no longer replicated once confounds were removed, which calls its validity into question. There were also no correlations between paradigms, and thus, no evidence for their convergent validity. In conclusion, findings from anticipatory-looking false-belief paradigms seem less reliable and valid than previously assumed, thus limiting the conclusions that can be drawn from them.

  14. Theory Meets Experiment: Metal Ion Effects in HCV Genomic RNA Kissing Complex Formation

    Directory of Open Access Journals (Sweden)

    Li-Zhen Sun

    2017-12-01

    Full Text Available The long-range base pairing between the 5BSL3. 2 and 3′X domains in hepatitis C virus (HCV genomic RNA is essential for viral replication. Experimental evidence points to the critical role of metal ions, especially Mg2+ ions, in the formation of the 5BSL3.2:3′X kissing complex. Furthermore, NMR studies suggested an important ion-dependent conformational switch in the kissing process. However, for a long time, mechanistic understanding of the ion effects for the process has been unclear. Recently, computational modeling based on the Vfold RNA folding model and the partial charge-based tightly bound ion (PCTBI model, in combination with the NMR data, revealed novel physical insights into the role of metal ions in the 5BSL3.2-3′X system. The use of the PCTBI model, which accounts for the ion correlation and fluctuation, gives reliable predictions for the ion-dependent electrostatic free energy landscape and ion-induced population shift of the 5BSL3.2:3′X kissing complex. Furthermore, the predicted ion binding sites offer insights about how ion-RNA interactions shift the conformational equilibrium. The integrated theory-experiment study shows that Mg2+ ions may be essential for HCV viral replication. Moreover, the observed Mg2+-dependent conformational equilibrium may be an adaptive property of the HCV genomic RNA such that the equilibrium is optimized to the intracellular Mg2+ concentration in liver cells for efficient viral replication.

  15. Set2 Methyltransferase Facilitates DNA Replication and Promotes Genotoxic Stress Responses through MBF-Dependent Transcription.

    Science.gov (United States)

    Pai, Chen-Chun; Kishkevich, Anastasiya; Deegan, Rachel S; Keszthelyi, Andrea; Folkes, Lisa; Kearsey, Stephen E; De León, Nagore; Soriano, Ignacio; de Bruin, Robertus Antonius Maria; Carr, Antony M; Humphrey, Timothy C

    2017-09-12

    Chromatin modification through histone H3 lysine 36 methylation by the SETD2 tumor suppressor plays a key role in maintaining genome stability. Here, we describe a role for Set2-dependent H3K36 methylation in facilitating DNA replication and the transcriptional responses to both replication stress and DNA damage through promoting MluI cell-cycle box (MCB) binding factor (MBF)-complex-dependent transcription in fission yeast. Set2 loss leads to reduced MBF-dependent ribonucleotide reductase (RNR) expression, reduced deoxyribonucleoside triphosphate (dNTP) synthesis, altered replication origin firing, and a checkpoint-dependent S-phase delay. Accordingly, prolonged S phase in the absence of Set2 is suppressed by increasing dNTP synthesis. Furthermore, H3K36 is di- and tri-methylated at these MBF gene promoters, and Set2 loss leads to reduced MBF binding and transcription in response to genotoxic stress. Together, these findings provide new insights into how H3K36 methylation facilitates DNA replication and promotes genotoxic stress responses in fission yeast. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  16. Set2 Methyltransferase Facilitates DNA Replication and Promotes Genotoxic Stress Responses through MBF-Dependent Transcription

    Directory of Open Access Journals (Sweden)

    Chen-Chun Pai

    2017-09-01

    Full Text Available Chromatin modification through histone H3 lysine 36 methylation by the SETD2 tumor suppressor plays a key role in maintaining genome stability. Here, we describe a role for Set2-dependent H3K36 methylation in facilitating DNA replication and the transcriptional responses to both replication stress and DNA damage through promoting MluI cell-cycle box (MCB binding factor (MBF-complex-dependent transcription in fission yeast. Set2 loss leads to reduced MBF-dependent ribonucleotide reductase (RNR expression, reduced deoxyribonucleoside triphosphate (dNTP synthesis, altered replication origin firing, and a checkpoint-dependent S-phase delay. Accordingly, prolonged S phase in the absence of Set2 is suppressed by increasing dNTP synthesis. Furthermore, H3K36 is di- and tri-methylated at these MBF gene promoters, and Set2 loss leads to reduced MBF binding and transcription in response to genotoxic stress. Together, these findings provide new insights into how H3K36 methylation facilitates DNA replication and promotes genotoxic stress responses in fission yeast.

  17. Chromosomal DNA replication of Vicia faba cells

    International Nuclear Information System (INIS)

    Ikushima, Takaji

    1976-01-01

    The chromosomal DNA replication of higher plant cells has been investigated by DNA fiber autoradiography. The nuclear DNA fibers of Vicia root meristematic cells are organized into many tandem arrays of replication units or replicons which exist as clusters with respect to replication. DNA is replicated bidirectionally from the initiation points at the average rate of 0.15 μm/min at 20 0 C, and the average interinitiation interval is about 16 μm. The manner of chromosomal DNA replication in this higher plant is similar to that found in other eukaryotic cells at a subchromosomal level. (auth.)

  18. New insights into HCV replication in original cells from Aedes mosquitoes.

    Science.gov (United States)

    Fallecker, Catherine; Caporossi, Alban; Rechoum, Yassine; Garzoni, Frederic; Larrat, Sylvie; François, Olivier; Fender, Pascal; Morand, Patrice; Berger, Imre; Petit, Marie-Anne; Drouet, Emmanuel

    2017-08-22

    The existing literature about HCV association with, and replication in mosquitoes is extremely poor. To fill this gap, we performed cellular investigations aimed at exploring (i) the capacity of HCV E1E2 glycoproteins to bind on Aedes mosquito cells and (ii) the ability of HCV serum particles (HCVsp) to replicate in these cell lines. First, we used purified E1E2 expressing baculovirus-derived HCV pseudo particles (bacHCVpp) so we could investigate their association with mosquito cell lines from Aedes aegypti (Aag-2) and Aedes albopictus (C6/36). We initiated a series of infections of both mosquito cells (Ae aegypti and Ae albopictus) with the HCVsp (Lat strain - genotype 3) and we observed the evolution dynamics of viral populations within cells over the course of infection via next-generation sequencing (NGS) experiments. Our binding assays revealed bacHCVpp an association with the mosquito cells, at comparable levels obtained with human hepatocytes (HepaRG cells) used as a control. In our infection experiments, the HCV RNA (+) were detectable by RT-PCR in the cells between 21 and 28 days post-infection (p.i.). In human hepatocytes HepaRG and Ae aegypti insect cells, NGS experiments revealed an increase of global viral diversity with a selection for a quasi-species, suggesting a structuration of the population with elimination of deleterious mutations. The evolutionary pattern in Ae albopictus insect cells is different (stability of viral diversity and polymorphism). These results demonstrate for the first time that natural HCV could really replicate within Aedes mosquitoes, a discovery which may have major consequences for public health as well as in vaccine development.

  19. Surface micro topography replication in injection moulding

    DEFF Research Database (Denmark)

    Arlø, Uffe Rolf

    Thermoplastic injection moulding is a widely used industrial process that involves surface generation by replication. The surface topography of injection moulded plastic parts can be important for aesthetical or technical reasons. With the emergence of microengineering and nanotechnology additional...... importance of surface topography follows. In general the replication is not perfect and the topography of the plastic part differs from the inverse topography of the mould cavity. It is desirable to be able to control the degree of replication perfection or replication quality. This requires an understanding...... of the physical mechanisms of replication. Such understanding can lead to improved process design and facilitate in-line process quality control with respect to surface properties. The purpose of the project is to identify critical factors that affect topography replication quality and to obtain an understanding...

  20. Inclusion bodies are a site of ebolavirus replication.

    Science.gov (United States)

    Hoenen, Thomas; Shabman, Reed S; Groseth, Allison; Herwig, Astrid; Weber, Michaela; Schudt, Gordian; Dolnik, Olga; Basler, Christopher F; Becker, Stephan; Feldmann, Heinz

    2012-11-01

    Inclusion bodies are a characteristic feature of ebolavirus infections in cells. They contain large numbers of preformed nucleocapsids, but their biological significance has been debated, and they have been suggested to be aggregates of viral proteins without any further biological function. However, recent data for other viruses that produce similar structures have suggested that inclusion bodies might be involved in genome replication and transcription. In order to study filovirus inclusion bodies, we fused mCherry to the ebolavirus polymerase L, which is found in inclusion bodies. The resulting L-mCherry fusion protein was functional in minigenome assays and incorporated into virus-like particles. Importantly, L-mCherry fluorescence in transfected cells was readily detectable and distributed in a punctate pattern characteristic for inclusion bodies. A recombinant ebolavirus encoding L-mCherry instead of L was rescued and showed virtually identical growth kinetics and endpoint titers to those for wild-type virus. Using this virus, we showed that the onset of inclusion body formation corresponds to the onset of viral genome replication, but that viral transcription occurs prior to inclusion body formation. Live-cell imaging further showed that inclusion bodies are highly dynamic structures and that they can undergo dramatic reorganization during cell division. Finally, by labeling nascent RNAs using click technology we showed that inclusion bodies are indeed the site of viral RNA synthesis. Based on these data we conclude that, rather than being inert aggregates of nucleocapsids, ebolavirus inclusion bodies are in fact complex and dynamic structures and an important site at which viral RNA replication takes place.