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Sample records for replication complex assembly

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

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    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.

  2. The kinase inhibitor SFV785 dislocates dengue virus envelope protein from the replication complex and blocks virus assembly.

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    Azlinda Anwar

    Full Text Available Dengue virus (DENV is the etiologic agent for dengue fever, for which there is no approved vaccine or specific anti-viral drug. As a remedy for this, we explored the use of compounds that interfere with the action of required host factors and describe here the characterization of a kinase inhibitor (SFV785, which has selective effects on NTRK1 and MAPKAPK5 kinase activity, and anti-viral activity on Hepatitis C, DENV and yellow fever viruses. SFV785 inhibited DENV propagation without inhibiting DENV RNA synthesis or translation. The compound did not cause any changes in the cellular distribution of non-structural 3, a protein critical for DENV RNA synthesis, but altered the distribution of the structural envelope protein from a reticulate network to enlarged discrete vesicles, which altered the co-localization with the DENV replication complex. Ultrastructural electron microscopy analyses of DENV-infected SFV785-treated cells showed the presence of viral particles that were distinctly different from viable enveloped virions within enlarged ER cisternae. These viral particles were devoid of the dense nucleocapsid. The secretion of the viral particles was not inhibited by SFV785, however a reduction in the amount of secreted infectious virions, DENV RNA and capsid were observed. Collectively, these observations suggest that SFV785 inhibited the recruitment and assembly of the nucleocapsid in specific ER compartments during the DENV assembly process and hence the production of infectious DENV. SFV785 and derivative compounds could be useful biochemical probes to explore the DENV lifecycle and could also represent a new class of anti-virals.

  3. The Role of the N-Terminal Domains of Bacterial Initiator DnaA in the Assembly and Regulation of the Bacterial Replication Initiation Complex

    Science.gov (United States)

    Zawilak-Pawlik, Anna; Nowaczyk, Małgorzata; Zakrzewska-Czerwińska, Jolanta

    2017-01-01

    The primary role of the bacterial protein DnaA is to initiate chromosomal replication. The DnaA protein binds to DNA at the origin of chromosomal replication (oriC) and assembles into a filament that unwinds double-stranded DNA. Through interaction with various other proteins, DnaA also controls the frequency and/or timing of chromosomal replication at the initiation step. Escherichia coli DnaA also recruits DnaB helicase, which is present in unwound single-stranded DNA and in turn recruits other protein machinery for replication. Additionally, DnaA regulates the expression of certain genes in E. coli and a few other species. Acting as a multifunctional factor, DnaA is composed of four domains that have distinct, mutually dependent roles. For example, C-terminal domain IV interacts with double-stranded DnaA boxes. Domain III drives ATP-dependent oligomerization, allowing the protein to form a filament that unwinds DNA and subsequently binds to and stabilizes single-stranded DNA in the initial replication bubble; this domain also interacts with multiple proteins that control oligomerization. Domain II constitutes a flexible linker between C-terminal domains III–IV and N-terminal domain I, which mediates intermolecular interactions between DnaA and binds to other proteins that affect DnaA activity and/or formation of the initiation complex. Of these four domains, the role of the N-terminus (domains I–II) in the assembly of the initiation complex is the least understood and appears to be the most species-dependent region of the protein. Thus, in this review, we focus on the function of the N-terminus of DnaA in orisome formation and the regulation of its activity in the initiation complex in different bacteria. PMID:28489024

  4. The human cytomegalovirus gene products essential for late viral gene expression assemble into prereplication complexes before viral DNA replication.

    Science.gov (United States)

    Isomura, Hiroki; Stinski, Mark F; Murata, Takayuki; Yamashita, Yoriko; Kanda, Teru; Toyokuni, Shinya; Tsurumi, Tatsuya

    2011-07-01

    The regulation of human cytomegalovirus (HCMV) late gene expression by viral proteins is poorly understood, and these viral proteins could be targets for novel antivirals. HCMV open reading frames (ORFs) UL79, -87, and -95 encode proteins with homology to late gene transcription factors of murine gammaherpesvirus 68 ORFs 18, 24, and 34, respectively. To determine whether these HCMV proteins are also essential for late gene transcription of a betaherpesvirus, we mutated HCMV ORFs UL79, -87, and -95. Cells were infected with the recombinant viruses at high and low multiplicities of infection (MOIs). While viral DNA was detected with the recombinant viruses, infectious virus was not detected unless the wild-type viral proteins were expressed in trans. At a high MOI, mutation of ORF UL79, -87, or -95 had no effect on the level of major immediate-early (MIE) gene expression or viral DNA replication, but late viral gene expression from the UL44, -75, and -99 ORFs was not detected. At a low MOI, preexpression of UL79 or -87, but not UL95, in human fibroblast cells negatively affected the level of MIE viral gene expression and viral DNA replication. The products of ORFs UL79, -87, and -95 were expressed as early viral proteins and recruited to prereplication complexes (pre-RCs), along with UL44, before the initiation of viral DNA replication. All three HCMV ORFs are indispensable for late viral gene expression and viral growth. The roles of UL79, -87, and -95 in pre-RCs for late viral gene expression are discussed.

  5. Electron microscopic analysis of rotavirus assembly-replication intermediates

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    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.

  6. 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

  7. Characteristics of the poliovirus replication complex.

    Science.gov (United States)

    Bienz, K; Egger, D; Pfister, T

    1994-01-01

    In the infected cell, the poliovirus replication complex (RC) is found in the center of a rosette formed by many virus-induced vesicles. The RC is attached to the vesicular membranes and contains a compact central part which encloses the replication forks of the replicative intermediate and all proteins necessary for strand elongation. The growing plus strands of the replicative intermediate protrude from the central part of the RC, but are still enclosed by membraneous structures of the rosette. After completion, progeny 36S RNA is set free at the surface of the rosette. In an in vitro transcription system, isolated replication complex-containing rosettes are active in initiation, elongation and maturation (release) of plus strand progeny RNA. Full functionality of the RC depends on an intact structural framework of all membraneous components of the rosette.

  8. Assembling large, complex environmental metagenomes

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    Howe, A. C. [Michigan State Univ., East Lansing, MI (United States). Microbiology and Molecular Genetics, Plant Soil and Microbial Sciences; Jansson, J. [USDOE Joint Genome Institute (JGI), Walnut Creek, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Earth Sciences Division; Malfatti, S. A. [USDOE Joint Genome Institute (JGI), Walnut Creek, CA (United States); Tringe, S. G. [USDOE Joint Genome Institute (JGI), Walnut Creek, CA (United States); Tiedje, J. M. [Michigan State Univ., East Lansing, MI (United States). Microbiology and Molecular Genetics, Plant Soil and Microbial Sciences; Brown, C. T. [Michigan State Univ., East Lansing, MI (United States). Microbiology and Molecular Genetics, Computer Science and Engineering

    2012-12-28

    The large volumes of sequencing data required to sample complex environments deeply pose new challenges to sequence analysis approaches. De novo metagenomic assembly effectively reduces the total amount of data to be analyzed but requires significant computational resources. We apply two pre-assembly filtering approaches, digital normalization and partitioning, to make large metagenome assemblies more computationaly tractable. Using a human gut mock community dataset, we demonstrate that these methods result in assemblies nearly identical to assemblies from unprocessed data. We then assemble two large soil metagenomes from matched Iowa corn and native prairie soils. The predicted functional content and phylogenetic origin of the assembled contigs indicate significant taxonomic differences despite similar function. The assembly strategies presented are generic and can be extended to any metagenome; full source code is freely available under a BSD license.

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

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    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.

  10. An Assembly Funnel Makes Biomolecular Complex Assembly Efficient

    Science.gov (United States)

    Zenk, John; Schulman, Rebecca

    2014-01-01

    Like protein folding and crystallization, the self-assembly of complexes is a fundamental form of biomolecular organization. While the number of methods for creating synthetic complexes is growing rapidly, most require empirical tuning of assembly conditions and/or produce low yields. We use coarse-grained simulations of the assembly kinetics of complexes to identify generic limitations on yields that arise because of the many simultaneous interactions allowed between the components and intermediates of a complex. Efficient assembly occurs when nucleation is fast and growth pathways are few, i.e. when there is an assembly “funnel”. For typical complexes, an assembly funnel occurs in a narrow window of conditions whose location is highly complex specific. However, by redesigning the components this window can be drastically broadened, so that complexes can form quickly across many conditions. The generality of this approach suggests assembly funnel design as a foundational strategy for robust biomolecular complex synthesis. PMID:25360818

  11. Peptide-Assembled Optically Responsive Nanoparticle Complexes

    National Research Council Canada - National Science Library

    Slocik, Joseph M; Tam, Felicia; Halas, Naomi J; Naik, Rajesh R

    2007-01-01

    .... Here we report two types of active nanoparticle complexes, with properties controlled by near-infrared illumination, resulting from the assembly of photothermally responsive plasmonic nanoparticle...

  12. Fidelity of a human cell DNA replication complex

    International Nuclear Information System (INIS)

    Roberts, J.D.; Kunkel, T.A.

    1988-01-01

    The authors have measured the fidelity of bidirectional, semiconservative DNA synthesis by a human DNA replication complex in vitro. Replication was performed by extracts of HeLa cells in the presence of simian virus 40 (SV40) large tumor antigen by using a double-stranded phage M13mp2 DNA template containing the SV40 origin of replication and either of two different target sequences for scoring mutations in the lacZα-complementation gene, which encodes the α region (specifying the amino-terminal portion) of β-galactosidase. Replicative synthesis was substantially more accurate than synthesis by the human DNA polymerase α-DNA primase complex purified from HeLa cell extracts by immunoaffinity chromatography, suggesting that additional factors or activities in the extract may increase fidelity during bidirectional replication. However, by using a sensitive opal codon reversion assay, single-base substitution errors were readily detected in the replication products at frequencies significantly higher than estimated spontaneous mutation rates in vivo. These data suggest that additional fidelity factors may be present during chromosomal replication in vivo and/or that the fidelity of replication alone does not account for the low spontaneous mutation rates in eukaryotes

  13. Fidelity of a human cell DNA replication complex

    Energy Technology Data Exchange (ETDEWEB)

    Roberts, J.D.; Kunkel, T.A. (National Institute of Environmental Health Sciences, Research Triangle Park, NC (USA))

    1988-10-01

    The authors have measured the fidelity of bidirectional, semiconservative DNA synthesis by a human DNA replication complex in vitro. Replication was performed by extracts of HeLa cells in the presence of simian virus 40 (SV40) large tumor antigen by using a double-stranded phage M13mp2 DNA template containing the SV40 origin of replication and either of two different target sequences for scoring mutations in the lacZ{alpha}-complementation gene, which encodes the {alpha} region (specifying the amino-terminal portion) of {beta}-galactosidase. Replicative synthesis was substantially more accurate than synthesis by the human DNA polymerase {alpha}-DNA primase complex purified from HeLa cell extracts by immunoaffinity chromatography, suggesting that additional factors or activities in the extract may increase fidelity during bidirectional replication. However, by using a sensitive opal codon reversion assay, single-base substitution errors were readily detected in the replication products at frequencies significantly higher than estimated spontaneous mutation rates in vivo. These data suggest that additional fidelity factors may be present during chromosomal replication in vivo and/or that the fidelity of replication alone does not account for the low spontaneous mutation rates in eukaryotes.

  14. 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.

  15. 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

  16. Structural Insights into the Coupling of Virion Assembly and Rotavirus Replication

    Science.gov (United States)

    Trask, Shane D.; McDonald, Sarah M.; Patton, John T.

    2013-01-01

    Preface Viral replication is rapid and robust, but it is far from a chaotic process. Instead, successful production of infectious progeny requires that events occur in the correct place and at the correct time. Rotavirus, a segmented double-stranded RNA virus of the Reoviridae family, seems to govern its replication through ordered disassembly and assembly of a triple-layered icosahedral capsid. In recent years, high-resolution structural data have provided unprecedented insight into these events. In this Review, we explore the current understanding of rotavirus replication and how it compares to other Reoviridae family members. PMID:22266782

  17. 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.

  18. Complex Commingling: Nucleoporins and the Spindle Assembly Checkpoint

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    Ikram Mossaid

    2015-11-01

    Full Text Available The segregation of the chromosomes during mitosis is an important process, in which the replicated DNA content is properly allocated into two daughter cells. To ensure their genomic integrity, cells present an essential surveillance mechanism known as the spindle assembly checkpoint (SAC, which monitors the bipolar attachment of the mitotic spindle to chromosomes to prevent errors that would result in chromosome mis-segregation and aneuploidy. Multiple components of the nuclear pore complex (NPC, a gigantic protein complex that forms a channel through the nuclear envelope to allow nucleocytoplasmic exchange of macromolecules, were shown to be critical for faithful cell division and implicated in the regulation of different steps of the mitotic process, including kinetochore and spindle assembly as well as the SAC. In this review, we will describe current knowledge about the interconnection between the NPC and the SAC in an evolutional perspective, which primarily relies on the two mitotic checkpoint regulators, Mad1 and Mad2. We will further discuss the role of NPC constituents, the nucleoporins, in kinetochore and spindle assembly and the formation of the mitotic checkpoint complex during mitosis and interphase.

  19. Tissue engineering and regenerative strategies to replicate biocomplexity of vascular elastic matrix assembly.

    Science.gov (United States)

    Bashur, Chris A; Venkataraman, Lavanya; Ramamurthi, Anand

    2012-06-01

    Cardiovascular tissues exhibit architecturally complex extracellular matrices, of which the elastic matrix forms a major component. The elastic matrix critically maintains native structural configurations of vascular tissues, determines their ability to recoil after stretch, and regulates cell signaling pathways involved in morphogenesis, injury response, and inflammation via biomechanical transduction. The ability to tissue engineer vascular replacements that incorporate elastic matrix superstructures unique to cardiac and vascular tissues is thus important to maintaining vascular homeostasis. However, the vascular elastic matrix is particularly difficult to tissue engineer due to the inherently poor ability of adult vascular cells to synthesize elastin precursors and organize them into mature structures in a manner that replicates the biocomplexity of elastic matrix assembly during development. This review discusses current tissue engineering materials (e.g., growth factors and scaffolds) and methods (e.g., dynamic stretch and contact guidance) used to promote cellular synthesis and assembly of elastic matrix superstructures, and the limitations of these approaches when applied to smooth muscle cells, the primary elastin-generating cell type in vascular tissues. The potential application of these methods for in situ regeneration of disrupted elastic matrix at sites of proteolytic vascular disease (e.g., abdominal aortic aneurysms) is also discussed. Finally, the review describes the potential utility of alternative cell types to elastic tissue engineering and regenerative matrix repair. Future progress in the field is contingent on developing a thorough understanding of developmental elastogenesis and then mimicking the spatiotemporal changes in the cellular microenvironment that occur during that phase. This will enable us to tissue engineer clinically applicable elastic vascular tissue replacements and to develop elastogenic therapies to restore homeostasis in

  20. Recruitment of Brd4 to the human papillomavirus type 16 DNA replication complex is essential for replication of viral DNA.

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    Wang, Xin; Helfer, Christine M; Pancholi, Neha; Bradner, James E; You, Jianxin

    2013-04-01

    Replication of the human papillomavirus (HPV) DNA genome relies on viral factors E1 and E2 and the cellular replication machinery. Bromodomain-containing protein 4 (Brd4) interacts with viral E2 protein to mediate papillomavirus (PV) genome maintenance and viral transcription. However, the functional role of Brd4 in the HPV life cycle remains to be clearly defined. In this study, we provide the first look into the E2-Brd4 interaction in the presence of other important viral factors, such as the HPV16 E1 protein and the viral genome. We show that Brd4 is recruited to actively replicating HPV16 origin foci together with HPV16 E1, E2, and a number of the cellular replication factors: replication protein A70 (RPA70), replication factor C1 (RFC1), and DNA polymerase δ. Mutagenesis disrupting the E2-Brd4 interaction abolishes the formation of the HPV16 replication complex and impairs HPV16 DNA replication in cells. Brd4 was further demonstrated to be necessary for HPV16 viral DNA replication using a cell-free replication system in which depletion of Brd4 by small interfering RNA (siRNA) silencing leads to impaired HPV16 viral DNA replication and recombinant Brd4 protein is able to rescue viral DNA replication. In addition, releasing endogenous Brd4 from cellular chromatin by using the bromodomain inhibitor JQ1(+) enhances HPV16 DNA replication, demonstrating that the role of Brd4 in HPV DNA replication could be uncoupled from its function in chromatin-associated transcriptional regulation and cell cycle control. Our study reveals a new role for Brd4 in HPV genome replication, providing novel insights into understanding the life cycle of this oncogenic DNA virus.

  1. Modeling the assembly order of multimeric heteroprotein complexes.

    Science.gov (United States)

    Peterson, Lenna X; Togawa, Yoichiro; Esquivel-Rodriguez, Juan; Terashi, Genki; Christoffer, Charles; Roy, Amitava; Shin, Woong-Hee; Kihara, Daisuke

    2018-01-01

    Protein-protein interactions are the cornerstone of numerous biological processes. Although an increasing number of protein complex structures have been determined using experimental methods, relatively fewer studies have been performed to determine the assembly order of complexes. In addition to the insights into the molecular mechanisms of biological function provided by the structure of a complex, knowing the assembly order is important for understanding the process of complex formation. Assembly order is also practically useful for constructing subcomplexes as a step toward solving the entire complex experimentally, designing artificial protein complexes, and developing drugs that interrupt a critical step in the complex assembly. There are several experimental methods for determining the assembly order of complexes; however, these techniques are resource-intensive. Here, we present a computational method that predicts the assembly order of protein complexes by building the complex structure. The method, named Path-LzerD, uses a multimeric protein docking algorithm that assembles a protein complex structure from individual subunit structures and predicts assembly order by observing the simulated assembly process of the complex. Benchmarked on a dataset of complexes with experimental evidence of assembly order, Path-LZerD was successful in predicting the assembly pathway for the majority of the cases. Moreover, when compared with a simple approach that infers the assembly path from the buried surface area of subunits in the native complex, Path-LZerD has the strong advantage that it can be used for cases where the complex structure is not known. The path prediction accuracy decreased when starting from unbound monomers, particularly for larger complexes of five or more subunits, for which only a part of the assembly path was correctly identified. As the first method of its kind, Path-LZerD opens a new area of computational protein structure modeling and will be

  2. Modeling the assembly order of multimeric heteroprotein complexes.

    Directory of Open Access Journals (Sweden)

    Lenna X Peterson

    2018-01-01

    Full Text Available Protein-protein interactions are the cornerstone of numerous biological processes. Although an increasing number of protein complex structures have been determined using experimental methods, relatively fewer studies have been performed to determine the assembly order of complexes. In addition to the insights into the molecular mechanisms of biological function provided by the structure of a complex, knowing the assembly order is important for understanding the process of complex formation. Assembly order is also practically useful for constructing subcomplexes as a step toward solving the entire complex experimentally, designing artificial protein complexes, and developing drugs that interrupt a critical step in the complex assembly. There are several experimental methods for determining the assembly order of complexes; however, these techniques are resource-intensive. Here, we present a computational method that predicts the assembly order of protein complexes by building the complex structure. The method, named Path-LzerD, uses a multimeric protein docking algorithm that assembles a protein complex structure from individual subunit structures and predicts assembly order by observing the simulated assembly process of the complex. Benchmarked on a dataset of complexes with experimental evidence of assembly order, Path-LZerD was successful in predicting the assembly pathway for the majority of the cases. Moreover, when compared with a simple approach that infers the assembly path from the buried surface area of subunits in the native complex, Path-LZerD has the strong advantage that it can be used for cases where the complex structure is not known. The path prediction accuracy decreased when starting from unbound monomers, particularly for larger complexes of five or more subunits, for which only a part of the assembly path was correctly identified. As the first method of its kind, Path-LZerD opens a new area of computational protein structure

  3. STRUCTURAL AND OPERATIONAL COMPLEXITY OF MANUAL ASSEMBLY SYSTEMS

    OpenAIRE

    Atiya Al-Zuheri

    2013-01-01

    Increased complexity is one of the biggest challenges in assembly industry today. Designing and implementing manual assembly systems require identifying operational and structural complexity sources inherent with these systems. Lack of this fact causes inaccuracy in the results from the system application. This study details sources of both types of complexity in manual assembly systems. Also, the paper will present the modeling techniques to address these complexities in the design approach ...

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

    NARCIS (Netherlands)

    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

  5. 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.

  6. Replication-Coupled Nucleosome Assembly and Positioning by ATP-Dependent Chromatin-Remodeling Enzymes

    Directory of Open Access Journals (Sweden)

    Tejas Yadav

    2016-04-01

    Full Text Available During DNA replication, chromatin must be disassembled and faithfully reassembled on newly synthesized genomes. The mechanisms that govern the assembly of chromatin structures following DNA replication are poorly understood. Here, we exploited Okazaki fragment synthesis and other assays to study how nucleosomes are deposited and become organized in S. cerevisiae. We observe that global nucleosome positioning is quickly established on newly synthesized DNA in vivo. Importantly, we find that ATP-dependent chromatin-remodeling enzymes, Isw1 and Chd1, collaborate with histone chaperones to remodel nucleosomes as they are loaded behind a replication fork. Using a whole-genome sequencing approach, we determine that the positioning of newly deposited nucleosomes in vivo is specified by the combined actions of ATP-dependent chromatin-remodeling enzymes and select DNA-binding proteins. Altogether, our data provide in vivo evidence for coordinated “loading and remodeling” of nucleosomes behind the replication fork, allowing for rapid organization of chromatin during S phase.

  7. Uracil Excision for Assembly of Complex Pathways

    DEFF Research Database (Denmark)

    Cavaleiro, Mafalda; Nielsen, Morten Thrane; Kim, Se Hyeuk

    2015-01-01

    Despite decreasing prices on synthetic DNA constructs, higher-order assembly of PCR-generated DNA continues to be an important exercise in molecular and synthetic biology. Simplicity and robustness are attractive features met by the uracil excision DNA assembly method, which is one of the most in...... genes into the genome, and a standardized assembly pipeline using benchmarked oligonucleotides for pathway assembly and multigene expression optimization....

  8. Peptide-Assembled Optically Responsive Nanoparticle Complexes (Preprint)

    National Research Council Canada - National Science Library

    Naik, Rajesh R; Slocik, Joseph M; Tam, Felicia; Halas, Naomi J

    2007-01-01

    .... Here we report two types of active nanoparticle complexes, with properties controlled by near infrared illumination, resulting from the assembly of photothermally responsive plasmonic nanoparticle...

  9. Localization of adenovirus morphogenesis players, together with visualization of assembly intermediates and failed products, favor a model where assembly and packaging occur concurrently at the periphery of the replication center.

    Directory of Open Access Journals (Sweden)

    Gabriela N Condezo

    2017-04-01

    Full Text Available Adenovirus (AdV morphogenesis is a complex process, many aspects of which remain unclear. In particular, it is not settled where in the nucleus assembly and packaging occur, and whether these processes occur in a sequential or a concerted manner. Here we use immunofluorescence and immunoelectron microscopy (immunoEM to trace packaging factors and structural proteins at late times post infection by either wildtype virus or a delayed packaging mutant. We show that representatives of all assembly factors are present in the previously recognized peripheral replicative zone, which therefore is the AdV assembly factory. Assembly intermediates and abortive products observed in this region favor a concurrent assembly and packaging model comprising two pathways, one for capsid proteins and another one for core components. Only when both pathways are coupled by correct interaction between packaging proteins and the genome is the viral particle produced. Decoupling generates accumulation of empty capsids and unpackaged cores.

  10. Localization of adenovirus morphogenesis players, together with visualization of assembly intermediates and failed products, favor a model where assembly and packaging occur concurrently at the periphery of the replication center.

    Science.gov (United States)

    Condezo, Gabriela N; San Martín, Carmen

    2017-04-01

    Adenovirus (AdV) morphogenesis is a complex process, many aspects of which remain unclear. In particular, it is not settled where in the nucleus assembly and packaging occur, and whether these processes occur in a sequential or a concerted manner. Here we use immunofluorescence and immunoelectron microscopy (immunoEM) to trace packaging factors and structural proteins at late times post infection by either wildtype virus or a delayed packaging mutant. We show that representatives of all assembly factors are present in the previously recognized peripheral replicative zone, which therefore is the AdV assembly factory. Assembly intermediates and abortive products observed in this region favor a concurrent assembly and packaging model comprising two pathways, one for capsid proteins and another one for core components. Only when both pathways are coupled by correct interaction between packaging proteins and the genome is the viral particle produced. Decoupling generates accumulation of empty capsids and unpackaged cores.

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

    Science.gov (United States)

    Shiomi, Yasushi; Nishitani, Hideo

    2017-01-26

    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.

  12. 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.

  13. Operon Gene Order Is Optimized for Ordered Protein Complex Assembly

    Science.gov (United States)

    Wells, Jonathan N.; Bergendahl, L. Therese; Marsh, Joseph A.

    2016-01-01

    Summary The assembly of heteromeric protein complexes is an inherently stochastic process in which multiple genes are expressed separately into proteins, which must then somehow find each other within the cell. Here, we considered one of the ways by which prokaryotic organisms have attempted to maximize the efficiency of protein complex assembly: the organization of subunit-encoding genes into operons. Using structure-based assembly predictions, we show that operon gene order has been optimized to match the order in which protein subunits assemble. Exceptions to this are almost entirely highly expressed proteins for which assembly is less stochastic and for which precisely ordered translation offers less benefit. Overall, these results show that ordered protein complex assembly pathways are of significant biological importance and represent a major evolutionary constraint on operon gene organization. PMID:26804901

  14. 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.

  15. E1-mediated recruitment of a UAF1-USP deubiquitinase complex facilitates human papillomavirus DNA replication.

    Science.gov (United States)

    Lehoux, Michaël; Gagnon, David; Archambault, Jacques

    2014-08-01

    The human papillomavirus (HPV) E1 helicase promotes viral DNA replication through its DNA unwinding activity and association with host factors. The E1 proteins from anogenital HPV types interact with the cellular WD repeat-containing factor UAF1 (formerly known as p80). Specific amino acid substitutions in E1 that impair this interaction inhibit maintenance of the viral episome in immortalized keratinocytes and reduce viral DNA replication by up to 70% in transient assays. In this study, we determined by affinity purification of UAF1 that it interacts with three deubiquitinating enzymes in C33A cervical carcinoma cells: USP1, a nuclear protein, and the two cytoplasmic enzymes USP12 and USP46. Coimmunoprecipitation experiments indicated that E1 assembles into a ternary complex with UAF1 and any one of these three USPs. Moreover, expression of E1 leads to a redistribution of USP12 and USP46 from the cytoplasm to the nucleus. Chromatin immunoprecipitation studies further revealed that E1 recruits these threes USPs to the viral origin in association with UAF1. The function of USP1, USP12, and USP46 in viral DNA replication was investigated by overproduction of catalytically inactive versions of these enzymes in transient assays. All three dominant negative USPs reduced HPV31 DNA replication by up to 60%, an effect that was specific, as it was not observed in assays performed with a truncated E1 lacking the UAF1-binding domain or with bovine papillomavirus 1 E1, which does not bind UAF1. These results highlight the importance of the USP1, USP12, and USP46 deubiquitinating enzymes in anogenital HPV DNA replication. Human papillomaviruses are small DNA tumor viruses that induce benign and malignant lesions of the skin and mucosa. HPV types that infect the anogenital tract are the etiological agents of cervical cancer, the majority of anal cancers, and a growing proportion of head-and-neck cancers. Replication of the HPV genome requires the viral protein E1, a DNA helicase

  16. Assembly factors for the membrane arm of human complex I.

    Science.gov (United States)

    Andrews, Byron; Carroll, Joe; Ding, Shujing; Fearnley, Ian M; Walker, John E

    2013-11-19

    Mitochondrial respiratory complex I is a product of both the nuclear and mitochondrial genomes. The integration of seven subunits encoded in mitochondrial DNA into the inner membrane, their association with 14 nuclear-encoded membrane subunits, the construction of the extrinsic arm from 23 additional nuclear-encoded proteins, iron-sulfur clusters, and flavin mononucleotide cofactor require the participation of assembly factors. Some are intrinsic to the complex, whereas others participate transiently. The suppression of the expression of the NDUFA11 subunit of complex I disrupted the assembly of the complex, and subcomplexes with masses of 550 and 815 kDa accumulated. Eight of the known extrinsic assembly factors plus a hydrophobic protein, C3orf1, were associated with the subcomplexes. The characteristics of C3orf1, of another assembly factor, TMEM126B, and of NDUFA11 suggest that they all participate in constructing the membrane arm of complex I.

  17. Nucleobase-mediated, photocatalytic production of amphiphiles to promote the self-assembly of a simple self-replicating protocell.

    Science.gov (United States)

    Monnard, Pierre-Alain; Maurer, Sarah, E.; Albertsen, Anders, N.; Boncella, James, M.; Cape, Jonathan, L.

    Living cells are in many respects the ultimate nanoscale chemical system. Within a very small volume they can produce highly specific useful products by extracting resources and free energy from the environment. They are also self-organized, self-controlled, and capable of self-repair and self-replication. Designing artificial chemical systems (artificial cells or protocells) that would be endowed with these powerful capabilities has been investigated extensively in the recent years. Chemical systems usually studied were based on the encapsulation of a set of genes along with catalytic protein machinery within the self-assembled boundaries of liposome/vesicles. The generated systems have many of the characteristics of a living system, but lack the regulation by genetic information of all protocell functions. Departing from these encapsulated models, we have been attempting to implement a simple, chemical system in which the regulation of the metabolism is truly mediated by information molecules. Our proposed system is composed of a chemical mixture composed of fatty acids that form bilayers (compartment), amphiphilic information molecules (nucleic acids -NA), and metabolic complexes (photosensitizers). Due to the intrinsic properties of all its components, a chemical system will self-assemble into aqueous, colloid mixtures that will be conducive to the metabolic steps, the non-enzymatic polymerization of the information, and the photochemical fatty acid production from its oil-like precursor. The reaction products (e.g., the container molecules) will in turn promote system growth and replication. In this scheme, the NA acts as an information molecule mediating the metabolic catalysis (electron donor/relay system) with a ruthenium metal complex as a cofactor and sensitizer, which is used to convert the hydrophobic precursor container molecules into amphiphiles, thus directly linking protocell metabolism with information. In a first experimental design, NA has been

  18. Replicating centromeric chromatin: Spatial and temporal control of CENP-A assembly

    International Nuclear Information System (INIS)

    Nechemia-Arbely, Yael; Fachinetti, Daniele; Cleveland, Don W.

    2012-01-01

    The centromere is the fundamental unit for insuring chromosome inheritance. This complex region has a distinct type of chromatin in which histone H3 is replaced by a structurally different homologue identified in humans as CENP-A. In metazoans, specific DNA sequences are neither required nor sufficient for centromere identity. Rather, an epigenetic mark comprised of CENP-A containing chromatin is thought to be the major determinant of centromere identity. In this view, CENP-A deposition and chromatin assembly are fundamental processes for the maintenance of centromeric identity across mitotic and meiotic divisions. Several lines of evidence support CENP-A deposition in metazoans occurring at only one time in the cell cycle. Such cell cycle-dependent loading of CENP-A is found in divergent species from human to fission yeast, albeit with differences in the cell cycle point at which CENP-A is assembled. Cell cycle dependent CENP-A deposition requires multiple assembly factors for its deposition and maintenance. This review discusses the regulation of new CENP-A deposition and its relevance to centromere identity and inheritance.

  19. Principles of assembly reveal a periodic table of protein complexes.

    Science.gov (United States)

    Ahnert, Sebastian E; Marsh, Joseph A; Hernández, Helena; Robinson, Carol V; Teichmann, Sarah A

    2015-12-11

    Structural insights into protein complexes have had a broad impact on our understanding of biological function and evolution. In this work, we sought a comprehensive understanding of the general principles underlying quaternary structure organization in protein complexes. We first examined the fundamental steps by which protein complexes can assemble, using experimental and structure-based characterization of assembly pathways. Most assembly transitions can be classified into three basic types, which can then be used to exhaustively enumerate a large set of possible quaternary structure topologies. These topologies, which include the vast majority of observed protein complex structures, enable a natural organization of protein complexes into a periodic table. On the basis of this table, we can accurately predict the expected frequencies of quaternary structure topologies, including those not yet observed. These results have important implications for quaternary structure prediction, modeling, and engineering. Copyright © 2015, American Association for the Advancement of Science.

  20. An efficient approach to BAC based assembly of complex genomes.

    Science.gov (United States)

    Visendi, Paul; Berkman, Paul J; Hayashi, Satomi; Golicz, Agnieszka A; Bayer, Philipp E; Ruperao, Pradeep; Hurgobin, Bhavna; Montenegro, Juan; Chan, Chon-Kit Kenneth; Staňková, Helena; Batley, Jacqueline; Šimková, Hana; Doležel, Jaroslav; Edwards, David

    2016-01-01

    There has been an exponential growth in the number of genome sequencing projects since the introduction of next generation DNA sequencing technologies. Genome projects have increasingly involved assembly of whole genome data which produces inferior assemblies compared to traditional Sanger sequencing of genomic fragments cloned into bacterial artificial chromosomes (BACs). While whole genome shotgun sequencing using next generation sequencing (NGS) is relatively fast and inexpensive, this method is extremely challenging for highly complex genomes, where polyploidy or high repeat content confounds accurate assembly, or where a highly accurate 'gold' reference is required. Several attempts have been made to improve genome sequencing approaches by incorporating NGS methods, to variable success. We present the application of a novel BAC sequencing approach which combines indexed pools of BACs, Illumina paired read sequencing, a sequence assembler specifically designed for complex BAC assembly, and a custom bioinformatics pipeline. We demonstrate this method by sequencing and assembling BAC cloned fragments from bread wheat and sugarcane genomes. We demonstrate that our assembly approach is accurate, robust, cost effective and scalable, with applications for complete genome sequencing in large and complex genomes.

  1. The homeotic protein HOXC13 is a member of human DNA replication complexes.

    Science.gov (United States)

    Comelli, Laura; Marchetti, Laura; Arosio, Daniele; Riva, Silvano; Abdurashidova, Gulnara; Beltram, Fabio; Falaschi, Arturo

    2009-02-01

    The homeotic (and oncogenic) HOXC13 protein was shown to have an affinity for a DNA fragment corresponding to the sequence covered by the pre-replicative complex of the human lamin B2 replication origin. We show here that HOXC13 is a member of human replicative complexes. Our fluorescent fusion-protein data demonstrate that it co-localizes with replication foci of early-S cells and that this peculiar behaviour is driven by the homeodomain. By ChIP analysis we also show that HOXC13 binds the lamin B2 replication origin and the origins located near the TOP1 and MCM4 genes in asynchronously growing cells, whereas it does not bind these origins in G(0) resting cells, consistently with its involvement in origin function.

  2. Directed self-assembly of DNA tiles into complex nanocages.

    Science.gov (United States)

    Tian, Cheng; Li, Xiang; Liu, Zhiyu; Jiang, Wen; Wang, Guansong; Mao, Chengde

    2014-07-28

    Tile-based self-assembly is a powerful method in DNA nanotechnology and has produced a wide range of well-defined nanostructures. But the resulting structures are relatively simple. Increasing the structural complexity and the scope of the accessible structures is an outstanding challenge in molecular self-assembly. A strategy to partially address this problem by introducing flexibility into assembling DNA tiles and employing directing agents to control the self-assembly process is presented. To demonstrate this strategy, a range of DNA nanocages have been rationally designed and constructed. Many of them can not be assembled otherwise. All of the resulting structures have been thoroughly characterized by gel electrophoresis and cryogenic electron microscopy. This strategy greatly expands the scope of accessible DNA nanostructures and would facilitate technological applications such as nanoguest encapsulation, drug delivery, and nanoparticle organization. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  3. Hierarchical self-assembly of complex polyhedral microcontainers

    International Nuclear Information System (INIS)

    Filipiak, David J; Leong, Timothy G; Gracias, David H; Azam, Anum

    2009-01-01

    The concept of self-assembly of a two-dimensional (2D) template to a three-dimensional (3D) structure has been suggested as a strategy to enable highly parallel fabrication of complex, patterned microstructures. We have previously studied the surface-tension-based self-assembly of patterned, microscale polyhedral containers (cubes, square pyramids and tetrahedral frusta). In this paper, we describe the observed hierarchical self-assembly of more complex, patterned polyhedral containers in the form of regular dodecahedra and octahedra. The hierarchical design methodology, combined with the use of self-correction mechanisms, was found to greatly reduce the propagation of self-assembly error that occurs in these more complex systems. It is a highly effective way to mass-produce patterned, complex 3D structures on the microscale and could also facilitate encapsulation of cargo in a parallel and cost-effective manner. Furthermore, the behavior that we have observed may be useful in the assembly of complex systems with large numbers of components

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

    Directory of Open Access Journals (Sweden)

    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.

  5. Purification of highly active alphavirus replication complexes demonstrates altered fractionation of multiple cellular membranes.

    Science.gov (United States)

    Pietilä, Maija K; van Hemert, Martijn J; Ahola, Tero

    2018-01-24

    Positive-strand RNA viruses replicate their genomes in membrane-associated structures; alphaviruses and many other groups induce membrane invaginations called spherules. Here, we established a protocol to purify these membranous replication complexes (RCs) from cells infected with Semliki Forest virus (SFV). We isolated SFV spherules located on the plasma membrane and further purified them using two consecutive density gradients. This revealed that SFV infection strongly modifies cellular membranes. We removed soluble proteins, the Golgi and most of the mitochondria, but plasma membrane, endoplasmic reticulum (ER) and late endosome markers enriched in the membrane fraction that contained viral RNA synthesizing activity, replicase proteins and minus- and plus-strand RNA. Electron microscopy revealed that the purified membranes displayed spherule-like structures with a narrow neck. This membrane enrichment was specific to viral replication as such a distribution of membrane markers was only observed after infection. Besides the plasma membrane, SFV infection remodeled the ER, and the co-fractionation of the RC-carrying plasma membrane and ER suggests that SFV may recruit ER proteins or membrane to the site of replication. The purified RCs were highly active in synthesizing both genomic and subgenomic RNA. Detergent solubilization destroyed the replication activity demonstrating that the membrane association of the complex is essential. Most of the newly made RNA was in double-stranded replicative molecules but the purified complexes also produced single-stranded RNA as well as released newly made RNA. This indicates that the purification established here maintained the functionality of RCs and thus enables further structural and functional studies of active RCs. IMPORTANCE Similar to all positive-strand RNA viruses, the arthropod-borne alphaviruses induce membranous genome factories but little is known about the arrangement of viral replicase proteins and the

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

    Science.gov (United States)

    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.

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

    Directory of Open Access Journals (Sweden)

    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.

  8. ACH-806, an NS4A antagonist, inhibits hepatitis C virus replication by altering the composition of viral replication complexes.

    Science.gov (United States)

    Yang, Wengang; Sun, Yongnian; Hou, Xiaohong; Zhao, Yongsen; Fabrycki, Joanne; Chen, Dawei; Wang, Xiangzhu; Agarwal, Atul; Phadke, Avinash; Deshpande, Milind; Huang, Mingjun

    2013-07-01

    Treatment of hepatitis C patients with direct-acting antiviral drugs involves the combination of multiple small-molecule inhibitors of distinctive mechanisms of action. ACH-806 (or GS-9132) is a novel, small-molecule inhibitor specific for hepatitis C virus (HCV). It inhibits viral RNA replication in HCV replicon cells and was active in genotype 1 HCV-infected patients in a proof-of-concept clinical trial (1). Here, we describe a potential mechanism of action (MoA) wherein ACH-806 alters viral replication complex (RC) composition and function. We found that ACH-806 did not affect HCV polyprotein translation and processing, the early events of the formation of HCV RC. Instead, ACH-806 triggered the formation of a homodimeric form of NS4A with a size of 14 kDa (p14) both in replicon cells and in Huh-7 cells where NS4A was expressed alone. p14 production was negatively regulated by NS3, and its appearance in turn was associated with reductions in NS3 and, especially, NS4A content in RCs due to their accelerated degradation. A previously described resistance substitution near the N terminus of NS3, where NS3 interacts with NS4A, attenuated the reduction of NS3 and NS4A conferred by ACH-806 treatment. Taken together, we show that the compositional changes in viral RCs are associated with the antiviral activity of ACH-806. Small molecules, including ACH-806, with this novel MoA hold promise for further development and provide unique tools for clarifying the functions of NS4A in HCV replication.

  9. Synthesis, characterization and self-assembly of Co 3 complexes ...

    Indian Academy of Sciences (India)

    Home; Journals; Journal of Chemical Sciences; Volume 126; Issue 5. Synthesis, characterization and self-assembly of Co3+ complexes appended with phenol and catechol groups. Afsar Ali Deepak Bansal Rajeev Gupt. Special issue on Chemical Crystallography Volume 126 Issue 5 September 2014 pp 1535-1546 ...

  10. Complex Colloidal Structures by Self-assembly in Electric Fields

    NARCIS (Netherlands)

    Vutukuri, H.R.

    2012-01-01

    The central theme of this thesis is exploiting the directed self-assembly of both isotropic and anisotropic colloidal particles to achieve the fabrication of one-, two-, and three-dimensional complex colloidal structures using external electric fields and/or a simple in situ thermal annealing

  11. DNA Virus Replication Compartments

    Science.gov (United States)

    Schmid, Melanie; Speiseder, Thomas; Dobner, Thomas

    2014-01-01

    Viruses employ a variety of strategies to usurp and control cellular activities through the orchestrated recruitment of macromolecules to specific cytoplasmic or nuclear compartments. Formation of such specialized virus-induced cellular microenvironments, which have been termed viroplasms, virus factories, or virus replication centers, complexes, or compartments, depends on molecular interactions between viral and cellular factors that participate in viral genome expression and replication and are in some cases associated with sites of virion assembly. These virus-induced compartments function not only to recruit and concentrate factors required for essential steps of the viral replication cycle but also to control the cellular mechanisms of antiviral defense. In this review, we summarize characteristic features of viral replication compartments from different virus families and discuss similarities in the viral and cellular activities that are associated with their assembly and the functions they facilitate for viral replication. PMID:24257611

  12. Uracil DNA glycosylase BKRF3 contributes to Epstein-Barr virus DNA replication through physical interactions with proteins in viral DNA replication complex.

    Science.gov (United States)

    Su, Mei-Tzu; Liu, I-Hua; Wu, Chia-Wei; Chang, Shu-Ming; Tsai, Ching-Hwa; Yang, Pei-Wen; Chuang, Yu-Chia; Lee, Chung-Pei; Chen, Mei-Ru

    2014-08-01

    Epstein-Barr virus (EBV) BKRF3 shares sequence homology with members of the uracil-N-glycosylase (UNG) protein family and has DNA glycosylase activity. Here, we explored how BKRF3 participates in the DNA replication complex and contributes to viral DNA replication. Exogenously expressed Flag-BKRF3 was distributed mostly in the cytoplasm, whereas BKRF3 was translocated into the nucleus and colocalized with the EBV DNA polymerase BALF5 in the replication compartment during EBV lytic replication. The expression level of BKRF3 increased gradually during viral replication, coupled with a decrease of cellular UNG2, suggesting BKRF3 enzyme activity compensates for UNG2 and ensures the fidelity of viral DNA replication. In immunoprecipitation-Western blotting, BKRF3 was coimmuno-precipitated with BALF5, the polymerase processivity factor BMRF1, and the immediate-early transactivator Rta. Coexpression of BMRF1 appeared to facilitate the nuclear targeting of BKRF3 in immunofluorescence staining. Residues 164 to 255 of BKRF3 were required for interaction with Rta and BALF5, whereas residues 81 to 166 of BKRF3 were critical for BMRF1 interaction in glutathione S-transferase (GST) pulldown experiments. Viral DNA replication was defective in cells harboring BKRF3 knockout EBV bacmids. In complementation assays, the catalytic mutant BKRF3(Q90L,D91N) restored viral DNA replication, whereas the leucine loop mutant BKRF3(H213L) only partially rescued viral DNA replication, coupled with a reduced ability to interact with the viral DNA polymerase and Rta. Our data suggest that BKRF3 plays a critical role in viral DNA synthesis predominantly through its interactions with viral proteins in the DNA replication compartment, while its enzymatic activity may be supplementary for uracil DNA glycosylase (UDG) function during virus replication. Catalytic activities of both cellular UDG UNG2 and viral UDGs contribute to herpesviral DNA replication. To ensure that the enzyme activity executes at

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

    Science.gov (United States)

    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

  14. Probing Interactions in Complex Molecular Systems through Ordered Assembly

    International Nuclear Information System (INIS)

    De Yoreo, J.J.; Bartelt, M.C.; Orme, C.A.; Villacampa, A.; Weeks, B.L.; Miller, A.E.

    2002-01-01

    Emerging from the machinery of epitaxial science and chemical synthesis, is a growing emphasis on development of self-organized systems of complex molecular species. The nature of self-organization in these systems spans the continuum from simple crystallization of large molecules such as dendrimers and proteins, to assembly into large organized networks of nanometer-scale structures such as quantum dots or nanoparticles. In truth, self-organization in complex molecular systems has always been a central feature of many scientific disciplines including fields as diverse as structural biology, polymer science and geochemistry. But over the past decade, changes in those fields have often been marked by the degree to which researchers are using molecular-scale approaches to understand the hierarchy of structures and processes driven by this ordered assembly. At the same time, physical scientists have begun to use their knowledge of simple atomic and molecular systems to fabricate synthetic self-organized systems. This increasing activity in the field of self-organization is testament to the success of the physical and chemical sciences in building a detailed understanding of crystallization and epitaxy in simple atomic and molecular systems, one that is soundly rooted in thermodynamics and chemical kinetics. One of the fundamental challenges of chemistry and materials science in the coming decades is to develop a similarly well-founded physical understanding of assembly processes in complex molecular systems. Over the past five years, we have successfully used in situ atomic force microscopy (AFM) to investigate the physical controls on single crystal epitaxy from solutions for a wide range of molecular species. More recently, we have combined this method with grazing incidence X-ray diffraction and kinetic Monte Carlo modeling in order to relate morphology to surface atomic structure and processes. The purpose of this proposal was to extend this approach to assemblies

  15. 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

  16. 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.

  17. Using lanthanoid complexes to phase large macromolecular assemblies

    International Nuclear Information System (INIS)

    Talon, Romain; Kahn, Richard; Durá, M. Asunción; Maury, Olivier; Vellieux, Frédéric M. D.; Franzetti, Bruno; Girard, Eric

    2011-01-01

    A lanthanoid complex, [Eu(DPA) 3 ] 3− , was used to obtain experimental phases at 4.0 Å resolution of PhTET1-12s, a large self-compartmentalized homo-dodecameric protease complex of 444 kDa. Lanthanoid ions exhibit extremely large anomalous X-ray scattering at their L III absorption edge. They are thus well suited for anomalous diffraction experiments. A novel class of lanthanoid complexes has been developed that combines the physical properties of lanthanoid atoms with functional chemical groups that allow non-covalent binding to proteins. Two structures of large multimeric proteins have already been determined by using such complexes. Here the use of the luminescent europium tris-dipicolinate complex [Eu(DPA) 3 ] 3− to solve the low-resolution structure of a 444 kDa homododecameric aminopeptidase, called PhTET1-12s from the archaea Pyrococcus horikoshii, is reported. Surprisingly, considering the low resolution of the data, the experimental electron density map is very well defined. Experimental phases obtained by using the lanthanoid complex lead to maps displaying particular structural features usually observed in higher-resolution maps. Such complexes open a new way for solving the structure of large molecular assemblies, even with low-resolution data

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

    Science.gov (United States)

    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.

  19. Double smectic self-assembly in block copolypeptide complexes

    KAUST Repository

    Haataja, Johannes S.

    2012-11-12

    We show double smectic-like self-assemblies in the solid state involving alternating layers of different polypeptide α-helices. We employed rod-coil poly(γ-benzyl l-glutamate)-block-poly(l-lysine) (PBLG-b-PLL) as the polymeric scaffold, where the PLL amino residues were ionically complexed to di-n-butyl phosphate (diC4P), di(2-ethylhexyl) phosphate (diC2/6P), di(2-octyldodecyl) phosphate (diC8/12P), or di-n-dodecyl phosphate (diC12P), forming PBLG-b-PLL(diC4P), PBLG-b-PLL(diC2/6P), PBLG-b-PLL(diC8/12P), and PBLG-b-PLL(diC12P) complexes, respectively. The complexes contain PBLG α-helices of fixed diameter and PLL-surfactant complexes adopting either α-helices of tunable diameters or β-sheets. For PBLG-b-PLL(diC4P), that is, using a surfactant with short n-butyl tails, both blocks were α-helical, of roughly equal diameter and thus with minor packing frustrations, leading to alternating PBLG and PLL(diC4P) smectic layers of approximately perpendicular alignment of both types of α-helices. Surfactants with longer and branched alkyl tails lead to an increased diameter of the PLL-surfactant α-helices. Smectic alternating PBLG and PLL(diC2/6P) layers involve larger packing frustration, which leads to poor overall order and suggests an arrangement of tilted PBLG α-helices. In PBLG-b-PLL(diC8/12P), the PLL(diC8/12P) α-helices are even larger and the overall structure is poor. Using a surfactant with two linear n-dodecyl tails leads to well-ordered β-sheet domains of PLL(diC12P), consisting of alternating PLL and alkyl chain layers. This dominates the whole assembly, and at the block copolypeptide length scale, the PBLG α-helices do not show internal order and have poor organization. Packing frustration becomes an important aspect to design block copolypeptide assemblies, even if frustration could be relieved by conformational imperfections. The results suggest pathways to control hierarchical liquid-crystalline assemblies by competing interactions and by

  20. Transient association of MCM complex proteins with the nuclear matrix during initiation of mammalian DNA replication.

    Science.gov (United States)

    Hesketh, Emma L; Knight, John R P; Wilson, Rosemary H C; Chong, James P J; Coverley, Dawn

    2015-01-01

    The minichromosome maintenance complex (MCM2-7) is the putative DNA helicase in eukaryotes, and essential for DNA replication. By applying serial extractions to mammalian cells synchronized by release from quiescence, we reveal dynamic changes to the sub-nuclear compartmentalization of MCM2 as cells pass through late G1 and early S phase, identifying a brief window when MCM2 becomes transiently attached to the nuclear-matrix. The data distinguish 3 states that correspond to loose association with chromatin prior to DNA replication, transient highly stable binding to the nuclear-matrix coincident with initiation, and a post-initiation phase when MCM2 remains tightly associated with chromatin but not the nuclear-matrix. The data suggests that functional MCM complex loading takes place at the nuclear-matrix.

  1. Transient association of MCM complex proteins with the nuclear matrix during initiation of mammalian DNA replication

    OpenAIRE

    Hesketh, Emma L; Knight, John RP; Wilson, Rosemary HC; Chong, James PJ; Coverley, Dawn

    2015-01-01

    The minichromosome maintenance complex (MCM2-7) is the putative DNA helicase in eukaryotes, and essential for DNA replication. By applying serial extractions to mammalian cells synchronized by release from quiescence, we reveal dynamic changes to the sub-nuclear compartmentalization of MCM2 as cells pass through late G1 and early S phase, identifying a brief window when MCM2 becomes transiently attached to the nuclear-matrix. The data distinguish 3 states that correspond to loose association ...

  2. Chl12 (Ctf18) Forms a Novel Replication Factor C-Related Complex and Functions Redundantly with Rad24 in the DNA Replication Checkpoint Pathway

    OpenAIRE

    Naiki, Takahiro; Kondo, Tae; Nakada, Daisuke; Matsumoto, Kunihiro; Sugimoto, Katsunori

    2001-01-01

    RAD24 has been identified as a gene essential for the DNA damage checkpoint in budding yeast. Rad24 is structurally related to subunits of the replication factor C (RFC) complex, and forms an RFC-related complex with Rfc2, Rfc3, Rfc4, and Rfc5. The rad24Δ mutation enhances the defect of rfc5-1 in the DNA replication block checkpoint, implicating RAD24 in this checkpoint. CHL12 (also called CTF18) encodes a protein that is structurally related to the Rad24 and RFC proteins. We show here that a...

  3. 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

  4. Assembly, destruction and manipulation of atomic, molecular and complex systems

    International Nuclear Information System (INIS)

    Le Padellec, Arnaud Pierre Frederic

    2003-04-01

    In this report for Accreditation to Supervise Researches (HDR), the author first indicates his professional curriculum (diplomas, teaching activities, responsibilities in the field of education and research, publications), and then proposes a presentation of his scientific works and researches. He notably proposes an overview of the different experimental techniques he implemented: CRYRING storage ring, confluent beams, flow post-discharge with mass spectrometry and Langmuir probe, crossed beams, and so on. He reports works dealing with the manipulation and destruction of atomic, molecular and complex systems: detachment of atomic anions by electronic impact, detachment and dissociation of small carbon aggregates by electronic impact, dissociative recombination, dissociative ionisation and excitation, creation of pairs of ions, manipulation of sodium fluoride aggregates. He finally presents research projects regarding the assembly of molecular and complex systems

  5. Modeling self-assembly and phase behavior in complex mixtures.

    Science.gov (United States)

    Balazs, Anna C

    2007-01-01

    Using a variety of computational techniques, I investigate how the self-assembly of complex mixtures can be guided by surfaces or external stimuli to form spatially regular or temporally periodic patterns. Focusing on mixtures in confined geometries, I examine how thermodynamic and hydrodynamic effects can be exploited to create regular arrays of nanowires or monodisperse, particle-filled droplets. I also show that an applied light source and chemical reaction can be harnessed to create hierarchically ordered patterns in ternary, phase-separating mixtures. Finally, I consider the combined effects of confining walls and a chemical reaction to demonstrate that a swollen polymer gel can be driven to form dynamically periodic structures. In addition to illustrating the effectiveness of external factors in directing the self-organization of multicomponent mixtures, the selected examples illustrate how coarse-grained models can be used to capture both the equilibrium phase behavior and the dynamics of these complex systems.

  6. Synthesis and self-assembly of complex hollow materials

    KAUST Repository

    Zeng, Hua Chun

    2011-01-01

    Hollow materials with interiors or voids and pores are a class of lightweight nanostructured matters that promise many future technological applications, and they have received significant research attention in recent years. On the basis of well-known physicochemical phenomena and principles, for example, several solution-based protocols have been developed for the general preparation of these complex materials under mild reaction conditions. This article is thus a short introductory review on the synthetic aspects of this field of development. The synthetic methodologies can be broadly divided into three major categories: (i) template-assisted synthesis, (ii) self-assembly with primary building blocks, and (iii) induced matter relocations. In most cases, both synthesis and self-assembly are involved in the above processes. Further combinations of these methodologies appear to be very important, as they will allow one to prepare functional materials at a higher level of complexity and precision. The synthetic strategies are introduced through some simple case studies with schematic illustrations. Salient features of the methods developed have been summarized, and some urgent issues of this field have also been indicated. © 2011 The Royal Society of Chemistry.

  7. The brome mosaic virus 3' untranslated sequence regulates RNA replication, recombination, and virion assembly.

    Science.gov (United States)

    Rao, A L N; Cheng Kao, C

    2015-08-03

    The 3' untranslated region in each of the three genomic RNAs of Brome mosaic virus (BMV) is highly homologous and contains a sequence that folds into a tRNA-like structure (TLS). Experiments performed over the past four decades revealed that the BMV 3' TLS regulates many important steps in BMV infection. This review summarizes in vitro and in vivo studies of the roles of the BMV 3' TLS functioning as a minus-strand promoter, in RNA recombination, and to nucleate virion assembly. Copyright © 2015 Elsevier B.V. All rights reserved.

  8. Recruitment of Brd4 to the Human Papillomavirus Type 16 DNA Replication Complex Is Essential for Replication of Viral DNA

    OpenAIRE

    Wang, Xin; Helfer, Christine M.; Pancholi, Neha; Bradner, James E.; You, Jianxin

    2013-01-01

    Replication of the human papillomavirus (HPV) DNA genome relies on viral factors E1 and E2 and the cellular replication machinery. Bromodomain-containing protein 4 (Brd4) interacts with viral E2 protein to mediate papillomavirus (PV) genome maintenance and viral transcription. However, the functional role of Brd4 in the HPV life cycle remains to be clearly defined. In this study, we provide the first look into the E2-Brd4 interaction in the presence of other important viral factors, such as t...

  9. Stearoyl coenzyme A desaturase 1 is associated with hepatitis C virus replication complex and regulates viral replication

    DEFF Research Database (Denmark)

    Nguyen, LN; Lim, YS; Pham, Long

    2014-01-01

    The hepatitis C virus (HCV) life cycle is tightly regulated by lipid metabolism of host cells. In order to identify host factors involved in HCV propagation, we have recently screened a small interfering RNA (siRNA) library targeting host genes that control lipid metabolism and lipid droplet...... formation using cell culture-grown HCV (HCVcc)-infected cells. We selected and characterized the gene encoding stearoyl coenzyme A (CoA) desaturase 1 (SCD1). siRNA-mediated knockdown or pharmacological inhibition of SCD1 abrogated HCV replication in both subgenomic replicon and Jc1-infected cells, while...... exogenous supplementation of either oleate or palmitoleate, products of SCD1 activity, resurrected HCV replication in SCD1 knockdown cells. SCD1 was coimmunoprecipitated with HCV nonstructural proteins and colocalized with both double-stranded RNA (dsRNA) and HCV nonstructural proteins, indicating that SCD1...

  10. Stable assembly of HIV-1 export complexes occurs cotranscriptionally

    DEFF Research Database (Denmark)

    Nawroth, Isabel; Mueller, Florian; Basyuk, Eugenia

    2014-01-01

    The HIV-1 Rev protein mediates export of unspliced and singly spliced viral transcripts by binding to the Rev response element (RRE) and recruiting the cellular export factor CRM1. Here, we investigated the recruitment of Rev to the transcription sites of HIV-1 reporters that splice either post......- or cotranscriptionally. In both cases, we observed that Rev localized to the transcription sites of the reporters and recruited CRM1. Rev and CRM1 remained at the reporter transcription sites when cells were treated with the splicing inhibitor Spliceostatin A (SSA), showing that the proteins associate with RNA prior...... to or during early spliceosome assembly. Fluorescence recovery after photobleaching (FRAP) revealed that Rev and CRM1 have similar kinetics as the HIV-1 RNA, indicating that Rev, CRM1, and RRE-containing RNAs are released from the site of transcription in one single export complex. These results suggest...

  11. Tissue Engineering and Regenerative Strategies to Replicate Biocomplexity of Vascular Elastic Matrix Assembly

    OpenAIRE

    Bashur, Chris A.; Venkataraman, Lavanya; Ramamurthi, Anand

    2012-01-01

    Cardiovascular tissues exhibit architecturally complex extracellular matrices, of which the elastic matrix forms a major component. The elastic matrix critically maintains native structural configurations of vascular tissues, determines their ability to recoil after stretch, and regulates cell signaling pathways involved in morphogenesis, injury response, and inflammation via biomechanical transduction. The ability to tissue engineer vascular replacements that incorporate elastic matrix super...

  12. Structural mechanisms of DREAM complex assembly and regulation.

    Science.gov (United States)

    Guiley, Keelan Z; Liban, Tyler J; Felthousen, Jessica G; Ramanan, Parameshwaran; Litovchick, Larisa; Rubin, Seth M

    2015-05-01

    The DREAM complex represses cell cycle genes during quiescence through scaffolding MuvB proteins with E2F4/5 and the Rb tumor suppressor paralog p107 or p130. Upon cell cycle entry, MuvB dissociates from p107/p130 and recruits B-Myb and FoxM1 for up-regulating mitotic gene expression. To understand the biochemical mechanisms underpinning DREAM function and regulation, we investigated the structural basis for DREAM assembly. We identified a sequence in the MuvB component LIN52 that binds directly to the pocket domains of p107 and p130 when phosphorylated on the DYRK1A kinase site S28. A crystal structure of the LIN52-p107 complex reveals that LIN52 uses a suboptimal LxSxExL sequence together with the phosphate at nearby S28 to bind the LxCxE cleft of the pocket domain with high affinity. The structure explains the specificity for p107/p130 over Rb in the DREAM complex and how the complex is disrupted by viral oncoproteins. Based on insights from the structure, we addressed how DREAM is disassembled upon cell cycle entry. We found that p130 and B-Myb can both bind the core MuvB complex simultaneously but that cyclin-dependent kinase phosphorylation of p130 weakens its association. Together, our data inform a novel target interface for studying MuvB and p130 function and the design of inhibitors that prevent tumor escape in quiescence. © 2015 Guiley et al.; Published by Cold Spring Harbor Laboratory Press.

  13. Near-atomic structural model for bacterial DNA replication initiation complex and its functional insights.

    Science.gov (United States)

    Shimizu, Masahiro; Noguchi, Yasunori; Sakiyama, Yukari; Kawakami, Hironori; Katayama, Tsutomu; Takada, Shoji

    2016-12-13

    Upon DNA replication initiation in Escherichia coli, the initiator protein DnaA forms higher-order complexes with the chromosomal origin oriC and a DNA-bending protein IHF. Although tertiary structures of DnaA and IHF have previously been elucidated, dynamic structures of oriC-DnaA-IHF complexes remain unknown. Here, combining computer simulations with biochemical assays, we obtained models at almost-atomic resolution for the central part of the oriC-DnaA-IHF complex. This complex can be divided into three subcomplexes; the left and right subcomplexes include pentameric DnaA bound in a head-to-tail manner and the middle subcomplex contains only a single DnaA. In the left and right subcomplexes, DnaA ATPases associated with various cellular activities (AAA+) domain III formed helices with specific structural differences in interdomain orientations, provoking a bend in the bound DNA. In the left subcomplex a continuous DnaA chain exists, including insertion of IHF into the DNA looping, consistent with the DNA unwinding function of the complex. The intervening spaces in those subcomplexes are crucial for DNA unwinding and loading of DnaB helicases. Taken together, this model provides a reasonable near-atomic level structural solution of the initiation complex, including the dynamic conformations and spatial arrangements of DnaA subcomplexes.

  14. Integrated quality assurance for assembly and testing of complex structures

    Science.gov (United States)

    von Kopylow, Christoph; Bothe, Thorsten; Elandaloussi, Frank; Kalms, Michael; Jüptner, Werner

    2005-11-01

    Modern production processes are directed by properties of the components to be manufactured. These components have different sizes, functionalities, high assembly complexity and high security requirements. The increasing requirements during the manufacturing of complex products like cars and aircrafts demand new solutions for the quality assurance - especially for the production at different places. The main focus is to find a measurement strategy that is cost effective, flexible and adaptive. That means a clear definition of the measurement problem, the measurement with adapted resolution, the data preparation and evaluation and support during measurement and utilisation of the results directly in the production. In this paper we describe flexible measurement devices on example of three different techniques: fringe projection, fringe reflection and shearography. These techniques allow the detection of surface and subsurface defects like bumps, dents and delaminations with high resolution. The defects can be optically mapped onto the object's surface. Results are demonstrated with big components taken from automotive and aircraft production. We will point out the most important adaptations of the systems to realize miniaturized, robust and mobile devices for the quality assurance in an industrial environment. Additionally the implementation into a Mobile Maintenance and Control structure is demonstrated.

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

    Science.gov (United States)

    Cui, Hongguang; Wang, Aiming

    2016-05-15

    The potyviral RNA genome encodes two polyproteins that are proteolytically processed by three viral protease domains into 11 mature proteins. Extensive molecular studies have identified functions for the majority of the viral proteins. For example, 6K2, one of the two smallest potyviral proteins, is an integral membrane protein and induces the endoplasmic reticulum (ER)-originated replication vesicles that target the chloroplast for robust viral replication. However, the functional role of 6K1, the other smallest protein, remains uncharacterized. In this study, we developed a series of recombinant full-length viral cDNA clones derived from a Canadian Plum pox virus (PPV) isolate. We found that deletion of any of the short motifs of 6K1 (each of which ranged from 5 to 13 amino acids), most of the 6K1 sequence (but with the conserved sequence of the cleavage sites being retained), or all of the 6K1 sequence in the PPV infectious clone abolished viral replication. The trans expression of 6K1 or the cis expression of a dislocated 6K1 failed to rescue the loss-of-replication phenotype, suggesting the temporal and spatial requirement of 6K1 for viral replication. Disruption of the N- or C-terminal cleavage site of 6K1, which prevented the release of 6K1 from the polyprotein, either partially or completely inhibited viral replication, suggesting the functional importance of the mature 6K1. We further found that green fluorescent protein-tagged 6K1 formed punctate inclusions at the viral early infection stage and colocalized with chloroplast-bound viral replicase elements 6K2 and NIb. Taken together, our results suggest that 6K1 is required for viral replication and is an important viral element of the viral replication complex at the early infection stage. Potyviruses account for more than 30% of known plant viruses and consist of many agriculturally important viruses. The genomes of potyviruses encode two polyproteins that are proteolytically processed into 11 mature

  16. Heat Shock Protein 90 Ensures Efficient Mumps Virus Replication by Assisting with Viral Polymerase Complex Formation.

    Science.gov (United States)

    Katoh, Hiroshi; Kubota, Toru; Nakatsu, Yuichiro; Tahara, Maino; Kidokoro, Minoru; Takeda, Makoto

    2017-03-15

    Paramyxoviral RNAs are synthesized by a viral RNA-dependent RNA polymerase (RdRp) consisting of the large (L) protein and its cofactor phosphoprotein (P protein). The L protein is a multifunctional protein that catalyzes RNA synthesis, mRNA capping, and mRNA polyadenylation. Growing evidence shows that the stability of several paramyxovirus L proteins is regulated by heat shock protein 90 (Hsp90). In this study, we demonstrated that Hsp90 activity was important for mumps virus (MuV) replication. The Hsp90 activity was required for L-protein stability and activity because an Hsp90-specific inhibitor, 17-allylamino-17-demethoxygeldanamycin (17-AAG), destabilized the MuV L protein and suppressed viral RNA synthesis. However, once the L protein formed a mature polymerase complex with the P protein, Hsp90 activity was no longer required for the stability and activity of the L protein. When the Hsp90 activity was inhibited, the MuV L protein was degraded through the CHIP (C terminus of Hsp70-interacting protein)-mediated proteasomal pathway. High concentrations of 17-AAG showed strong cytotoxicity to certain cell types, but combined use of an Hsp70 inhibitor, VER155008, potentiated degradation of the L protein, allowing a sufficient reduction of 17-AAG concentration to block MuV replication with minimum cytotoxicity. Regulation of the L protein by Hsp90 and Hsp70 chaperones was also demonstrated for another paramyxovirus, the measles virus. Collectively, our data show that the Hsp90/Hsp70 chaperone machinery assists in the maturation of the paramyxovirus L protein and thereby in the formation of a mature RdRp complex and efficient viral replication. IMPORTANCE Heat shock protein 90 (Hsp90) is nearly universally required for viral protein homeostasis. Here, we report that Hsp90 activity is required for efficient propagation of mumps virus (MuV). Hsp90 functions in the maintenance of the catalytic subunit of viral polymerase, the large (L) protein, prior to formation of a

  17. Isolation of Endogenously Assembled RNA-Protein Complexes Using Affinity Purification Based on Streptavidin Aptamer S1

    Directory of Open Access Journals (Sweden)

    Yangchao Dong

    2015-09-01

    Full Text Available Efficient isolation of endogenously assembled viral RNA-protein complexes is essential for understanding virus replication mechanisms. We have developed an affinity purification strategy based on an RNA affinity tag that allows large-scale preparation of native viral RNA-binding proteins (RBPs. The streptavidin-binding aptamer S1 sequence was inserted into the 3′ end of dengue virus (DENV 5′–3′ UTR RNA, and the DENV RNA UTR fused to the S1 RNA aptamer was expressed in living mammalian cells. This allowed endogenous viral ribonucleoprotein (RNP assembly and isolation of RNPs from whole cell extract, through binding the S1 aptamer to streptavidin magnetic beads. Several novel host DENV RBPs were subsequently identified by liquid chromatography with tandem mass spectrometry (LC-MS/MS, including RPS8, which we further implicate in DENV replication. We proposed efficient S1 aptamer-based isolation of viral assembled RNPs from living mammalian cells will be generally applicable to the purification of high- and low-affinity RBPs and RNPs under endogenous conditions.

  18. 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.

  19. 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.

  20. The 20S proteasome as an assembly platform for the 19S regulatory complex

    DEFF Research Database (Denmark)

    Hendil, Klaus Aksel Bjørner; Kriegenburg, Franziska; Tanaka, Keiji

    2009-01-01

    26S proteasomes consist of cylindrical 20S proteasomes with 19S regulatory complexes attached to the ends. Treatment with high concentrations of salt causes the regulatory complexes to separate into two sub-complexes, the base, which is in contact with the 20S proteasome, and the lid, which...... is the distal part of the 19S complex. Here, we describe two assembly intermediates of the human regulatory complex. One is a dimer of the two ATPase subunits, Rpt3 and Rpt6. The other is a complex of nascent Rpn2, Rpn10, Rpn11, Rpn13, and Txnl1, attached to preexisting 20S proteasomes. This early assembly...... complex does not yet contain Rpn1 or any of the ATPase subunits of the base. Thus, assembly of 19S regulatory complexes takes place on preexisting 20S proteasomes, and part of the lid is assembled before the base....

  1. Proteomics reveals dynamic assembly of repair complexes during bypass of DNA cross-links

    DEFF Research Database (Denmark)

    Räschle, Markus; Smeenk, Godelieve; Hansen, Rebecca K

    2015-01-01

    a technique called chromatin mass spectrometry (CHROMASS) to study protein recruitment dynamics during perturbed DNA replication in Xenopus egg extracts. Using CHROMASS, we systematically monitored protein assembly and disassembly on ICL-containing chromatin. Among numerous prospective DNA repair factors, we...... to damaged DNA in vertebrate cells....

  2. Fabrication of complex structures or assemblies by Hot Isostatic Pressure (HIP) welding

    Science.gov (United States)

    Ashurst, A. N.; Goldstein, M.; Ryan, M. J.; Lessmann, G. G.; Bryant, W. A.

    1974-01-01

    HIP welding is effective method for fabricating complex structures or assemblies such as alternator rotors, regeneratively-cooled rocket-motor thrust chambers, and jet engine turbine blades. It can be applied to fabrication of many assemblies which require that component parts be welded together along complex interfaces.

  3. Rab11A-controlled assembly of the inner membrane complex is required for completion of apicomplexan cytokinesis.

    Directory of Open Access Journals (Sweden)

    Carolina Agop-Nersesian

    2009-01-01

    Full Text Available The final step during cell division is the separation of daughter cells, a process that requires the coordinated delivery and assembly of new membrane to the cleavage furrow. While most eukaryotic cells replicate by binary fission, replication of apicomplexan parasites involves the assembly of daughters (merozoites/tachyzoites within the mother cell, using the so-called Inner Membrane Complex (IMC as a scaffold. After de novo synthesis of the IMC and biogenesis or segregation of new organelles, daughters bud out of the mother cell to invade new host cells. Here, we demonstrate that the final step in parasite cell division involves delivery of new plasma membrane to the daughter cells, in a process requiring functional Rab11A. Importantly, Rab11A can be found in association with Myosin-Tail-Interacting-Protein (MTIP, also known as Myosin Light Chain 1 (MLC1, a member of a 4-protein motor complex called the glideosome that is known to be crucial for parasite invasion of host cells. Ablation of Rab11A function results in daughter parasites having an incompletely formed IMC that leads to a block at a late stage of cell division. A similar defect is observed upon inducible expression of a myosin A tail-only mutant. We propose a model where Rab11A-mediated vesicular traffic driven by an MTIP-Myosin motor is necessary for IMC maturation and to deliver new plasma membrane to daughter cells in order to complete cell division.

  4. Mouse norovirus 1 utilizes the cytoskeleton network to establish localization of the replication complex proximal to the microtubule organizing center.

    Science.gov (United States)

    Hyde, Jennifer L; Gillespie, Leah K; Mackenzie, Jason M

    2012-04-01

    Human noroviruses (family Caliciviridae) are the leading cause of nonbacterial gastroenteritis worldwide. Although Human noroviruses are significant enteric pathogens, there exists no reliable vaccine or therapy to treat infected individuals. To date, attempts to cultivate Human noroviruses within the laboratory have met with little success; however, the related murine norovirus mouse norovirus 1 (MNV-1) has provided an ideal model system to study norovirus replication due to the ease with which the virus is cultivated and the ability to infect a small animal model with this virus. Previously we have identified the association between MNV-1 and components of the host secretory pathway and proposed a role for the viral open reading frame 1 proteins in the replication cycle. Here we describe for the first time a role for cytoskeletal components in early MNV-1 replication events. We show that the MNV-1 utilizes microtubules to position the replication complex adjacent to the microtubule organizing center. Chemical disruption of the microtubule network disperses the sites of MNV-1 replication throughout the cell and impairs production of viral protein and infectious virus. Furthermore, we demonstrate the ability of MNV-1 to redistribute acetylated tubulin to the replication complex and that this association is potentially mediated via the MNV-1 major structural protein, VP1. Transient expression of MNV-1 VP1 exhibited extensive colocalization with both α-tubulin and acetylated tubulin and was observed to alter the distribution of acetylated tubulin in transfected cells. This study highlights the role of the cytoskeleton in early virus replication events and demonstrates the importance of this interaction in establishing the intracellular location of MNV-1 replication complexes.

  5. 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.

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

    Directory of Open Access Journals (Sweden)

    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. Human mitochondrial complex I assembles through the combination of evolutionary conserved modules: a framework to interpret complex I deficiencies.

    NARCIS (Netherlands)

    Ugalde, C.; Vogel, R.O.; Huijbens, R.J.F.; Heuvel, L.P.W.J. van den; Smeitink, J.A.M.; Nijtmans, L.G.J.

    2004-01-01

    With 46 subunits, human mitochondrial complex I is the largest enzyme of the oxidative phosphorylation system. We have studied the assembly of complex I in cultured human cells. This will provide essential information about the nature of complex I deficiencies and will enhance our understanding of

  8. Moleculo Long-Read Sequencing Facilitates Assembly and Genomic Binning from Complex Soil Metagenomes

    Energy Technology Data Exchange (ETDEWEB)

    White, Richard Allen; Bottos, Eric M.; Roy Chowdhury, Taniya; Zucker, Jeremy D.; Brislawn, Colin J.; Nicora, Carrie D.; Fansler, Sarah J.; Glaesemann, Kurt R.; Glass, Kevin; Jansson, Janet K.; Langille, Morgan

    2016-06-28

    ABSTRACT

    Soil metagenomics has been touted as the “grand challenge” for metagenomics, as the high microbial diversity and spatial heterogeneity of soils make them unamenable to current assembly platforms. Here, we aimed to improve soil metagenomic sequence assembly by applying the Moleculo synthetic long-read sequencing technology. In total, we obtained 267 Gbp of raw sequence data from a native prairie soil; these data included 109.7 Gbp of short-read data (~100 bp) from the Joint Genome Institute (JGI), an additional 87.7 Gbp of rapid-mode read data (~250 bp), plus 69.6 Gbp (>1.5 kbp) from Moleculo sequencing. The Moleculo data alone yielded over 5,600 reads of >10 kbp in length, and over 95% of the unassembled reads mapped to contigs of >1.5 kbp. Hybrid assembly of all data resulted in more than 10,000 contigs over 10 kbp in length. We mapped three replicate metatranscriptomes derived from the same parent soil to the Moleculo subassembly and found that 95% of the predicted genes, based on their assignments to Enzyme Commission (EC) numbers, were expressed. The Moleculo subassembly also enabled binning of >100 microbial genome bins. We obtained via direct binning the first complete genome, that of “CandidatusPseudomonas sp. strain JKJ-1” from a native soil metagenome. By mapping metatranscriptome sequence reads back to the bins, we found that several bins corresponding to low-relative-abundanceAcidobacteriawere highly transcriptionally active, whereas bins corresponding to high-relative-abundanceVerrucomicrobiawere not. These results demonstrate that Moleculo sequencing provides a significant advance for resolving complex soil microbial communities.

    IMPORTANCESoil microorganisms carry out key processes for life on our planet, including cycling of carbon and other nutrients and supporting growth of plants. However, there is poor molecular-level understanding of their

  9. Degradation of the mitochondrial complex I assembly factor TMEM126B under chronic hypoxia.

    Science.gov (United States)

    Fuhrmann, Dominik C; Wittig, Ilka; Dröse, Stefan; Schmid, Tobias; Dehne, Nathalie; Brüne, Bernhard

    2018-02-20

    Cell stress such as hypoxia elicits adaptive responses, also on the level of mitochondria, and in part is mediated by the hypoxia-inducible factor (HIF) 1α. Adaptation of mitochondria towards acute hypoxic conditions is reasonably well understood, while regulatory mechanisms, especially of respiratory chain assembly factors, under chronic hypoxia remains elusive. One of these assembly factors is transmembrane protein 126B (TMEM126B). This protein is part of the mitochondrial complex I assembly machinery. We identified changes in complex I abundance under chronic hypoxia, in association with impaired substrate-specific mitochondrial respiration. Complexome profiling of isolated mitochondria of the human leukemia monocytic cell line THP-1 revealed HIF-1α-dependent deficits in complex I assembly and mitochondrial complex I assembly complex (MCIA) abundance. Of all mitochondrial MCIA members, we proved a selective HIF-1-dependent decrease of TMEM126B under chronic hypoxia. Mechanistically, HIF-1α induces the E3-ubiquitin ligase F-box/WD repeat-containing protein 1A (β-TrCP1), which in turn facilitates the proteolytic degradation of TMEM126B. Attenuating a functional complex I assembly appears critical for cellular adaptation towards chronic hypoxia and is linked to destruction of the mitochondrial assembly factor TMEM126B.

  10. Giant capsids from lattice self-assembly of cyclodextrin complexes

    NARCIS (Netherlands)

    Yang, Shenyu; Yan, Yun; Huang, Jianbin; Petukhov, Andrei V.; Kroon - Batenburg, Loes M. J.; Drechsler, Markus; Zhou, Chengcheng; Tu, Mei; Granick, Steve; Jiang, Lingxiang

    2017-01-01

    Proteins can readily assemble into rigid, crystalline and functional structures such as viral capsids and bacterial compartments. Despite ongoing advances, it is still a fundamental challenge to design and synthesize protein-mimetic molecules to form crystalline structures. Here we report the

  11. 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...

  12. Self-Assembly of Mono- and Dinuclear Metal Complexes; Oxidation Catalysis and Metalloenzyme Models

    NARCIS (Netherlands)

    Gelling, Onko-Jan; Rispens, Minze T.; Lubben, Marcel; Feringa, Bernard

    1994-01-01

    In this chapter several approaches to achieve assembly of mono- and dinuclear metal complexes, which can be considered structural and functional models for metalloenzymes, are described. The emphasis lies on oxidation chemistry, summarizing O2 binding, hydroxylation, demethylation, dehalogenation

  13. Mutation of C20orf7 Disrupts Complex I Assembly and Causes Lethal Neonatal Mitochondrial Disease

    NARCIS (Netherlands)

    Sugiana, Canny; Pagliarini, David J.; McKenzie, Matthew; Kirby, Denise M.; Salemi, Renato; Abu-Amero, Khaled K.; Dahl, Hans-Henrik M.; Hutchison, Wendy M.; Vascotto, Katherine A.; Smith, Stacey M.; Newbold, Robert F.; Christodoulou, John; Calvo, Sarah; Mootha, Vamsi K.; Ryan, Michael T.; Thorburn, David R.

    2008-01-01

    Complex I (NADH:ubiquinone oxidoreductase) is the first and largest multimeric complex of the mitochondrial respiratory chain. Human complex I comprises seven Subunits encoded by mitochondrial DNA and 38 nuclear-encoded subunits that are assembled together in a process that is only partially

  14. Directed assembly of functional light harvesting antenna complexes onto chemically patterned surfaces

    International Nuclear Information System (INIS)

    Escalante, Maryana; Maury, Pascale; Bruinink, Christiaan M; Werf, Kees van der; Olsen, John D; Timney, John A; Huskens, Jurriaan; Hunter, C Neil; Subramaniam, Vinod; Otto, Cees

    2008-01-01

    We report the directed assembly of the photosynthetic membrane proteins LH1 and LH2 isolated from the purple bacterium Rhodobacter sphaeroides onto chemically patterned substrates. Nanoimprint lithography was used to pattern discrete regions of amino- and fluoro-terminated or poly(ethylene glycol) self-assembled monolayers onto a glass substrate. Densely packed layers of assembled protein complexes were observed with atomic force microscopy. The protein complexes attached selectively to the amino-terminated regions by electrostatic interactions. Spectral images generated with a hybrid scanning probe and fluorescence microscope confirmed that the patterned proteins retained their native optical signatures

  15. The Ctf18 RFC-like complex positions yeast telomeres but does not specify their replication time.

    Science.gov (United States)

    Hiraga, Shin-ichiro; Robertson, E Douglas; Donaldson, Anne D

    2006-04-05

    Chromosome ends in Saccharomyces cerevisiae are positioned in clusters at the nuclear rim. We report that Ctf18, Ctf8, and Dcc1, the subunits of a Replication Factor C (RFC)-like complex, are essential for the perinuclear positioning of telomeres. In both yeast and mammalian cells, peripheral nuclear positioning of chromatin during G1 phase correlates with late DNA replication. We find that the mislocalized telomeres of ctf18 cells still replicate late, showing that late DNA replication does not require peripheral positioning during G1. The Ku and Sir complexes have been shown to act through separate pathways to position telomeres, but in the absence of Ctf18 neither pathway can act fully to maintain telomere position. Surprisingly CTF18 is not required for Ku or Sir4-mediated peripheral tethering of a nontelomeric chromosome locus. Our results suggest that the Ctf18 RFC-like complex modifies telomeric chromatin to make it competent for normal localization to the nuclear periphery.

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

    Directory of Open Access Journals (Sweden)

    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.

  17. 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.

  18. Dissecting the sequential assembly and localization of intraflagellar transport particle complex B in Chlamydomonas.

    Directory of Open Access Journals (Sweden)

    Elizabeth A Richey

    Full Text Available Intraflagellar transport (IFT, the key mechanism for ciliogenesis, involves large protein particles moving bi-directionally along the entire ciliary length. IFT particles contain two large protein complexes, A and B, which are constructed with proteins in a core and several peripheral proteins. Prior studies have shown that in Chlamydomonas reinhardtii, IFT46, IFT52, and IFT88 directly interact with each other and are in a subcomplex of the IFT B core. However, ift46, bld1, and ift88 mutants differ in phenotype as ift46 mutants are able to form short flagella, while the other two lack flagella completely. In this study, we investigated the functional differences of these individual IFT proteins contributing to complex B assembly, stability, and basal body localization. We found that complex B is completely disrupted in bld1 mutant, indicating an essential role of IFT52 for complex B core assembly. Ift46 mutant cells are capable of assembling a relatively intact complex B, but such complex is highly unstable and prone to degradation. In contrast, in ift88 mutant cells the complex B core still assembles and remains stable, but the peripheral proteins no longer attach to the B core. Moreover, in ift88 mutant cells, while complex A and the anterograde IFT motor FLA10 are localized normally to the transition fibers, complex B proteins instead are accumulated at the proximal ends of the basal bodies. In addition, in bld2 mutant, the IFT complex B proteins still localize to the proximal ends of defective centrioles which completely lack transition fibers. Taken together, these results revealed a step-wise assembly process for complex B, and showed that the complex first localizes to the proximal end of the centrioles and then translocates onto the transition fibers via an IFT88-dependent mechanism.

  19. Extremophile Poeciliidae: multivariate insights into the complexity of speciation along replicated ecological gradients.

    Science.gov (United States)

    Riesch, Rüdiger; Tobler, Michael; Lerp, Hannes; Jourdan, Jonas; Doumas, Tess; Nosil, Patrik; Langerhans, R Brian; Plath, Martin

    2016-06-22

    Replicate population pairs that diverge in response to similar selective regimes allow for an investigation of (a) whether phenotypic traits diverge in a similar and predictable fashion, (b) whether there is gradual variation in phenotypic divergence reflecting variation in the strength of natural selection among populations, (c) whether the extent of this divergence is correlated between multiple character suites (i.e., concerted evolution), and (d) whether gradual variation in phenotypic divergence predicts the degree of reproductive isolation, pointing towards a role for adaptation as a driver of (ecological) speciation. Here, we use poeciliid fishes of the genera Gambusia and Poecilia that have repeatedly evolved extremophile lineages able to tolerate high and sustained levels of toxic hydrogen sulfide (H2S) to answer these questions. We investigated evolutionary divergence in response to H2S in Gambusia spp. (and to a lesser extent Poecilia spp.) using a multivariate approach considering the interplay of life history, body shape, and population genetics (nuclear miscrosatellites to infer population genetic differentiation as a proxy for reproductive isolation). We uncovered both shared and unique patterns of evolution: most extremophile Gambusia predictably evolved larger heads and offspring size, matching a priori predictions for adaptation to sulfidic waters, while variation in adult life histories was idiosyncratic. When investigating patterns for both genera (Gambusia and Poecilia), we found that divergence in offspring-related life histories and body shape were positively correlated across populations, but evidence for individual-level associations between the two character suites was limited, suggesting that genetic linkage, developmental interdependencies, or pleiotropic effects do not explain patterns of concerted evolution. We further found that phenotypic divergence was positively correlated with both environmental H2S-concentration and neutral

  20. Plasticity of TOM complex assembly in skeletal muscle mitochondria in response to chronic contractile activity.

    Science.gov (United States)

    Joseph, Anna-Maria; Hood, David A

    2012-03-01

    We investigated the assembly of the TOM complex within skeletal muscle under conditions of chronic contractile activity-induced mitochondrial biogenesis. Tom40 import into mitochondria was increased by chronic contractile activity, as was its time-dependent assembly into the TOM complex. These changes coincided with contractile activity-induced augmentations in the expression of key protein import machinery components Tim17, Tim23, and Tom22, as well as the cytosolic chaperone Hsp90. These data indicate the adaptability of the TOM protein import complex and suggest a regulatory role for the assembly of this complex in exercise-induced mitochondrial biogenesis. Copyright © 2011 Elsevier B.V. and Mitochondria Research Society. All rights reserved. All rights reserved.

  1. A Bottleneck Detection Algorithm for Complex Product Assembly Line Based on Maximum Operation Capacity

    Directory of Open Access Journals (Sweden)

    Dongping Zhao

    2014-01-01

    Full Text Available Because of the complex constraints in complex product assembly line, existing algorithms not always detect bottleneck correctly and they have a low convergence rate. In order to solve this problem, a hybrid algorithm of adjacency matrix and improved genetic algorithm (GA was proposed. First, complex assembly network model (CANM was defined based on operation capacity of each workstation. Second, adjacency matrix was proposed to convert bottleneck detection of complex assembly network (CAN into a combinatorial optimization problem of max-flow. Third, an improved GA was proposed to solve this max-flow problem by retaining the best chromosome. Finally, the min-cut sets of CAN were obtained after calculation, and bottleneck workstations were detected according to the analysis of min-cut sets. A case study shows that this algorithm can detect bottlenecks correctly and its convergence rate is high.

  2. Synthesis, characterization and self-assembly of Co complexes ...

    Indian Academy of Sciences (India)

    [Co(H2O)6](ClO4)2. The subsequent deprotection with. BBr3 provides complexes 1-3 with free phenol and cat- echol groups.3 Interestingly, during deprotection; one of the bidentate ligands was protonated and coordi- nated via neutral Oamide atom with intact N-H group. (scheme 1). We believe that the generation of HBr.

  3. A chiral Mn (IV) complex and its supramolecular assembly ...

    Indian Academy of Sciences (India)

    Singlecrystal X-ray analysis revealed that compound 1 crystallises in the monoclinic 21 space group with six mononuclear [MnIVL2] units in the asymmetric unit along with three solvent DMF molecules. In the crystal structure, each Mn(IV) complex, acting as the building unit, undergoes supramolecular linking through C-H ...

  4. Conserved residues in the delta subunit help the E. coli clamp loader, gamma complex, target primer-template DNA for clamp assembly.

    Science.gov (United States)

    Chen, Siying; Coman, Maria Magdalena; Sakato, Miho; O'Donnell, Michael; Hingorani, Manju M

    2008-06-01

    The Escherichia coli clamp loader, gamma complex (gamma(3)deltadelta'lambdapsi), catalyzes ATP-driven assembly of beta clamps onto primer-template DNA (p/tDNA), enabling processive replication. The mechanism by which gamma complex targets p/tDNA for clamp assembly is not resolved. According to previous studies, charged/polar amino acids inside the clamp loader chamber interact with the double-stranded (ds) portion of p/tDNA. We find that dsDNA, not ssDNA, can trigger a burst of ATP hydrolysis by gamma complex and clamp assembly, but only at far higher concentrations than p/tDNA. Thus, contact between gamma complex and dsDNA is necessary and sufficient, but not optimal, for the reaction, and additional contacts with p/tDNA likely facilitate its selection as the optimal substrate for clamp assembly. We investigated whether a conserved sequence-HRVW(279)QNRR--in delta subunit contributes to such interactions, since Tryptophan-279 specifically cross-links to the primer-template junction. Mutation of delta-W279 weakens gamma complex binding to p/tDNA, hampering its ability to load clamps and promote proccessive DNA replication, and additional mutations in the sequence (delta-R277, delta-R283) worsen the interaction. These data reveal a novel location in the C-terminal domain of the E. coli clamp loader that contributes to DNA binding and helps define p/tDNA as the preferred substrate for the reaction.

  5. Proline residues in the HIV-1 NH2-terminal capsid domain: structure determinants for proper core assembly and subsequent steps of early replication.

    Science.gov (United States)

    Fitzon, T; Leschonsky, B; Bieler, K; Paulus, C; Schröder, J; Wolf, H; Wagner, R

    2000-03-15

    Recent analyses suggest that the p24 capsid (p24(CA)) domain of the HIV-1 group-specific antigen (Gag) may be divided into two structurally and functionally distinct moieties: (i) an amino-terminal portion, previously shown to bind the cellular chaperone cyclophilin A, and (ii) a carboxy-terminal domain, known to contribute to the interaction of the Gag and Gag-Pol precursors during the early assembly process. In order to gain deeper insight into the role of the amino-terminal domain of the p24(CA) protein during viral replication, eight highly conserved proline residues known to promote turns and to terminate alpha-helices within the p24 tertiary structure were replaced by a leucine residue (P-position-L). Following transfection of the proviral constructs in COS7 cells, the majority of the mutants resembled wild-type viruses with respect to the assembly and release of virions. However, although the released particles contained wild-type levels of genomic viral RNA, the mature products of the Gag and Gag-Pol polyproteins as well as the Env glycoproteins-all of them, except mutant P225L-were either noninfectious or severely affected in their replicative capacity. Entry assays monitoring the process of viral DNA synthesis led to the classification of selected provirus mutants into four different phenotypes: (i) mutant P225L was infectious and allowed complete reverse transcription including formation of 2-LTR circles; (ii) mutants P149L, P170L, and P217L failed to form 2-LTR circles; (iii) mutant P222L displayed a severe defect in binding and incorporating cyclophilin A into virions, was delayed with respect to DNA polymerization, and failed to form a 2-LTR replication intermediate; and (iv) mutant P133L was unable even to synthesize a first-strand cDNA product. All replication-defective mutants were characterized by severe alterations in the stability of virion cores, which were in two cases reflected by visible changes in the core morphology. These results suggest

  6. Folding and assembly of large macromolecular complexes monitored by hydrogen-deuterium exchange and mass spectrometry

    Directory of Open Access Journals (Sweden)

    Tuma Roman

    2008-04-01

    Full Text Available Abstract Recent advances in protein mass spectrometry (MS have enabled determinations of hydrogen deuterium exchange (HDX in large macromolecular complexes. HDX-MS became a valuable tool to follow protein folding, assembly and aggregation. The methodology has a wide range of applications in biotechnology ranging from quality control for over-expressed proteins and their complexes to screening of potential ligands and inhibitors. This review provides an introduction to protein folding and assembly followed by the principles of HDX and MS detection, and concludes with selected examples of applications that might be of interest to the biotechnology community.

  7. Self-assembly of biomimetic light-harvesting complexes capable of hydrogen evolution

    Directory of Open Access Journals (Sweden)

    Kai Liu

    2017-01-01

    Full Text Available Biomimetics provides us a new perspective to understand complex biological process and strategy to fabricate functional materials. However, a great challenge still remains to design and fabricate biomimetic materials using a facile but effective method. Here, we develop a biomimetic light harvesting architecture based on one-step co-assembly of amphiphilic amino acid and porphyrin. Amphiphilic amino acid can self-assemble into nanofibers via π-stacking and hydrogen binding interactions. Negatively charged porphyrin adsorbs on the surface of the assembled nanofibers through electrostatic force, and the nanofibers further organize into porous urchin-like microspheres induced presumably by hydrophobic interaction. The assembled amphiphilic amino acid nanofibers work as a template to tune the organization of porphyrin with an architecture principle analogous to natural light harvesting complex. The co-assembled microspheres exhibit enhanced light capture due to the light reflection in the porous structure. Reaction center (platinum nanoparticles can be effectively coupled with the light harvesting microspheres via photoreduction. After visible light illumination, hydrogen evolution occurs on the hybrid microspheres. Keywords: Light-harvesting, Amino acid, Porphyrin, Co-assembly, Hydrogen evolution

  8. NDUFAF5 Hydroxylates NDUFS7 at an Early Stage in the Assembly of Human Complex I*

    Science.gov (United States)

    Rhein, Virginie F.; Carroll, Joe; Ding, Shujing; Fearnley, Ian M.; Walker, John E.

    2016-01-01

    Complex I (NADH ubiquinone oxidoreductase) in mammalian mitochondria is an L-shaped assembly of 45 proteins. One arm lies in the inner membrane, and the other extends about 100 Å into the matrix of the organelle. The extrinsic arm contains binding sites for NADH, the primary electron acceptor FMN, and seven iron-sulfur clusters that form a pathway for electrons linking FMN to the terminal electron acceptor, ubiquinone, which is bound in a tunnel in the region of the junction between the arms. The membrane arm contains four antiporter-like domains, energetically coupled to the quinone site and involved in pumping protons from the matrix into the intermembrane space contributing to the proton motive force. Seven of the subunits, forming the core of the membrane arm, are translated from mitochondrial genes, and the remaining subunits, the products of nuclear genes, are imported from the cytosol. Their assembly is coordinated by at least thirteen extrinsic assembly factor proteins that are not part of the fully assembled complex. They assist in insertion of co-factors and in building up the complex from smaller sub-assemblies. One such factor, NDUFAF5, belongs to the family of seven-β-strand S-adenosylmethionine-dependent methyltransferases. However, similar to another family member, RdmB, it catalyzes the introduction of a hydroxyl group, in the case of NDUFAF5, into Arg-73 in the NDUFS7 subunit of human complex I. This modification occurs early in the pathway of assembly of complex I, before the formation of the juncture between peripheral and membrane arms. PMID:27226634

  9. NDUFAF5 Hydroxylates NDUFS7 at an Early Stage in the Assembly of Human Complex I.

    Science.gov (United States)

    Rhein, Virginie F; Carroll, Joe; Ding, Shujing; Fearnley, Ian M; Walker, John E

    2016-07-08

    Complex I (NADH ubiquinone oxidoreductase) in mammalian mitochondria is an L-shaped assembly of 45 proteins. One arm lies in the inner membrane, and the other extends about 100 Å into the matrix of the organelle. The extrinsic arm contains binding sites for NADH, the primary electron acceptor FMN, and seven iron-sulfur clusters that form a pathway for electrons linking FMN to the terminal electron acceptor, ubiquinone, which is bound in a tunnel in the region of the junction between the arms. The membrane arm contains four antiporter-like domains, energetically coupled to the quinone site and involved in pumping protons from the matrix into the intermembrane space contributing to the proton motive force. Seven of the subunits, forming the core of the membrane arm, are translated from mitochondrial genes, and the remaining subunits, the products of nuclear genes, are imported from the cytosol. Their assembly is coordinated by at least thirteen extrinsic assembly factor proteins that are not part of the fully assembled complex. They assist in insertion of co-factors and in building up the complex from smaller sub-assemblies. One such factor, NDUFAF5, belongs to the family of seven-β-strand S-adenosylmethionine-dependent methyltransferases. However, similar to another family member, RdmB, it catalyzes the introduction of a hydroxyl group, in the case of NDUFAF5, into Arg-73 in the NDUFS7 subunit of human complex I. This modification occurs early in the pathway of assembly of complex I, before the formation of the juncture between peripheral and membrane arms. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  10. Living supramolecular polymerization achieved by collaborative assembly of platinum(II) complexes and block copolymers.

    Science.gov (United States)

    Zhang, Kaka; Yeung, Margaret Ching-Lam; Leung, Sammual Yu-Lut; Yam, Vivian Wing-Wah

    2017-11-07

    An important feature of biological systems to achieve complexity and precision is the involvement of multiple components where each component plays its own role and collaborates with other components. Mimicking this, we report living supramolecular polymerization achieved by collaborative assembly of two structurally dissimilar components, that is, platinum(II) complexes and poly(ethylene glycol)- b -poly(acrylic acid) (PEG- b -PAA). The PAA blocks neutralize the charges of the platinum(II) complexes, with the noncovalent metal-metal and π-π interactions directing the longitudinal growth of the platinum(II) complexes into 1D crystalline nanostructures, and the PEG blocks inhibiting the transverse growth of the platinum(II) complexes and providing the whole system with excellent solubility. The ends of the 1D crystalline nanostructures have been found to be active during the assembly and remain active after the assembly. One-dimensional segmented nanostructures with heterojunctions have been produced by sequential growth of two types of platinum(II) complexes. The PAA blocks act as adapters at the heterojunctions for lattice matching between chemically and crystallographically different platinum(II) complexes, achieving heterojunctions with a lattice mismatch as large as 21%. Published under the PNAS license.

  11. Mechanical Link between Cohesion Establishment and DNA Replication: Ctf7p/Eco1p, a Cohesion Establishment Factor, Associates with Three Different Replication Factor C Complexes

    OpenAIRE

    Kenna, Margaret A.; Skibbens, Robert V.

    2003-01-01

    CTF7/ECO1 is an essential yeast gene required for the establishment of sister chromatid cohesion. The findings that CTF7/ECO1, POL30 (PCNA), and CHL12/CTF18 (a replication factor C [RFC] homolog) genetically interact provided the first evidence that the processes of cohesion establishment and DNA replication are intimately coupled—a link now confirmed by other studies. To date, however, it is unknown how Ctf7p/Eco1p function is coupled to DNA replication or whether Ctf7p/Eco1p physically asso...

  12. Complex shapes self-assembled from single-stranded DNA tiles.

    Science.gov (United States)

    Wei, Bryan; Dai, Mingjie; Yin, Peng

    2012-05-30

    Programmed self-assembly of strands of nucleic acid has proved highly effective for creating a wide range of structures with desired shapes. A particularly successful implementation is DNA origami, in which a long scaffold strand is folded by hundreds of short auxiliary strands into a complex shape. Modular strategies are in principle simpler and more versatile and have been used to assemble DNA or RNA tiles into periodic and algorithmic two-dimensional lattices, extended ribbons and tubes, three-dimensional crystals, polyhedra and simple finite two-dimensional shapes. But creating finite yet complex shapes from a large number of uniquely addressable tiles remains challenging. Here we solve this problem with the simplest tile form, a 'single-stranded tile' (SST) that consists of a 42-base strand of DNA composed entirely of concatenated sticky ends and that binds to four local neighbours during self-assembly. Although ribbons and tubes with controlled circumferences have been created using the SST approach, we extend it to assemble complex two-dimensional shapes and tubes from hundreds (in some cases more than one thousand) distinct tiles. Our main design feature is a self-assembled rectangle that serves as a molecular canvas, with each of its constituent SST strands--folded into a 3 nm-by-7 nm tile and attached to four neighbouring tiles--acting as a pixel. A desired shape, drawn on the canvas, is then produced by one-pot annealing of all those strands that correspond to pixels covered by the target shape; the remaining strands are excluded. We implement the strategy with a master strand collection that corresponds to a 310-pixel canvas, and then use appropriate strand subsets to construct 107 distinct and complex two-dimensional shapes, thereby establishing SST assembly as a simple, modular and robust framework for constructing nanostructures with prescribed shapes from short synthetic DNA strands.

  13. Promotion of mitochondrial membrane complex assembly by a proteolytically inactive yeast Lon

    NARCIS (Netherlands)

    Rep, M; van Dijl, J M; Suda, K; Schatz, G; Grivell, L A; Suzuki, C K

    1996-01-01

    Afg3p and Rca1p are adenosine triphosphate (ATP)-dependent metalloproteases in yeast mitochondria. Cells lacking both proteins exhibit defects in respiration-dependent growth, degradation of mitochondrially synthesized proteins, and assembly of inner-membrane complexes. Defects in growth and protein

  14. Self-assembly of α-helical polypeptides driven by complex coacervation.

    Science.gov (United States)

    Priftis, Dimitrios; Leon, Lorraine; Song, Ziyuan; Perry, Sarah L; Margossian, Khatcher O; Tropnikova, Anna; Cheng, Jianjun; Tirrell, Matthew

    2015-09-14

    Reported is the ability of α-helical polypeptides to self-assemble with oppositely-charged polypeptides to form liquid complexes while maintaining their α-helical secondary structure. Coupling the α-helical polypeptide to a neutral, hydrophilic polymer and subsequent complexation enables the formation of nanoscale coacervate-core micelles. While previous reports on polypeptide complexation demonstrated a critical dependence of the nature of the complex (liquid versus solid) on chirality, the α-helical structure of the positively charged polypeptide prevents the formation of β-sheets, which would otherwise drive the assembly into a solid state, thereby, enabling coacervate formation between two chiral components. The higher charge density of the assembly, a result of the folding of the α-helical polypeptide, provides enhanced resistance to salts known to inhibit polypeptide complexation. The unique combination of properties of these materials can enhance the known potential of fluid polypeptide complexes for delivery of biologically relevant molecules. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  15. Genetic Analysis of Serum-Derived Defective Hepatitis C Virus Genomes Revealed Novel ViralcisElements for Virus Replication and Assembly.

    Science.gov (United States)

    Li, Qingchao; Tong, Yimin; Xu, Yongfen; Niu, Junqi; Zhong, Jin

    2018-04-01

    Defective viral genomes (DVGs) of hepatitis C virus (HCV) exist, but their biological significances have not been thoroughly investigated. Here, we analyzed HCV DVGs circulating in patient sera that possess deletions in the structural protein-encoding region. About 30% of 41 HCV clinical isolates possess DVGs that originated from the full-length genome in the same patients. No correlation between DVGs, viremia, and alanine aminotransferase (ALT) levels was found. Sequencing analysis of DVGs revealed the existence of deletion hot spots, with upstream sites in E1 and downstream sites in E2 and NS2. Interestingly, the coding sequences for the core protein and the C-terminal protease domain of NS2 were always intact in DVGs despite the fact that both proteins are dispensable for HCV genome replication. Mechanistic studies showed that transmembrane segment 3 (TMS3) of NS2, located immediately upstream of its protease domain, was required for the cleavage of NS2-NS3 and the replication of DVGs. Moreover, we identified a highly conserved secondary structure (SL750) within the core domain 2-coding region that is critical for HCV genome packaging. In summary, our analysis of serum-derived HCV DVGs revealed novel viral cis elements that play important roles in virus replication and assembly. IMPORTANCE HCV DVGs have been identified in vivo and in vitro , but their biogenesis and physiological significances remain elusive. In addition, a conventional packaging signal has not yet been identified on the HCV RNA genome, and mechanisms underlying the specificity in the encapsidation of the HCV genome into infectious particles remain to be uncovered. Here, we identified new viral cis elements critical for the HCV life cycle by determining genetic constraints that define the boundary of serum-derived HCV DVGs. We found that transmembrane segment 3 of NS2, located immediately upstream of its protease domain, was required for the cleavage of NS2-NS3 and the replication of DVGs. We

  16. Thermodynamic and Kinetic Control of Charged Triblock Copolymer Assembly into Complex Nanostructures

    Science.gov (United States)

    Cui, Honggang; Pochan, Darrin; Chen, Zhiyun; Wooley, Karen

    2007-03-01

    Self-assembly of poly (acrylic acid)-block-poly (methyl acrylate)-block-polystyrene triblock copolymers produces various ordered nano-domains in THF/water solution through the interaction with organic counterions. These assembled structures include classic micelles (spheres, cylinders and vesicles), and non-classic micelles (disks, toroids, branched micelles and segmented micelles). Each micelle structure is stable and reproducible at different assembly conditions depending on not only solution components (thermodynamics) but also mixing procedure and consequent self-assembly pathway (kinetics). The key factors that determine the thermodynamic interactions that help define the assembled structures and the kinetic assembly process include THF/water ratio, PS block length, the type and amount of organic counterions, and the mixing pathway. The complex phase behavior and controlled morphology production have been studied via in-situ cryogenic transmission electron microscopy in combination with scattering techniques (small angle neutron scattering and light scattering). Delicate control of the interplay of thermodynamics with slow chain kinetics of block copolymers in solution offers a new strategy to create unique, functional nanostructures.

  17. In vitro assembly of Ebola virus nucleocapsid-like complex expressed in E. coli

    Directory of Open Access Journals (Sweden)

    Ruchao Peng

    2016-09-01

    Full Text Available Abstract Ebola virus (EBOV harbors an RNA genome encapsidated by nucleoprotein (NP along with other viral proteins to form a nucleocapsid complex. Previous Cryo-eletron tomography and biochemical studies have shown the helical structure of EBOV nucleocapsid at nanometer resolution and the first 450 amino-acid of NP (NPΔ451–739 alone is capable of forming a helical nucleocapsid-like complex (NLC. However, the structural basis for NP-NP interaction and the dynamic procedure of the nucleocapsid assembly is yet poorly understood. In this work, we, by using an E. coli expression system, captured a series of images of NPΔ451–739 conformers at different stages of NLC assembly by negative-stain electron microscopy, which allowed us to picture the dynamic procedure of EBOV nucleocapsid assembly. Along with further biochemical studies, we showed the assembly of NLC is salt-sensitive, and also established an indispensible role of RNA in this process. We propose the diverse modes of NLC elongation might be the key determinants shaping the plasticity of EBOV virions. Our findings provide a new model for characterizing the self-oligomerization of viral nucleoproteins and studying the dynamic assembly process of viral nucleocapsid in vitro.

  18. Layer-by-layer self-assembly of ceramic particles for complex shape coating synthesis

    Science.gov (United States)

    Qiu, Hongwei

    Layer-by-layer (LbL) self-assembly was explored as a non-line-of-sight method for uniform infiltration and deposition of a multilayer of ceramic particles into complex structures. Key parameters for controlling the LbL self-assembly process were studied using a model system which consisted of a silicon substrate, 100 nm and 500 nm silica particles, and a polycation/polyanion combination. We correlated the surface coverage of the silica particles to the NaCl concentration used in deposition of the polyelectrolyte layers and to the number of the polyelectrolyte layers deposited. The effect of particle size on the surface coverage was rationally explained based on the screening length. We found that the effects of particle size, polydispersity, and electrolyte concentration in the particle suspension on the surface coverage and morphology of the first silica particle layer deposited on the polyelectrolyte layer surface were highly coupled, and resolving these effects was important for infiltrating a uniform coating of multilayer silica particle assemblies into a cellular structure as an ultimate complex substrate. Based on this understanding, the Lbl, self-assembly method was applied as a method of assembling, infiltrating, and immobilizing a 4-layer coating of negatively charged ˜3 mum Pd/NaAI(Si)O catalyst particles in the confined space of the cellular structure with ˜400 mum interconnected cells. The 4-layer coating deposited on the inner wall of a stainless steel capillary tube was mechanically stable under water flow rate up to 10 ml/min over the pH range of 3 to 11. Scotch tape peeling evaluation suggested that failure locations were mostly within the catalyst particle assembly, but near the assembly-PEM interface region.

  19. 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

  20. The structure and function of replication protein A in DNA replication.

    Science.gov (United States)

    Prakash, Aishwarya; Borgstahl, Gloria E O

    2012-01-01

    In all organisms from bacteria and archaea to eukarya, single-stranded DNA binding proteins play an essential role in most, if not all, nuclear metabolism involving single-stranded DNA (ssDNA). Replication protein A (RPA), the major eukaryotic ssDNA binding protein, has two important roles in DNA metabolism: (1) in binding ssDNA to protect it and to keep it unfolded, and (2) in coordinating the assembly and disassembly of numerous proteins and protein complexes during processes such as DNA replication. Since its discovery as a vital player in the process of replication, RPAs roles in recombination and DNA repair quickly became evident. This chapter summarizes the current understanding of RPA's roles in replication by reviewing the available structural data, DNA-binding properties, interactions with various replication proteins, and interactions with DNA repair proteins when DNA replication is stalled.

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  2. Self-Assembly of Discrete Metal Complexes in Aqueous Solution via Block Copolypeptide Amphiphiles

    Directory of Open Access Journals (Sweden)

    Timothy J. Deming

    2013-01-01

    Full Text Available The integration of discrete metal complexes has been attracting significant interest due to the potential of these materials for soft metal-metal interactions and supramolecular assembly. Additionally, block copolypeptide amphiphiles have been investigated concerning their capacity for self-assembly into structures such as nanoparticles, nanosheets and nanofibers. In this study, we combined these two concepts by investigating the self-assembly of discrete metal complexes in aqueous solution using block copolypeptides. Normally, discrete metal complexes such as [Au(CN2]−, when molecularly dispersed in water, cannot interact with one another. Our results demonstrated, however, that the addition of block copolypeptide amphiphiles such as K183L19 to [Au(CN2]− solutions induced one-dimensional integration of the discrete metal complex, resulting in photoluminescence originating from multinuclear complexes with metal-metal interactions. Transmission electron microscopy (TEM showed a fibrous nanostructure with lengths and widths of approximately 100 and 20 nm, respectively, which grew to form advanced nanoarchitectures, including those resembling the weave patterns of Waraji (traditional Japanese straw sandals. This concept of combining block copolypeptide amphiphiles with discrete coordination compounds allows the design of flexible and functional supramolecular coordination systems in water.

  3. The dynamic assembly of distinct RNA polymerase I complexes modulates rDNA transcription.

    Science.gov (United States)

    Torreira, Eva; Louro, Jaime Alegrio; Pazos, Irene; González-Polo, Noelia; Gil-Carton, David; Duran, Ana Garcia; Tosi, Sébastien; Gallego, Oriol; Calvo, Olga; Fernández-Tornero, Carlos

    2017-03-06

    Cell growth requires synthesis of ribosomal RNA by RNA polymerase I (Pol I). Binding of initiation factor Rrn3 activates Pol I, fostering recruitment to ribosomal DNA promoters. This fundamental process must be precisely regulated to satisfy cell needs at any time. We present in vivo evidence that, when growth is arrested by nutrient deprivation, cells induce rapid clearance of Pol I-Rrn3 complexes, followed by the assembly of inactive Pol I homodimers. This dual repressive mechanism reverts upon nutrient addition, thus restoring cell growth. Moreover, Pol I dimers also form after inhibition of either ribosome biogenesis or protein synthesis. Our mutational analysis, based on the electron cryomicroscopy structures of monomeric Pol I alone and in complex with Rrn3, underscores the central role of subunits A43 and A14 in the regulation of differential Pol I complexes assembly and subsequent promoter association.

  4. The assembly of the plasmodial PLP synthase complex follows a defined course.

    Directory of Open Access Journals (Sweden)

    Ingrid B Müller

    Full Text Available BACKGROUND: Plants, fungi, bacteria and the apicomplexan parasite Plasmodium falciparum are able to synthesize vitamin B6 de novo, whereas mammals depend upon the uptake of this essential nutrient from their diet. The active form of vitamin B6 is pyridoxal 5-phosphate (PLP. For its synthesis two enzymes, Pdx1 and Pdx2, act together, forming a multimeric complex consisting of 12 Pdx1 and 12 Pdx2 protomers. METHODOLOGY/PRINCIPAL FINDINGS: Here we report amino acid residues responsible for stabilization of the structural and enzymatic integrity of the plasmodial PLP synthase, identified by using distinct mutational analysis and biochemical approaches. Residues R85, H88 and E91 (RHE are located at the Pdx1:Pdx1 interface and play an important role in Pdx1 complex assembly. Mutation of these residues to alanine impedes both Pdx1 activity and Pdx2 binding. Furthermore, changing D26, K83 and K151 (DKK, amino acids from the active site of Pdx1, to alanine obstructs not only enzyme activity but also formation of the complex. In contrast to the monomeric appearance of the RHE mutant, alteration of the DKK residues results in a hexameric assembly, and does not affect Pdx2 binding or its activity. While the modelled position of K151 is distal to the Pdx1:Pdx1 interface, it affects the assembly of hexameric Pdx1 into a functional dodecamer, which is crucial for PLP synthesis. CONCLUSIONS/SIGNIFICANCE: Taken together, our data suggest that the assembly of a functional Pdx1:Pdx2 complex follows a defined pathway and that inhibition of this assembly results in an inactive holoenzyme.

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

    Science.gov (United States)

    de Biasio, Alfredo; de Opakua, Alain Ibáñez; Mortuza, Gulnahar B.; Molina, Rafael; Cordeiro, Tiago N.; Castillo, Francisco; Villate, Maider; Merino, Nekane; Delgado, Sandra; Gil-Cartón, David; Luque, Irene; Diercks, Tammo; Bernadó, Pau; Montoya, Guillermo; Blanco, Francisco J.

    2015-03-01

    The intrinsically disordered protein p15PAF regulates DNA replication and repair by binding to the proliferating cell nuclear antigen (PCNA) sliding clamp. We present the structure of the human p15PAF-PCNA complex. Crystallography and NMR show the central PCNA-interacting protein motif (PIP-box) of p15PAF tightly bound to the front-face of PCNA. In contrast to other PCNA-interacting proteins, p15PAF also contacts the inside of, and passes through, the PCNA ring. The disordered p15PAF termini emerge at opposite faces of the ring, but remain protected from 20S proteasomal degradation. Both free and PCNA-bound p15PAF 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 p15PAF acts as a flexible drag that regulates PCNA sliding along the DNA and facilitates the switch from replicative to translesion synthesis polymerase binding.

  6. Structure of p15(PAF)-PCNA complex and implications for clamp sliding during DNA replication and repair.

    Science.gov (United States)

    De Biasio, Alfredo; de Opakua, Alain Ibáñez; Mortuza, Gulnahar B; Molina, Rafael; Cordeiro, Tiago N; Castillo, Francisco; Villate, Maider; Merino, Nekane; Delgado, Sandra; Gil-Cartón, David; Luque, Irene; Diercks, Tammo; Bernadó, Pau; Montoya, Guillermo; Blanco, Francisco J

    2015-03-12

    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-box) of p15(PAF) tightly bound to the front-face of PCNA. In contrast to other PCNA-interacting proteins, p15(PAF) also contacts the inside of, and passes through, the PCNA ring. The disordered p15(PAF) termini emerge at opposite faces of the ring, but remain protected from 20S proteasomal degradation. Both 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 the DNA and facilitates the switch from replicative to translesion synthesis polymerase binding.

  7. Programming molecular self-assembly of intrinsically disordered proteins containing sequences of low complexity

    Science.gov (United States)

    Simon, Joseph R.; Carroll, Nick J.; Rubinstein, Michael; Chilkoti, Ashutosh; López, Gabriel P.

    2017-06-01

    Dynamic protein-rich intracellular structures that contain phase-separated intrinsically disordered proteins (IDPs) composed of sequences of low complexity (SLC) have been shown to serve a variety of important cellular functions, which include signalling, compartmentalization and stabilization. However, our understanding of these structures and our ability to synthesize models of them have been limited. We present design rules for IDPs possessing SLCs that phase separate into diverse assemblies within droplet microenvironments. Using theoretical analyses, we interpret the phase behaviour of archetypal IDP sequences and demonstrate the rational design of a vast library of multicomponent protein-rich structures that ranges from uniform nano-, meso- and microscale puncta (distinct protein droplets) to multilayered orthogonally phase-separated granular structures. The ability to predict and program IDP-rich assemblies in this fashion offers new insights into (1) genetic-to-molecular-to-macroscale relationships that encode hierarchical IDP assemblies, (2) design rules of such assemblies in cell biology and (3) molecular-level engineering of self-assembled recombinant IDP-rich materials.

  8. Complex assembly, crystallization and preliminary X-ray crystallographic analysis of the human Rod–Zwilch–ZW10 (RZZ) complex

    International Nuclear Information System (INIS)

    Altenfeld, Anika; Wohlgemuth, Sabine; Wehenkel, Annemarie; Vetter, Ingrid R.; Musacchio, Andrea

    2015-01-01

    The 800 kDa complex of the human Rod, Zwilch and ZW10 proteins (the RZZ complex) was reconstituted in insect cells, purified, crystallized and subjected to preliminary X-ray diffraction analysis. The spindle-assembly checkpoint (SAC) monitors kinetochore–microtubule attachment during mitosis. In metazoans, the three-subunit Rod–Zwilch–ZW10 (RZZ) complex is a crucial SAC component that interacts with additional SAC-activating and SAC-silencing components, including the Mad1–Mad2 complex and cytoplasmic dynein. The RZZ complex contains two copies of each subunit and has a predicted molecular mass of ∼800 kDa. Given the low abundance of the RZZ complex in natural sources, its recombinant reconstitution was attempted by co-expression of its subunits in insect cells. The RZZ complex was purified to homogeneity and subjected to systematic crystallization attempts. Initial crystals containing the entire RZZ complex were obtained using the sitting-drop method and were subjected to optimization to improve the diffraction resolution limit. The crystals belonged to space group P3 1 (No. 144) or P3 2 (No. 145), with unit-cell parameters a = b = 215.45, c = 458.7 Å, α = β = 90.0, γ = 120.0°

  9. Complex assembly, crystallization and preliminary X-ray crystallographic analysis of the human Rod–Zwilch–ZW10 (RZZ) complex

    Energy Technology Data Exchange (ETDEWEB)

    Altenfeld, Anika; Wohlgemuth, Sabine [Max Planck Institute of Molecular Physiology, Otto Hahn Strasse 11, 44227 Dortmund (Germany); Wehenkel, Annemarie [Institut Curie, CNRS UMR 3348/INSERM U1005, Bâtiment 110, Centre Universitaire, 91405 Orsay CEDEX (France); Vetter, Ingrid R. [Max Planck Institute of Molecular Physiology, Otto Hahn Strasse 11, 44227 Dortmund (Germany); Musacchio, Andrea, E-mail: andrea.musacchio@mpi-dortmund.mpg.de [Max Planck Institute of Molecular Physiology, Otto Hahn Strasse 11, 44227 Dortmund (Germany); University of Duisburg-Essen, Universitätstrasse 1, 45141 Essen (Germany)

    2015-03-20

    The 800 kDa complex of the human Rod, Zwilch and ZW10 proteins (the RZZ complex) was reconstituted in insect cells, purified, crystallized and subjected to preliminary X-ray diffraction analysis. The spindle-assembly checkpoint (SAC) monitors kinetochore–microtubule attachment during mitosis. In metazoans, the three-subunit Rod–Zwilch–ZW10 (RZZ) complex is a crucial SAC component that interacts with additional SAC-activating and SAC-silencing components, including the Mad1–Mad2 complex and cytoplasmic dynein. The RZZ complex contains two copies of each subunit and has a predicted molecular mass of ∼800 kDa. Given the low abundance of the RZZ complex in natural sources, its recombinant reconstitution was attempted by co-expression of its subunits in insect cells. The RZZ complex was purified to homogeneity and subjected to systematic crystallization attempts. Initial crystals containing the entire RZZ complex were obtained using the sitting-drop method and were subjected to optimization to improve the diffraction resolution limit. The crystals belonged to space group P3{sub 1} (No. 144) or P3{sub 2} (No. 145), with unit-cell parameters a = b = 215.45, c = 458.7 Å, α = β = 90.0, γ = 120.0°.

  10. Assembly and function of the major histocompatibility complex (MHC) I peptide-loading complex are conserved across higher vertebrates.

    Science.gov (United States)

    Hinz, Andreas; Jedamzick, Johanna; Herbring, Valentina; Fischbach, Hanna; Hartmann, Jessica; Parcej, David; Koch, Joachim; Tampé, Robert

    2014-11-28

    Antigen presentation to cytotoxic T lymphocytes via major histocompatibility complex class I (MHC I) molecules depends on the heterodimeric transporter associated with antigen processing (TAP). For efficient antigen supply to MHC I molecules in the ER, TAP assembles a macromolecular peptide-loading complex (PLC) by recruiting tapasin. In evolution, TAP appeared together with effector cells of adaptive immunity at the transition from jawless to jawed vertebrates and diversified further within the jawed vertebrates. Here, we compared TAP function and interaction with tapasin of a range of species within two classes of jawed vertebrates. We found that avian and mammalian TAP1 and TAP2 form heterodimeric complexes across taxa. Moreover, the extra N-terminal domain TMD0 of mammalian TAP1 and TAP2 as well as avian TAP2 recruits tapasin. Strikingly, however, only TAP1 and TAP2 from the same taxon can form a functional heterodimeric translocation complex. These data demonstrate that the dimerization interface between TAP1 and TAP2 and the tapasin docking sites for PLC assembly are conserved in evolution, whereas elements of antigen translocation diverged later in evolution and are thus taxon specific. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  11. Assembly/disassembly of a complex icosahedral virus to incorporate heterologous nucleic acids

    Science.gov (United States)

    Pascual, Elena; Mata, Carlos P.; Carrascosa, José L.; Castón, José R.

    2017-12-01

    Hollow protein containers are widespread in nature, and include virus capsids as well as eukaryotic and bacterial complexes. Protein cages are studied extensively for applications in nanotechnology, nanomedicine and materials science. Their inner and outer surfaces can be modified chemically or genetically, and the internal cavity can be used to template, store and/or arrange molecular cargos. Virus capsids and virus-like particles (VLP, noninfectious particles) provide versatile platforms for nanoscale bioengineering. Study of capsid protein self-assembly into monodispersed particles, and of VLP structure and biophysics is necessary not only to understand natural processes, but also to infer how these platforms can be redesigned to furnish novel functional VLP. Here we address the assembly dynamics of infectious bursal disease virus (IBDV), a complex icosahedral virus. IBDV has a ~70 nm-diameter T  =  13 capsid with VP2 trimers as the only structural subunits. During capsid assembly, VP2 is synthesized as a precursor (pVP2) whose C terminus is cleaved. The pVP2 C terminus has an amphipathic helix that controls VP2 polymorphism. In the absence of the VP3 scaffolding protein, necessary for control of assembly, 466/456-residue pVP2 intermediates bearing this helix assemble into VLP only when expressed with an N-terminal His6 tag (the HT-VP2-466 protein). HT-VP2-466 capsids are optimal for genetic insertion of proteins (cargo space ~78 000 nm3). We established an in vitro assembly/disassembly system of HT-VP2-466-based VLP for heterologous nucleic acid packaging and/or encapsulation of drugs and other molecules. HT-VP2-466 (empty) capsids were disassembled and reassembled by dialysis against low-salt/basic pH and high-salt/acid pH buffers, respectively, thus illustrating the reversibility in vitro of IBDV capsid assembly. HT-VP2-466 VLP also packed heterologous DNA by non-specific confinement during assembly. These and previous results establish the bases

  12. Fatigue analysis of assembled marine floating platform for special purposes under complex water environments

    Science.gov (United States)

    Ma, Guang-ying; Yao, Yun-long

    2018-03-01

    In this paper, the fatigue lives of a new type of assembled marine floating platform for special purposes were studied. Firstly, by using ANSYS AQWA software, the hydrodynamic model of the platform was established. Secondly, the structural stresses under alternating change loads were calculated under complex water environments, such as wind, wave, current and ice. The minimum fatigue lives were obtained under different working conditions. The analysis results showed that the fatigue life of the platform structure can meet the requirements

  13. 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

    that new histones incorporated during DNA replication provide a signature of post-replicative chromatin, read by the human TONSL–MMS22L 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...

  14. 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.

  15. Structural basis of the pH-dependent assembly of a botulinum neurotoxin complex.

    Science.gov (United States)

    Matsui, Tsutomu; Gu, Shenyan; Lam, Kwok-Ho; Carter, Lester G; Rummel, Andreas; Mathews, Irimpan I; Jin, Rongsheng

    2014-11-11

    Botulinum neurotoxins (BoNTs) are among the most poisonous biological substances known. They assemble with non-toxic non-hemagglutinin (NTNHA) protein to form the minimally functional progenitor toxin complexes (M-PTC), which protects BoNT in the gastrointestinal tract and releases it upon entry into the circulation. Here we provide molecular insight into the assembly between BoNT/A and NTNHA-A using small-angle X-ray scattering. We found that the free form BoNT/A maintains a pH-independent conformation with limited domain flexibility. Intriguingly, the free form NTNHA-A adopts pH-dependent conformational changes due to a torsional motion of its C-terminal domain. Once forming a complex at acidic pH, they each adopt a stable conformation that is similar to that observed in the crystal structure of the M-PTC. Our results suggest that assembly of the M-PTC depends on the environmental pH and that the complex form of BoNT/A is induced by interacting with NTNHA-A at acidic pH. Copyright © 2014 Elsevier Ltd. All rights reserved.

  16. Complex shapes self–assembled from single–stranded DNA tiles

    Science.gov (United States)

    Wei, Bryan; Dai, Mingjie; Yin, Peng

    2014-01-01

    Programmed self-assembly of strands of nucleic acid has proved highly effective for creating a wide range of structures with desired shapes1–25. A particularly successful implementation is DNA origami, in which a long scaffold strand is folded by hundreds of short auxiliary strands into a complex shape9, 14–16,18–21,25. Modular strategies are in principle simpler and more versatile and have been used to assemble DNA2–5,8,10–13,17,23 or RNA7,22 tiles into periodic3,4,7,22 and algorithmic5 two-dimensional lattices, extended ribbons10,12 and tubes4,12,13, three-dimensional crystals17, polyhedra11 and simple finite two-dimensional shapes7,8. But creating finite yet complex shapes from a large number of uniquely addressable tiles remains challenging. Here we solve this problem with the simplest tile form, a ‘single-stranded tile’ (SST) that consists of a 42-base strand of DNA composed entirely of concatenated sticky ends and that binds to four local neighbours during self-assembly12. Although ribbons and tubes with controlled circumferences12 have been created using the SST approach, we extend it to assemble complex two-dimensional shapes and tubes from hundreds (in some cases more than one thousand) distinct tiles. Our main design feature is a self-assembled rectangle that serves as a molecular canvas, with each of its constituent SST strands—folded into a 3nm-by-7 nm tile and attached to four neighbouring tiles—acting as a pixel. A desired shape, drawn on the canvas, is then produced by one-pot annealing of all those strands that correspond to pixels covered by the target shape; the remaining strands are excluded. We implement the strategy with a master strand collection that corresponds to a 310-pixel canvas, and then use appropriate strand subsets to construct 107 distinct and complex two-dimensional shapes, thereby establishing SST assembly as a simple, modular and robust framework for constructing nanostructures with prescribed shapes from short

  17. Bub1 in Complex with LANA Recruits PCNA To Regulate Kaposi's Sarcoma-Associated Herpesvirus Latent Replication and DNA Translesion Synthesis.

    Science.gov (United States)

    Sun, Zhiguo; Jha, Hem Chandra; Robertson, Erle S

    2015-10-01

    Latent DNA replication of Kaposi's sarcoma-associated herpesvirus (KSHV) initiates at the terminal repeat (TR) element and requires trans-acting elements, both viral and cellular, such as ORCs, MCMs, and latency-associated nuclear antigen (LANA). However, how cellular proteins are recruited to the viral genome is not very clear. Here, we demonstrated that the host cellular protein, Bub1, is involved in KSHV latent DNA replication. We show that Bub1 constitutively interacts with proliferating cell nuclear antigen (PCNA) via a highly conserved PIP box motif within the kinase domain. Furthermore, we demonstrated that Bub1 can form a complex with LANA and PCNA in KSHV-positive cells. This strongly indicated that Bub1 serves as a scaffold or molecular bridge between LANA and PCNA. LANA recruited PCNA to the KSHV genome via Bub1 to initiate viral replication in S phase and interacted with PCNA to promote its monoubiquitination in response to UV-induced damage for translesion DNA synthesis. This resulted in increased survival of KSHV-infected cells. During latency in KSHV-infected cells, the viral episomal DNA replicates once each cell cycle. KSHV does not express DNA replication proteins during latency. Instead, KSHV LANA recruits the host cell DNA replication machinery to the replication origin. However, the mechanism by which LANA mediates replication is uncertain. Here, we show that LANA is able to form a complex with PCNA, a critical protein for viral DNA replication. Furthermore, our findings suggest that Bub1, a spindle checkpoint protein, serves as a scaffold or molecular bridge between LANA and PCNA. Our data further support a role for Bub1 and LANA in PCNA-mediated cellular DNA replication processes as well as monoubiquitination of PCNA in response to UV damage. These data reveal a therapeutic target for inhibition of KSHV persistence in malignant cells. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  18. Complex assembly, crystallization and preliminary X-ray crystallographic analysis of the human Rod-Zwilch-ZW10 (RZZ) complex.

    Science.gov (United States)

    Altenfeld, Anika; Wohlgemuth, Sabine; Wehenkel, Annemarie; Vetter, Ingrid R; Musacchio, Andrea

    2015-04-01

    The spindle-assembly checkpoint (SAC) monitors kinetochore-microtubule attachment during mitosis. In metazoans, the three-subunit Rod-Zwilch-ZW10 (RZZ) complex is a crucial SAC component that interacts with additional SAC-activating and SAC-silencing components, including the Mad1-Mad2 complex and cytoplasmic dynein. The RZZ complex contains two copies of each subunit and has a predicted molecular mass of ∼800 kDa. Given the low abundance of the RZZ complex in natural sources, its recombinant reconstitution was attempted by co-expression of its subunits in insect cells. The RZZ complex was purified to homogeneity and subjected to systematic crystallization attempts. Initial crystals containing the entire RZZ complex were obtained using the sitting-drop method and were subjected to optimization to improve the diffraction resolution limit. The crystals belonged to space group P3₁ (No. 144) or P3₂ (No. 145), with unit-cell parameters a = b = 215.45, c = 458.7 Å, α = β = 90.0, γ = 120.0°.

  19. Design and construction of self-assembling supramolecular protein complexes using artificial and fusion proteins as nanoscale building blocks.

    Science.gov (United States)

    Kobayashi, Naoya; Arai, Ryoichi

    2017-08-01

    The central goal of nanobiotechnology is to design and construct novel biomaterials of nanometer sizes. In this short review, we describe recent progress of several approaches for designing and creating artificial self-assembling protein complexes and primarily focus on the following biotechnological strategies for using artificial and fusion proteins as nanoscale building blocks: fusion proteins designed for symmetrical self-assembly; three-dimensional domain-swapped oligomers; self-assembling designed coiled-coil peptide modules; metal-directed self-assembling engineered proteins; computationally designed self-assembling de novo proteins; and self-assembling protein nanobuilding blocks (PN-Blocks) using an intermolecularly folded dimeric de novo protein. These state-of-the-art nanobiotechnologies for designing supramolecular protein complexes will facilitate the development of novel functional nanobiomaterials. Copyright © 2017 Elsevier Ltd. All rights reserved.

  20. Synthesis and Self-Assembly of Chiral Cylindrical Molecular Complexes: Functional Heterogeneous Liquid-Solid Materials Formed by Helicene Oligomers

    Directory of Open Access Journals (Sweden)

    Nozomi Saito

    2018-01-01

    Full Text Available Chiral cylindrical molecular complexes of homo- and hetero-double-helices derived from helicene oligomers self-assemble in solution, providing functional heterogeneous liquid-solid materials. Gels and liotropic liquid crystals are formed by fibril self-assembly in solution; molecular monolayers and fibril films are formed by self-assembly on solid surfaces; gels containing gold nanoparticles emit light; silica nanoparticles aggregate and adsorb double-helices. Notable dynamics appears during self-assembly, including multistep self-assembly, solid surface catalyzed double-helix formation, sigmoidal and stairwise kinetics, molecular recognition of nanoparticles, discontinuous self-assembly, materials clocking, chiral symmetry breaking and homogeneous-heterogeneous transitions. These phenomena are derived from strong intercomplex interactions of chiral cylindrical molecular complexes.

  1. Self-assembly of complex two-dimensional shapes from single-stranded DNA tiles.

    Science.gov (United States)

    Wei, Bryan; Vhudzijena, Michelle K; Robaszewski, Joanna; Yin, Peng

    2015-05-08

    Current methods in DNA nano-architecture have successfully engineered a variety of 2D and 3D structures using principles of self-assembly. In this article, we describe detailed protocols on how to fabricate sophisticated 2D shapes through the self-assembly of uniquely addressable single-stranded DNA tiles which act as molecular pixels on a molecular canvas. Each single-stranded tile (SST) is a 42-nucleotide DNA strand composed of four concatenated modular domains which bind to four neighbors during self-assembly. The molecular canvas is a rectangle structure self-assembled from SSTs. A prescribed complex 2D shape is formed by selecting the constituent molecular pixels (SSTs) from a 310-pixel molecular canvas and then subjecting the corresponding strands to one-pot annealing. Due to the modular nature of the SST approach we demonstrate the scalability, versatility and robustness of this method. Compared with alternative methods, the SST method enables a wider selection of information polymers and sequences through the use of de novo designed and synthesized short DNA strands.

  2. Predicting Chiral Nanostructures, Lattices and Superlattices in Complex Multicomponent Nanoparticle Self-Assembly

    KAUST Repository

    Hur, Kahyun

    2012-06-13

    "Bottom up" type nanoparticle (NP) self-assembly is expected to provide facile routes to nanostructured materials for various, for example, energy related, applications. Despite progress in simulations and theories, structure prediction of self-assembled materials beyond simple model systems remains challenging. Here we utilize a field theory approach for predicting nanostructure of complex and multicomponent hybrid systems with multiple types of short- and long-range interactions. We propose design criteria for controlling a range of NP based nanomaterial structures. In good agreement with recent experiments, the theory predicts that ABC triblock terpolymer directed assemblies with ligand-stabilized NPs can lead to chiral NP network structures. Furthermore, we predict that long-range Coulomb interactions between NPs leading to simple NP lattices, when applied to NP/block copolymer (BCP) assemblies, induce NP superlattice formation within the phase separated BCP nanostructure, a strategy not yet realized experimentally. We expect such superlattices to be of increasing interest to communities involved in research on, for example, energy generation and storage, metamaterials, as well as microelectronics and information storage. © 2012 American Chemical Society.

  3. Size estimation of complexes consisting of hairpin DNAs bound to an assembler-strand. [Abstract only

    Science.gov (United States)

    Baumann, Ulrich

    1994-01-01

    In connection with a model of a primitive translation based entirely on nucleic acids the structural requirements and interactions of small nucleic acids were studied. As shown previously hairpins bind to a complementary single-strand by base pairs of their loop nucleotides if the loop contains at least five nucleotides (Baumann et al 1987). Here, the question is approached to which degree a single-strand, the assembler-strand, is occupied with hairpin molecules. A gapless occupation would allow the close proximity necessary for the assumed peptide bond formation by hairpins bearing an amino acid at their 3'-end. Oligo(dC)(sub n), n = 12, 15, 18, and the investigated hairpins form complexes which are detectable under nondenaturing electrophoretic conditions. The size range is estimated to be 3-4 hairpin molecules to one assembler molecule when oligo(dC)(sub n), n = 15, 18, is used. Due to the limitation of the method, dependence of the migration velocity on the shape which is unique and thus limits the use of marker nucleic acids, and due to the uncertainty if the ends of the assembler form base pairs, a gapless occupation of the assembler is conceivable but not proven.

  4. MEGAHIT: an ultra-fast single-node solution for large and complex metagenomics assembly via succinct de Bruijn graph.

    Science.gov (United States)

    Li, Dinghua; Liu, Chi-Man; Luo, Ruibang; Sadakane, Kunihiko; Lam, Tak-Wah

    2015-05-15

    MEGAHIT is a NGS de novo assembler for assembling large and complex metagenomics data in a time- and cost-efficient manner. It finished assembling a soil metagenomics dataset with 252 Gbps in 44.1 and 99.6 h on a single computing node with and without a graphics processing unit, respectively. MEGAHIT assembles the data as a whole, i.e. no pre-processing like partitioning and normalization was needed. When compared with previous methods on assembling the soil data, MEGAHIT generated a three-time larger assembly, with longer contig N50 and average contig length; furthermore, 55.8% of the reads were aligned to the assembly, giving a fourfold improvement. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  5. Solution structure of the Atg1 complex: implications for the architecture of the phagophore assembly site.

    Science.gov (United States)

    Köfinger, Jürgen; Ragusa, Michael J; Lee, Il-Hyung; Hummer, Gerhard; Hurley, James H

    2015-05-05

    The biogenesis of autophagosomes commences at the phagophore assembly site (PAS), a protein-vesicle ultrastructure that is organized by the Atg1 complex. The Atg1 complex consists of the Atg1 protein kinase, the intrinsically disordered region-rich Atg13, and the dimeric double crescent-shaped Atg17-Atg31-Atg29 subcomplex. We show that the PAS contains a relatively uniform ∼28 copies of Atg17, and upon autophagy induction, similar numbers of Atg1 and Atg13 molecules. We then apply ensemble refinement of small-angle X-ray scattering to determine the solution structures of the Atg1-Atg13 and Atg17-Atg31-Atg29 subcomplexes and the Atg1 complex, using a trimmed minipentamer tractable to biophysical studies. We observe tetramers of Atg1 pentamers that assemble via Atg17-Atg31-Atg29. This leads to a model for the higher organization of the Atg1 complex in PAS scaffolding. Copyright © 2015 Elsevier Ltd. All rights reserved.

  6. Monitoring ligand-dependent assembly of receptor ternary complexes in live cells by BRETFect.

    Science.gov (United States)

    Cotnoir-White, David; El Ezzy, Mohamed; Boulay, Pierre-Luc; Rozendaal, Marieke; Bouvier, Michel; Gagnon, Etienne; Mader, Sylvie

    2018-03-13

    There is currently an unmet need for versatile techniques to monitor the assembly and dynamics of ternary complexes in live cells. Here we describe bioluminescence resonance energy transfer with fluorescence enhancement by combined transfer (BRETFect), a high-throughput technique that enables robust spectrometric detection of ternary protein complexes based on increased energy transfer from a luciferase to a fluorescent acceptor in the presence of a fluorescent intermediate. Its unique donor-intermediate-acceptor relay system is designed so that the acceptor can receive energy either directly from the donor or indirectly via the intermediate in a combined transfer, taking advantage of the entire luciferase emission spectrum. BRETFect was used to study the ligand-dependent cofactor interaction properties of the estrogen receptors ERα and ERβ, which form homo- or heterodimers whose distinctive regulatory properties are difficult to dissect using traditional methods. BRETFect uncovered the relative capacities of hetero- vs. homodimers to recruit receptor-specific cofactors and regulatory proteins, and to interact with common cofactors in the presence of receptor-specific ligands. BRETFect was also used to follow the assembly of ternary complexes between the V2R vasopressin receptor and two different intracellular effectors, illustrating its use for dissection of ternary protein-protein interactions engaged by G protein-coupled receptors. Our results indicate that BRETFect represents a powerful and versatile technique to monitor the dynamics of ternary interactions within multimeric complexes in live cells.

  7. Nuclear Pores Regulate Muscle Development and Maintenance by Assembling a Localized Mef2C Complex.

    Science.gov (United States)

    Raices, Marcela; Bukata, Lucas; Sakuma, Stephen; Borlido, Joana; Hernandez, Leanora S; Hart, Daniel O; D'Angelo, Maximiliano A

    2017-06-05

    Nuclear pore complexes (NPCs) are multiprotein channels connecting the nucleus with the cytoplasm. NPCs have been shown to have tissue-specific composition, suggesting that their function can be specialized. However, the physiological roles of NPC composition changes and their impacts on cellular processes remain unclear. Here we show that the addition of the Nup210 nucleoporin to NPCs during myoblast differentiation results in assembly of an Mef2C transcriptional complex required for efficient expression of muscle structural genes and microRNAs. We show that this NPC-localized complex is essential for muscle growth, myofiber maturation, and muscle cell survival and that alterations in its activity result in muscle degeneration. Our findings suggest that NPCs regulate the activity of functional gene groups by acting as scaffolds that promote the local assembly of tissue-specific transcription complexes and show how nuclear pore composition changes can be exploited to regulate gene expression at the nuclear periphery. Copyright © 2017 Elsevier Inc. All rights reserved.

  8. Complex assembly, crystallization and preliminary X-ray crystallographic studies of duck MHC class I molecule

    International Nuclear Information System (INIS)

    Zhang, Jianhua; Chen, Yong; Gao, Feng; Chen, Weihong; Qi, Jianxun; Xia, Chun

    2009-01-01

    Using a peptide derived from H5N1, a complex of duck MHC class I molecule (DuMHC I) with duck β 2 -microglobulin (Duβ 2 m) was assembled and crystallized. Initial structure analysis indicated that the crystals did not contain the complete DuMHC I complex but instead contained DuMHC I α3-domain and Duβ 2 m subunits. In order to understand the biological properties of the immune systems of waterfowl and to establish a system for structural studies of duck class I major histocompatibility complex (DuMHC I), a complex of DuMHC I with duck β 2 -microglobulin (Duβ 2 m) and the peptide AEIEDLIF (AF8) derived from H5N1 NP residues 251–258 was assembled. The complex was crystallized; the crystals belonged to space group C222 1 , with unit-cell parameters a = 54.7, b = 72.4, c = 102.2 Å, and diffracted to 2.3 Å resolution. Matthews coefficient calculation and initial structure determination by molecular replacement showed that the crystals did not contain the whole DuMHC I complex, but instead contained the DuMHC I α3 domain and a Duβ2m molecule (DuMHC I α3+β2m). Another complex of DuMHC I with the peptide IDWFDGKE derived from a chicken fusion protein also generated the same results. The stable structure of DuMHC I α3+β2m may reflect some unique characteristics of DuMHC I and pave the way for novel MHC structure-related studies in the future

  9. Molecular suction pads: self-assembled monolayers of subphthalocyaninatoboron complexes on gold.

    Science.gov (United States)

    Glebe, Ulrich; Baio, Joe E; Árnadóttir, Líney; Siemeling, Ulrich; Weidner, Tobias

    2013-04-15

    Subphthalocyaninatoboron complexes with six long-chain alkylthio substituents in their periphery are applicable for the formation of self-assembled monolayers (SAMs) on gold. Such films are prepared from solution with the axially chlorido-substituted derivatives and characterised by near-edge X-ray absorption fine structure (NEXAFS) spectroscopy, X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (ToF-SIMS). The results are in accord with the formation of SAMs assembled by the chemisorption of both covalently bound thiolate-type as well as coordinatively bound thioether units. The adsorbate molecules adopt an essentially flat adsorption geometry on the substrate, resembling a suction pad on a surface. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  10. Identification and assembly of genomes and genetic elements in complex metagenomic samples without using reference genomes

    DEFF Research Database (Denmark)

    Nielsen, Henrik Bjørn; Almeida, Mathieu; Juncker, Agnieszka

    2014-01-01

    , such as particular bacterial strains or viruses, remains a largely unsolved problem. Here we present a method, based on binning co-abundant genes across a series of metagenomic samples, that enables comprehensive discovery of new microbial organisms, viruses and co-inherited genetic entities and aids assembly...... of microbial genomes without the need for reference sequences. We demonstrate the method on data from 396 human gut microbiome samples and identify 7,381 co-abundance gene groups (CAGs), including 741 metagenomic species (MGS). We use these to assemble 238 high-quality microbial genomes and identify...... affiliations between MGS and hundreds of viruses or genetic entities. Our method provides the means for comprehensive profiling of the diversity within complex metagenomic samples....

  11. SARS-coronavirus replication/transcription complexes are membrane-protected and need a host factor for activity in vitro.

    Directory of Open Access Journals (Sweden)

    Martijn J van Hemert

    2008-05-01

    Full Text Available SARS-coronavirus (SARS-CoV replication and transcription are mediated by a replication/transcription complex (RTC of which virus-encoded, non-structural proteins (nsps are the primary constituents. The 16 SARS-CoV nsps are produced by autoprocessing of two large precursor polyproteins. The RTC is believed to be associated with characteristic virus-induced double-membrane structures in the cytoplasm of SARS-CoV-infected cells. To investigate the link between these structures and viral RNA synthesis, and to dissect RTC organization and function, we isolated active RTCs from infected cells and used them to develop the first robust assay for their in vitro activity. The synthesis of genomic RNA and all eight subgenomic mRNAs was faithfully reproduced by the RTC in this in vitro system. Mainly positive-strand RNAs were synthesized and protein synthesis was not required for RTC activity in vitro. All RTC activity, enzymatic and putative membrane-spanning nsps, and viral RNA cosedimented with heavy membrane structures. Furthermore, the pelleted RTC required the addition of a cytoplasmic host factor for reconstitution of its in vitro activity. Newly synthesized subgenomic RNA appeared to be released, while genomic RNA remained predominantly associated with the RTC-containing fraction. RTC activity was destroyed by detergent treatment, suggesting an important role for membranes. The RTC appeared to be protected by membranes, as newly synthesized viral RNA and several replicase/transcriptase subunits were protease- and nuclease-resistant and became susceptible to degradation only upon addition of a non-ionic detergent. Our data establish a vital functional dependence of SARS-CoV RNA synthesis on virus-induced membrane structures.

  12. DNA Polymerase III, but Not Polymerase IV, Must Be Bound to a τ-Containing DnaX Complex to Enable Exchange into Replication Forks.

    Science.gov (United States)

    Yuan, Quan; Dohrmann, Paul R; Sutton, Mark D; McHenry, Charles S

    2016-05-27

    Examples of dynamic polymerase exchange have been previously characterized in model systems provided by coliphages T4 and T7. Using a dominant negative D403E polymerase (Pol) III α that can form initiation complexes and sequester primer termini but not elongate, we investigated the possibility of exchange at the Escherichia coli replication fork on a rolling circle template. Unlike other systems, addition of polymerase alone did not lead to exchange. Only when D403E Pol III was bound to a τ-containing DnaX complex did exchange occur. In contrast, addition of Pol IV led to rapid exchange in the absence of bound DnaX complex. Examination of Pol III* with varying composition of τ or the alternative shorter dnaX translation product γ showed that τ-, τ2-, or τ3-DnaX complexes supported equivalent levels of synthesis, identical Okazaki fragment size, and gaps between fragments, possessed the ability to challenge pre-established replication forks, and displayed equivalent susceptibility to challenge by exogenous D403E Pol III*. These findings reveal that redundant interactions at the replication fork must stabilize complexes containing only one τ. Previously, it was thought that at least two τs in the trimeric DnaX complex were required to couple the leading and lagging strand polymerases at the replication fork. Possible mechanisms of exchange are discussed. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  13. Uracil DNA glycosylase interacts with the p32 subunit of the replication protein A complex to modulate HIV-1 reverse transcription for optimal virus dissemination.

    Science.gov (United States)

    Herate, Cecile; Vigne, Clarisse; Guenzel, Carolin A; Lambele, Marie; Rouyez, Marie-Christine; Benichou, Serge

    2016-04-12

    Through incorporation into virus particles, the HIV-1 Vpr protein participates in the early steps of the virus life cycle by influencing the reverse transcription process. We previously showed that this positive impact on reverse transcription was related to Vpr binding to the uracil DNA glycosylase 2 enzyme (UNG2), leading to enhancement of virus infectivity in established CD4-positive cell lines via a nonenzymatic mechanism. We report here that Vpr can form a trimolecular complex with UNG2 and the p32 subunit (RPA32) of the replication protein A (RPA) complex and we explore how these cellular proteins can influence virus replication and dissemination in the primary target cells of HIV-1, which express low levels of both proteins. Virus infectivity and replication in peripheral blood mononuclear cells and monocyte-derived macrophages (MDMs), as well as the efficiency of the viral DNA synthesis, were significantly reduced when viruses were produced from cells depleted of endogenous UNG2 or RPA32. Moreover, viruses produced in macrophages failed to replicate efficiently in UNG2- and RPA32-depleted T lymphocytes. Reciprocally, viruses produced in UNG2-depleted T cells did not replicate efficiently in MDMs confirming the positive role of UNG2 for virus dissemination. Our data show the positive effect of UNG2 and RPA32 on the reverse transcription process leading to optimal virus replication and dissemination between the primary target cells of HIV-1.

  14. Complex self-assembled patterns using sparse commensurate templates with locally varying motifs.

    Science.gov (United States)

    Yang, Joel K W; Jung, Yeon Sik; Chang, Jae-Byum; Mickiewicz, R A; Alexander-Katz, A; Ross, C A; Berggren, Karl K

    2010-04-01

    Templated self-assembly of block copolymer thin films can generate periodic arrays of microdomains within a sparse template, or complex patterns using 1:1 templates. However, arbitrary pattern generation directed by sparse templates remains elusive. Here, we show that an array of carefully spaced and shaped posts, prepared by electron-beam patterning of an inorganic resist, can be used to template complex patterns in a cylindrical-morphology block copolymer. We use two distinct methods: making the post spacing commensurate with the equilibrium periodicity of the polymer, which controls the orientation of the linear features, and making local changes to the shape or distribution of the posts, which direct the formation of bends, junctions and other aperiodic features in specific locations. The first of these methods permits linear patterns to be directed by a sparse template that occupies only a few percent of the area of the final self-assembled pattern, while the second method can be used to selectively and locally template complex linear patterns.

  15. Retinal cone photoreceptors require phosducin-like protein 1 for G protein complex assembly and signaling.

    Directory of Open Access Journals (Sweden)

    Christopher M Tracy

    Full Text Available G protein β subunits (Gβ play essential roles in phototransduction as part of G protein βγ (Gβγ and regulator of G protein signaling 9 (RGS9-Gβ5 heterodimers. Both are obligate dimers that rely on the cytosolic chaperone CCT and its co-chaperone PhLP1 to form complexes from their nascent polypeptides. The importance of PhLP1 in the assembly process was recently demonstrated in vivo in a retinal rod-specific deletion of the Phlp1 gene. To test whether this is a general mechanism that also applies to other cell types, we disrupted the Phlp1 gene specifically in mouse cones and measured the effects on G protein expression and cone visual signal transduction. In PhLP1-deficient cones, expression of cone transducin (Gt2 and RGS9-Gβ5 subunits was dramatically reduced, resulting in a 27-fold decrease in sensitivity and a 38-fold delay in cone photoresponse recovery. These results demonstrate the essential role of PhLP1 in cone G protein complex formation. Our findings reveal a common mechanism of Gβγ and RGS9-Gβ5 assembly in rods and cones, highlighting the importance of PhLP1 and CCT-mediated Gβ complex formation in G protein signaling.

  16. Structural basis for the subunit assembly of the anaphase-promoting complex.

    Science.gov (United States)

    Schreiber, Anne; Stengel, Florian; Zhang, Ziguo; Enchev, Radoslav I; Kong, Eric H; Morris, Edward P; Robinson, Carol V; da Fonseca, Paula C A; Barford, David

    2011-02-10

    The anaphase-promoting complex or cyclosome (APC/C) is an unusually large E3 ubiquitin ligase responsible for regulating defined cell cycle transitions. Information on how its 13 constituent proteins are assembled, and how they interact with co-activators, substrates and regulatory proteins is limited. Here, we describe a recombinant expression system that allows the reconstitution of holo APC/C and its sub-complexes that, when combined with electron microscopy, mass spectrometry and docking of crystallographic and homology-derived coordinates, provides a precise definition of the organization and structure of all essential APC/C subunits, resulting in a pseudo-atomic model for 70% of the APC/C. A lattice-like appearance of the APC/C is generated by multiple repeat motifs of most APC/C subunits. Three conserved tetratricopeptide repeat (TPR) subunits (Cdc16, Cdc23 and Cdc27) share related superhelical homo-dimeric architectures that assemble to generate a quasi-symmetrical structure. Our structure explains how this TPR sub-complex, together with additional scaffolding subunits (Apc1, Apc4 and Apc5), coordinate the juxtaposition of the catalytic and substrate recognition module (Apc2, Apc11 and Apc10 (also known as Doc1)), and TPR-phosphorylation sites, relative to co-activator, regulatory proteins and substrates.

  17. 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.

  18. Molecular Assembly of Clostridium botulinum progenitor M complex of type E.

    Science.gov (United States)

    Eswaramoorthy, Subramaniam; Sun, Jingchuan; Li, Huilin; Singh, Bal Ram; Swaminathan, Subramanyam

    2015-12-07

    Clostridium botulinum neurotoxin (BoNT) is released as a progenitor complex, in association with a non-toxic-non-hemagglutinin protein (NTNH) and other associated proteins. We have determined the crystal structure of M type Progenitor complex of botulinum neurotoxin E [PTC-E(M)], a heterodimer of BoNT and NTNH. The crystal structure reveals that the complex exists as a tight, interlocked heterodimer of BoNT and NTNH. The crystal structure explains the mechanism of molecular assembly of the complex and reveals several acidic clusters at the interface responsible for association at low acidic pH and disassociation at basic/neutral pH. The similarity of the general architecture between the PTC-E(M) and the previously determined PTC-A(M) strongly suggests that the progenitor M complexes of all botulinum serotypes may have similar molecular arrangement, although the neurotoxins apparently can take very different conformation when they are released from the M complex.

  19. A Self-Assembled DNA Origami-Gold Nanorod Complex for Cancer Theranostics.

    Science.gov (United States)

    Jiang, Qiao; Shi, Yuefeng; Zhang, Qian; Li, Na; Zhan, Pengfei; Song, Linlin; Dai, Luru; Tian, Jie; Du, Yang; Cheng, Zhen; Ding, Baoquan

    2015-10-01

    A self-assembled DNA origami (DO)-gold nanorod (GNR) complex, which is a dual-functional nanotheranostics constructed by decorating GNRs onto the surface of DNA origami, is demonstrated. After 24 h incubation of two structured DO-GNR complexes with human MCF7 breast cancer cells, significant enhancement of cell uptake is achieved compared to bare GNRs by two-photon luminescence imaging. Particularly, the triangle shaped DO-GNR complex exhibits optimal cellular accumulation. Compared to GNRs, improved photothermolysis against tumor cells is accomplished for the triangle DO-GNR complex by two-photon laser or NIR laser irradiation. Moreover, the DO-GNR complex exhibits enhanced antitumor efficacy compared with bare GNRs in nude mice bearing breast tumor xenografts. The results demonstrate that the DO-GNR complex can achieve optimal two-photon cell imaging and photothermal effect, suggesting a promising candidate for cancer diagnosis and therapy both in vitro and in vivo. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  20. Sensitization effects of supramolecular assemblies on the luminescence of terbium-ion prulifloxacin complexes

    Energy Technology Data Exchange (ETDEWEB)

    Wang Hong; Yi Chongyue; Li Xue; Fang Fang [School of Chemistry and Chemical Engineering, Huazhong University of Science and Technology, Wuhan 430074 (China); Yang Yajiang, E-mail: yjyang@mail.hust.edu.c [School of Chemistry and Chemical Engineering, Huazhong University of Science and Technology, Wuhan 430074 (China)

    2011-04-15

    Luminescence enhancement of terbium-ion prulifloxacin complexes (Tb(III)-PUFX) in supramolecular hydrogels formed by assembly of 1,3:2,4-di-O-benzylidene-D-sorbitol (DBS) was investigated by steady-state fluorescence, varying temperature fluorescence and time-resolved fluorescence. The luminescence images show that Tb(III)-PUFX were dispersed in the DBS gels. The luminescence intensity of Tb(III)-PUFX in the DBS gels was significantly increased in comparison with that in corresponding aqueous solutions. The varying temperature fluorescent spectra show that the luminescence intensity of Tb(III)-PUFX decreased with an increase in the temperature. This implies that the luminescence enhancement of Tb(III)-PUFX is related to the dissociation and the formation of the DBS assemblies. Time-resolved fluorescence measurements show slower rotational motion in DBS gels in comparison with that in the corresponding aqueous solutions. This may be ascribed to a unique microstructure of three-dimensional network formed by DBC aggregates, resulting in deactivation of the nonradiative relaxation. The images of field emission scanning electron microscopy and polarized optical microscopy indicate that the morphology of the DBS assemblies was not influenced upon addition of Tb(III)-PUFX to the DBS gels.

  1. Mechanical assembly of complex, 3D mesostructures from releasable multilayers of advanced materials.

    Science.gov (United States)

    Yan, Zheng; Zhang, Fan; Liu, Fei; Han, Mengdi; Ou, Dapeng; Liu, Yuhao; Lin, Qing; Guo, Xuelin; Fu, Haoran; Xie, Zhaoqian; Gao, Mingye; Huang, Yuming; Kim, JungHwan; Qiu, Yitao; Nan, Kewang; Kim, Jeonghyun; Gutruf, Philipp; Luo, Hongying; Zhao, An; Hwang, Keh-Chih; Huang, Yonggang; Zhang, Yihui; Rogers, John A

    2016-09-01

    Capabilities for assembly of three-dimensional (3D) micro/nanostructures in advanced materials have important implications across a broad range of application areas, reaching nearly every class of microsystem technology. Approaches that rely on the controlled, compressive buckling of 2D precursors are promising because of their demonstrated compatibility with the most sophisticated planar technologies, where materials include inorganic semiconductors, polymers, metals, and various heterogeneous combinations, spanning length scales from submicrometer to centimeter dimensions. We introduce a set of fabrication techniques and design concepts that bypass certain constraints set by the underlying physics and geometrical properties of the assembly processes associated with the original versions of these methods. In particular, the use of releasable, multilayer 2D precursors provides access to complex 3D topologies, including dense architectures with nested layouts, controlled points of entanglement, and other previously unobtainable layouts. Furthermore, the simultaneous, coordinated assembly of additional structures can enhance the structural stability and drive the motion of extended features in these systems. The resulting 3D mesostructures, demonstrated in a diverse set of more than 40 different examples with feature sizes from micrometers to centimeters, offer unique possibilities in device design. A 3D spiral inductor for near-field communication represents an example where these ideas enable enhanced quality ( Q ) factors and broader working angles compared to those of conventional 2D counterparts.

  2. An Epstein-Barr Virus-Encoded Protein Complex Requires an Origin of Lytic Replication In Cis to Mediate Late Gene Transcription

    Science.gov (United States)

    Djavadian, Reza; Chiu, Ya-Fang; Johannsen, Eric

    2016-01-01

    Epstein-Barr virus lytic replication is accomplished by an intricate cascade of gene expression that integrates viral DNA replication and structural protein synthesis. Most genes encoding structural proteins exhibit “true” late kinetics–their expression is strictly dependent on lytic DNA replication. Recently, the EBV BcRF1 gene was reported to encode a TATA box binding protein homolog, which preferentially recognizes the TATT sequence found in true late gene promoters. BcRF1 is one of seven EBV genes with homologs found in other β- and γ-, but not in α-herpesviruses. Using EBV BACmids, we systematically disrupted each of these “βγ” genes. We found that six of them, including BcRF1, exhibited an identical phenotype: intact viral DNA replication with loss of late gene expression. The proteins encoded by these six genes have been found by other investigators to form a viral protein complex that is essential for activation of TATT-containing reporters in EBV-negative 293 cells. Unexpectedly, in EBV infected 293 cells, we found that TATT reporter activation was weak and non-specific unless an EBV origin of lytic replication (OriLyt) was present in cis. Using two different replication-defective EBV genomes, we demonstrated that OriLyt-mediated DNA replication is required in cis for TATT reporter activation and for late gene expression from the EBV genome. We further demonstrate by fluorescence in situ hybridization that the late BcLF1 mRNA localizes to EBV DNA replication factories. These findings support a model in which EBV true late genes are only transcribed from newly replicated viral genomes. PMID:27348612

  3. Replication Clamps and Clamp Loaders

    Science.gov (United States)

    Hedglin, Mark; Kumar, Ravindra; Benkovic, Stephen J.

    2013-01-01

    To achieve the high degree of processivity required for DNA replication, DNA polymerases associate with ring-shaped sliding clamps that encircle the template DNA and slide freely along it. The closed circular structure of sliding clamps necessitates an enzyme-catalyzed mechanism, which not only opens them for assembly and closes them around DNA, but specifically targets them to sites where DNA synthesis is initiated and orients them correctly for replication. Such a feat is performed by multisubunit complexes known as clamp loaders, which use ATP to open sliding clamp rings and place them around the 3′ end of primer–template (PT) junctions. Here we discuss the structure and composition of sliding clamps and clamp loaders from the three domains of life as well as T4 bacteriophage, and provide our current understanding of the clamp-loading process. PMID:23545418

  4. Constructing Asymmetric Polyion Complex Vesicles via Template Assembling Strategy: Formulation Control and Tunable Permeability

    Directory of Open Access Journals (Sweden)

    Junbo Li

    2017-11-01

    Full Text Available A strategy for constructing polyion complex vesicles (PICsomes with asymmetric structure is described. Poly(methylacrylic acid-block-poly(N-isopropylacrylamide modified gold nanoparticles (PMAA-b-PNIPAm-@-Au NPs were prepared and then assembled with poly(ethylene glycol-block-poly[1-methyl-3-(2-methacryloyloxy propylimidazolium bromine] (PEG-b-PMMPImB via polyion complex of PMMA and PMMPImB. After removing the Au NPs template, asymmetric PICsomes composed of a PNIPAm inner-shell, PIC wall, and PEG outer-corona were obtained. These PICsomes have low protein absorption and thermally tunable permeability, provided by the PEG outer-corona and the PNIPAm inner-shell, respectively. Moreover, PICsome size can be tailored by using templates of predetermined sizes. This novel strategy for constructing asymmetric PICsomes with well-defined properties and controllable size is valuable for applications such as drug delivery, catalysis and monitoring of chemical reactions, and biomimetics.

  5. Dimeric Self-assembling via Hydrogen Bonding and Emissive Behavior of a New Copper (I Complex

    Directory of Open Access Journals (Sweden)

    Juciely M. dos Reis

    2017-04-01

    Full Text Available This work describes the synthesis, structural characterization and emissive behavior of a new copper (I complex based on 1-thiocarbamoyl-5-(4-methoxiphenyl-3-phenyl-4,5-dihydro-1H-pyrazole ligand. A dimeric self-assembling via hydrogen bonding was determined by analyzing the short contacts present in the solid-state structure by means of X-ray crystallography. The spectroscopic properties were determined using UV-Vis and fluorescence experiments and an interesting behavior as bluish luminescence was assigned mainly to the mixed (MLCT + IL electronic transitions of the Cu(Id10 ® (S=C–Nligand type. The complete characterization of the new copper (I complex also included elemental analyses and IR spectroscopy. DOI: http://dx.doi.org/10.17807/orbital.v9i1.952

  6. Dissecting the molecular assembly of theToxoplasma gondiiMyoA motility complex.

    Science.gov (United States)

    Powell, Cameron J; Jenkins, Meredith L; Parker, Michelle L; Ramaswamy, Raghavendran; Kelsen, Anne; Warshaw, David M; Ward, Gary E; Burke, John E; Boulanger, Martin J

    2017-11-24

    Apicomplexan parasites such as Toxoplasma gondii rely on a unique form of locomotion known as gliding motility. Generating the mechanical forces to support motility are divergent class XIV myosins (MyoA) coordinated by accessory proteins known as light chains. Although the importance of the MyoA-light chain complex is well-established, the detailed mechanisms governing its assembly and regulation are relatively unknown. To establish a molecular blueprint of this dynamic complex, we first mapped the adjacent binding sites of light chains MLC1 and ELC1 on the MyoA neck (residues 775-818) using a combination of hydrogen-deuterium exchange mass spectrometry and isothermal titration calorimetry. We then determined the 1.85 Å resolution crystal structure of MLC1 in complex with its cognate MyoA peptide. Structural analysis revealed a bilobed architecture with MLC1 clamping tightly around the helical MyoA peptide, consistent with the stable 10 nm K d measured by isothermal titration calorimetry. We next showed that coordination of calcium by an EF-hand in ELC1 and prebinding of MLC1 to the MyoA neck enhanced the affinity of ELC1 for the MyoA neck 7- and 8-fold, respectively. When combined, these factors enhanced ELC1 binding 49-fold (to a K d of 12 nm). Using the full-length MyoA motor (residues 1-831), we then showed that, in addition to coordinating the neck region, ELC1 appears to engage the MyoA converter subdomain, which couples the motor domain to the neck. These data support an assembly model where staged binding events cooperate to yield high-affinity complexes that are able to maximize force transduction. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  7. Ori-somes, NP complexes descending from origin regions of animal chromosomal DNA replication. A micromorphological study

    Czech Academy of Sciences Publication Activity Database

    Korb, Jan; Štokrová, Jitka; Říman, Josef

    2001-01-01

    Roč. 19, č. 2 (2001), s. 343-350 ISSN 0739-1102 Institutional research plan: CEZ:AV0Z5052915 Keywords : origin regions of DNA replication * replicative structure s * electron microscopy Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 1.243, year: 2001

  8. Assembly and stoichiometry of the core structure of the bacterial flagellar type III export gate complex.

    Science.gov (United States)

    Fukumura, Takuma; Makino, Fumiaki; Dietsche, Tobias; Kinoshita, Miki; Kato, Takayuki; Wagner, Samuel; Namba, Keiichi; Imada, Katsumi; Minamino, Tohru

    2017-08-01

    The bacterial flagellar type III export apparatus, which is required for flagellar assembly beyond the cell membranes, consists of a transmembrane export gate complex and a cytoplasmic ATPase complex. FlhA, FlhB, FliP, FliQ, and FliR form the gate complex inside the basal body MS ring, although FliO is required for efficient export gate formation in Salmonella enterica. However, it remains unknown how they form the gate complex. Here we report that FliP forms a homohexameric ring with a diameter of 10 nm. Alanine substitutions of conserved Phe-137, Phe-150, and Glu-178 residues in the periplasmic domain of FliP (FliPP) inhibited FliP6 ring formation, suppressing flagellar protein export. FliO formed a 5-nm ring structure with 3 clamp-like structures that bind to the FliP6 ring. The crystal structure of FliPP derived from Thermotoga maritia, and structure-based photo-crosslinking experiments revealed that Phe-150 and Ser-156 of FliPP are involved in the FliP-FliP interactions and that Phe-150, Arg-152, Ser-156, and Pro-158 are responsible for the FliP-FliO interactions. Overexpression of FliP restored motility of a ∆fliO mutant to the wild-type level, suggesting that the FliP6 ring is a functional unit in the export gate complex and that FliO is not part of the final gate structure. Copurification assays revealed that FlhA, FlhB, FliQ, and FliR are associated with the FliO/FliP complex. We propose that the assembly of the export gate complex begins with FliP6 ring formation with the help of the FliO scaffold, followed by FliQ, FliR, and FlhB and finally FlhA during MS ring formation.

  9. 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

  10. PolyC-binding protein 1 interacts with 5'-untranslated region of enterovirus 71 RNA in membrane-associated complex to facilitate viral replication.

    Directory of Open Access Journals (Sweden)

    Zhen Luo

    Full Text Available Enterovirus 71 (EV71 is one causative agent of hand, foot, and mouth disease (HFMD, which may lead to severe neurological disorders and mortality in children. EV71 genome is a positive single-stranded RNA containing a single open reading frame (ORF flanked by 5'-untranslated region (5'UTR and 3'UTR. The 5'UTR is fundamentally important for virus replication by interacting with cellular proteins. Here, we revealed that poly(C-binding protein 1 (PCBP1 specifically binds to the 5'UTR of EV71. Detailed studies indicated that the RNA-binding K-homologous 1 (KH1 domain of PCBP1 is responsible for its binding to the stem-loop I and IV of EV71 5'UTR. Interestingly, we revealed that PCBP1 is distributed in the nucleus and cytoplasm of uninfected cells, but mainly localized in the cytoplasm of EV71-infected cells due to interaction and co-localization with the viral RNA. Furthermore, sub-cellular distribution analysis showed that PCBP1 is located in ER-derived membrane, in where virus replication occurred in the cytoplasm of EV71-infected cells, suggesting PCBP1 is recruited in a membrane-associated replication complex. In addition, we found that the binding of PCBP1 to 5'UTR resulted in enhancing EV71 viral protein expression and virus production so as to facilitate viral replication. Thus, we revealed a novel mechanism in which PCBP1 as a positive regulator involved in regulation of EV71 replication in the host specialized membrane-associated replication complex, which provides an insight into cellular factors involved in EV71 replication.

  11. Computational complexity of algorithms for sequence comparison, short-read assembly and genome alignment.

    Science.gov (United States)

    Baichoo, Shakuntala; Ouzounis, Christos A

    A multitude of algorithms for sequence comparison, short-read assembly and whole-genome alignment have been developed in the general context of molecular biology, to support technology development for high-throughput sequencing, numerous applications in genome biology and fundamental research on comparative genomics. The computational complexity of these algorithms has been previously reported in original research papers, yet this often neglected property has not been reviewed previously in a systematic manner and for a wider audience. We provide a review of space and time complexity of key sequence analysis algorithms and highlight their properties in a comprehensive manner, in order to identify potential opportunities for further research in algorithm or data structure optimization. The complexity aspect is poised to become pivotal as we will be facing challenges related to the continuous increase of genomic data on unprecedented scales and complexity in the foreseeable future, when robust biological simulation at the cell level and above becomes a reality. Copyright © 2017 Elsevier B.V. All rights reserved.

  12. The origin of the supernumerary subunits and assembly factors of complex I: A treasure trove of pathway evolution

    NARCIS (Netherlands)

    Elurbe, D.M.; Huynen, M.A.

    2016-01-01

    We review and document the evolutionary origin of all complex I assembly factors and nine supernumerary subunits from protein families. Based on experimental data and the conservation of critical residues we identify a spectrum of protein function conservation between the complex I representatives

  13. Insight into structure and assembly of the nuclear pore complex by utilizing the genome of a eukaryotic thermophile

    DEFF Research Database (Denmark)

    Amlacher, Stefan; Sarges, Phillip; Flemming, Dirk

    2011-01-01

    Despite decades of research, the structure and assembly of the nuclear pore complex (NPC), which is composed of ~30 nucleoporins (Nups), remain elusive. Here, we report the genome of the thermophilic fungus Chaetomium thermophilum (ct) and identify the complete repertoire of Nups therein. The the...... of a thermophilic eukaryote for studying complex molecular machines....

  14. 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...... increased DNA replication stress....

  15. When lithography meets self-assembly: a review of recent advances in the directed assembly of complex metal nanostructures on planar and textured surfaces

    Science.gov (United States)

    Hughes, Robert A.; Menumerov, Eredzhep; Neretina, Svetlana

    2017-07-01

    One of the foremost challenges in nanofabrication is the establishment of a processing science that integrates wafer-based materials, techniques, and devices with the extraordinary physicochemical properties accessible when materials are reduced to nanoscale dimensions. Such a merger would allow for exacting controls on nanostructure positioning, promote cooperative phenomenon between adjacent nanostructures and/or substrate materials, and allow for electrical contact to individual or groups of nanostructures. With neither self-assembly nor top-down lithographic processes being able to adequately meet this challenge, advancements have often relied on a hybrid strategy that utilizes lithographically-defined features to direct the assembly of nanostructures into organized patterns. While these so-called directed assembly techniques have proven viable, much of this effort has focused on the assembly of periodic arrays of spherical or near-spherical nanostructures comprised of a single element. Work directed toward the fabrication of more complex nanostructures, while still at a nascent stage, has nevertheless demonstrated the possibility of forming arrays of nanocubes, nanorods, nanoprisms, nanoshells, nanocages, nanoframes, core-shell structures, Janus structures, and various alloys on the substrate surface. In this topical review, we describe the progress made in the directed assembly of periodic arrays of these complex metal nanostructures on planar and textured substrates. The review is divided into three broad strategies reliant on: (i) the deterministic positioning of colloidal structures, (ii) the reorganization of deposited metal films at elevated temperatures, and (iii) liquid-phase chemistry practiced directly on the substrate surface. These strategies collectively utilize a broad range of techniques including capillary assembly, microcontact printing, chemical surface modulation, templated dewetting, nanoimprint lithography, and dip-pen nanolithography and

  16. Luminescent lanthanide complexes with 4-acetamidobenzoate: Synthesis, supramolecular assembly via hydrogen bonds, crystal structures and photoluminescence

    International Nuclear Information System (INIS)

    Yin Xia; Fan Jun; Wang Zhihong; Zheng Shengrun; Tan Jingbo; Zhang Weiguang

    2011-01-01

    Four new luminescent complexes, namely, [Eu(aba) 2 (NO 3 )(C 2 H 5 OH) 2 ] (1), [Eu(aba) 3 (H 2 O) 2 ].0.5 (4, 4'-bpy).2H 2 O (2), [Eu 2 (aba) 4 (2, 2'-bpy) 2 (NO 3 ) 2 ].4H 2 O (3) and [Tb 2 (aba) 4 (phen) 2 (NO 3 ) 2 ].2C 2 H 5 OH (4) were obtained by treating Ln(NO 3 ) 3 .6H 2 O and 4-acetamidobenzoic acid (Haba) with different coligands (4, 4'-bpy=4, 4'-bipyridine, 2, 2'-bpy=2, 2'-bipyridine, and phen=1, 10-phenanthroline). They exhibit 1D chains (1-2) and dimeric structures (3-4), respectively. This structural variation is mainly attributed to the change of coligands and various coordination modes of aba molecules. Moreover, the coordination units are further connected via hydrogen bonds to form 2D even 3D supramolecular networks. These complexes show characteristic emissions in the visible region at room temperature. In addition, thermal behaviors of four complexes have been investigated under air atmosphere. The relationship between the structures and physical properties has been discussed. - Graphical abstract: Structure variation of four complexes is attributed to the change of coligands and various coordination modes of aba molecules. Moreover, they show characteristic emissions in the visible region. Highlights: → Auxiliary ligands have played the crucial roles on the structures of the resulting complexes. → Isolated structure units are further assembled via H-bonds to form supramolecular networks. → These solid-state complexes exhibit strong, characteristic emissions in the visible region.

  17. Effects of physical, chemical or biological factors on DNA replication in mammalian cells. Part 2. Recovery of DNA complex, nucleoid and DNA replication after gamma irradiation

    Energy Technology Data Exchange (ETDEWEB)

    Synzynys, B.I.; Kiseleva, V.I.; Trofimova, S.F.

    1984-11-01

    Murine LL cell line was employed in studies on the effects of gamma irradiation (6 Gy, 8 Gy/min from Co-60 source) on the kinetics and repair of DNA superstructure and recovery of DNA synthesis. Comparison of the kinetic data for the postradiation repair of the DNA-membrane complex, nucleoid, and recovery of DNA synthesis demonstrated that functional recovery of the former two factors preceded DNA synthesis by at least 2 h. In view of this lag, it appears that additional factors are involved in radiation damage that must be repaired before DNA synthesis commences. 10 references, 3 figures.

  18. HTP-1 coordinates synaptonemal complex assembly with homolog alignment during meiosis in C. elegans.

    Science.gov (United States)

    Couteau, Florence; Zetka, Monique

    2005-11-15

    During meiosis, the mechanisms responsible for homolog alignment, synapsis, and recombination are precisely coordinated to culminate in the formation of crossovers capable of directing accurate chromosome segregation. An outstanding question is how the cell ensures that the structural hallmark of meiosis, the synaptonemal complex (SC), forms only between aligned pairs of homologous chromosomes. In the present study, we find that two closely related members of the him-3 gene family in Caenorhabditis elegans function as regulators of synapsis. HTP-1 functionally couples homolog alignment to its stabilization by synapsis by preventing the association of SC components with unaligned and immature chromosome axes; in the absence of the protein, nonhomologous contacts between chromosomes are inappropriately stabilized, resulting in extensive nonhomologous synapsis and a drastic decline in chiasma formation. In the absence of both HTP-1 and HTP-2, synapsis is abrogated per se and the early association of SC components with chromosomes observed in htp-1 mutants does not occur, suggesting a function for the proteins in licensing SC assembly. Furthermore, our results suggest that early steps of recombination occur in a narrow window of opportunity in early prophase that ends with SC assembly, resulting in a mechanistic coupling of the two processes to promote crossing over.

  19. The linear ubiquitin assembly complex (LUBAC) is essential for NLRP3 inflammasome activation

    Science.gov (United States)

    Rodgers, Mary A.; Bowman, James W.; Fujita, Hiroaki; Orazio, Nicole; Shi, Mude; Liang, Qiming; Amatya, Rina; Kelly, Thomas J.; Iwai, Kazuhiro; Ting, Jenny

    2014-01-01

    Linear ubiquitination is a newly discovered posttranslational modification that is currently restricted to a small number of known protein substrates. The linear ubiquitination assembly complex (LUBAC), consisting of HOIL-1L, HOIP, and Sharpin, has been reported to activate NF-κB–mediated transcription in response to receptor signaling by ligating linear ubiquitin chains to Nemo and Rip1. Despite recent advances, the detailed roles of LUBAC in immune cells remain elusive. We demonstrate a novel HOIL-1L function as an essential regulator of the activation of the NLRP3/ASC inflammasome in primary bone marrow–derived macrophages (BMDMs) independently of NF-κB activation. Mechanistically, HOIL-1L is required for assembly of the NLRP3/ASC inflammasome and the linear ubiquitination of ASC, which we identify as a novel LUBAC substrate. Consequently, we find that HOIL-1L−/− mice have reduced IL-1β secretion in response to in vivo NLRP3 stimulation and survive lethal challenge with LPS. Together, these data demonstrate that linear ubiquitination is required for NLRP3 inflammasome activation, defining the molecular events of NLRP3 inflammasome activation and expanding the role of LUBAC as an innate immune regulator. Furthermore, our observation is clinically relevant because patients lacking HOIL-1L expression suffer from pyogenic bacterial immunodeficiency, providing a potential new therapeutic target for enhancing inflammation in immunodeficient patients. PMID:24958845

  20. Deterministic Assembly of Complex Bacterial Communities in Guts of Germ-Free Cockroaches.

    Science.gov (United States)

    Mikaelyan, Aram; Thompson, Claire L; Hofer, Markus J; Brune, Andreas

    2016-02-15

    The gut microbiota of termites plays important roles in the symbiotic digestion of lignocellulose. However, the factors shaping the microbial community structure remain poorly understood. Because termites cannot be raised under axenic conditions, we established the closely related cockroach Shelfordella lateralis as a germ-free model to study microbial community assembly and host-microbe interactions. In this study, we determined the composition of the bacterial assemblages in cockroaches inoculated with the gut microbiota of termites and mice using pyrosequencing analysis of their 16S rRNA genes. Although the composition of the xenobiotic communities was influenced by the lineages present in the foreign inocula, their structure resembled that of conventional cockroaches. Bacterial taxa abundant in conventional cockroaches but rare in the foreign inocula, such as Dysgonomonas and Parabacteroides spp., were selectively enriched in the xenobiotic communities. Donor-specific taxa, such as endomicrobia or spirochete lineages restricted to the gut microbiota of termites, however, either were unable to colonize germ-free cockroaches or formed only small populations. The exposure of xenobiotic cockroaches to conventional adults restored their normal microbiota, which indicated that autochthonous lineages outcompete foreign ones. Our results provide experimental proof that the assembly of a complex gut microbiota in insects is deterministic. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  1. Chlorophyll biosynthesis and assembly into chlorophyll-protein complexes in isolated developing chloroplasts

    International Nuclear Information System (INIS)

    Bhaya, D.; Castelfranco, P.A.

    1985-01-01

    Isolated developing plastids from greening cucumber cotyledons or from photoperiodically grown pea seedlings incorporated 14 C-labeled 5-aminolevulinic acid (ALA) into chlorophyll (Chl). Incorporation was light dependent, enhanced by S-adenosylmethionine, and linear for 1 hr. The in vitro rate of Chl synthesis from ALA was comparable to the in vivo rate of Chl accumulation. Levulinic acid and dioxoheptanoic acid strongly inhibited Chl synthesis but not plastid protein synthesis. Neither chloramphenicol nor spectinomycin affected Chl synthesis, although protein synthesis was strongly inhibited. Components of thylakoid membranes from plastids incubated with [ 14 C]ALA were resolved by electrophoresis and then subjected to autoradiography. This work showed that (i) newly synthesized Chl was assembled into Chl-protein complexes and (ii) the inhibition of protein synthesis during the incubation did not alter the labeling pattern. Thus, there was no observable short-term coregulation between Chl synthesis (from ALA) and the synthesis of membrane proteins in isolated plastids

  2. Metal complexation and monolayer self-assembly of the bio-organic semiconductor Alizarin

    Energy Technology Data Exchange (ETDEWEB)

    Uppal, Neeti [Dept. Earth and Environmental Sciences, Ludwig-Maximilians-Universitaet Muenchen (LMU) and Center for NanoSciences (CeNS), Muenchen (Germany); Institut fuer Physik, Universitaet Augsburg (Germany); Gast, Norbert [Dept. Earth and Environmental Sciences, Ludwig-Maximilians-Universitaet Muenchen (LMU) and Center for NanoSciences (CeNS), Muenchen (Germany); Zentrum Neue Technologien, Deutsches Museum, Muenchen (Germany); Bueno, Martin [Fakultaet Feinwerk- und Mikrotechnik, Physikalische Technik, Hochschule Muenchen (Germany); Heckl, Wolfgang M. [Dept. of Physics, Technische Universitaet Muenchen (TUM), Garching (Germany); Zentrum Neue Technologien, Deutsches Museum, Muenchen (Germany); Trixler, Frank [Dept. Earth and Environmental Sciences, Ludwig-Maximilians-Universitaet Muenchen (LMU) and Center for NanoSciences (CeNS), Muenchen (Germany); Dept. of Physics, Technische Universitaet Muenchen (TUM), Garching (Germany); Zentrum Neue Technologien, Deutsches Museum, Muenchen (Germany)

    2010-07-01

    Organic Solid/Solid Wetting Deposition (OSWD) (Trixler et al.: Chem.Eur.J. 13 (2007), 7785) enables to deposit insoluble molecules such as organic pigments and semiconductors on substrate surfaces under ambient conditions. We explore the potential of OSWD to grow and manipulate monolayers of biomolecules and their chelates on graphite and use Alizarin as a model system - a natural organic compound which occurs mainly as an anthraquinone glycoside in plants. Our investigations via Scanning Tunneling Microscopy (STM), Tunneling Spectroscopy (TS) and Molecular Modelling reveal that OSWD works also with bio-organic molecules and chelate complexes and show that the advantages of OSWD (self-assembly under ambient conditions in a non-solvent environment, nanomanipulation via molecular extraction) can all be tapped.

  3. Polyion complex micelles prepared by self-assembly of block-graft polycation and hyperbranched polyanion

    Science.gov (United States)

    Dai, Yu; Wang, Hongquan; Zhang, Xiaojin

    2017-09-01

    Polyion complex (PIC) micelles were prepared by self-assembly of block-graft polycation monomethoxy poly(ethylene glycol)- block-(poly(ɛ-caprolactone)- graft-polyethylenimine) (PEG- b-(PCL- g-PEI)) and hyperbranched polyanion sodium carboxyl-modified hyperbranched polyesters (Hx-COONa, x = 20, 30, 40). The results from commonly used MTT assay indicated that PIC micelles had good biocompatibility. PIC micelles with N/COO- of 8/3 had appropriate size (sub-110 nm) and moderate zeta potential ( 3 mV). PIC micelles were nano-sized spheres, and the average size was about 50 nm. PIC micelles had high drug loading capacity for hydrophilic drugs such as doxorubicin (DOX) hydrochloride and released the drugs under the influence of pH and ionic strength.

  4. Assembly of Francisella novicida Cpf1 endonuclease in complex with guide RNA and target DNA.

    Science.gov (United States)

    Alcón, Pablo; Montoya, Guillermo; Stella, Stefano

    2017-07-01

    Bacteria and archaea use the CRISPR-Cas system as an adaptive response against infection by foreign nucleic acids. Owing to its remarkable flexibility, this mechanism has been harnessed and adopted as a powerful tool for genome editing. The CRISPR-Cas system includes two classes that are subdivided into six types and 19 subtypes according to conservation of the cas gene and loci organization. Recently, a new protein with endonuclease activity belonging to class 2 type V has been identified. This endonuclease, termed Cpf1, in complex with a single CRISPR RNA (crRNA) is able to recognize and cleave a target DNA preceded by a 5'-TTN-3' protospacer-adjacent motif (PAM) complementary to the RNA guide. To obtain structural insight into the inner workings of Cpf1, the crystallization of an active complex containing the full extent of the crRNA and a 31-nucleotide dsDNA target was attempted. The gene encoding Cpf1 from Francisella novicida was cloned, overexpressed and purified. The crRNA was transcribed and purified in vitro. Finally, the ternary FnCpf1-crRNA-DNA complex was assembled and purified by preparative electrophoresis before crystallization. Crystals belonging to space group C222 1 , with unit-cell parameters a = 85.2, b = 137.6, c = 320.5 Å, were obtained and subjected to preliminary diffraction experiments.

  5. The UL8 subunit of the helicase-primase complex of herpes simplex virus promotes DNA annealing and has a high affinity for replication forks.

    Science.gov (United States)

    Bermek, Oya; Weller, Sandra K; Griffith, Jack D

    2017-09-22

    During lytic infection, herpes simplex virus (HSV) DNA is replicated by a mechanism involving DNA recombination. For instance, replication of the HSV-1 genome produces X- and Y-branched structures, reminiscent of recombination intermediates. HSV-1's replication machinery includes a trimeric helicase-primase composed of helicase (UL5) and primase (UL52) subunits and a third subunit, UL8. UL8 has been reported to stimulate the helicase and primase activities of the complex in the presence of ICP8, an HSV-1 protein that functions as an annealase, a protein that binds complementary single-stranded DNA (ssDNA) and facilitates its annealing to duplex DNA. UL8 also influences the intracellular localization of the UL5/UL52 subunits, but UL8's catalytic activities are not known. In this study we used a combination of biochemical techniques and transmission electron microscopy. First, we report that UL8 alone forms protein filaments in solution. Moreover, we also found that UL8 binds to ssDNAs >50-nucletides long and promotes the annealing of complementary ssDNA to generate highly branched duplex DNA structures. Finally, UL8 has a very high affinity for replication fork structures containing a gap in the lagging strand as short as 15 nucleotides, suggesting that UL8 may aid in directing or loading the trimeric complex onto a replication fork. The properties of UL8 uncovered here suggest that UL8 may be involved in the generation of the X- and Y-branched structures that are the hallmarks of HSV replication. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  6. The origin of the supernumerary subunits and assembly factors of complex I: A treasure trove of pathway evolution.

    Science.gov (United States)

    Elurbe, Dei M; Huynen, Martijn A

    2016-07-01

    We review and document the evolutionary origin of all complex I assembly factors and nine supernumerary subunits from protein families. Based on experimental data and the conservation of critical residues we identify a spectrum of protein function conservation between the complex I representatives and their non-complex I homologs. This spectrum ranges from proteins that have retained their molecular function but in which the substrate specificity may have changed or have become more specific, like NDUFAF5, to proteins that have lost their original molecular function and critical catalytic residues like NDUFAF6. In between are proteins that have retained their molecular function, which however appears unrelated to complex I, like ACAD9, or proteins in which amino acids of the active site are conserved but for which no enzymatic activity has been reported, like NDUFA10. We interpret complex I evolution against the background of molecular evolution theory. Complex I supernumerary subunits and assembly factors appear to have been recruited from proteins that are mitochondrial and/or that are expressed when complex I is active. Within the evolution of complex I and its assembly there are many cases of neofunctionalization after gene duplication, like ACAD9 and TMEM126B, one case of subfunctionalization: ACPM1 and ACPM2 in Yarrowia lipolytica, and one case in which a complex I protein itself appears to have been the source of a new protein from another complex: NDUFS6 gave rise to cytochrome c oxidase subunit COX4/COX5b. Complex I and its assembly can therewith be regarded as a treasure trove for pathway evolution. This article is part of a Special Issue entitled Respiratory complex I, edited by Volker Zickermann and Ulrich Brandt. Copyright © 2016 Elsevier B.V. All rights reserved.

  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. Self-Assembly of Complex DNA Tessellations by Using Low-Symmetry Multi-arm DNA Tiles.

    Science.gov (United States)

    Zhang, Fei; Jiang, Shuoxing; Li, Wei; Hunt, Ashley; Liu, Yan; Yan, Hao

    2016-07-25

    Modular DNA tile-based self-assembly is a versatile way to engineer basic tessellation patterns on the nanometer scale, but it remains challenging to achieve high levels of structural complexity. We introduce a set of general design principles to create intricate DNA tessellations by employing multi-arm DNA motifs with low symmetry. We achieved two novel Archimedean tiling patterns, (4.8.8) and (3.6.3.6), and one pattern with higher-order structures beyond the complexity observed in Archimedean tiling. Our success in assembling complicated DNA tessellations demonstrates the broad design space of DNA structural motifs, enriching the toolbox of DNA tile-based self-assembly and expanding the complexity boundaries of DNA tile-based tessellation. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  9. Combined Respiratory Chain Deficiency and UQCC2 Mutations in Neonatal Encephalomyopathy: Defective Supercomplex Assembly in Complex III Deficiencies

    Directory of Open Access Journals (Sweden)

    René G. Feichtinger

    2017-01-01

    Full Text Available Vertebrate respiratory chain complex III consists of eleven subunits. Mutations in five subunits either mitochondrial (MT-CYB or nuclear (CYC1, UQCRC2, UQCRB, and UQCRQ encoded have been reported. Defects in five further factors for assembly (TTC19, UQCC2, and UQCC3 or iron-sulphur cluster loading (BCS1L and LYRM7 cause complex III deficiency. Here, we report a second patient with UQCC2 deficiency. This girl was born prematurely; pregnancy was complicated by intrauterine growth retardation and oligohydramnios. She presented with respiratory distress syndrome, developed epileptic seizures progressing to status epilepticus, and died at day 33. She had profound lactic acidosis and elevated urinary pyruvate. Exome sequencing revealed two homozygous missense variants in UQCC2, leading to a severe reduction of UQCC2 protein. Deficiency of complexes I and III was found enzymatically and on the protein level. A review of the literature on genetically distinct complex III defects revealed that, except TTC19 deficiency, the biochemical pattern was very often a combined respiratory chain deficiency. Besides complex III, typically, complex I was decreased, in some cases complex IV. In accordance with previous observations, the presence of assembled complex III is required for the stability or assembly of complexes I and IV, which might be related to respirasome/supercomplex formation.

  10. Assembly of functional photosystem complexes in Rhodobacter sphaeroides incorporating carotenoids from the spirilloxanthin pathway

    Science.gov (United States)

    Chi, Shuang C.; Mothersole, David J.; Dilbeck, Preston; Niedzwiedzki, Dariusz M.; Zhang, Hao; Qian, Pu; Vasilev, Cvetelin; Grayson, Katie J.; Jackson, Philip J.; Martin, Elizabeth C.; Li, Ying; Holten, Dewey; Neil Hunter, C.

    2015-01-01

    Carotenoids protect the photosynthetic apparatus against harmful radicals arising from the presence of both light and oxygen. They also act as accessory pigments for harvesting solar energy, and are required for stable assembly of many light-harvesting complexes. In the phototrophic bacterium Rhodobacter (Rba.) sphaeroides phytoene desaturase (CrtI) catalyses three sequential desaturations of the colourless carotenoid phytoene, extending the number of conjugated carbon–carbon double bonds, N, from three to nine and producing the yellow carotenoid neurosporene; subsequent modifications produce the yellow/red carotenoids spheroidene/spheroidenone (N = 10/11). Genomic crtI replacements were used to swap the native three-step Rba. sphaeroides CrtI for the four-step Pantoea agglomerans enzyme, which re-routed carotenoid biosynthesis and culminated in the production of 2,2′-diketo-spirilloxanthin under semi-aerobic conditions. The new carotenoid pathway was elucidated using a combination of HPLC and mass spectrometry. Premature termination of this new pathway by inactivating crtC or crtD produced strains with lycopene or rhodopin as major carotenoids. All of the spirilloxanthin series carotenoids are accepted by the assembly pathways for LH2 and RC–LH1–PufX complexes. The efficiency of carotenoid-to-bacteriochlorophyll energy transfer for 2,2′-diketo-spirilloxanthin (15 conjugated C 000000000000 000000000000 000000000000 111111111111 000000000000 111111111111 000000000000 000000000000 000000000000 C bonds; N = 15) in LH2 complexes is low, at 35%. High energy transfer efficiencies were obtained for neurosporene (N = 9; 94%), spheroidene (N = 10; 96%) and spheroidenone (N = 11; 95%), whereas intermediate values were measured for lycopene (N = 11; 64%), rhodopin (N = 11; 62%) and spirilloxanthin (N = 13; 39%). The variety and stability of these novel Rba. sphaeroides antenna complexes make them useful experimental models for investigating the

  11. Respiratory Chain Supercomplexes associate with the Cysteine Desulfurase Complex of the Iron-Sulfur Cluster Assembly Machinery.

    Science.gov (United States)

    Böttinger, Lena; Mårtensson, Christoph U; Song, Jiyao; Zufall, Nicole; Wiedemann, Nils; Becker, Thomas

    2018-01-31

    Mitochondria are the powerhouses of eukaryotic cells. The activity of the respiratory chain complexes generates a proton gradient across the inner membrane, which is used by the F 1 F O -ATP synthase to produce ATP for cellular metabolism. In baker's yeast Saccharomyces cerevisiae the cytochrome bc1 complex (complex III) and cytochrome c oxidase (complex IV) associate in respiratory chain supercomplexes. Iron-sulfur clusters (ISC) form reactive centers of respiratory chain complexes. The assembly of ISC occurs in the mitochondrial matrix and is essential for cell viability. The cysteine desulfurase Nfs1 provides sulfur for ISC assembly and forms with partner proteins the ISC-biogenesis desulfurase complex (ISD complex). Here, we report an unexpected interaction of the active ISD complex with the cytochrome bc1 complex and cytochrome c oxidase. The individual deletion of complex III or complex IV blocks the association of the ISD complex with respiratory chain components. We conclude that the ISD complex binds selectively to respiratory chain supercomplexes. We propose that this molecular link contributes to coordination of iron-sulfur cluster formation with respiratory activity. © 2018 by The American Society for Cell Biology.

  12. Metal Halide Perovskite Supercrystals: Gold-Bromide Complex Triggered Assembly of CsPbBr3 Nanocubes.

    Science.gov (United States)

    Wang, Kun-Hua; Yang, Jun-Nan; Ni, Qian-Kun; Yao, Hong-Bin; Yu, Shu-Hong

    2018-01-16

    Using nanocrystals as "artificial atoms" to construct supercrystals is an interesting process to explore the stacking style of nanoscale building blocks and corresponding collective properties. Various types of semiconducting supercrystals have been constructed via the assembly of nanocrystals driven by the entropic, electrostatic, or van der Waals interactions. We report a new type of metal halide perovskite supercrystals via the gold-bromide complex triggered assembly of newly emerged attractive CsPbBr 3 nanocubes. Through introducing gold-bromide (Au-Br) complexes into CsPbBr 3 nanocubes suspension, the self-assembly process of CsPbBr 3 nanocubes to form supercrystals was investigated with the different amount of Au-Br complexes added to the suspensions, which indicates that the driven force of the formation of CsPbBr 3 supercrystals included the van der Waals interactions among carbon chains and electrostatic interactions between Au-Br complexes and surfactants. Accordingly, the optical properties change with the assembly of CsPbBr 3 nanocubes and the variation of mesoscale structures of supercrystals with heating treatment was revealed as well, demonstrating the ionic characteristics of CsPbBr 3 nanocrystals. The fabricated CsPbBr 3 supercrystal presents a novel type of semiconducting supercrystals that will open an avenue for the assembly of ionic nanocrystals.

  13. The Rieske Iron-Sulfur Protein: Import and Assembly into the Cytochrome bc 1 Complex of Yeast Mitochondria

    Science.gov (United States)

    Conte, Laura; Zara, Vincenzo

    2011-01-01

    The Rieske iron-sulfur protein, one of the catalytic subunits of the cytochrome bc 1 complex, is involved in electron transfer at the level of the inner membrane of yeast mitochondria. The Rieske iron-sulfur protein is encoded by nuclear DNA and, after being synthesized in the cytosol, is imported into mitochondria with the help of a cleavable N-terminal presequence. The imported protein, besides incorporating the 2Fe-2S cluster, also interacts with other catalytic and non-catalytic subunits of the cytochrome bc 1 complex, thereby assembling into the mature and functional respiratory complex. In this paper, we summarize the most recent findings on the import and assembly of the Rieske iron-sulfur protein into Saccharomyces cerevisiae mitochondria, also discussing a possible role of this protein both in the dimerization of the cytochrome bc 1 complex and in the interaction of this homodimer with other complexes of the mitochondrial respiratory chain. PMID:21716720

  14. The Rieske Iron-Sulfur Protein: Import and Assembly into the Cytochrome bc(1) Complex of Yeast Mitochondria.

    Science.gov (United States)

    Conte, Laura; Zara, Vincenzo

    2011-01-01

    The Rieske iron-sulfur protein, one of the catalytic subunits of the cytochrome bc(1) complex, is involved in electron transfer at the level of the inner membrane of yeast mitochondria. The Rieske iron-sulfur protein is encoded by nuclear DNA and, after being synthesized in the cytosol, is imported into mitochondria with the help of a cleavable N-terminal presequence. The imported protein, besides incorporating the 2Fe-2S cluster, also interacts with other catalytic and non-catalytic subunits of the cytochrome bc(1) complex, thereby assembling into the mature and functional respiratory complex. In this paper, we summarize the most recent findings on the import and assembly of the Rieske iron-sulfur protein into Saccharomyces cerevisiae mitochondria, also discussing a possible role of this protein both in the dimerization of the cytochrome bc(1) complex and in the interaction of this homodimer with other complexes of the mitochondrial respiratory chain.

  15. NUCLEAR MYOSIN II REGULATES THE ASSEMBLY OF PREINITIATION COMPLEX FOR ICAM-1 GENE TRANSCRIPTION

    Science.gov (United States)

    Li, Qingjie; Sarna, Sushil K.

    2009-01-01

    Background and Aims Actin-myosin II motor converts chemical energy into force/motion in muscle and non-muscle cells. The phosphorylation of regulatory light chain (MLC20) is critical to the cytoplasmic functions of these motors. We do not know whether myosin II and actins in the nucleus function as motors to generate relative motion, such as that between RNA polymerase II holoenzyme and DNA, for assembly of the preinitiation complex. Methods The experiments were performed on primary cultures of human colonic circular smooth muscle cells (HCCSMCs) and rat colonic circular muscle strips. Results We show that myosin II and α- and β-actins are present in the nuclei of colonic smooth muscle cells. The nuclear myosin II is tethered to recognition sequence AGCTCC (−39/−34) in the ICAM-1 core promoter region. The actins are known to complex with RNA polymerase II and they are tethered to the nucleoskeleton. The dephosphorylation of MLC20 increases the transcription of ICAM-1, whereas its phosphorylation decreases it. Colonic inflammation suppresses nuclear MLCK, which increases the unphosphorylated form of nuclear MLC20, resulting in enhanced transcription of ICAM-1. Conclusions 1) Myosin II is a core transcription factor; 2) the phosphorylation/dephosphorylation of nuclear MLC20 results in the sliding of myosin and actin molecules past each other producing relative motion between the DNA bound to the myosin II and RNA polymerase II holoenzyme bound to actins and nucleoskeleton. PMID:19328794

  16. Molecular Self-Assembly of Group 11 Pyrazolate Complexes as Phosphorescent Chemosensors for Detection of Benzene

    Science.gov (United States)

    Ghazalli, N. F.; Yuliati, L.; Lintang, H. O.

    2018-01-01

    We highlight the systematic study on vapochromic sensing of aromatic vapors such as benzene using phosphorescent trinuclear pyrazolate complexes (2) with supramolecular assembly of a weak intermolecular metal-metal interaction consisting of 4-(3,5-dimethoxybenzyl)-3,5-dimethyl pyrazole ligand (1) and group 11 metal ions (Cu(I), Ag(I), Au(I)). The resulting chemosensor 2(Cu) revealed positive response to benzene vapors in 5 mins by blue-shifting its emission band in 44 nm (from 616 to 572 nm) and emitted bright orange to green, where this change cannot be recovered even with external stimuli. Comparing to 2(Ag) with longer metal-metal distance (473 nm) with same sensing time and quenching in 37%, 2(Au) gave quenching in 81% from its original intensity at 612 nm with reusability in 82% without external stimuli and emitted less emissive of red-orange from its original color. The shifting phenomenon in 2(Cu) suggests diffusion of benzene vapors to inside molecules for formation of intermolecular interaction with Cu(I)-Cu(I) interaction while quenching phenomenon in 2(Au) suggests diffusion of benzene vapors to between the Au(I)-Au(I) interaction. These results indicate that suitable molecular structure of ligand and metal ion in pyrazolate complex is important for designing chemosensor in the detection of benzene vapors.

  17. Nephrin regulates lamellipodia formation by assembling a protein complex that includes Ship2, filamin and lamellipodin.

    Directory of Open Access Journals (Sweden)

    Madhusudan Venkatareddy

    Full Text Available Actin dynamics has emerged at the forefront of podocyte biology. Slit diaphragm junctional adhesion protein Nephrin is necessary for development of the podocyte morphology and transduces phosphorylation-dependent signals that regulate cytoskeletal dynamics. The present study extends our understanding of Nephrin function by showing in cultured podocytes that Nephrin activation induced actin dynamics is necessary for lamellipodia formation. Upon activation Nephrin recruits and regulates a protein complex that includes Ship2 (SH2 domain containing 5' inositol phosphatase, Filamin and Lamellipodin, proteins important in regulation of actin and focal adhesion dynamics, as well as lamellipodia formation. Using the previously described CD16-Nephrin clustering system, Nephrin ligation or activation resulted in phosphorylation of the actin crosslinking protein Filamin in a p21 activated kinase dependent manner. Nephrin activation in cell culture results in formation of lamellipodia, a process that requires specialized actin dynamics at the leading edge of the cell along with focal adhesion turnover. In the CD16-Nephrin clustering model, Nephrin ligation resulted in abnormal morphology of actin tails in human podocytes when Ship2, Filamin or Lamellipodin were individually knocked down. We also observed decreased lamellipodia formation and cell migration in these knock down cells. These data provide evidence that Nephrin not only initiates actin polymerization but also assembles a protein complex that is necessary to regulate the architecture of the generated actin filament network and focal adhesion dynamics.

  18. Role of the Pif1-PCNA Complex in Pol δ-Dependent Strand Displacement DNA Synthesis and Break-Induced Replication.

    Science.gov (United States)

    Buzovetsky, Olga; Kwon, Youngho; Pham, Nhung Tuyet; Kim, Claire; Ira, Grzegorz; Sung, Patrick; Xiong, Yong

    2017-11-14

    The S. cerevisiae Pif1 helicase functions with DNA polymerase (Pol) δ in DNA synthesis during break-induced replication (BIR), a conserved pathway responsible for replication fork repair and telomere recombination. Pif1 interacts with the DNA polymerase processivity clamp PCNA, but the functional significance of the Pif1-PCNA complex remains to be elucidated. Here, we solve the crystal structure of PCNA in complex with a non-canonical PCNA-interacting motif in Pif1. The structure guides the construction of a Pif1 mutant that is deficient in PCNA interaction. This mutation impairs the ability of Pif1 to enhance DNA strand displacement synthesis by Pol δ in vitro and also the efficiency of BIR in cells. These results provide insights into the role of the Pif1-PCNA-Pol δ ensemble during DNA break repair by homologous recombination. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  19. Chromatin replication and histone dynamics

    DEFF Research Database (Denmark)

    Alabert, Constance; Jasencakova, Zuzana; Groth, Anja

    2017-01-01

    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...

  20. Biogenesis of the mitochondrial TOM complex: Mim1 promotes insertion and assembly of signal-anchored receptors.

    Science.gov (United States)

    Becker, Thomas; Pfannschmidt, Sylvia; Guiard, Bernard; Stojanovski, Diana; Milenkovic, Dusanka; Kutik, Stephan; Pfanner, Nikolaus; Meisinger, Chris; Wiedemann, Nils

    2008-01-04

    The translocase of the outer membrane (TOM complex) is the central entry gate for nuclear-encoded mitochondrial precursor proteins. All Tom proteins are also encoded by nuclear genes and synthesized as precursors in the cytosol. The channel-forming beta-barrel protein Tom40 is targeted to mitochondria via Tom receptors and inserted into the outer membrane by the sorting and assembly machinery (SAM complex). A further outer membrane protein, Mim1, plays a less defined role in assembly of Tom40 into the TOM complex. The three receptors Tom20, Tom22, and Tom70 are anchored in the outer membrane by a single transmembrane alpha-helix, located at the N terminus in the case of Tom20 and Tom70 (signal-anchored) or in the C-terminal portion in the case of Tom22 (tail-anchored). Insertion of the precursor of Tom22 into the outer membrane requires pre-existing Tom receptors while the import pathway of the precursors of Tom20 and Tom70 is only poorly understood. We report that Mim1 is required for efficient membrane insertion and assembly of Tom20 and Tom70, but not Tom22. We show that Mim1 associates with SAM(core) components to a large SAM complex, explaining its role in late steps of the assembly pathway of Tom40. We conclude that Mim1 is not only required for biogenesis of the beta-barrel protein Tom40 but also for membrane insertion and assembly of signal-anchored Tom receptors. Thus, Mim1 plays an important role in the efficient assembly of the mitochondrial TOM complex.

  1. Conserved SMP domains of the ERMES complex bind phospholipids and mediate tether assembly.

    Science.gov (United States)

    AhYoung, Andrew P; Jiang, Jiansen; Zhang, Jiang; Khoi Dang, Xuan; Loo, Joseph A; Zhou, Z Hong; Egea, Pascal F

    2015-06-23

    Membrane contact sites (MCS) between organelles are proposed as nexuses for the exchange of lipids, small molecules, and other signals crucial to cellular function and homeostasis. Various protein complexes, such as the endoplasmic reticulum-mitochondrial encounter structure (ERMES), function as dynamic molecular tethers between organelles. Here, we report the reconstitution and characterization of subcomplexes formed by the cytoplasm-exposed synaptotagmin-like mitochondrial lipid-binding protein (SMP) domains present in three of the five ERMES subunits--the soluble protein Mdm12, the endoplasmic reticulum (ER)-resident membrane protein Mmm1, and the mitochondrial membrane protein Mdm34. SMP domains are conserved lipid-binding domains found exclusively in proteins at MCS. We show that the SMP domains of Mdm12 and Mmm1 associate into a tight heterotetramer with equimolecular stoichiometry. Our 17-Å-resolution EM structure of the complex reveals an elongated crescent-shaped particle in which two Mdm12 subunits occupy symmetric but distal positions at the opposite ends of a central ER-anchored Mmm1 homodimer. Rigid body fitting of homology models of these SMP domains in the density maps reveals a distinctive extended tubular structure likely traversed by a hydrophobic tunnel. Furthermore, these two SMP domains bind phospholipids and display a strong preference for phosphatidylcholines, a class of phospholipids whose exchange between the ER and mitochondria is essential. Last, we show that the three SMP-containing ERMES subunits form a ternary complex in which Mdm12 bridges Mmm1 to Mdm34. Our findings highlight roles for SMP domains in ERMES assembly and phospholipid binding and suggest a structure-based mechanism for the facilitated transport of phospholipids between organelles.

  2. Genome Replication in Thermococcus kodakarensis Independent of Cdc6 and an Origin of Replication

    Directory of Open Access Journals (Sweden)

    Alexandra M. Gehring

    2017-10-01

    Full Text Available The initiation of DNA replication is typically tightly regulated by proteins that form initiation complexes at specific sequences known as replication origins. In Archaea and Eukaryotes, Cdc6, a near-universally conserved protein binds and facilitates the origin-dependent assembly of the replicative apparatus. TK1901 encodes Cdc6 in Thermococcus kodakarensis but, as we report here, TK1901 and the presumed origin of replication can be deleted from the genome of this hyperthermophilic Archaeon without any detectable effects on growth, genetic competence or the ability to support autonomous plasmid replication. All regions of the genome were equally represented in the sequences generated by whole genome sequencing of DNA isolated from T. kodakarensis strains with or without TK1901, inconsistent with DNA initiation occurring at one or few origins, and instead suggestive of replication initiating at many sites distributed throughout the genome. We were unable to generate strains lacking the recombination factors, RadA or RadB, consistent with T. kodakarensis cells, that are oligoploid (7–19 genomes per cell, employing a recombination-based mechanism of DNA replication. Deletion of the previously presumed origin region reduced the long-term viability of cultures supporting the possibility that retaining an origin-based mechanism of DNA initiation provides a survival mechanism for stationary phase cells with only one genome.

  3. Nanoparticle bioconjugates as "bottom-up" assemblies of artifical multienzyme complexes

    Science.gov (United States)

    Keighron, Jacqueline D.

    2010-11-01

    The sequential enzymes of several metabolic pathways have been shown to exist in close proximity with each other in the living cell. Although not proven in all cases, colocalization may have several implications for the rate of metabolite formation. Proximity between the sequential enzymes of a metabolic pathway has been proposed to have several benefits for the overall rate of metabolite formation. These include reduced diffusion distance for intermediates, sequestering of intermediates from competing pathways and the cytoplasm. Restricted diffusion in the vicinity of an enzyme can also cause the pooling of metabolites, which can alter reaction equilibria to control the rate of reaction through inhibition. Associations of metabolic enzymes are difficult to isolate ex vivo due to the weak interactions believed to colocalize sequential enzymes within the cell. Therefore model systems in which the proximity and diffusion of intermediates within the experiment system are controlled are attractive alternatives to explore the effects of colocalization of sequential enzymes. To this end three model systems for multienzyme complexes have been constructed. Direct adsorption enzyme:gold nanoparticle bioconjugates functionalized with malate dehydrogenase (MDH) and citrate synthase (CS) allow for proximity between to the enzymes to be controlled from the nanometer to micron range. Results show that while the enzymes present in the colocalized and non-colocalized systems compared here behaved differently overall the sequential activity of the pathway was improved by (1) decreasing the diffusion distance between active sites, (2) decreasing the diffusion coefficient of the reaction intermediate to prevent escape into the bulk solution, and (3) decreasing the overall amount of bioconjugate in the solution to prevent the pathway from being inhibited by the buildup of metabolite over time. Layer-by-layer (LBL) assemblies of MDH and CS were used to examine the layering effect of

  4. Characterization of receptor-associated protein complex assembly in interleukin (IL)-2- and IL-15-activated T-cell lines

    DEFF Research Database (Denmark)

    Osinalde, Nerea; Sánchez-Quiles, Virginia; Akimov, Vyacheslav

    2017-01-01

    to their functional dichotomy. In this study, we aimed to decipher the receptor complex assembly in IL-2- and IL-15-activated T-lymphocytes that is highly orchestrated by site-specific phosphorylation events. Comparing the cytokine-induced interactome of the interleukin receptor beta and gamma subunits shared...

  5. A self-assembled, π-stacked complex as a finely-tunable magnetic aligner for biomolecular NMR applications.

    Science.gov (United States)

    Sato, Sota; Takeuchi, Ryosuke; Yagi-Utsumi, Maho; Yamaguchi, Takumi; Yamaguchi, Yoshiki; Kato, Koichi; Fujita, Makoto

    2015-02-14

    The number of molecules constituting 1D intermolecular aggregates of the π-stacked self-assembled complex was controlled by altering the concentration and temperature. Enhanced magnetic orientation was observed for the aggregates with larger aggregation numbers. It was demonstrated that the designable complex aggregates serve as magnetic aligners to induce magnetic alignment upon an analyte protein coexisting in the solution, resulting in the observation of residual dipolar coupling (RDC) of the analyte.

  6. Assembly of Complex Nano-Structure from Single Atoms —Chemical Identification, Manipulation and Assembly by AFM—

    Science.gov (United States)

    Morita, Seizo; Sugimoto, Yoshiaki; Ooyabu, Noriaki; Custance, Óscar; Abe, Masayuki; Pou, Pablo; Jelinek, Pavel; Pérez, Rubén

    An atomic force microscope (AFM) under noncontact and nearcontact regions operated at room-temperature (RT) in ultrahigh vacuum, is used as a tool for topography-based atomic discrimination and atomic-interchange manipulations of two intermixed atomic species on semiconductor surfaces. Noncontact AFM topography based site-specific force curves provide the chemical covalent bonding forces between the tip apex and the atoms at the surface. Here, we introduced an example related to topography-based atomic discrimination using selected Sn and Si adatoms in Sn/Si(111)-(√3 ×√3 ) surface. Recently, under nearcontact region, we found a lateral atom-interchange manipulation phenomenon at RT in Sn/Ge(111)-c(2×8) intermixed sample. This phenomenon can interchange an embedded Sn atom with a neighbor Ge atom at RT. Using the vector scan method under nearcontact region, we constructed “Atom Inlay”, that is, atom letters “Sn” consisted of 19 Sn atoms embedded in Ge(111)-c(2×8) substrate. Using these methods, now we can assemble compound semiconductor nanostructures atom-by-atom.

  7. Self-assemble nanoparticles based on polypeptides containing C-terminal luminescent Pt-cysteine complex

    Science.gov (United States)

    Vlakh, E. G.; Grachova, E. V.; Zhukovsky, D. D.; Hubina, A. V.; Mikhailova, A. S.; Shakirova, J. R.; Sharoyko, V. V.; Tunik, S. P.; Tennikova, T. B.

    2017-02-01

    The growing attention to the luminescent nanocarriers is strongly stimulated by their potential application as drug delivery systems and by the necessity to monitor their distribution in cells and tissues. In this communication we report on the synthesis of amphiphilic polypeptides bearing C-terminal phosphorescent label together with preparation of nanoparticles using the polypeptides obtained. The approach suggested is based on a unique and highly technological process where the new phosphorescent Pt-cysteine complex serves as initiator of the ring-opening polymerization of α-amino acid N-carboxyanhydrides to obtain the polypeptides bearing intact the platinum chromophore covalently bound to the polymer chain. It was established that the luminescent label retains unchanged its emission characteristics not only in the polypeptides but also in more complicated nanoaggregates such as the polymer derived amphiphilic block-copolymers and self-assembled nanoparticles. The phosphorescent nanoparticles display no cytotoxicity and hemolytic activity in the tested range of concentrations and easily internalize into living cells that makes possible in vivo cell visualization, including prospective application in time resolved imaging and drug delivery monitoring.

  8. Synthesis and Self-Assembly of Amphiphilic Triblock Terpolymers with Complex Macromolecular Architecture

    KAUST Repository

    Polymeropoulos, George

    2015-11-25

    Two star triblock terpolymers (PS-b-P2VP-b-PEO)3 and one dendritic-like terpolymer [PS-b-P2VP-b-(PEO)2]3 of PS (polystyrene), P2VP (poly(2-vinylpyridine)), and PEO (poly(ethylene oxide)), never reported before, were synthesized by combining atom transfer radical and anionic polymerizations. The synthesis involves the transformation of the -Br groups of the previously reported Br-terminated 3-arm star diblock copolymers to one or two -OH groups, followed by anionic polymerization of ethylene oxide to afford the star or dendritic structure, respectively. The well-defined structure of the terpolymers was confirmed by static light scattering, size exclusion chromatography, and NMR spectroscopy. The self-assembly in solution and the morphology in bulk of the terpolymers, studied by dynamic light scattering and transmission electron microscopy, respectively, reveal new insights in the phase separation of these materials with complex macromolecular architecture. © 2015 American Chemical Society.

  9. 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......A mutant lacking the alternative sigma factor sigma(28) was reduced, which demonstrated that bacterial factors are required. Accumulation of biomass coincided with an increase in the optical density at 600 nm and ceased when the bacteria reached the stationary growth phase. L. pneumophila neither grew nor......, and no sizeable patches of clonally growing bacteria were observed. Our findings indicate that biofilm formation by L. pneumophila in a rich medium is due to growth of planktonic bacteria rather than to growth of sessile bacteria. In agreement with this conclusion, GFP-labeled L. pneumophila initially adhered...

  10. 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.

  11. Nuclear pore complex assembly and maintenance in POM121- and gp210-deficient cells

    DEFF Research Database (Denmark)

    Stavru, Fabrizia; Nautrup-Pedersen, Gitte; Cordes, Volker C

    2006-01-01

    nucleoporins, such as Nup62 or Nup358, to ectopic assembly sites. It thus appears to act as a nucleation site for the assembly of NPC substructures. Nonetheless, we observed functional NPCs and intact NEs in severely POM121-depleted cells. Double knockdowns of gp210 and POM121 in HeLa cells, as well...

  12. Fluorinated liquid crystals: design of soft nanostructures and increased complexity of self-assembly by perfluorinated segments.

    Science.gov (United States)

    Tschierske, Carsten

    2012-01-01

    The effects of perfluorinated and semiperfluorinated hydrocarbon units on the self-assembly of rod-like, disc-like, polycatenar, taper- and star-shaped, dendritic, and bent-core liquid crystalline (LC) materials is reviewed. The influence of fluorinated segments is analyzed on the basis of their contributions to the cohesive energy density, molecular shape, conformational flexibility, micro-segregation, space filling, and interface curvature. Though the focus is on recent progress in the last decade, previous main contributions, general aspects of perfluorinated organic molecules, and the basics of LC self-assembly are also briefly discussed to provide a complete overall picture. The main focus is on structure-property-relations and the use of micro-segregation to tailor mesophase morphologies. Especially polyphilic molecules with perfluorinated segments provide new modes of LC self-assembly, leading to ordered fluids with periodic multi-compartment structures and enhanced complexity compared to previously known systems.

  13. Photoresponsive Molecular Memory Films Composed of Sequentially Assembled Heterolayers Containing Ruthenium Complexes.

    Science.gov (United States)

    Nagashima, Takumi; Ozawa, Hiroaki; Suzuki, Takashi; Nakabayashi, Takuya; Kanaizuka, Katsuhiko; Haga, Masa-Aki

    2016-01-26

    Photoresponsive molecular memory films were fabricated by a layer-by-layer (LbL) assembling of two dinuclear Ru complexes with tetrapodal phosphonate anchors, containing either 2,3,5,6-tetra(2-pyridyl)pyrazine or 1,2,4,5-tetra(2-pyridyl)benzene as a bridging ligand (Ru-NP and Ru-CP, respectively), using zirconium phosphonate to link the layers. Various types of multilayer homo- and heterostructures were constructed. In the multilayer heterofilms such as ITO||(Ru-NP)m |(Ru-CP)n , the difference in redox potentials between Ru-NP and Ru-CP layers was approximately 0.7 V, which induced a potential gradient determined by the sequence of the layers. In the ITO||(Ru-NP)m |(Ru-CP)n multilayer heterofilms, the direct electron transfer (ET) from the outer Ru-CP layers to the ITO were observed to be blocked for m>2, and charge trapping in the outer Ru-CP layers became evident from the appearance of an intervalence charge transfer (IVCT) band at 1140 nm from the formation of the mixed-valent state of Ru-CP units, resulting from the reductive ET mediation of the inner Ru-NP layers. Therefore, the charging/discharging ("1"and "0") states in the outer Ru-CP layers could be addressed and interconverted by applying potential pulses between -0.5 and +0.7 V. The two states could be read out by the direction of the photocurrent (anodic or cathodic). The molecular heterolayer films thus represent a typical example of a photoresponsive memory device; that is, the writing process may be achieved by the applied potential (-0.5 or +0.7 V), while the readout process is achieved by measuring the direction of the photocurrent (anodic or cathodic). Sequence-sensitive multilayer heterofilms, using redox-active complexes as building blocks, thus demonstrate great potential for the design of molecular functional devices. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  14. Targeting and Assembly of Components of the TOC Protein Import Complex at the Chloroplast Outer Envelope Membrane

    Directory of Open Access Journals (Sweden)

    Lynn G.L. Richardson

    2014-06-01

    Full Text Available The translocon at the outer envelope membrane of chloroplasts (TOC initiates the import of thousands of nuclear encoded preproteins required for chloroplast biogenesis and function. The multimeric TOC complex contains two GTP-regulated receptors, Toc34 and Toc159, which recognize the transit peptides of preproteins and initiate protein import through a β–barrel membrane channel, Toc75. Different isoforms of Toc34 and Toc159 assemble with Toc75 to form structurally and functionally diverse translocons, and the composition and levels of TOC translocons is required for the import of specific subsets of coordinately expressed proteins during plant growth and development. Consequently, the proper assembly of the TOC complexes is key to ensuring organelle homeostasis. This review will focus on our current knowledge of the targeting and assembly of TOC components to form functional translocons at the outer membrane. Our analyses reveal that the targeting of TOC components involves elements common to the targeting of other outer membrane proteins, but also include unique features that appear to have evolved to specifically facilitate assembly of the import apparatus.

  15. Targeting and assembly of components of the TOC protein import complex at the chloroplast outer envelope membrane.

    Science.gov (United States)

    Richardson, Lynn G L; Paila, Yamuna D; Siman, Steven R; Chen, Yi; Smith, Matthew D; Schnell, Danny J

    2014-01-01

    The translocon at the outer envelope membrane of chloroplasts (TOC) initiates the import of thousands of nuclear encoded preproteins required for chloroplast biogenesis and function. The multimeric TOC complex contains two GTP-regulated receptors, Toc34 and Toc159, which recognize the transit peptides of preproteins and initiate protein import through a β-barrel membrane channel, Toc75. Different isoforms of Toc34 and Toc159 assemble with Toc75 to form structurally and functionally diverse translocons, and the composition and levels of TOC translocons is required for the import of specific subsets of coordinately expressed proteins during plant growth and development. Consequently, the proper assembly of the TOC complexes is key to ensuring organelle homeostasis. This review will focus on our current knowledge of the targeting and assembly of TOC components to form functional translocons at the outer membrane. Our analyses reveal that the targeting of TOC components involves elements common to the targeting of other outer membrane proteins, but also include unique features that appear to have evolved to specifically facilitate assembly of the import apparatus.

  16. 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...... synthesis is perturbed, cells can suffer loss of both genome and epigenome integrity with severe consequences for the organism....

  17. Human cytomegalovirus-infected cells have unstable assembly of major histocompatibility complex class I complexes and are resistant to lysis by cytotoxic T lymphocytes.

    OpenAIRE

    Warren, A P; Ducroq, D H; Lehner, P J; Borysiewicz, L K

    1994-01-01

    Viruses which cause persistence in the naturally infected host are predicted to have evolved immune evasion mechanisms. Human cytomegalovirus (HCMV) causes significant morbidity and mortality in immunocompromised patients yet persists without clinical manifestations in seropositive individuals who have normal immune function. We report that HCMV infection in vitro impairs major histocompatibility complex class I (MHC-I) assembly accompanied by resistance to killing by cytotoxic CD8+ T lymphoc...

  18. Human cytomegalovirus-infected cells have unstable assembly of major histocompatibility complex class I complexes and are resistant to lysis by cytotoxic T lymphocytes.

    Science.gov (United States)

    Warren, A P; Ducroq, D H; Lehner, P J; Borysiewicz, L K

    1994-05-01

    Viruses which cause persistence in the naturally infected host are predicted to have evolved immune evasion mechanisms. Human cytomegalovirus (HCMV) causes significant morbidity and mortality in immunocompromised patients yet persists without clinical manifestations in seropositive individuals who have normal immune function. We report that HCMV infection in vitro impairs major histocompatibility complex class I (MHC-I) assembly accompanied by resistance to killing by cytotoxic CD8+ T lymphocytes. Pulse-chase metabolic labelling experiments show that MHC-I complexes continue to be assembled by both uninfected and HCMV-infected cells. However, MHC-I molecules are unstable in HCMV-infected cells and are rapidly broken down. Endoglycosidase H treatment of immunoprecipitates indicates that the breakdown of MHC-I complexes in HCMV-infected cells occurs primarily in a pre-Golgi compartment. Interference with normal MHC-I assembly and expression, if relevant in vivo, may have implications for the restriction of the diversity of the CD8+ cytotoxic T lymphocyte repertoire directed against HCMV antigens and may be an important mechanism of viral persistence.

  19. 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

  20. Increased spread and replication efficiency of Listeria monocytogenes in organotypic brain-slices is related to multilocus variable number of tandem repeat analysis (MLVA) complex.

    Science.gov (United States)

    Guldimann, Claudia; Bärtschi, Michelle; Frey, Joachim; Zurbriggen, Andreas; Seuberlich, Torsten; Oevermann, Anna

    2015-07-03

    Listeria (L.) monocytogenes causes fatal infections in many species including ruminants and humans. In ruminants, rhombencephalitis is the most prevalent form of listeriosis. Using multilocus variable number tandem repeat analysis (MLVA) we recently showed that L. monocytogenes isolates from ruminant rhombencephalitis cases are distributed over three genetic complexes (designated A, B and C). However, the majority of rhombencephalitis strains and virtually all those isolated from cattle cluster in MLVA complex A, indicating that strains of this complex may have increased neurotropism and neurovirulence. The aim of this study was to investigate whether ruminant rhombencephalitis strains have an increased ability to propagate in the bovine hippocampal brain-slice model and can be discriminated from strains of other sources. For this study, forty-seven strains were selected and assayed on brain-slice cultures, a bovine macrophage cell line (BoMac) and a human colorectal adenocarcinoma cell line (Caco-2). They were isolated from ruminant rhombencephalitis cases (n = 21) and other sources including the environment, food, human neurolisteriosis cases and ruminant/human non-encephalitic infection cases (n = 26). All but one L. monocytogenes strain replicated in brain slices, irrespectively of the source of the isolate or MLVA complex. The replication of strains from MLVA complex A was increased in hippocampal brain-slice cultures compared to complex C. Immunofluorescence revealed that microglia are the main target cells for L. monocytogenes and that strains from MLVA complex A caused larger infection foci than strains from MLVA complex C. Additionally, they caused larger plaques in BoMac cells, but not CaCo-2 cells. Our brain slice model data shows that all L. monocytogenes strains should be considered potentially neurovirulent. Secondly, encephalitis strains cannot be conclusively discriminated from non-encephalitis strains with the bovine organotypic brain slice

  1. Database Replication

    Directory of Open Access Journals (Sweden)

    Marius Cristian MAZILU

    2010-12-01

    Full Text Available For someone who has worked in an environment in which the same database is used for data entry and reporting, or perhaps managed a single database server that was utilized by too many users, the advantages brought by data replication are clear. The main purpose of this paper is to emphasize those advantages as well as presenting the different types of Database Replication and the cases in which their use is recommended.

  2. 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.

  3. Recombination-dependent concatemeric viral DNA replication.

    Science.gov (United States)

    Lo Piano, Ambra; Martínez-Jiménez, María I; Zecchi, Lisa; Ayora, Silvia

    2011-09-01

    The initiation of viral double stranded (ds) DNA replication involves proteins that recruit and load the replisome at the replication origin (ori). Any block in replication fork progression or a programmed barrier may act as a factor for ori-independent remodelling and assembly of a new replisome at the stalled fork. Then replication initiation becomes dependent on recombination proteins, a process called recombination-dependent replication (RDR). RDR, which is recognized as being important for replication restart and stability in all living organisms, plays an essential role in the replication cycle of many dsDNA viruses. The SPP1 virus, which infects Bacillus subtilis cells, serves as a paradigm to understand the links between replication and recombination in circular dsDNA viruses. SPP1-encoded initiator and replisome assembly proteins control the onset of viral replication and direct the recruitment of host-encoded replisomal components at viral oriL. SPP1 uses replication fork reactivation to switch from ori-dependent θ-type (circle-to-circle) replication to σ-type RDR. Replication fork arrest leads to a double strand break that is processed by viral-encoded factors to generate a D-loop into which a new replisome is assembled, leading to σ-type viral replication. SPP1 RDR proteins are compared with similar proteins encoded by other viruses and their possible in vivo roles are discussed. Copyright © 2011 Elsevier B.V. All rights reserved.

  4. Replication and transcription activities of ribonucleoprotein complexes reconstituted from avian H5N1, H1N1pdm09 and H3N2 influenza A viruses.

    Directory of Open Access Journals (Sweden)

    Karry L K Ngai

    Full Text Available Avian influenza viruses pose a serious pandemic threat to humans. Better knowledge on cross-species adaptation is important. This study examined the replication and transcription efficiency of ribonucleoprotein complexes reconstituted by plasmid co-transfection between H5N1, H1N1pdm09 and H3N2 influenza A viruses, and to identify mutations in the RNA polymerase subunit that affect human adaptation. Viral RNA polymerase subunits PB1, PB2, PA and NP derived from influenza viruses were co-expressed with pPolI-vNP-Luc in human cells, and with its function evaluated by luciferase reporter assay. A quantitative RT-PCR was used to measure vRNA, cRNA, and mRNA levels for assessing the replication and transcription efficiency. Mutations in polymerase subunit were created to identify signature of increased human adaptability. H5N1 ribonucleoprotein complexes incorporated with PB2 derived from H1N1pdm09 and H3N2 viruses increased the polymerase activity in human cells. Furthermore, single amino acid substitutions at PB2 of H5N1 could affect polymerase activity in a temperature-dependent manner. By using a highly sensitive quantitative reverse transcription-polymerase chain reaction, an obvious enhancement in replication and transcription activities of ribonucleoproteins was observed by the introduction of lysine at residue 627 in the H5N1 PB2 subunit. Although less strongly in polymerase activity, E158G mutation appeared to alter the accumulation of H5N1 RNA levels in a temperature-dependent manner, suggesting a temperature-dependent mechanism in regulating transcription and replication exists. H5N1 viruses can adapt to humans either by acquisition of PB2 from circulating human-adapted viruses through reassortment, or by mutations at critical sites in PB2. This information may help to predict the pandemic potential of newly emerged influenza strains, and provide a scientific basis for stepping up surveillance measures and vaccine production.

  5. C11orf83, a mitochondrial cardiolipin-binding protein involved in bc1 complex assembly and supercomplex stabilization.

    Science.gov (United States)

    Desmurs, Marjorie; Foti, Michelangelo; Raemy, Etienne; Vaz, Frédéric Maxime; Martinou, Jean-Claude; Bairoch, Amos; Lane, Lydie

    2015-04-01

    Mammalian mitochondria may contain up to 1,500 different proteins, and many of them have neither been confidently identified nor characterized. In this study, we demonstrated that C11orf83, which was lacking experimental characterization, is a mitochondrial inner membrane protein facing the intermembrane space. This protein is specifically associated with the bc1 complex of the electron transport chain and involved in the early stages of its assembly by stabilizing the bc1 core complex. C11orf83 displays some overlapping functions with Cbp4p, a yeast bc1 complex assembly factor. Therefore, we suggest that C11orf83, now called UQCC3, is the functional human equivalent of Cbp4p. In addition, C11orf83 depletion in HeLa cells caused abnormal crista morphology, higher sensitivity to apoptosis, a decreased ATP level due to impaired respiration and subtle, but significant, changes in cardiolipin composition. We showed that C11orf83 binds to cardiolipin by its α-helices 2 and 3 and is involved in the stabilization of bc1 complex-containing supercomplexes, especially the III2/IV supercomplex. We also demonstrated that the OMA1 metalloprotease cleaves C11orf83 in response to mitochondrial depolarization, suggesting a role in the selection of cells with damaged mitochondria for their subsequent elimination by apoptosis, as previously described for OPA1. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  6. Association of polioviral proteins of the P2 genomic region with the viral replication complex and virus-induced membrane synthesis as visualized by electron microscopic immunocytochemistry and autoradiography.

    Science.gov (United States)

    Bienz, K; Egger, D; Pasamontes, L

    1987-09-01

    Using high resolution electron microscopic autoradiography and immunocytochemistry with monoclonal antibodies against poliovirus proteins of the P2 genomic region, the location of these proteins in respect to the virus-induced vesicle formation and the viral RNA synthesis was followed during the viral replication cycle. It was found that P2 proteins become rER associated soon after their synthesis. At the site of protein and rER interaction, electron-dense patches appear. Simultaneously, membrane protrusions grow and form vesicles which finally budd off, carrying the patches on their outer surface. As shown by autoradiography, these patches are the site of viral RNA replication and, therefore, they represent the poliovirus replication complex. The vesicles with the replication complex, including replicating and replicated viral RNA, move away from the rER to form a continuously growing vesiculated area in the center of the infected cell, where virus maturation takes place. A likely function of the 2C protein is to attach the replication complex, or some of its components, to the vesicular membranes.

  7. 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.

  8. 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.

  9. Engineering the cell surface display of cohesins for assembly of cellulosome-inspired enzyme complexes on Lactococcus lactis

    Directory of Open Access Journals (Sweden)

    Wieczorek Andrew S

    2010-09-01

    Full Text Available Abstract Background The assembly and spatial organization of enzymes in naturally occurring multi-protein complexes is of paramount importance for the efficient degradation of complex polymers and biosynthesis of valuable products. The degradation of cellulose into fermentable sugars by Clostridium thermocellum is achieved by means of a multi-protein "cellulosome" complex. Assembled via dockerin-cohesin interactions, the cellulosome is associated with the cell surface during cellulose hydrolysis, forming ternary cellulose-enzyme-microbe complexes for enhanced activity and synergy. The assembly of recombinant cell surface displayed cellulosome-inspired complexes in surrogate microbes is highly desirable. The model organism Lactococcus lactis is of particular interest as it has been metabolically engineered to produce a variety of commodity chemicals including lactic acid and bioactive compounds, and can efficiently secrete an array of recombinant proteins and enzymes of varying sizes. Results Fragments of the scaffoldin protein CipA were functionally displayed on the cell surface of Lactococcus lactis. Scaffolds were engineered to contain a single cohesin module, two cohesin modules, one cohesin and a cellulose-binding module, or only a cellulose-binding module. Cell toxicity from over-expression of the proteins was circumvented by use of the nisA inducible promoter, and incorporation of the C-terminal anchor motif of the streptococcal M6 protein resulted in the successful surface-display of the scaffolds. The facilitated detection of successfully secreted scaffolds was achieved by fusion with the export-specific reporter staphylococcal nuclease (NucA. Scaffolds retained their ability to associate in vivo with an engineered hybrid reporter enzyme, E. coli β-glucuronidase fused to the type 1 dockerin motif of the cellulosomal enzyme CelS. Surface-anchored complexes exhibited dual enzyme activities (nuclease and β-glucuronidase, and were

  10. Human Cytomegalovirus pUL47 Modulates Tegumentation and Capsid Accumulation at the Viral Assembly Complex

    Science.gov (United States)

    Cappadona, Ilaria; Villinger, Clarissa; Schutzius, Gabi; Mertens, Thomas

    2015-01-01

    ABSTRACT Human cytomegalovirus (HCMV) tegument protein pUL47 is an interaction partner of pUL48 and highly conserved among herpesviruses. It is closely associated with the capsid and has an important function early in infection. Here, we report a specific role of pUL47 in the tegumentation of capsids in the cytoplasm. A newly generated mutant virus (TB-47stop), in which expression of pUL47 is blocked, exhibited a severe impairment in cell-to-cell spread and release of infectivity from infected cells. Ultrastructural analysis of TB-47stop-infected cells clearly showed cytoplasmic accumulations of nonenveloped capsids that were only partially tegumented, indicating that these capsids failed to complete tegumentation. Nevertheless, these accumulations were positive for HCMV inner tegument proteins pp150 and pUL48, suggesting that their attachment to capsids occurs independently of pUL47. Despite these morphological alterations, fully enveloped virus particles were found in the extracellular space and at the viral assembly complex (vAC) of TB-47stop-infected cells, indicating that pUL47 is not essential for the generation of virions. We confirmed findings that incorporation of pUL48 into virions is impaired in the absence of pUL47. Interestingly, pUL47 exhibited a strong nuclear localization in transfected cells, whereas it was found exclusively at the vAC in the context of virus infection. Colocalization of pUL47 and pUL48 at the vAC is consistent with their interaction. We also found a shift to a more nuclear localization of pUL47 when the expression of pUL48 was reduced. Summarizing our results, we hypothesize that pUL48 directs pUL47 to the vAC to promote tegumentation and secondary envelopment of capsids. IMPORTANCE Generation of infectious HCMV particles requires an organized and multistep process involving the action of several viral and cellular proteins as well as protein-protein interactions. A better understanding of these processes is important for

  11. 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

  12. Structure of a helicase–helicase loader complex reveals insights into the mechanism of bacterial primosome assembly

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Bin; Eliason, William K.; Steitz, Thomas A.

    2013-09-19

    During the assembly of the bacterial loader-dependent primosome, helicase loader proteins bind to the hexameric helicase ring, deliver it onto the oriC DNA and then dissociate from the complex. Here, to provide a better understanding of this key process, we report the crystal structure of the ~570-kDa prepriming complex between the Bacillus subtilis loader protein and the Bacillus stearothermophilus helicase, as well as the helicase-binding domain of primase with a molar ratio of 6:6:3 at 7.5 Å resolution. The overall architecture of the complex exhibits a three-layered ring conformation. Moreover, the structure combined with the proposed model suggests that the shift from the ‘open-ring’ to the ‘open-spiral’ and then the ‘closed-spiral’ state of the helicase ring due to the binding of single-stranded DNA may be the cause of the loader release.

  13. Molecular and electronic structure of osmium complexes confined to Au(111) surfaces using a self-assembled molecular bridge

    Energy Technology Data Exchange (ETDEWEB)

    Llave, Ezequiel de la; Herrera, Santiago E.; Adam, Catherine; Méndez De Leo, Lucila P.; Calvo, Ernesto J.; Williams, Federico J., E-mail: fwilliams@qi.fcen.uba.ar [INQUIMAE-CONICET, Departamento de Química Inorgánica, Analítica y Química-Física, Facultad Ciencias Exactas y Naturales, Universidad de Buenos Aires, Ciudad Universitaria, Pabellón 2, Buenos Aires C1428EHA (Argentina)

    2015-11-14

    The molecular and electronic structure of Os(II) complexes covalently bonded to self-assembled monolayers (SAMs) on Au(111) surfaces was studied by means of polarization modulation infrared reflection absorption spectroscopy, photoelectron spectroscopies, scanning tunneling microscopy, scanning tunneling spectroscopy, and density functional theory calculations. Attachment of the Os complex to the SAM proceeds via an amide covalent bond with the SAM alkyl chain 40° tilted with respect to the surface normal and a total thickness of 26 Å. The highest occupied molecular orbital of the Os complex is mainly based on the Os(II) center located 2.2 eV below the Fermi edge and the LUMO molecular orbital is mainly based on the bipyridine ligands located 1.5 eV above the Fermi edge.

  14. The human RNase MRP complex : composition, assembly and role in human disease

    NARCIS (Netherlands)

    Eenennaam, Hans van

    2002-01-01

    Not all RNA molecules in human cells are being translated into proteins. Some of them function in binding proteins, thereby forming so-called RNA-protein complexes. The RNase MRP complex is an example of such an RNA-protein complex. In this thesis two new protein components of the human RNase MRP

  15. The Assembly and Intermolecular Properties of the Hsp70-Tomm34-Hsp90 Molecular Chaperone Complex

    Czech Academy of Sciences Publication Activity Database

    Trčka, F.; Durech, M.; Hernychová, L.; Man, Petr; Müller, P.; Vojtěšek, B.

    2014-01-01

    Roč. 289, č. 14 (2014), s. 9887-9901 ISSN 0021-9258 R&D Projects: GA ČR(CZ) P301/11/1678; GA MZd(CZ) 00209805 Grant - others:Regional Center for Applied Molecular Oncology(CZ) CZ.1.05/2.1.00/03.0101 Institutional support: RVO:61388971 Keywords : HSP90 * chaperone * protein assembly Subject RIV: EE - Microbiology, Virology Impact factor: 4.573, year: 2014

  16. DNA origami: a quantum leap for self-assembly of complex structures

    DEFF Research Database (Denmark)

    Tørring, Thomas; Voigt, Niels Vinther; Nangreave, Jeanette

    2011-01-01

    The spatially controlled positioning of functional materials by self-assembly is one of the fundamental visions of nanotechnology. Major steps towards this goal have been achieved using DNA as a programmable building block. This tutorial review will focus on one of the most promising methods: DNA...... origami. The basic design principles, organization of a variety of functional materials and recent implementation of DNA robotics are discussed together with future challenges and opportunities....

  17. A D53 repression motif induces oligomerization of TOPLESS corepressors and promotes assembly of a corepressor-nucleosome complex.

    Science.gov (United States)

    Ma, Honglei; Duan, Jingbo; Ke, Jiyuan; He, Yuanzheng; Gu, Xin; Xu, Ting-Hai; Yu, Hong; Wang, Yonghong; Brunzelle, Joseph S; Jiang, Yi; Rothbart, Scott B; Xu, H Eric; Li, Jiayang; Melcher, Karsten

    2017-06-01

    TOPLESS are tetrameric plant corepressors of the conserved Tup1/Groucho/TLE (transducin-like enhancer of split) family. We show that they interact through their TOPLESS domains (TPDs) with two functionally important ethylene response factor-associated amphiphilic repression (EAR) motifs of the rice strigolactone signaling repressor D53: the universally conserved EAR-3 and the monocot-specific EAR-2. We present the crystal structure of the monocot-specific EAR-2 peptide in complex with the TOPLESS-related protein 2 (TPR2) TPD, in which the EAR-2 motif binds the same TPD groove as jasmonate and auxin signaling repressors but makes additional contacts with a second TPD site to mediate TPD tetramer-tetramer interaction. We validated the functional relevance of the two TPD binding sites in reporter gene assays and in transgenic rice and demonstrate that EAR-2 binding induces TPD oligomerization. Moreover, we demonstrate that the TPD directly binds nucleosomes and the tails of histones H3 and H4. Higher-order assembly of TPD complexes induced by EAR-2 binding markedly stabilizes the nucleosome-TPD interaction. These results establish a new TPD-repressor binding mode that promotes TPD oligomerization and TPD-nucleosome interaction, thus illustrating the initial assembly of a repressor-corepressor-nucleosome complex.

  18. Vipp1 is required for basic thylakoid membrane formation but not for the assembly of thylakoid protein complexes.

    Science.gov (United States)

    Aseeva, Elena; Ossenbühl, Friederich; Sippel, Claudia; Cho, Won K; Stein, Bernhard; Eichacker, Lutz A; Meurer, Jörg; Wanner, Gerhard; Westhoff, Peter; Soll, Jürgen; Vothknecht, Ute C

    2007-02-01

    Vipp1 (vesicle inducing protein in plastids 1) is found in cyanobacteria and chloroplasts where it is essential for thylakoid formation. Arabidopsis thaliana mutant plants with a reduction of Vipp1 to about 20% of wild type content become albinotic at an early stage. We propose that this drastic phenotype results from an inability of the remaining Vipp1 protein to assemble into a homo-oligomeric complex, indicating that oligomerization is a prerequisite for Vipp1 function. A Vipp1-ProteinA fusion protein, expressed in the Deltavipp1 mutant background, is able to reinstate oligomerization and restore photoautotrophic growth. Plants containing Vipp1-ProteinA in amounts comparable to Vipp1 in the wild type exhibit a wild type phenotype. However, plants with a reduced amount of Vipp1-ProteinA protein are growth-retarded and significantly paler than the wild type. This phenotype is caused by a decrease in thylakoid membrane content and a concomitant reduction in photosynthetic activity. To the extent that thylakoid membranes are made in these plants they are properly assembled with protein-pigment complexes and are photosynthetically active. This strongly supports a function of Vipp1 in basic thylakoid membrane formation and not in the functional assembly of thylakoid protein complexes. Intriguingly, electron microscopic analysis shows that chloroplasts in the mutant plants are not equally affected by the Vipp1 shortage. Indeed, a wide range of different stages of thylakoid development ranging from wild-type-like chloroplasts to plastids nearly devoid of thylakoids can be observed in organelles of one and the same cell.

  19. Non-destructive failure analysis and measurement for molded devices and complex assemblies with X-ray CT and 3D image processing techniques

    International Nuclear Information System (INIS)

    Yin, Xiaoming; Liew, Seaw Jia; Jiang, Ting Ying; Xu, Jian; Kakarala, Ramakrishna

    2013-01-01

    In both automotive and healthcare sectors, reliable failure analysis and accurate measurement of molded devices and complex assemblies are important. Current methods of failure analysis and measurement require these molded parts to be cross-sectioned so that internal features or dimensions can be accessible. As a result, the parts are deemed unusable and additional failure introduced by sectioning may cause misinterpretation of the results. X-ray CT and 3D image processing techniques provide a new nondestructive solution for failure analysis and measurement of molded devices and complex assemblies. These techniques simplify failure analysis and measurement of molded devices and assemblies, and improve the productivity of molding manufacturing significantly.

  20. In vitro production and characterization of partly assembled human CD3 complexes

    DEFF Research Database (Denmark)

    Kastrup, J; Pedersen, L Ø; Dietrich, J

    2002-01-01

    in Golgi apparatus before the fully assembled T-cell receptor is transported to the cell surface. To study the structural properties of the human CD3 chains, we have developed new methods to produce and fold the extracellular domains of CD3 gamma, CD3 delta and CD3 epsilon. Proteins were expressed...... in Escherichia coli as denatured chains and de novo folded in vitro. CD3 gamma and CD3 epsilon folded as soluble monomers, whereas CD3 delta did not yield any soluble proteins. When folding the chains pairwise, soluble CD3 gamma epsilon and CD3 delta epsilon heterodimers could be isolated, whereas CD3 gamma...

  1. Automated Glycan Assembly of Complex Oligosaccharides Related to Blood Group Determinants.

    Science.gov (United States)

    Hahm, Heung Sik; Liang, Chien-Fu; Lai, Chian-Hui; Fair, Richard J; Schuhmacher, Frank; Seeberger, Peter H

    2016-07-15

    Lactotetraosyl (Lc4) and neo-lactotetraosyl (nLc4) are backbones that are common to many glycans. Using automated glycan assembly, these common core structures were constructed and elaborated to access synthetically challenging glycans of biological relevance. The incorporation of α-fucoses is demonstrated for H-type I and II; α(1,3)-galactose epitopes were prepared, and the pentasaccharide HNK-1 required incorporation of a 3-O-sulfate. In addition to preparing the target structures, essential insights were gained regarding the relationships of glycosylating agents and nucleophiles as well as the linker stability.

  2. 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.

  3. 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.

  4. C. elegans PAT-6/actopaxin plays a critical role in the assembly of integrin adhesion complexes in vivo.

    Science.gov (United States)

    Lin, Xinyi; Qadota, Hiroshi; Moerman, Donald G; Williams, Benjamin D

    2003-05-27

    The novel focal adhesion protein actopaxin includes tandem unconventional calponin homology (CH) domains and a less well-conserved N-terminal stretch. Dominant-negative studies have implicated actopaxin in focal adhesion formation. PAT-6/actopaxin, the sole actopaxin homolog in C. elegans, is located in body wall muscle attachments that are in vivo homologs of focal adhesions. We show using pat-6 protein null alleles that PAT-6/actopaxin has critical nonredundant roles during attachment maturation. It is required to recruit UNC-89 and myofilaments to newly forming attachments, and also to reposition the attachments so that they form the highly ordered array of dense body and M line attachments that are characteristic of mature muscle cells. PAT-6/actopaxin is not required for the deposition of UNC-52/perlecan in the basal lamina, nor for the initiation of attachment assembly, including the clustering of integrin into foci and the recruitment of attachment proteins PAT-4/ILK, UNC-112, and DEB-1/vinculin from the cytosol. PAT-6/actopaxin, PAT-4/ILK, and UNC-112 are each required for the same steps during attachment assembly in vivo, consistent with the notion that they work together in multiprotein complex. Supporting this idea, PAT-4/ILK can simultaneously bind to PAT-6/actopaxin and UNC-112, forming a ternary complex, in yeast three-hybrid assays. Finally, we show that both calponin homology domains are required for PAT-6/actopaxin's critical functions during attachment assembly in vivo. We show directly by loss-of-function genetics that PAT-6/actopaxin plays essential roles during the maturation of integrin-mediated muscle attachments in vivo.

  5. Nef enhances HIV-1 infectivity via association with the virus assembly complex

    International Nuclear Information System (INIS)

    Qi Mingli; Aiken, Christopher

    2008-01-01

    The HIV-1 accessory protein Nef enhances virus infectivity by facilitating an early post-entry step of infection. Nef acts in the virus producer cell, leading to a beneficial modification to HIV-1 particles. Nef itself is incorporated into HIV-1 particles, where it is cleaved by the viral protease during virion maturation. To probe the role of virion-associated Nef in HIV-1 infection, we generated a fusion protein consisting of the host protein cyclophilin A (CypA) linked to the amino terminus of Nef. The resulting CypA-Nef protein enhanced the infectivity of Nef-defective HIV-1 particles and was specifically incorporated into the virions via association with Gag during particle assembly. Pharmacologic or genetic inhibition of CypA-Nef binding to Gag prevented incorporation of CypA-Nef into virions and inhibited infectivity enhancement. Our results indicate that infectivity enhancement by Nef requires its association with a component of the assembling HIV-1 particle

  6. Controlled release of astaxanthin from nanoporous silicified-phospholipids assembled boron nitride complex for cosmetic applications

    Science.gov (United States)

    Lee, Hye Sun; Sung, Dae Kyung; Kim, Sung Hyun; Choi, Won Il; Hwang, Ee Tag; Choi, Doo Jin; Chang, Jeong Ho

    2017-12-01

    Nanoporous silicified-phospholipids assembled boron nitride (nSPLs@BN) powder was prepared and demonstrated for use in controlled release of anti-oxidant astaxanthin (AX) as a cosmetic application. The nanoporous silicified phospholipids (nSPLs) were obtained by the silicification with tetraethyl orthosilicate (TEOS) of the hydrophilic region of phospholipid bilayers. This process involved the co-assembly of chemically active phospholipid bilayers within the porous silica matrix. In addition, nSPLs@BN was characterized using several analytical techniques and tested to assess their efficiency as drug delivery systems. We calculated the maximum release amounts as a function of time and various pH. The release rate of AX from the nSPLs@BN for the initial 24 h was 10.7 μmol/(h mg) at pH 7.4. Furthermore, we determined the antioxidant activity (KD) for the released AX with DPPH (1,1-diphenyl-2-picryl-hydrazyl) radical and the result was 34.6%.

  7. Visual Perception-Based Statistical Modeling of Complex Grain Image for Product Quality Monitoring and Supervision on Assembly Production Line.

    Directory of Open Access Journals (Sweden)

    Jinping Liu

    Full Text Available Computer vision as a fast, low-cost, noncontact, and online monitoring technology has been an important tool to inspect product quality, particularly on a large-scale assembly production line. However, the current industrial vision system is far from satisfactory in the intelligent perception of complex grain images, comprising a large number of local homogeneous fragmentations or patches without distinct foreground and background. We attempt to solve this problem based on the statistical modeling of spatial structures of grain images. We present a physical explanation in advance to indicate that the spatial structures of the complex grain images are subject to a representative Weibull distribution according to the theory of sequential fragmentation, which is well known in the continued comminution of ore grinding. To delineate the spatial structure of the grain image, we present a method of multiscale and omnidirectional Gaussian derivative filtering. Then, a product quality classifier based on sparse multikernel-least squares support vector machine is proposed to solve the low-confidence classification problem of imbalanced data distribution. The proposed method is applied on the assembly line of a food-processing enterprise to classify (or identify automatically the production quality of rice. The experiments on the real application case, compared with the commonly used methods, illustrate the validity of our method.

  8. Visual Perception-Based Statistical Modeling of Complex Grain Image for Product Quality Monitoring and Supervision on Assembly Production Line.

    Science.gov (United States)

    Liu, Jinping; Tang, Zhaohui; Zhang, Jin; Chen, Qing; Xu, Pengfei; Liu, Wenzhong

    2016-01-01

    Computer vision as a fast, low-cost, noncontact, and online monitoring technology has been an important tool to inspect product quality, particularly on a large-scale assembly production line. However, the current industrial vision system is far from satisfactory in the intelligent perception of complex grain images, comprising a large number of local homogeneous fragmentations or patches without distinct foreground and background. We attempt to solve this problem based on the statistical modeling of spatial structures of grain images. We present a physical explanation in advance to indicate that the spatial structures of the complex grain images are subject to a representative Weibull distribution according to the theory of sequential fragmentation, which is well known in the continued comminution of ore grinding. To delineate the spatial structure of the grain image, we present a method of multiscale and omnidirectional Gaussian derivative filtering. Then, a product quality classifier based on sparse multikernel-least squares support vector machine is proposed to solve the low-confidence classification problem of imbalanced data distribution. The proposed method is applied on the assembly line of a food-processing enterprise to classify (or identify) automatically the production quality of rice. The experiments on the real application case, compared with the commonly used methods, illustrate the validity of our method.

  9. Replication, refinement & reachability

    DEFF Research Database (Denmark)

    Debois, Søren; Hildebrandt, Thomas T.; Slaats, Tijs

    2018-01-01

    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 “Dynamic...... Condition Response (DCR) graphs” declarative process model for analysis, execution and safe run-time refinement of process-aware information systems; including replication of sub-processes. We prove that event-reachability and refinement are np-hard for DCR? processes without replication...

  10. Molecular Processes Underlying the Structure and Assembly of Thin Films and Nanoparticles at Complex interfaces

    Energy Technology Data Exchange (ETDEWEB)

    Richmond, Geraldine [Univ. of Oregon, Eugene, OR (United States)

    2016-06-03

    differences in how water behaves at hydrophobic self-assembled monolayer (SAMS)/water interfaces relative to the organic liquid/water interfaces. Several monolayer films have been examined in these studies using a combination of vibrational sum frequency spectroscopy (VSFS), contact angle measurements and AFM. At the hydrocarbon monolayer/water interface we find that water has a weak bonding interaction with the monolayer film that results in an orientation of water at the terminus of these hydrocarbon chains. The water-film interaction is still present for fluorinated films but it is found to be considerably weaker. Hydration and Surfactant Adsorption at Salt/Water Interfaces This set of studies has examined the molecular characteristics of the CaF2/water interface using VSFS. Our first studies detailed the structure and orientation of water molecules adsorbed at this mineral surfaces including studies of the surface in the presence of aqueous solutions of salts. These studies have been followed by a series of static and time-resolved studies of the adsorption of carboxylic acid containing organics at this surface, specifically carboxylic acid surfactants and acetic acid. In the latter we have developed a new method for time resolved studies that involve sequential wavelength tuning and automated control of spatial beam overlap at the target can probe amplitude changes of sum-frequency resonances in widely spaced infrared regions. This offers great advantages for the study of the synchronism of molecular processes at interfaces. This approach is particularly suitable to investigate the synchronization of interfacial processes such as surfactant adsorption at charged mineral surfaces. Macromolecular Assembly at Liquid/Liquid Interfaces Macromolecular assembly at the interface between water and a hydrophobic surface underlies some of the most important biological and environmental processes on the planet. Our work has examined polymer adsorption and assembly of

  11. PROTEOLYTIC REMOVAL OF THE CARBOXYL TERMINUS OF THE T4 GENE 32 HELIX-DESTABILIZING PROTEIN ALTERS THE T4 IN VITRO REPLICATION COMPLEX

    Energy Technology Data Exchange (ETDEWEB)

    Burke, R.L.; Alberts, B.M.; Hosoda, J.

    1980-07-01

    The proteolytic removal of about 60 amino acids from the COOH terminus of the bacteriophage T4 helix-destabilizing protein (gene 32 protein) produces 32*I, a 27,000-dalton fragment which still binds tightly and cooperatively to single-stranded DNA. The substitution of 32*I protein for intact 32 protein in the seven-protein T4 replication complex results in dramatic changes in some of the reactions catalyzed by this in vitro DNA replication system, while leaving others largely unperturbed. (1) Like intact 32 protein, the 32*I protein promotes DNA synthesis by the DNA polymerase when the T4 polymerase accessory proteins (gene 44/62 and 45 proteins) are also present. The host helix-destabilizing protein (Escherichia coli ssb protein) cannot replace the 32*I protein for this synthesis. (2) Unlike intact 32 protein, 32*I protein strongly inhibits DNA synthesis catalyzed by the T4 DNA polymerase alone on a primed single-stranded DNA template. (3) Unlike intact 32 protein, the 32*I protein strongly inhibits RNA primer synthesis catalyzed by the T4 gene 41 and 61 proteins and also reduces the efficiency of RNA primer utilization. As a result, de novo DNA chain starts are blocked completely in the complete T4 replication system, and no lagging strand DNA synthesis occurs. (4) The 32*I protein does not bind to either the T4 DNA polymerase or to the T4 gene 61 protein in the absence of DNA; these associations (detected with intact 32 protein) would therefore appear to be essential for the normal control of 32 protein activity, and to account at least in part for observations 2 and 3, above. We propose that the COOH-terminal domain of intact 32 protein functions to guide its interactions with the T4 DNA polymerase and the T4 gene 61 RNA-priming protein. When this domain is removed, as in 32*I protein, the helix destabilization induced by the protein is controlled inadequately, so that polymerizing enzymes tend to be displaced from the growing 3{prime}-OH end of a

  12. Structural Biology of Proteins of the Multi-enzyme Assembly Human Pyruvate Dehydrogenase Complex

    Science.gov (United States)

    2003-01-01

    Objectives and research challenges of this effort include: 1. Need to establish Human Pyruvate Dehydrogenase Complex protein crystals; 2. Need to test value of microgravity for improving crystal quality of Human Pyruvate Dehydrogenase Complex protein crystals; 3. Need to improve flight hardware in order to control and understand the effects of microgravity on crystallization of Human Pyruvate Dehydrogenase Complex proteins; 4. Need to integrate sets of national collaborations with the restricted and specific requirements of flight experiments; 5. Need to establish a highly controlled experiment in microgravity with a rigor not yet obtained; 6. Need to communicate both the rigor of microgravity experiments and the scientific value of results obtained from microgravity experiments to the national community; and 7. Need to advance the understanding of Human Pyruvate Dehydrogenase Complex structures so that scientific and commercial advance is identified for these proteins.

  13. A Capping Step During Automated Glycan Assembly Enables Access to Complex Glycans in High Yield.

    Science.gov (United States)

    Yu, Yang; Kononov, Andrew; Delbianco, Martina; Seeberger, Peter H

    2018-04-20

    The products of multi-step automated solid phase syntheses are purified after release from the resin. Capping of unreacted nucleophiles is commonplace in automated oligonucleotide synthesis to minimize accumulation of deletion sequences. To date, capping was not used routinely during automated glycan assembly (AGA) since previous capping protocols suffered from long reaction times and conditions incompatible with some protective groups. Here, a method using methanesulfonic acid and acetic anhydride for the fast and quantitative capping of hydroxyl groups that failed to be glycosylated is reported. Commonly used protective groups in AGA are stable under these capping conditions. The introduction of a capping step into the coupling cycle drastically improved overall yields by decreasing side-products and simplifying purification, while reducing building block consumption. To illustrate the method, the biologically important tetrasaccharide Lc4, as well as a 50-mer polymannoside were prepared. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  14. Characterization of a subunit of the outer dynein arm docking complex necessary for correct flagellar assembly in Leishmania donovani.

    Directory of Open Access Journals (Sweden)

    Simone Harder

    Full Text Available BACKGROUND: In order to proceed through their life cycle, Leishmania parasites switch between sandflies and mammals. The flagellated promastigote cells transmitted by the insect vector are phagocytized by macrophages within the mammalian host and convert into the amastigote stage, which possesses a rudimentary flagellum only. During an earlier proteomic study of the stage differentiation of the parasite we identified a component of the outer dynein arm docking complex, a structure of the flagellar axoneme. The 70 kDa subunit of the outer dynein arm docking complex consists of three subunits altogether and is essential for the assembly of the outer dynein arm onto the doublet microtubule of the flagella. According to the nomenclature of the well-studied Chlamydomonas reinhardtii complex we named the Leishmania protein LdDC2. METHODOLOGY/PRINCIPAL FINDINGS: This study features a characterization of the protein over the life cycle of the parasite. It is synthesized exclusively in the promastigote stage and localizes to the flagellum. Gene replacement mutants of lddc2 show reduced growth rates and diminished flagellar length. Additionally, the normally spindle-shaped promastigote parasites reveal a more spherical cell shape giving them an amastigote-like appearance. The mutants lose their motility and wiggle in place. Ultrastructural analyses reveal that the outer dynein arm is missing. Furthermore, expression of the amastigote-specific A2 gene family was detected in the deletion mutants in the absence of a stage conversion stimulus. In vitro infectivity is slightly increased in the mutant cell line compared to wild-type Leishmania donovani parasites. CONCLUSIONS/SIGNIFICANCE: Our results indicate that the correct assembly of the flagellum has a great influence on the investigated characteristics of Leishmania parasites. The lack of a single flagellar protein causes an aberrant morphology, impaired growth and altered infectiousness of the parasite.

  15. Assembly, intracellular processing, and expression at the cell surface of the human alpha beta T cell receptor/CD3 complex. Function of the CD3-zeta chain

    DEFF Research Database (Denmark)

    Geisler, C; Kuhlmann, J; Rubin, B

    1989-01-01

    complex, the role of the CD3 chains for the TCR/CD3 expression have not been experimentally addressed in human T cells. In this study the function of the CD3-zeta chain for the assembly, intracellular processing, and expression of the TCR/CD3 complex in the human leukemic T cell line Jurkat...

  16. Endoplasmic reticulum resident protein of 90 kilodaltons associates with the T- and B-cell antigen receptors and major histocompatibility complex antigens during their assembly

    NARCIS (Netherlands)

    Hochstenbach, F.; DAVID, V.; WATKINS, S.; Brenner, M. B.

    1992-01-01

    In the endoplasmic reticulum (ER), newly synthesized subunits of the T-cell antigen receptor (TCR), membrane-bound immunoglobulin (mIg), and major histocompatibility complex (MHC) class I antigens must fold correctly and assemble completely into multimeric protein complexes prior to transport to the

  17. Platinum Complex Assemblies as Luminescent Probes and Tags for Drugs and Toxins in Water.

    Science.gov (United States)

    Sinn, Stephan; Biedermann, Frank; De Cola, Luisa

    2017-02-03

    A reactive phosphorescent probe for aza-heterocyclic drugs and toxins was developed, affording a supramolecular emission-switch-on chemosensor in water. Complex formation of the heterocycles with a platinum(II) precursor proceeds readily at ambient conditions, allowing for facile analyte screening. Fifty-two structurally diverse compounds were tested, out of which 23 pyridines, imidazoles, and triazoles formed strongly emissive complex aggregates. Importantly, they all can be clearly distinguished from each other through a principal component analysis, but often also by simple visual inspection, for example, by their emission color differences (large shifts from blue to red). Also, kinetic reaction profiles and time-resolved emission features can provide valuable information for analyte distinction. The Pt II complexes can be applied as emissive labels for drugs and biomolecules, owing to their advantageous photophysical properties and chemical stability in biological media such as blood. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  18. Succinate dehydrogenase assembly factor 2 is needed for assembly and activity of mitochondrial complex II and for normal root elongation in Arabidopsis.

    Science.gov (United States)

    Huang, Shaobai; Taylor, Nicolas L; Ströher, Elke; Fenske, Ricarda; Millar, A Harvey

    2013-02-01

    Mitochondria complex II (succinate dehydrogenase, SDH) plays a central role in respiratory metabolism as a component of both the electron transport chain and the tricarboxylic acid cycle. We report the identification of an SDH assembly factor by analysis of T-DNA insertions in At5g51040, a protein with unknown function that was identified by mass spectrometry analysis as a low abundance mitochondrial protein. This gene is co-expressed with a number of genes encoding mitochondrial proteins, including SDH1-1, and has low partial sequence similarity to human SDHAF2, a protein required for flavin-adenine dinucleotide (FAD) insertion into SDH. In contrast to observations of other SDH deficient lines in Arabidopsis, the sdhaf2 line did not affect photosynthetic rate or stomatal conductance, but instead showed inhibition of primary root elongation with early lateral root emergence, presumably due to the low SDH activity caused by the reduced abundance of SDHAF2. Both roots and leaves showed succinate accumulation but different responses in the abundance of other organic acids and amino acids assayed. Isolated mitochondria showed lowered SDH1 protein abundance, lowered maximal SDH activity and less protein-bound flavin-adenine dinucleotide (FAD) at the molecular mass of SDH1 in the gel separation. The short root phenotype and SDH function of sdhaf2 was fully complemented by transformation with SDHAF2. Application of the SDH inhibitor, malonate, phenocopied the sdhaf2 root architecture in WT. Whole root respiratory assays showed no difference between WT and sdhaf2, but micro-respirometry of the tips of roots clearly showed low oxygen consumption in sdhaf2 which could explain a metabolic deficit responsible for root tip growth. © 2012 The Authors The Plant Journal © 2012 Blackwell Publishing Ltd.

  19. The assembly of rotaxane-like dye/cyclodextrin/surface complexes on aluminium trihydroxide or goethite.

    Science.gov (United States)

    Cooper, Rachel J; Camp, Philip J; Gordon, Ross J; Henderson, David K; Henry, Dorothy C R; McNab, Hamish; De Silva, Sonali S; Tackley, Daniel; Tasker, Peter A; Wight, Paul

    2006-06-21

    Simple azo-dyes carrying phosphonic acid and arsonic acid substituents such as 4-(4-hydroxyphenyl azo)phenylphosphonic acid (5) and 4-(4-hydroxyphenylazo)phenylarsonic acid (6) bind more strongly to high surface area oxides such as aluminium trihydroxide and goethite than their carboxylic and sulfonic acid analogues and the phosphonate-functionalized dyes have been shown to have greater humidity fastness when printed onto commercial alumina-coated papers. Adsorption isotherm measurements provide evidence for the formation of ternary dye/cyclodextrin/surface complexes. Dyes which form such ternary complexes show higher light fastness when printed onto alumina coated papers in an ink formulation containing alpha-cyclodextrin.

  20. Bcs1p can rescue a large and productive cytochrome bc(1) complex assembly intermediate in the inner membrane of yeast mitochondria.

    Science.gov (United States)

    Conte, Laura; Trumpower, Bernard L; Zara, Vincenzo

    2011-01-01

    The yeast cytochrome bc(1) complex, a component of the mitochondrial respiratory chain, is composed of ten distinct protein subunits. In the assembly of the bc(1) complex, some ancillary proteins, such as the chaperone Bcs1p, are actively involved. The deletion of the nuclear gene encoding this chaperone caused the arrest of the bc(1) assembly and the formation of a functionally inactive bc(1) core structure of about 500-kDa. This immature bc(1) core structure could represent, on the one hand, a true assembly intermediate or, on the other hand, a degradation product and/or an incorrect product of assembly. The experiments here reported show that the gradual expression of Bcs1p in the yeast strain lacking this protein was progressively able to rescue the bc(1) core structure leading to the formation of the functional homodimeric bc(1) complex. Following Bcs1p expression, the mature bc(1) complex was also progressively converted into two supercomplexes with the cytochrome c oxidase complex. The capability of restoring the bc(1) complex and the supercomplexes was also possessed by the mutated yeast R81C Bcsp1. Notably, in the human ortholog BCS1L, the corresponding point mutation (R45C) was instead the cause of a severe bc(1) complex deficiency. Differently from the yeast R81C Bcs1p, two other mutated Bcs1p's (K192P and F401I) were unable to recover the bc(1) core structure in yeast. This study identifies for the first time a productive assembly intermediate of the yeast bc(1) complex and gives new insights into the molecular mechanisms involved in the last steps of bc(1) assembly. Copyright © 2010 Elsevier B.V. All rights reserved.

  1. Reconstitution of membrane protein complexes involved in pneumococcal septal cell wall assembly.

    Directory of Open Access Journals (Sweden)

    Marjolaine Noirclerc-Savoye

    Full Text Available The synthesis of peptidoglycan, the major component of the bacterial cell wall, is essential to cell survival, yet its mechanism remains poorly understood. In the present work, we have isolated several membrane protein complexes consisting of the late division proteins of Streptococcus pneumoniae: DivIB, DivIC, FtsL, PBP2x and FtsW, or subsets thereof. We have co-expressed membrane proteins from S. pneumoniae in Escherichia coli. By combining two successive affinity chromatography steps, we obtained membrane protein complexes with a very good purity. These complexes are functional, as indicated by the retained activity of PBP2x to bind a fluorescent derivative of penicillin and to hydrolyze the substrate analogue S2d. Moreover, we have evidenced the stabilizing role of protein-protein interactions within each complex. This work paves the way for a complete reconstitution of peptidoglycan synthesis in vitro, which will be critical to the elucidation of its intricate regulation mechanisms.

  2. Structural requirements for the assembly of LINC complexes and their function in cellular mechanical stiffness

    International Nuclear Information System (INIS)

    Stewart-Hutchinson, P.J.; Hale, Christopher M.; Wirtz, Denis; Hodzic, Didier

    2008-01-01

    The evolutionary-conserved interactions between KASH and SUN domain-containing proteins within the perinuclear space establish physical connections, called LINC complexes, between the nucleus and the cytoskeleton. Here, we show that the KASH domains of Nesprins 1, 2 and 3 interact promiscuously with luminal domains of Sun1 and Sun2. These constructs disrupt endogenous LINC complexes as indicated by the displacement of endogenous Nesprins from the nuclear envelope. We also provide evidence that KASH domains most probably fit a pocket provided by SUN domains and that post-translational modifications are dispensable for that interaction. We demonstrate that the disruption of endogenous LINC complexes affect cellular mechanical stiffness to an extent that compares to the loss of mechanical stiffness previously reported in embryonic fibroblasts derived from mouse lacking A-type lamins, a mouse model of muscular dystrophies and cardiomyopathies. These findings support a model whereby physical connections between the nucleus and the cytoskeleton are mediated by interactions between diverse combinations of Sun proteins and Nesprins through their respective evolutionary-conserved domains. Furthermore, they emphasize, for the first time, the relevance of LINC complexes in cellular mechanical stiffness suggesting a possible involvement of their disruption in various laminopathies, a group of human diseases linked to mutations of A-type lamins

  3. Assembly of the Major Light-Harvesting Complex II in Lipid Nanodiscs.

    NARCIS (Netherlands)

    Pandit, A.; Shirzad-Wasei, N.; Wlodarczyk, L.M.; Roon, H. van; Boekema, E.J.; Dekker, J.P.; Grip, W.J. de

    2011-01-01

    Self-aggregation of isolated plant light-harvesting complexes (LHCs) upon detergent extraction is associated with fluorescence quenching and is used as an in vitro model to study the photophysical processes of nonphotochemical quenching (NPQ). In the NPQ state, in vivo induced under excess solar

  4. Assembly of the Major Light-Harvesting Complex II in Lipid Nanodiscs

    NARCIS (Netherlands)

    Pandit, Anjali; Shirzad-Wasei, Nazhat; Wlodarczyk, Lucyna M.; van Roon, Henny; Boekema, Egbert J.; Dekker, Jan P.; de Grip, Willem J.; Brown, Leonid S.

    2011-01-01

    Self-aggregation of isolated plant light-harvesting complexes (LHCs) upon detergent extraction is associated with fluorescence quenching and is used as an in vitro model to study the photophysical processes of nonphotochemical quenching (NPQ). In the NPQ state, in vivo induced under excess solar

  5. A heterodimeric complex that promotes the assembly of mammalian 20S proteasomes

    DEFF Research Database (Denmark)

    Hirano, Yoko; Hendil, Klavs B.; Yashiroda, Hideki

    2005-01-01

    The 26S proteasome is a multisubunit protease responsible for regulated proteolysis in eukaryotic cells 1, 2 . It comprises one catalytic 20S proteasome and two axially positioned 19S regulatory complexes 3 . The 20S proteasome is composed of 28 subunits arranged in a cylindrical particle as four...

  6. Self-Assembled Complexes of Horseradish Peroxidase with Magnetic Nanoparticles Showing Enhanced Peroxidase Activity

    KAUST Repository

    Corgié, Stéphane C.

    2012-02-15

    Bio-nanocatalysts (BNCs) consisting of horseradish peroxidase (HRP) self-assembled with magnetic nanoparticles (MNPs) enhance enzymatic activity due to the faster turnover and lower inhibition of the enzyme. The size and magnetization of the MNPs affect the formation of the BNCs, and ultimately control the activity of the bound enzymes. Smaller MNPs form small clusters with a low affinity for the HRP. While the turnover for the bound fraction is drastically increased, there is no difference in the H 2O 2 inhibitory concentration. Larger MNPs with a higher magnetization aggregate in larger clusters and have a higher affinity for the enzyme and a lower substrate inhibition. All of the BNCs are more active than the free enzyme or the MNPs (BNCs > HRP ≤laquo; MNPs). Since the BNCs show surprising resilience in various reaction conditions, they may pave the way towards new hybrid biocatalysts with increased activities and unique catalytic properties for magnetosensitive enzymatic reactions. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  7. PucC and LhaA direct efficient assembly of the light-harvesting complexes in Rhodobacter sphaeroides

    DEFF Research Database (Denmark)

    Mothersole, David; Jackson, Philip J.; Vasilev, Cvetelin

    2016-01-01

    interactions between pigments newly arriving from BchG and nascent proteins within the SecYEG-SecDF-YajC-YidC assembly machinery, thereby co-ordinating pigment delivery, the co-translational insertion of LH polypeptides and their folding and assembly to form photosynthetic complexes.......The mature architecture of the photosynthetic membrane of the purple phototroph Rhodobacter sphaeroides has been characterised to a level where an atomic-level membrane model is available, but the roles of the putative assembly proteins LhaA and PucC in establishing this architecture are unknown...

  8. Application of X-ray digital radiography to online automated inspection of interior assembly structures of complex products

    Energy Technology Data Exchange (ETDEWEB)

    Han Yueping [National Key Laboratory for Electronic Measurement Technology, North University of China, Taiyuan 030051 (China) and Key Laboratory of Instrumentation Science and Dynamic Measurement (North University of China), Ministry of Education, Taiyuan 030051 (China)], E-mail: yuepinghan@163.com; Han Yan [Key Laboratory of Instrumentation Science and Dynamic Measurement (North University of China), Ministry of Education, Taiyuan 030051 (China); Li Ruihong [National Key Laboratory for Electronic Measurement Technology, North University of China, Taiyuan 030051 (China); Wang Liming [Key Laboratory of Instrumentation Science and Dynamic Measurement (North University of China), Ministry of Education, Taiyuan 030051 (China)

    2009-06-11

    The paper proposes an application of X-ray digital radiography to online automated inspection and recognition of the interior assembly structures of complex products by means of the multiple views techniques. First, a vertical hybrid projection function (VHPF) is proposed as the recognition feature of a two-dimensional image. VHPF combines an integral projection function and a standard deviation function so that it can reflect the mean and the variance of the pixels in the vertical direction in an image. Secondly, by considering the different importance grades of objects inside the product and the independence of these objects along the circumference, the paper presents a hierarchical recognition method and uses a neural network system to speed up the computation process with parallel operations. Thirdly, using the whole-orientation features of one standard swatch and by extracting its maximal system of linear independence as the feature basis, the issue of blind areas for recognition is resolved. Based on this approach, the first domestic X-ray multi-view digital detection system has been developed and applied to the online detection of objects containing complicated assembly structures.

  9. Application of X-ray digital radiography to online automated inspection of interior assembly structures of complex products

    International Nuclear Information System (INIS)

    Han Yueping; Han Yan; Li Ruihong; Wang Liming

    2009-01-01

    The paper proposes an application of X-ray digital radiography to online automated inspection and recognition of the interior assembly structures of complex products by means of the multiple views techniques. First, a vertical hybrid projection function (VHPF) is proposed as the recognition feature of a two-dimensional image. VHPF combines an integral projection function and a standard deviation function so that it can reflect the mean and the variance of the pixels in the vertical direction in an image. Secondly, by considering the different importance grades of objects inside the product and the independence of these objects along the circumference, the paper presents a hierarchical recognition method and uses a neural network system to speed up the computation process with parallel operations. Thirdly, using the whole-orientation features of one standard swatch and by extracting its maximal system of linear independence as the feature basis, the issue of blind areas for recognition is resolved. Based on this approach, the first domestic X-ray multi-view digital detection system has been developed and applied to the online detection of objects containing complicated assembly structures.

  10. Application of X-ray digital radiography to online automated inspection of interior assembly structures of complex products

    Science.gov (United States)

    Han, Yueping; Han, Yan; Li, Ruihong; Wang, Liming

    2009-06-01

    The paper proposes an application of X-ray digital radiography to online automated inspection and recognition of the interior assembly structures of complex products by means of the multiple views techniques. First, a vertical hybrid projection function (VHPF) is proposed as the recognition feature of a two-dimensional image. VHPF combines an integral projection function and a standard deviation function so that it can reflect the mean and the variance of the pixels in the vertical direction in an image. Secondly, by considering the different importance grades of objects inside the product and the independence of these objects along the circumference, the paper presents a hierarchical recognition method and uses a neural network system to speed up the computation process with parallel operations. Thirdly, using the whole-orientation features of one standard swatch and by extracting its maximal system of linear independence as the feature basis, the issue of blind areas for recognition is resolved. Based on this approach, the first domestic X-ray multi-view digital detection system has been developed and applied to the online detection of objects containing complicated assembly structures.

  11. pH-controlled self-assembling of meso-tetrakis(4-sulfonatophenyl)porphyrin-chitosan complexes

    Czech Academy of Sciences Publication Activity Database

    Synytsya, A.; Synytsya, Andriy.; Blafková, P.; Ederová, J.; Spěváček, Jiří; Slepička, P.; Král, V.; Volka, K.

    2009-01-01

    Roč. 10, č. 5 (2009), s. 1067-1076 ISSN 1525-7797 R&D Projects: GA ČR GA525/05/0273 Grant - others:GA AV ČR(CZ) KAN400480701; GA AV ČR(CZ) KAN200100801; GA AV ČR(CZ) KAN200200651; GA MŠk(CZ) LC06041; GA ČR(CZ) GA203/02/0420 Program:KA; KA; KA; LC; GA Institutional research plan: CEZ:AV0Z40500505 Keywords : self-assembling * meso-tetrakis(4-sulfonatophenyl)porphyrin- chitosan complex * spectroscopy Subject RIV: CD - Macromolecular Chemistry Impact factor: 4.502, year: 2009

  12. ToF-SIMS investigation of FIB-patterning of lactoferrin by using self-assembled monolayers of iron complexes

    International Nuclear Information System (INIS)

    Tuccitto, N.; Giamblanco, N.; Marletta, G.; Licciardello, A.

    2008-01-01

    Geometrically well-defined patterns of surface-immobilized proteins can be produced with several methods. We developed a method for patterning of proteins by means of specific, non-covalent interactions between a protein and a metal complex immobilized at the surface. In particular, reproducible patterns of lactoferrin have been obtained by exploiting the different adsorption properties of this protein on a OH-terminated self-assembled monolayer (SAM) or onto an iron-containing SAM present in certain regions of the pattern. The OH-terminated SAM was etched with a focused ion beam (FIB) in order to produce square regions of bare gold. These regions were selectively covered with a SAM of iron-terpyridine complex, formed via a stepwise procedure involving the initial formation of a mixed component SAM (containing the terpyridine ligand) and the subsequent reaction with an iron(II) salt in order to produce the complex. The patterned substrate was finally allowed to interact with a lactoferrin solution. It is shown that lactoferrin selectively and stably adsorbs on iron-containing layers, whereas it is not retained on the OH-terminated regions of the surface. The use of ToF-SIMS was crucial for obtaining this information, as well as for monitoring each sequential step necessary for the preparation of the patterns.

  13. Self-assembly of a 3d-5f trinuclear single-molecule magnet from a pentavalent uranyl complex

    International Nuclear Information System (INIS)

    Chatelain, Lucile; Pecaut, Jacques; Walsh, James P.S.; Tuna, Floriana; Mazzanti, Marinella

    2014-01-01

    Mixed-metal uranium compounds are very attractive candidates in the design of single-molecule magnets (SMMs), but only one 3d-5f hetero-polymetallic SMM containing a uranium center is known. Herein, we report two trimeric heterodimetallic 3d-5f complexes self-assembled by cation-cation interactions between a uranyl(V) complex and a TPA-capped M II complex (M=Mn (1), Cd (2); TPA=tris(2-pyridylmethyl)amine). The metal centers were strategically chosen to promote the formation of discrete molecules rather than extended chains. Compound 1, which contains an almost linear {Mn-O=U=O-Mn} core, exhibits SMM behavior with a relaxation barrier of 81±0.5 K - the highest reported for a mono-uranium system - arising from intramolecular Mn-U exchange interactions combined with the high Ising anisotropy of the uranyl(V) moiety. Compound 1 also exhibits an open magnetic hysteresis loop at temperatures less than 3 K, with a significant coercive field of 1.9 T at 1.8 K.

  14. Implementing the LIM code: the structural basis for cell type-specific assembly of LIM-homeodomain complexes

    Energy Technology Data Exchange (ETDEWEB)

    Bhati, Mugdha; Lee, Christopher; Nancarrow, Amy L.; Lee, Mihwa; Craig, Vanessa J.; Bach, Ingolf; Guss, J. Mitchell; Mackay, Joel P.; Matthews, Jacqueline M. (UMASS, MED); (Sydney)

    2008-09-03

    LIM-homeodomain (LIM-HD) transcription factors form a combinatorial 'LIM code' that contributes to the specification of cell types. In the ventral spinal cord, the binary LIM homeobox protein 3 (Lhx3)/LIM domain-binding protein 1 (Ldb1) complex specifies the formation of V2 interneurons. The additional expression of islet-1 (Isl1) in adjacent cells instead specifies the formation of motor neurons through assembly of a ternary complex in which Isl1 contacts both Lhx3 and Ldb1, displacing Lhx3 as the binding partner of Ldb1. However, little is known about how this molecular switch occurs. Here, we have identified the 30-residue Lhx3-binding domain on Isl1 (Isl1{sub LBD}). Although the LIM interaction domain of Ldb1 (Ldb1{sub LID}) and Isl1{sub LBD} share low levels of sequence homology, X-ray and NMR structures reveal that they bind Lhx3 in an identical manner, that is, Isl1{sub LBD} mimics Ldb1{sub LID}. These data provide a structural basis for the formation of cell type-specific protein-protein interactions in which unstructured linear motifs with diverse sequences compete to bind protein partners. The resulting alternate protein complexes can target different genes to regulate key biological events.

  15. Self-assembly of a 3d-5f trinuclear single-molecule magnet from a pentavalent uranyl complex

    Energy Technology Data Exchange (ETDEWEB)

    Chatelain, Lucile; Pecaut, Jacques [CEA-Grenoble (France). Lab. de Reconnaissance Ionique et Chimie de Coordination SCIB; Walsh, James P.S.; Tuna, Floriana [Manchester Univ. (United Kingdom). School of Chemistry and Photon Science Inst.; Mazzanti, Marinella [Ecole Polytechnique Federale de Lausanne (EPFL) (Switzerland). Inst. de Sciences et Ingenierie Chimiques

    2014-12-01

    Mixed-metal uranium compounds are very attractive candidates in the design of single-molecule magnets (SMMs), but only one 3d-5f hetero-polymetallic SMM containing a uranium center is known. Herein, we report two trimeric heterodimetallic 3d-5f complexes self-assembled by cation-cation interactions between a uranyl(V) complex and a TPA-capped M{sup II} complex (M=Mn (1), Cd (2); TPA=tris(2-pyridylmethyl)amine). The metal centers were strategically chosen to promote the formation of discrete molecules rather than extended chains. Compound 1, which contains an almost linear {Mn-O=U=O-Mn} core, exhibits SMM behavior with a relaxation barrier of 81±0.5 K - the highest reported for a mono-uranium system - arising from intramolecular Mn-U exchange interactions combined with the high Ising anisotropy of the uranyl(V) moiety. Compound 1 also exhibits an open magnetic hysteresis loop at temperatures less than 3 K, with a significant coercive field of 1.9 T at 1.8 K.

  16. Luminescent lanthanide complexes with 4-acetamidobenzoate: Synthesis, supramolecular assembly via hydrogen bonds, crystal structures and photoluminescence

    Science.gov (United States)

    Yin, Xia; Fan, Jun; Wang, Zhi Hong; Zheng, Sheng Run; Tan, Jing Bo; Zhang, Wei Guang

    2011-07-01

    Four new luminescent complexes, namely, [Eu(aba) 2(NO 3)(C 2H 5OH) 2] ( 1), [Eu(aba) 3(H 2O) 2]·0.5 (4, 4'-bpy)·2H 2O ( 2), [Eu 2(aba) 4(2, 2'-bpy) 2(NO 3) 2]·4H 2O ( 3) and [Tb 2(aba) 4(phen) 2(NO 3) 2]·2C 2H 5OH ( 4) were obtained by treating Ln(NO 3) 3·6H 2O and 4-acetamidobenzoic acid (Haba) with different coligands (4, 4'-bpy=4, 4'-bipyridine, 2, 2'-bpy=2, 2'-bipyridine, and phen=1, 10-phenanthroline). They exhibit 1D chains ( 1- 2) and dimeric structures ( 3- 4), respectively. This structural variation is mainly attributed to the change of coligands and various coordination modes of aba molecules. Moreover, the coordination units are further connected via hydrogen bonds to form 2D even 3D supramolecular networks. These complexes show characteristic emissions in the visible region at room temperature. In addition, thermal behaviors of four complexes have been investigated under air atmosphere. The relationship between the structures and physical properties has been discussed.

  17. GAPDH--a recruits a plant virus movement protein to cortical virus replication complexes to facilitate viral cell-to-cell movement.

    Directory of Open Access Journals (Sweden)

    Masanori Kaido

    2014-11-01

    Full Text Available The formation of virus movement protein (MP-containing punctate structures on the cortical endoplasmic reticulum is required for efficient intercellular movement of Red clover necrotic mosaic virus (RCNMV, a bipartite positive-strand RNA plant virus. We found that these cortical punctate structures constitute a viral replication complex (VRC in addition to the previously reported aggregate structures that formed adjacent to the nucleus. We identified host proteins that interacted with RCNMV MP in virus-infected Nicotiana benthamiana leaves using a tandem affinity purification method followed by mass spectrometry. One of these host proteins was glyceraldehyde 3-phosphate dehydrogenase-A (NbGAPDH-A, which is a component of the Calvin-Benson cycle in chloroplasts. Virus-induced gene silencing of NbGAPDH-A reduced RCNMV multiplication in the inoculated leaves, but not in the single cells, thereby suggesting that GAPDH-A plays a positive role in cell-to-cell movement of RCNMV. The fusion protein of NbGAPDH-A and green fluorescent protein localized exclusively to the chloroplasts. In the presence of RCNMV RNA1, however, the protein localized to the cortical VRC as well as the chloroplasts. Bimolecular fluorescence complementation assay and GST pulldown assay confirmed in vivo and in vitro interactions, respectively, between the MP and NbGAPDH-A. Furthermore, gene silencing of NbGAPDH-A inhibited MP localization to the cortical VRC. We discuss the possible roles of NbGAPDH-A in the RCNMV movement process.

  18. Homeotic proteins participate in the function of human-DNA replication origins.

    Science.gov (United States)

    Marchetti, Laura; Comelli, Laura; D'Innocenzo, Barbara; Puzzi, Luca; Luin, Stefano; Arosio, Daniele; Calvello, Mariantonietta; Mendoza-Maldonado, Ramiro; Peverali, Fiorenzo; Trovato, Fabio; Riva, Silvano; Biamonti, Giuseppe; Abdurashidova, Gulnara; Beltram, Fabio; Falaschi, Arturo

    2010-12-01

    Recent evidence points to homeotic proteins as actors in the crosstalk between development and DNA replication. The present work demonstrates that HOXC13, previously identified as a new member of human DNA replicative complexes, is a stable component of early replicating chromatin in living cells: it displays a slow nuclear dynamics due to its anchoring to the DNA minor groove via the arginine-5 residue of the homeodomain. HOXC13 binds in vivo to the lamin B2 origin in a cell-cycle-dependent manner consistent with origin function; the interaction maps with nucleotide precision within the replicative complex. HOXC13 displays in vitro affinity for other replicative complex proteins; it interacts also in vivo with the same proteins in a cell-cycle-dependent fashion. Chromatin-structure modifying treatments, disturbing origin function, reduce also HOXC13-origin interaction. The described interactions are not restricted to a single origin nor to a single homeotic protein (also HOXC10 binds the lamin B2 origin in vivo). Thus, HOX complexes probably contribute in a general, structure-dependent manner, to origin identification and assembly of replicative complexes thereon, in presence of specific chromatin configurations.

  19. 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.

  20. Proteomic analysis of Entamoeba histolytica in vivo assembled pre-mRNA splicing complexes.

    Science.gov (United States)

    Valdés, Jesús; Nozaki, Tomoyoshi; Sato, Emi; Chiba, Yoko; Nakada-Tsukui, Kumiko; Villegas-Sepúlveda, Nicolás; Winkler, Robert; Azuara-Liceaga, Elisa; Mendoza-Figueroa, María Saraí; Watanabe, Natsuki; Santos, Herbert J; Saito-Nakano, Yumiko; Galindo-Rosales, José Manuel

    2014-12-05

    The genome of the human intestinal parasite Entamoeba histolytica contains nearly 3000 introns and bioinformatic predictions indicate that major and minor spliceosomes occur in Entamoeba. However, except for the U2-, U4-, U5- and U6 snRNAs, no other splicing factor has been cloned and characterized. Here, we HA-tagged cloned the snRNP component U1A and assessed its expression and nuclear localization. Because the snRNP-free U1A form interacts with polyadenylate-binding protein, HA-U1A immunoprecipitates could identify early and late splicing complexes. Avoiding Entamoeba's endonucleases and ensuring the precipitation of RNA-binding proteins, parasite cultures were UV cross-linked prior to nuclear fraction immunoprecipitations with HA antibodies, and precipitates were subjected to tandem mass spectrometry (MS/MS) analyses. To discriminate their nuclear roles (chromatin-, co-transcriptional-, splicing-related), MS/MS analyses were carried out with proteins eluted with MS2-GST-sepharose from nuclear extracts of an MS2 aptamer-tagged Rabx13 intron amoeba transformant. Thus, we probed thirty-six Entamoeba proteins corresponding to 32 cognate splicing-specific factors, including 13 DExH/D helicases required for all stages of splicing, and 12 different splicing-related helicases were identified also. Furthermore 50 additional proteins, possibly involved in co-transcriptional processes were identified, revealing the complexity of co-transcriptional splicing in Entamoeba. Some of these later factors were not previously found in splicing complex analyses. Numerous facts about the splicing of the nearly 3000 introns of the Entamoeba genome have not been unraveled, particularly the splicing factors and their activities. Considering that many of such introns are located in metabolic genes, the knowledge of the splicing cues has the potential to be used to attack or control the parasite. We have found numerous new splicing-related factors which could have therapeutic benefit. We

  1. CENTRIOLE REPLICATION

    Science.gov (United States)

    Mizukami, Ikuko; Gall, Joseph

    1966-01-01

    Sperm formation was studied in the fern, Marsilea, and the cycad, Zamia, with particular emphasis on the centrioles. In Marsilea, the mature sperm possesses over 100 flagella, the basal bodies of which have the typical cylindrical structure of centrioles. Earlier observations by light microscopy suggested that these centrioles arise by fragmentation of a body known as the blepharoplast. In the youngest spermatids the blepharoplast is a hollow sphere approximately 0.8 µ in diameter. Its wall consists of closely packed immature centrioles, or procentrioles. The procentrioles are short cylinders which progressively lengthen during differentiation of the spermatid. At the same time they migrate to the surface of the cell, where each of them puts out a flagellum. A blepharoplast is found at each pole of the spindle during the last antheridial mitosis, and two blepharoplasts are found in the cytoplasm before this mitosis. Blepharoplasts are also found in the preceding cell generation, but their ultimate origin is obscure. Before the last mitosis the blepharoplasts are solid, consisting of a cluster of radially arranged tubules which bear some structural similarity to centrioles. In Zamia, similar stages are found during sperm formation, although here the number of flagella on each sperm is close to 20,000 and the blepharoplast measures about 10 µ in diameter. These observations are discussed in relation to theories of centriole replication. PMID:5950730

  2. Rapid Genome Mapping in Nanochannel Arrays for Highly Complete and Accurate De Novo Sequence Assembly of the Complex Aegilops tauschii Genome

    Science.gov (United States)

    Hastie, Alex R.; Dong, Lingli; Smith, Alexis; Finklestein, Jeff; Lam, Ernest T.; Huo, Naxin; Cao, Han; Kwok, Pui-Yan; Deal, Karin R.; Dvorak, Jan; Luo, Ming-Cheng; Gu, Yong; Xiao, Ming

    2013-01-01

    Next-generation sequencing (NGS) technologies have enabled high-throughput and low-cost generation of sequence data; however, de novo genome assembly remains a great challenge, particularly for large genomes. NGS short reads are often insufficient to create large contigs that span repeat sequences and to facilitate unambiguous assembly. Plant genomes are notorious for containing high quantities of repetitive elements, which combined with huge genome sizes, makes accurate assembly of these large and complex genomes intractable thus far. Using two-color genome mapping of tiling bacterial artificial chromosomes (BAC) clones on nanochannel arrays, we completed high-confidence assembly of a 2.1-Mb, highly repetitive region in the large and complex genome of Aegilops tauschii, the D-genome donor of hexaploid wheat (Triticum aestivum). Genome mapping is based on direct visualization of sequence motifs on single DNA molecules hundreds of kilobases in length. With the genome map as a scaffold, we anchored unplaced sequence contigs, validated the initial draft assembly, and resolved instances of misassembly, some involving contigs <2 kb long, to dramatically improve the assembly from 75% to 95% complete. PMID:23405223

  3. Structure of a novel shoulder-to-shoulder p24 dimer in complex with the broad-spectrum antibody A10F9 and its implication in capsid assembly.

    Directory of Open Access Journals (Sweden)

    Ying Gu

    Full Text Available Mature HIV-1 viral particles assemble as a fullerene configuration comprising p24 capsid hexamers, pentamers and dimers. In this paper, we report the X-ray crystal structures of the p24 protein from natural HIV-1 strain (BMJ4 in complex with Fab A10F9, which recognizes a conserved epitope in the C-terminal domain of the BMJ4 p24 protein. Our structures reveal a novel shoulder-to-shoulder p24 dimerization mode that is mediated by an S-S bridge at C177. Consistent with these structures, the shoulder-to-shoulder dimer that was obtained from the BMJ4 strain was also observed in p24 proteins from other strains by the introduction of a cysteine residue at position 177. The potential biological significance was further validated by the introduction of a C177A mutation in the BMJ4 strain, which then displays a low infectivity. Our data suggest that this novel shoulder-to-shoulder dimer interface trapped by this unique S-S bridge could represent a physiologically relevant mode of HIV-1 capsid assembly during virus maturation, although Cys residue itself may not be critical for HIV-I replication.

  4. Metal ion-assisted self-assembly of complexes for controlled and sustained release of minocycline for biomedical applications

    International Nuclear Information System (INIS)

    Zhang, Zhiling; Wang, Zhicheng; Nong, Jia; Nix, Camilla A; Zhong, Yinghui; Ji, Hai-Feng

    2015-01-01

    This study reports the development of novel drug delivery complexes self-assembled by divalent metal ion-assisted coacervation for controlled and sustained release of a hydrophilic small drug molecule minocycline hydrochloride (MH). MH is a multifaceted agent that has demonstrated therapeutic effects in infection, inflammation, tumor, as well as cardiovascular, renal, and neurological disorders due to its anti-microbial, anti-inflammatory, and cytoprotective properties. However, the inability to translate the high doses used in experimental animals to tolerable doses in human patients limits its clinical application. Localized delivery can potentially expose the diseased tissue to high concentrations of MH that systemic delivery cannot achieve, while minimizing the side effects from systemic exposure. The strong metal ion binding-assisted interaction enabled high drug entrapment and loading efficiency, and stable long term release for more than 71 d. Released MH demonstrated potent anti-biofilm, anti-inflammatory, and neuroprotective activities. Furthermore, MH release from the complexes is pH-sensitive as the chelation between minocycline and metal ions decreases with pH, allowing ‘smart’ drug release in response to the severity of pathology-induced tissue acidosis. This novel metal ion binding-mediated drug delivery mechanism can potentially be applied to other drugs that have high binding affinity for metal ions and may lead to the development of new delivery systems for a variety of drugs. (paper)

  5. Supramolecular Self-Assembly and Dual-Switch Vapochromic, Vapoluminescent, and Resistive Memory Behaviors of Amphiphilic Platinum(II) Complexes.

    Science.gov (United States)

    Li, Yongguang; Chen, Ling; Ai, Yeye; Hong, Eugene Yau-Hin; Chan, Alan Kwun-Wa; Yam, Vivian Wing-Wah

    2017-10-04

    A series of amphiphilic platinum(II) complexes with tridentate N-donor ligands has been synthesized and characterized. Different supramolecular architectures are constructed using the amphiphilic molecules as the building blocks through the formation of Pt···Pt and π-π stacking interactions in aqueous media. The aggregation-deaggregation-aggregation self-assembly behavior together with obvious spectroscopic changes could be fine-tuned by the addition of THF in aqueous media. More interestingly, one of the complexes is found to show fast response and high selectivity toward alcohol and water vapors with good reversibility, leading to drastic color and luminescence changes, and hence unique dual switching behavior, with the water molecules readily displaced by the alcohol vapor. Rapid writing and erasure have been realized via the control of a jet or a stream of alcohol vapor flow. In addition, it has been employed as active materials in the fabrication of small-molecule solution-processable resistive memory devices, exhibiting stable and promising binary memory performance with threshold voltages of ca. 3.4 V, high ON/OFF ratios of up to 10 5 and long retention times of over 10 4 s. The vapochromic and vapoluminescent materials are demonstrated to have potential applications in chemosensing, logic gates, VOC monitoring, and memory functions.

  6. Component-Level Electronic-Assembly Repair (CLEAR) Spacecraft Circuit Diagnostics by Analog and Complex Signature Analysis

    Science.gov (United States)

    Oeftering, Richard C.; Wade, Raymond P.; Izadnegahdar, Alain

    2011-01-01

    The Component-Level Electronic-Assembly Repair (CLEAR) project at the NASA Glenn Research Center is aimed at developing technologies that will enable space-flight crews to perform in situ component-level repair of electronics on Moon and Mars outposts, where there is no existing infrastructure for logistics spares. These technologies must provide effective repair capabilities yet meet the payload and operational constraints of space facilities. Effective repair depends on a diagnostic capability that is versatile but easy to use by crew members that have limited training in electronics. CLEAR studied two techniques that involve extensive precharacterization of "known good" circuits to produce graphical signatures that provide an easy-to-use comparison method to quickly identify faulty components. Analog Signature Analysis (ASA) allows relatively rapid diagnostics of complex electronics by technicians with limited experience. Because of frequency limits and the growing dependence on broadband technologies, ASA must be augmented with other capabilities. To meet this challenge while preserving ease of use, CLEAR proposed an alternative called Complex Signature Analysis (CSA). Tests of ASA and CSA were used to compare capabilities and to determine if the techniques provided an overlapping or complementary capability. The results showed that the methods are complementary.

  7. DNA damage response clamp 9-1-1 promotes assembly of ZMM proteins for formation of crossovers and synaptonemal complex

    Science.gov (United States)

    Shinohara, Miki; Hayashihara, Kayoko; Grubb, Jennifer T.; Bishop, Douglas K.; Shinohara, Akira

    2015-01-01

    Formation of crossovers between homologous chromosomes during meiosis is positively regulated by the ZMM proteins (also known as SIC proteins). DNA damage checkpoint proteins also promote efficient formation of interhomolog crossovers. Here, we examined, in budding yeast, the meiotic role of the heterotrimeric DNA damage response clamp composed of Rad17, Ddc1 and Mec3 (known as ‘9-1-1’ in other organisms) and a component of the clamp loader, Rad24 (known as Rad17 in other organisms). Cytological analysis indicated that the 9-1-1 clamp and its loader are not required for the chromosomal loading of RecA homologs Rad51 or Dmc1, but are necessary for the efficient loading of ZMM proteins. Interestingly, the loading of ZMM proteins onto meiotic chromosomes was independent of the checkpoint kinase Mec1 (the homolog of ATR) as well as Rad51. Furthermore, the ZMM member Zip3 (also known as Cst9) bound to the 9-1-1 complex in a cell-free system. These data suggest that, in addition to promoting interhomolog bias mediated by Rad51–Dmc1, the 9-1-1 clamp promotes crossover formation through a specific role in the assembly of ZMM proteins. Thus, the 9-1-1 complex functions to promote two crucial meiotic recombination processes, the regulation of interhomolog recombination and crossover formation mediated by ZMM. PMID:25736290

  8. Human Mitochondrial DNA Replication

    Science.gov (United States)

    Holt, Ian J.; Reyes, Aurelio

    2012-01-01

    Elucidation of the process of DNA replication in mitochondria is in its infancy. For many years, maintenance of the mitochondrial genome was regarded as greatly simplified compared to the nucleus. Mammalian mitochondria were reported to lack all DNA repair systems, to eschew DNA recombination, and to possess but a single DNA polymerase, polymerase γ. Polγ was said to replicate mitochondrial DNA exclusively via one mechanism, involving only two priming events and a handful of proteins. In this “strand-displacement model,” leading strand DNA synthesis begins at a specific site and advances approximately two-thirds of the way around the molecule before DNA synthesis is initiated on the “lagging” strand. Although the displaced strand was long-held to be coated with protein, RNA has more recently been proposed in its place. Furthermore, mitochondrial DNA molecules with all the features of products of conventional bidirectional replication have been documented, suggesting that the process and regulation of replication in mitochondria is complex, as befits a genome that is a core factor in human health and longevity. PMID:23143808

  9. TOM9.2 Is a Calmodulin-Binding Protein Critical for TOM Complex Assembly but Not for Mitochondrial Protein Import in Arabidopsis thaliana.

    Science.gov (United States)

    Parvin, Nargis; Carrie, Chris; Pabst, Isabelle; Läßer, Antonia; Laha, Debabrata; Paul, Melanie V; Geigenberger, Peter; Heermann, Ralf; Jung, Kirsten; Vothknecht, Ute C; Chigri, Fatima

    2017-04-03

    The translocon on the outer membrane of mitochondria (TOM) facilitates the import of nuclear-encoded proteins. The principal machinery of mitochondrial protein transport seems conserved in eukaryotes; however, divergence in the composition and structure of TOM components has been observed between mammals, yeast, and plants. TOM9, the plant homolog of yeast Tom22, is significantly smaller due to a truncation in the cytosolic receptor domain, and its precise function is not understood. Here we provide evidence showing that TOM9.2 from Arabidopsis thaliana is involved in the formation of mature TOM complex, most likely by influencing the assembly of the pore-forming subunit TOM40. Dexamethasone-induced RNAi gene silencing of TOM9.2 results in a severe reduction in the mature TOM complex, and the assembly of newly imported TOM40 into the complex is impaired. Nevertheless, mutant plants are fully viable and no obvious downstream effects of the loss of TOM complex, i.e., on mitochondrial import capacity, were observed. Furthermore, we found that TOM9.2 can bind calmodulin (CaM) in vitro and that CaM impairs the assembly of TOM complex in the isolated wild-type mitochondria, suggesting a regulatory role of TOM9.2 and a possible integration of TOM assembly into the cellular calcium signaling network. Copyright © 2017 The Author. Published by Elsevier Inc. All rights reserved.

  10. CmRBP50 protein phosphorylation is essential for assembly of a stable phloem-mobile high-affinity ribonucleoprotein complex.

    Science.gov (United States)

    Li, Pingfang; Ham, Byung-Kook; Lucas, William J

    2011-07-01

    RNA-binding proteins (RBPs) form ribonucleoprotein (RNP) complexes that play crucial roles in RNA processing for gene regulation. The angiosperm sieve tube system contains a unique population of transcripts, some of which function as long-distance signaling agents involved in regulating organ development. These phloem-mobile mRNAs are translocated as RNP complexes. One such complex is based on a phloem RBP named Cucurbita maxima RNA-binding protein 50 (CmRBP50), a member of the polypyrimidine track binding protein family. The core of this RNP complex contains six additional phloem proteins. Here, requirements for assembly of this CmRBP50 RNP complex are reported. Phosphorylation sites on CmRBP50 were mapped, and then coimmunoprecipitation and protein overlay studies established that the phosphoserine residues, located at the C terminus of CmRBP50, are critical for RNP complex assembly. In vitro pull-down experiments revealed that three phloem proteins, C. maxima phloem protein 16, C. maxima GTP-binding protein, and C. maxima phosphoinositide-specific phospholipase-like protein, bind directly with CmRBP50. This interaction required CmRBP50 phosphorylation. Gel mobility-shift assays demonstrated that assembly of the CmRBP50-based protein complex results in a system having enhanced binding affinity for phloem-mobile mRNAs carrying polypyrimidine track binding motifs. This property would be essential for effective long-distance translocation of bound mRNA to the target tissues.

  11. 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

  12. A One-Pot Self-Assembly Reaction to Prepare a Supramolecular Palladium(II) Cyclometalated Complex: An Undergraduate Organometallic Laboratory Experiment

    Science.gov (United States)

    Fernandez, Alberto; Lopez-Torres, Margarita; Fernandez, Jesus J.; Vazquez-Garcia, Digna; Vila, Jose M.

    2012-01-01

    A laboratory experiment for students in advanced inorganic chemistry is described. Students prepare palladium(II) cyclometalated complexes. A terdentate [C,N,O] Schiff base ligand is doubly deprotonated upon reaction with palladium(II) acetate in a self-assembly process to give a palladacycle with a characteristic tetranuclear structure. This…

  13. ATM and ATR Activities Maintain Replication Fork Integrity during SV40 Chromatin Replication

    Science.gov (United States)

    Sowd, Gregory A.; Li, Nancy Yan; Fanning, Ellen

    2013-01-01

    Mutation of DNA damage checkpoint signaling kinases ataxia telangiectasia-mutated (ATM) or ATM- and Rad3-related (ATR) results in genomic instability disorders. However, it is not well understood how the instability observed in these syndromes relates to DNA replication/repair defects and failed checkpoint control of cell cycling. As a simple model to address this question, we have studied SV40 chromatin replication in infected cells in the presence of inhibitors of ATM and ATR activities. Two-dimensional gel electrophoresis and southern blotting of SV40 chromatin replication products reveal that ATM activity prevents accumulation of unidirectional replication products, implying that ATM promotes repair of replication-associated double strand breaks. ATR activity alleviates breakage of a functional fork as it converges with a stalled fork. The results suggest that during SV40 chromatin replication, endogenous replication stress activates ATM and ATR signaling, orchestrating the assembly of genome maintenance machinery on viral replication intermediates. PMID:23592994

  14. Kinetics of the CRISPR-Cas9 effector complex assembly and the role of 3′-terminal segment of guide RNA

    Science.gov (United States)

    Mekler, Vladimir; Minakhin, Leonid; Semenova, Ekaterina; Kuznedelov, Konstantin; Severinov, Konstantin

    2016-01-01

    CRISPR-Cas9 is widely applied for genome engineering in various organisms. The assembly of single guide RNA (sgRNA) with the Cas9 protein may limit the Cas9/sgRNA effector complex function. We developed a FRET-based assay for detection of CRISPR–Cas9 complex binding to its targets and used this assay to investigate the kinetics of Cas9 assembly with a set of structurally distinct sgRNAs. We find that Cas9 and isolated sgRNAs form the effector complex efficiently and rapidly. Yet, the assembly process is sensitive to the presence of moderate concentrations of non-specific RNA competitors, which considerably delay the Cas9/sgRNA complex formation, while not significantly affecting already formed complexes. This observation suggests that the rate of sgRNA loading into Cas9 in cells can be determined by competition between sgRNA and intracellular RNA molecules for the binding to Cas9. Non-specific RNAs exerted particularly large inhibitory effects on formation of Cas9 complexes with sgRNAs bearing shortened 3′-terminal segments. This result implies that the 3′-terminal segment confers sgRNA the ability to withstand competition from non-specific RNA and at least in part may explain the fact that use of sgRNAs truncated for the 3′-terminal stem loops leads to reduced activity during genomic editing. PMID:26945042

  15. Assembling Metal Ions Induced Cyanide-Bridged Heterometallic 1D and Ion-Pair Complexes: Synthesis, Crystal Structures and Magnetic Properties

    Energy Technology Data Exchange (ETDEWEB)

    Kong, Lingqian [Liaocheng Univ., Liaocheng (China); Zhao, Zengdian; Chen, Kexun; Wang, Ping; Zhang, Daopeng [Shandong Univ. of Technology, Zibo (China)

    2013-07-15

    We obtained a heterobimetallic one-dimensional cyanide-bridged Mn(II)-Ni(II) complex and an Co(III)-Ni(II) ion-pair complex with [Ni(CN){sub 4}]{sup 2-} as building block and M(II)-phenanthroline (M = Mn, Co) compounds as assembling segment. The different structural types of complexes 1 and 2 indicate that the property of the metal ions the assembling segment contained have obvious influence on the structure of the cyanide-bridged complex. Investigation over the magnetic properties of complex 1 reveals an overall weak antiferromagnetic coupling between the adjacent Mn(II) ions bridged by the antiferromagnetic [-NC-Ni-CN-] unit. Among of all the molecular magnetism systems, for the well known reasons, cyanide-containing complexes have been widely employed as bridges to assemble homo/hetero-metallic molecular magnetic materials by using the cyanide bridge transferring magnetic coupling between the neighboring paramagnetic ions, in whichsome showed interesting magnetic properties, such as high-Tc magnets, spin crossover materials, single-molecule magnets (SMMs) and single-chain magnets (SCMs)

  16. Synthesis of Inorganic Nanocomposites by Selective Introduction of Metal Complexes into a Self-Assembled Block Copolymer Template

    Directory of Open Access Journals (Sweden)

    Hiroaki Wakayama

    2015-01-01

    Full Text Available Inorganic nanocomposites have characteristic structures that feature expanded interfaces, quantum effects, and resistance to crack propagation. These structures are promising for the improvement of many materials including thermoelectric materials, photocatalysts, and structural materials. Precise control of the inorganic nanocomposites’ morphology, size, and chemical composition is very important for these applications. Here, we present a novel fabrication method to control the structures of inorganic nanocomposites by means of a self-assembled block copolymer template. Different metal complexes were selectively introduced into specific polymer blocks of the block copolymer, and subsequent removal of the block copolymer template by oxygen plasma treatment produced hexagonally packed porous structures. In contrast, calcination removal of the block copolymer template yielded nanocomposites consisting of metallic spheres in a matrix of a metal oxide. These results demonstrate that different nanostructures can be created by selective use of processes to remove the block copolymer templates. The simple process of first mixing block copolymers and magnetic nanomaterial precursors and then subsequently removing the block copolymer template enables structural control of magnetic nanomaterials, which will facilitate their applicability in patterned media, including next-generation perpendicular magnetic recording media.

  17. Fabrication and characterization of magnesium and calcium trimesate complexes via ion-exchange and one-pot self-assembly reaction

    Science.gov (United States)

    Ozer, Demet; Oztas, Nursen Altuntas; Köse, Dursun A.; Şahin, Onur

    2018-03-01

    Using two different synthesis methods, two diversified magnesium and calcium complexes were successfully prepared. When the ion exchange method was used, C9H14MgO11.H2O and C18H30Ca3O24 complexes were obtained. When the one-pot self-assembly reaction was used, C18H34Mg3O26.4H2O and C9H12CaO10 complexes were produced. The structural characterizations were performed by using X-ray diffraction, FT-IR and elemental analyses. Thermal behavior of complexes were also determined via TGA method. The both complexes of magnesium and calcium trimesate have micro and mesoporosity with low porosity because of hydrogen bonds. Then hydrogen storage capacities of complexes were also determined. The differences in synthesis method result in the differences on complexes structure, morphology (shape, particle size and specific surface area) and hydrogen storage capacities.

  18. Uncoupling of Sister Replisomes during Eukaryotic DNA Replication

    NARCIS (Netherlands)

    Yardimci, Hasan; Loveland, Anna B.; Habuchi, Satoshi; van Oijen, Antoine M.; Walter, Johannes C.

    2010-01-01

    The duplication of eukaryotic genomes involves the replication of DNA from multiple origins of replication. In S phase, two sister replisomes assemble at each active origin, and they replicate DNA in opposite directions. Little is known about the functional relationship between sister replisomes.

  19. 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 replication....... After the assembly of a double heterohexameric MCM2-7 complex at replication origins in G1, the 2 heterohexamers separate from each other and associate with Cdc45 and GINS proteins in a CMG complex that is capable of unwinding dsDNA during S phase. Here, we have reconstituted and characterized...... the purified human MCM2-7 (hMCM2-7) hexameric complex by co-expression of its 6 different subunits in insect cells. The conformational variability of the complex has been analyzed by single particle electron microscopy in the presence of different nucleotide analogs and DNA. The interaction with nucleotide...

  20. Assembly of functional proton-translocating ATPase complex in yeast mitochondria with cytoplasmically synthesized subunit 8, a polypeptide normally encoded within the organelle.

    OpenAIRE

    Nagley, P; Farrell, L B; Gearing, D P; Nero, D; Meltzer, S; Devenish, R J

    1988-01-01

    A mitochondrial gene from Saccharomyces cerevisiae encoding a hydrophobic membrane protein, subunit 8 of the F0/F1-type mitochondrial ATPase complex, has been functionally replaced by an artificial nuclear gene specifying an imported version of this protein. The experiments reported here utilized a multicopy expression vector (pLF1) that replicates in the nucleus of yeast cells and that carries an inserted DNA segment, specifying a precursor protein (N9/Y8) consisting of subunit 8 fused to an...

  1. Light-triggered dissociation of self-assembled β-amyloid aggregates into small, nontoxic fragments by ruthenium (II) complex.

    Science.gov (United States)

    Son, Giyeong; Lee, Byung Il; Chung, You Jung; Park, Chan Beum

    2018-02-01

    The self-assembly of β-amyloid (Aβ) peptides into highly stable plaques is a major hallmark of Alzheimer's disease. Here, we report visible light-driven dissociation of β-sheet-rich Aβ aggregates into small, nontoxic fragments using ruthenium (II) complex {[Ru(bpy) 3 ] 2+ } that functions as a highly sensitive, biocompatible, photoresponsive anti-Aβ agent. According to our multiple analyses using thioflavin T, bicinchoninic acid, dynamic light scattering, atomic force microscopy, circular dichroism, and Fourier transform infrared spectroscopy, [Ru(bpy) 3 ] 2+ successfully disassembled Aβ aggregates by destabilizing the β-sheet secondary structure under illumination of white light-emitting diode light. We validated that photoexcited [Ru(bpy) 3 ] 2+ causes oxidative damages of Aβ peptides, resulting in the dissociation of Aβ aggregates. The efficacy of [Ru(bpy) 3 ] 2+ is attributed to reactive oxygen species, such as singlet oxygen, generated from [Ru(bpy) 3 ] 2+ that absorbed photon energy in the visible range. Furthermore, photoexcited [Ru(bpy) 3 ] 2+ strongly inhibited the self-assembly of Aβ monomers even at concentrations as low as 1 nM and reduced the cytotoxicity of Aβ aggregates. Alzheimer's disease is the most common progressive neurodegenerative disease, affecting more than 13% of the population over age 65. Over the last decades, researchers have focused on understanding the mechanism of amyloid formation, the hallmark of various amyloid diseases including Alzheimer's and Parkinson's. In this paper, we successfully demonstrate the dissociation of β-Amyloid (Aβ) aggregates into small, less-amyloidic fragments by photoexcited [Ru(bpy) 3 ] 2+ through destabilization of β-sheet secondary structure. We validated the light-triggered dissociation of amyloid structure using multiple analytical tools. Furthermore, we confirmed that photoexcited [Ru(bpy) 3 ] 2+ reduces cytotoxicity of Aβ aggregates. Our work should open a new horizon in the study of

  2. Subunit composition of CP43-less photosystem II complexes of Synechocystis sp. PCC 6803: implications for the assembly and repair of photosystem II.

    Science.gov (United States)

    Boehm, M; Yu, J; Reisinger, V; Beckova, M; Eichacker, L A; Schlodder, E; Komenda, J; Nixon, P J

    2012-12-19

    Photosystem II (PSII) mutants are useful experimental tools to trap potential intermediates involved in the assembly of the oxygen-evolving PSII complex. Here, we focus on the subunit composition of the RC47 assembly complex that accumulates in a psbC null mutant of the cyanobacterium Synechocystis sp. PCC 6803 unable to make the CP43 apopolypeptide. By using native gel electrophoresis, we showed that RC47 is heterogeneous and mainly found as a monomer of 220 kDa. RC47 complexes co-purify with small Cab-like proteins (ScpC and/or ScpD) and with Psb28 and its homologue Psb28-2. Analysis of isolated His-tagged RC47 indicated the presence of D1, D2, the CP47 apopolypeptide, plus nine of the 13 low-molecular-mass (LMM) subunits found in the PSII holoenzyme, including PsbL, PsbM and PsbT, which lie at the interface between the two momomers in the dimeric holoenzyme. Not detected were the LMM subunits (PsbK, PsbZ, Psb30 and PsbJ) located in the vicinity of CP43 in the holoenzyme. The photochemical activity of isolated RC47-His complexes, including the rate of reduction of P680(+), was similar to that of PSII complexes lacking the Mn(4)CaO(5) cluster. The implications of our results for the assembly and repair of PSII in vivo are discussed.

  3. Assembly of the novel five-component apicomplexan multi-aminoacyl-tRNA synthetase complex is driven by the hybrid scaffold protein Tg-p43.

    Directory of Open Access Journals (Sweden)

    Jason M van Rooyen

    Full Text Available In Toxoplasma gondii, as in other eukaryotes, a subset of the amino-acyl-tRNA synthetases are arranged into an abundant cytoplasmic multi-aminoacyl-tRNA synthetase (MARS complex. Through a series of genetic pull-down assays, we have identified the enzymes of this complex as: methionyl-, glutaminyl-, glutamyl-, and tyrosyl-tRNA synthetases, and we show that the N-terminal GST-like domain of a partially disordered hybrid scaffold protein, Tg-p43, is sufficient for assembly of the intact complex. Our gel filtration studies revealed significant heterogeneity in the size and composition of isolated MARS complexes. By targeting the tyrosyl-tRNA synthetases subunit, which was found exclusively in the complete 1 MDa complex, we were able to directly visualize MARS particles in the electron microscope. Image analyses of the negative stain data revealed the observed heterogeneity and instability of these complexes to be driven by the intrinsic flexibility of the domain arrangements within the MARS complex. These studies provide unique insights into the assembly of these ubiquitous but poorly understood eukaryotic complexes.

  4. Aberrant Assembly Complexes of the Reaction Center Light-harvesting 1 PufX (RC-LH1-PufX) Core Complex of Rhodobacter sphaeroides Imaged by Atomic Force Microscopy*

    Science.gov (United States)

    Olsen, John D.; Adams, Peter G.; Jackson, Philip J.; Dickman, Mark J.; Qian, Pu; Hunter, C. Neil

    2014-01-01

    In the purple phototrophic bacterium Rhodobacter sphaeroides, many protein complexes congregate within the membrane to form operational photosynthetic units consisting of arrays of light-harvesting LH2 complexes and monomeric and dimeric reaction center (RC)-light-harvesting 1 (LH1)-PufX “core” complexes. Each half of a dimer complex consists of a RC surrounded by 14 LH1 αβ subunits, with two bacteriochlorophylls (Bchls) sandwiched between each αβ pair of transmembrane helices. We used atomic force microscopy (AFM) to investigate the assembly of single molecules of the RC-LH1-PufX complex using membranes prepared from LH2-minus mutants. When the RC and PufX components were also absent, AFM revealed a series of LH1 variants where the repeating α1β1(Bchl)2 units had formed rings of variable size, ellipses, and spirals and also arcs that could be assembly products. The spiral complexes occur when the LH1 ring has failed to close, and short arcs are suggestive of prematurely terminated LH1 complex assembly. In the absence of RCs, we occasionally observed captive proteins enclosed by the LH1 ring. When production of LH1 units was restricted by lowering the relative levels of the cognate pufBA transcript, we imaged a mixture of complete RC-LH1 core complexes, empty LH1 rings, and isolated RCs, leading us to conclude that once a RC associates with the first α1β1(Bchl)2 subunit, cooperative associations between subsequent subunits and the RC tend to drive LH1 ring assembly to completion. PMID:25193660

  5. Aberrant assembly complexes of the reaction center light-harvesting 1 PufX (RC-LH1-PufX) core complex of Rhodobacter sphaeroides imaged by atomic force microscopy.

    Science.gov (United States)

    Olsen, John D; Adams, Peter G; Jackson, Philip J; Dickman, Mark J; Qian, Pu; Hunter, C Neil

    2014-10-24

    In the purple phototrophic bacterium Rhodobacter sphaeroides, many protein complexes congregate within the membrane to form operational photosynthetic units consisting of arrays of light-harvesting LH2 complexes and monomeric and dimeric reaction center (RC)-light-harvesting 1 (LH1)-PufX "core" complexes. Each half of a dimer complex consists of a RC surrounded by 14 LH1 αβ subunits, with two bacteriochlorophylls (Bchls) sandwiched between each αβ pair of transmembrane helices. We used atomic force microscopy (AFM) to investigate the assembly of single molecules of the RC-LH1-PufX complex using membranes prepared from LH2-minus mutants. When the RC and PufX components were also absent, AFM revealed a series of LH1 variants where the repeating α(1)β(1)(Bchl)2 units had formed rings of variable size, ellipses, and spirals and also arcs that could be assembly products. The spiral complexes occur when the LH1 ring has failed to close, and short arcs are suggestive of prematurely terminated LH1 complex assembly. In the absence of RCs, we occasionally observed captive proteins enclosed by the LH1 ring. When production of LH1 units was restricted by lowering the relative levels of the cognate pufBA transcript, we imaged a mixture of complete RC-LH1 core complexes, empty LH1 rings, and isolated RCs, leading us to conclude that once a RC associates with the first α1β1(Bchl)2 subunit, cooperative associations between subsequent subunits and the RC tend to drive LH1 ring assembly to completion. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  6. DNA-PK dependent targeting of DNA-ends to a protein complex assembled on matrix attachment region DNA sequences

    International Nuclear Information System (INIS)

    Mauldin, S.K.; Getts, R.C.; Perez, M.L.; DiRienzo, S.; Stamato, T.D.

    2003-01-01

    Full text: We find that nuclear protein extracts from mammalian cells contain an activity that allows DNA ends to associate with circular pUC18 plasmid DNA. This activity requires the catalytic subunit of DNA-PK (DNA-PKcs) and Ku since it was not observed in mutants lacking Ku or DNA-PKcs but was observed when purified Ku/DNA-PKcs was added to these mutant extracts. Competition experiments between pUC18 and pUC18 plasmids containing various nuclear matrix attachment region (MAR) sequences suggest that DNA ends preferentially associate with plasmids containing MAR DNA sequences. At a 1:5 mass ratio of MAR to pUC18, approximately equal amounts of DNA end binding to the two plasmids were observed, while at a 1:1 ratio no pUC18 end-binding was observed. Calculation of relative binding activities indicates that DNA-end binding activities to MAR sequences was 7 to 21 fold higher than pUC18. Western analysis of proteins bound to pUC18 and MAR plasmids indicates that XRCC4, DNA ligase IV, scaffold attachment factor A, topoisomerase II, and poly(ADP-ribose) polymerase preferentially associate with the MAR plasmid in the absence or presence of DNA ends. In contrast, Ku and DNA-PKcs were found on the MAR plasmid only in the presence of DNA ends. After electroporation of a 32P-labeled DNA probe into human cells and cell fractionation, 87% of the total intercellular radioactivity remained in nuclei after a 0.5M NaCl extraction suggesting the probe was strongly bound in the nucleus. The above observations raise the possibility that DNA-PK targets DNA-ends to a repair and/or DNA damage signaling complex which is assembled on MAR sites in the nucleus

  7. Stable interaction between the human proliferating cell nuclear antigen loader complex Ctf18-replication factor C (RFC) and DNA polymerase {epsilon} is mediated by the cohesion-specific subunits, Ctf18, Dcc1, and Ctf8.

    Science.gov (United States)

    Murakami, Takeshi; Takano, Ryuji; Takeo, Satoshi; Taniguchi, Rina; Ogawa, Kaori; Ohashi, Eiji; Tsurimoto, Toshiki

    2010-11-05

    One of the proliferating cell nuclear antigen loader complexes, Ctf18-replication factor C (RFC), is involved in sister chromatid cohesion. To examine its relationship with factors involved in DNA replication, we performed a proteomics analysis of Ctf18-interacting proteins. We found that Ctf18 interacts with a replicative DNA polymerase, DNA polymerase ε (pol ε). Co-immunoprecipitation with recombinant Ctf18-RFC and pol ε demonstrated that their binding is direct and mediated by two distinct interactions, one weak and one stable. Three subunits that are specifically required for cohesion in yeast, Ctf18, Dcc1, and Ctf8, formed a trimeric complex (18-1-8) and together enabled stable binding with pol ε. The C-terminal 23-amino acid stretch of Ctf18 was necessary for the trimeric association of 18-1-8 and was required for the stable interaction. The weak interaction was observed with alternative loader complexes including Ctf18-RFC(5), which lacks Dcc1 and Ctf8, suggesting that the common loader structures, including the RFC small subunits (RFC2-5), are responsible for the weak interaction. The two interaction modes, mediated through distinguishable structures of Ctf18-RFC, both occurred through the N-terminal half of pol ε, which includes the catalytic domain. The addition of Ctf18-RFC or Ctf18-RFC(5) to the DNA synthesis reaction caused partial inhibition and stimulation, respectively. Thus, Ctf18-RFC has multiple interactions with pol ε that promote polymorphic modulation of DNA synthesis. We propose that their interaction alters the DNA synthesis mode to enable the replication fork to cooperate with the establishment of cohesion.

  8. 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.

  9. SUMO and KSHV Replication

    Energy Technology Data Exchange (ETDEWEB)

    Chang, Pei-Ching [Institute of Microbiology and Immunology, National Yang-Ming University, Taipei 112, Taiwan (China); Kung, Hsing-Jien, E-mail: hkung@nhri.org.tw [Institute for Translational Medicine, College of Medical Science and Technology, Taipei Medical University, Taipei 110, Taiwan (China); Department of Biochemistry and Molecular Medicine, University of California, Davis, CA 95616 (United States); UC Davis Cancer Center, University of California, Davis, CA 95616 (United States); Division of Molecular and Genomic Medicine, National Health Research Institutes, 35 Keyan Road, Zhunan, Miaoli County 35053, Taiwan (China)

    2014-09-29

    Small Ubiquitin-related MOdifier (SUMO) modification was initially identified as a reversible post-translational modification that affects the regulation of diverse cellular processes, including signal transduction, protein trafficking, chromosome segregation, and DNA repair. Increasing evidence suggests that the SUMO system also plays an important role in regulating chromatin organization and transcription. It is thus not surprising that double-stranded DNA viruses, such as Kaposi’s sarcoma-associated herpesvirus (KSHV), have exploited SUMO modification as a means of modulating viral chromatin remodeling during the latent-lytic switch. In addition, SUMO regulation allows the disassembly and assembly of promyelocytic leukemia protein-nuclear bodies (PML-NBs), an intrinsic antiviral host defense, during the viral replication cycle. Overcoming PML-NB-mediated cellular intrinsic immunity is essential to allow the initial transcription and replication of the herpesvirus genome after de novo infection. As a consequence, KSHV has evolved a way as to produce multiple SUMO regulatory viral proteins to modulate the cellular SUMO environment in a dynamic way during its life cycle. Remarkably, KSHV encodes one gene product (K-bZIP) with SUMO-ligase activities and one gene product (K-Rta) that exhibits SUMO-targeting ubiquitin ligase (STUbL) activity. In addition, at least two viral products are sumoylated that have functional importance. Furthermore, sumoylation can be modulated by other viral gene products, such as the viral protein kinase Orf36. Interference with the sumoylation of specific viral targets represents a potential therapeutic strategy when treating KSHV, as well as other oncogenic herpesviruses. Here, we summarize the different ways KSHV exploits and manipulates the cellular SUMO system and explore the multi-faceted functions of SUMO during KSHV’s life cycle and pathogenesis.

  10. Effect of self-assembled peptide–mesenchymal stem cell complex on the progression of osteoarthritis in a rat model

    Directory of Open Access Journals (Sweden)

    Kim JE

    2014-05-01

    metalloproteinases 1, and matrix metalloproteinase 9 were diffusely stained in controls, whereas localized or minimal staining was observed in other groups. Modified Mankin scores were significantly lower in the SAP and SAP-MSC groups than in controls (P=0.001 and 0.013. Although not statistically significant, synovial inflammation scores were lower in the SAP (1.3±0.3 and SAP-MSC (1.3±0.2 groups than in controls (2.6±0.2. However, neither the cytokine level nor the behavioral score was significantly different between groups.Conclusion: Injection of SAP-MSC hydrogels showed evidence of chondroprotection, as measured by the histologic grading and decreased expression of biochemical markers of inflammation and apoptosis. It also lowered subchondral bone mineral density, which can be increased by OA. This suggests that the SAP-MSC complex may have clinical potential to inhibit OA progression.Keywords: self-assembled peptide, mesenchymal stem cell, osteoarthritis, apoptosis, chondrogenesis

  11. Two-dimensional layer architecture assembled by Keggin polyoxotungstate, Cu(II)-EDTA complex and sodium linker: Synthesis, crystal structures, and magnetic properties

    International Nuclear Information System (INIS)

    Liu Hong; Xu Lin; Gao Guanggang; Li Fengyan; Yang Yanyan; Li Zhikui; Sun Yu

    2007-01-01

    Reaction of Keggin polyoxotungstate with copper(II)-EDTA (EDTA=ethylenediamine tetraacetate) complex under mild conditions led to the formation of hybrid inorganic-organic compounds Na 4 (OH)[(Cu 2 EDTA)PW 12 O 40 ].17H 2 O (1) and Na 4 [(Cu 2 EDTA)SiW 12 O 40 ].19H 2 O (2). The single-crystal X-ray diffraction analyses reveal their two structural features: (1) one-dimensional chain structure consisting of Keggin polyoxotungstate and copper(II)-EDTA complex; (2) Two-dimensional layer architecture assembled by the one-dimensional chain structure and sodium linker. The results of magnetic measurements in the temperature range 300-2 K indicated the existence of ferromagnetic exchange interactions between the Cu II ions for both compounds. In addition, TGA analysis, IR spectra, and electrochemical properties were also investigated to well characterize these two compounds. - Graphical abstract: Two new polyoxometalate-based hybrids, Na 4 (OH)[Cu 2 (EDTA)PW 12 O 40 ].17H 2 O (1) and Na 4 [Cu 2 (EDTA)SiW 12 O 40 ].19H 2 O (2), have been synthesized and structurally characterized, which consist of one-dimensional chain structure assembled by Keggin polyoxotungstate and copper(II)-EDTA complex. The chains are further connected to form two-dimensional layer architecture assembled by the one-dimensional chain structure and sodium linker

  12. VIRUS instrument collimator assembly

    Science.gov (United States)

    Marshall, Jennifer L.; DePoy, Darren L.; Prochaska, Travis; Allen, Richard D.; Williams, Patrick; Rheault, Jean-Philippe; Li, Ting; Nagasawa, Daniel Q.; Akers, Christopher; Baker, David; Boster, Emily; Campbell, Caitlin; Cook, Erika; Elder, Alison; Gary, Alex; Glover, Joseph; James, Michael; Martin, Emily; Meador, Will; Mondrik, Nicholas; Rodriguez-Patino, Marisela; Villanueva, Steven; Hill, Gary J.; Tuttle, Sarah; Vattiat, Brian; Lee, Hanshin; Chonis, Taylor S.; Dalton, Gavin B.; Tacon, Mike

    2014-07-01

    The Visual Integral-Field Replicable Unit Spectrograph (VIRUS) instrument is a baseline array 150 identical fiber fed optical spectrographs designed to support observations for the Hobby-Eberly Telescope Dark Energy Experiment (HETDEX). The collimator subassemblies of the instrument have been assembled in a production line and are now complete. Here we review the design choices and assembly practices used to produce a suite of identical low-cost spectrographs in a timely fashion using primarily unskilled labor.

  13. Dynamic and reversible self-assembly of photoelectrochemical complexes based on lipid bilayer disks, photosynthetic reaction centers, and single-walled carbon nanotubes.

    Science.gov (United States)

    Boghossian, Ardemis A; Choi, Jong Hyun; Ham, Moon-Ho; Strano, Michael S

    2011-03-01

    An aqueous solution containing photosynthetic reaction centers (RCs), membrane scaffold proteins (MSPs), phospholipids, and single-walled carbon nanotubes (SWCNTs) solubilized with the surfactant sodium cholate (SC) reversibly self-assembles into a highly ordered structure upon dialysis of the latter. The resulting structure is photoelectrochemically active and consists of 4-nm-thick lipid bilayer disks (nanodisks, NDs) arranged parallel to the surface of the SWCNT with the RC housed within the bilayer such that its hole injecting site faces the nanotube surface. The structure can be assembled and disassembled autonomously with the addition or removal of surfactant. We model the kinetic and thermodynamic forces that drive the dynamics of this reversible self-assembly process. The assembly is monitored using spectrofluorimetry during dialysis and subsequent surfactant addition and used to fit a kinetic model to determine the forward and reverse rate constants of ND and ND-SWCNT formation. The calculated ND and ND-SWCNT forward rate constants are 79 mM(-1) s(-1) and 5.4 × 10(2) mM(-1) s(-1), respectively, and the reverse rate constants are negligible over the dialysis time scale. We find that the reaction is not diffusion-controlled since the ND-SWCNT reaction, which consists of entities with smaller diffusion coefficients, has a larger reaction rate constant. Using these rate parameters, we were able to develop a kinetic phase diagram for the formation of ND-SWCNT complexes, which indicates an optimal dialysis rate of approximately 8 × 10(-4) s(-1). We also fit the model to cyclic ND-SWCNT assembly and disassembly experiments and hence mimic the thermodynamic forces used in regeneration processes detailed previously. Such forces may form the basis of both synthetic and natural photoelectrochemical complexes capable of dynamic component replacement and repair.

  14. Self assembled rotaxane and pseudo-rotaxanes based on {beta}-cyclodextrin inclusion compounds with trans-1,4-bis[(4-pyridyl)ethenyl]benzene-pentacyanoferrate(II) complexes

    Energy Technology Data Exchange (ETDEWEB)

    Toma, Sergio H.; Toma, Henrique E. [Sao Paulo Univ., SP (Brazil). Inst. de Quimica]. E-mail: henetoma@iq.usp.br

    2007-03-15

    Inclusion compounds of trans-1,4-bis[(4-pyridyl)ethenyl]benzene (BPEB) and their corresponding pentacyanoferrate(II) complexes with {beta}-cyclodextrin have been studied in aqueous solution by {sup 1}H NMR and UV-Visible spectroscopy. All the inclusion compounds exhibit 1:1 stoichiometry is aqueous solution. In the presence of {beta}-cyclodextrin, the binuclear {l_brace}[Fe(CN){sub 5}]{sub 2}(BPEB)]{sup 6-} complex is gradually converted into rotaxane species bearing [Fe(CN){sub 5}]{sup 3-} end groups, by a self assembly inclusion mechanism, as confirmed by {sup 1}H NMR spectroscopy. (author)

  15. Evidence that the assembly of the yeast cytochrome bc1 complex involves the formation of a large core structure in the inner mitochondrial membrane.

    Science.gov (United States)

    Zara, Vincenzo; Conte, Laura; Trumpower, Bernard L

    2009-04-01

    The assembly status of the cytochrome bc(1) complex has been analyzed in distinct yeast deletion strains in which genes for one or more of the bc(1) subunits were deleted. In all the yeast strains tested, a bc(1) sub-complex of approximately 500 kDa was found when the mitochondrial membranes were analyzed by blue native electrophoresis. The subsequent molecular characterization of this sub-complex, carried out in the second dimension by SDS/PAGE and immunodecoration, revealed the presence of the two catalytic subunits, cytochrome b and cytochrome c(1), associated with the noncatalytic subunits core protein 1, core protein 2, Qcr7p and Qcr8p. Together, these bc(1) subunits build up the core structure of the cytochrome bc(1) complex, which is then able to sequentially bind the remaining subunits, such as Qcr6p, Qcr9p, the Rieske iron-sulfur protein and Qcr10p. This bc(1) core structure may represent a true assembly intermediate during the maturation of the bc(1) complex; first, because of its wide distribution in distinct yeast deletion strains and, second, for its characteristics of stability, which resemble those of the intact homodimeric bc(1) complex. By contrast, the bc(1) core structure is unable to interact with the cytochrome c oxidase complex to form respiratory supercomplexes. The characterization of this novel core structure of the bc(1) complex provides a number of new elements clarifying the molecular events leading to the maturation of the yeast cytochrome bc(1) complex in the inner mitochondrial membrane.

  16. Evidence that assembly of the yeast cytochrome bc1 complex involves formation of a large core structure in the inner mitochondrial membrane

    Science.gov (United States)

    Zara, Vincenzo; Conte, Laura; Trumpower, Bernard L.

    2009-01-01

    The assembly status of the cytochrome bc1 complex has been analyzed in distinct yeast deletion strains in which genes for one or more of the bc1 subunits had been deleted. In all the yeast strains tested a bc1 sub-complex of about 500 kDa was found when the mitochondrial membranes were analyzed by blue native electrophoresis. The subsequent molecular characterization of this sub-complex, carried out in the second dimension by SDS-PAGE and immunodecoration, revealed the presence of the two catalytic subunits cytochrome b and cytochrome c1, associated with the non catalytic subunits core protein 1, core protein 2, Qcr7p and Qcr8p. Altogether these bc1 subunits build up the core structure of the cytochrome bc1 complex which is then able to sequentially bind the remaining subunits, such as Qcr6p, Qcr9p, the Rieske iron-sulfur protein and Qcr10p. This bc1 core structure may represent a true assembly intermediate during the maturation of the bc1 complex, first because of its wide distribution in distinct yeast deletion strains and second for its characteristics of stability which resemble those of the intact homodimeric bc1 complex. Differently from this latter, however, the bc1 core structure is not able to interact with the cytochrome c oxidase complex to form respiratory supercomplexes. The characterization of this novel core structure of the bc1 complex provides a number of new elements for clarification of the molecular events leading to the maturation of the yeast cytochrome bc1 complex in the inner mitochondrial membrane. PMID:19236481

  17. The yeast SAS (something about silencing) protein complex contains a MYST-type putative acetyltransferase and functions with chromatin assembly factor ASF1

    Science.gov (United States)

    Osada, Shigehiro; Sutton, Ann; Muster, Nemone; Brown, Christine E.; Yates, John R.; Sternglanz, Rolf; Workman, Jerry L.

    2001-01-01

    It is well established that acetylation of histone and nonhistone proteins is intimately linked to transcriptional activation. However, loss of acetyltransferase activity has also been shown to cause silencing defects, implicating acetylation in gene silencing. The something about silencing (Sas) 2 protein of Saccharomyces cerevisiae, a member of the MYST (MOZ, Ybf2/Sas3, Sas2, and TIP60) acetyltransferase family, promotes silencing at HML and telomeres. Here we identify a ∼450-kD SAS complex containing Sas2p, Sas4p, and the tf2f-related Sas5 protein. Mutations in the conserved acetyl-CoA binding motif of Sas2p are shown to disrupt the ability of Sas2p to mediate the silencing at HML and telomeres, providing evidence for an important role for the acetyltransferase activity of the SAS complex in silencing. Furthermore, the SAS complex is found to interact with chromatin assembly factor Asf1p, and asf1 mutants show silencing defects similar to mutants in the SAS complex. Thus, ASF1-dependent chromatin assembly may mediate the role of the SAS complex in silencing. PMID:11731479

  18. Eukaryotic Replisome Components Cooperate to Process Histones During Chromosome Replication

    Directory of Open Access Journals (Sweden)

    Magdalena Foltman

    2013-03-01

    Full Text Available DNA unwinding at eukaryotic replication forks displaces parental histones, which must be redeposited onto nascent DNA in order to preserve chromatin structure. By screening systematically for replisome components that pick up histones released from chromatin into a yeast cell extract, we found that the Mcm2 helicase subunit binds histones cooperatively with the FACT (facilitiates chromatin transcription complex, which helps to re-establish chromatin during transcription. FACT does not associate with the Mcm2-7 helicase at replication origins during G1 phase but is subsequently incorporated into the replisome progression complex independently of histone binding and uniquely among histone chaperones. The amino terminal tail of Mcm2 binds histones via a conserved motif that is dispensable for DNA synthesis per se but helps preserve subtelomeric chromatin, retain the 2 micron minichromosome, and support growth in the absence of Ctf18-RFC. Our data indicate that the eukaryotic replication and transcription machineries use analogous assemblies of multiple chaperones to preserve chromatin integrity.

  19. Cytotoxic potency of self-assembled Ruthenium(II-NHC complexes with pincer type 2, 6-bis(N-methylimidazolylidene/benzimidazolylidenepyrazine ligands

    Directory of Open Access Journals (Sweden)

    Gourisannkar Roymahapatra

    2015-01-01

    Full Text Available Objective: To study the cytotoxic potency of self-assembled Ruthenium(II-NHC complexes with 2,6-di-(N-methylimidazolylidene/benzimidazolylidenepyrazine ligands. Materials and Methods: Ru(II-N-heterocyclic (Ru-NHC complexes, Bis-[2,6-di-(N-methylimidazol-2-ylidenepyrazine]ruthenium(II hexaflurophosphate (3, Bis-[2,6-di-(N-methylbenzimidazol-2-ylidenepyrazine]ruthenium(II hexaflurophosphate (4 have been synthesized from corresponding ligands 2,6-di-(N-methylimidazoliumpyrazine dichloride (1; 2,6-di-(N-methylbenzimidazoliumpyrazine dichloride (2. Complexes were studied to determine their pro-apoptotic activity against HCT15 and Hep2 cell lines, and antimicrobial activity against Pseudomonas aeruginosa, Staphylococcus epidermidis and Candida albicans. Results: Both, complex 3 and 4, formed a nanosphere structure in aqueous growth medium. Cytotoxicity study revealed that complex 3 was more effective than complex 4. Complexes mainly target cellular DNA and bacterial cell wall. Conclusion: This is the first report on the formation of nanoball structure of Ru(II-NHC complexes. Thus, complex 3 provides a new insight to develop antitumor or antimicrobial drug.

  20. E2F-Rb complexes assemble and inhibit cdc25A transcription in cervical carcinoma cells following repression of human papillomavirus oncogene expression

    DEFF Research Database (Denmark)

    Wu, L; Goodwin, E C; Naeger, L K

    2000-01-01

    in the absence of E2 expression. Expression of the E2 protein also led to posttranscriptional increase in the level of E2F4, p105(Rb), and p130 and induced the formation of nuclear E2F4-p130 and E2F4-p105(Rb) complexes. This resulted in marked rearrangement of the protein complexes that formed at the distal E2F...... site in the cdc25A promoter, including the replacement of free E2F complexes with E2F4-p105(Rb) complexes. These experiments indicated that repression of E2F-responsive promoters following HPV E6/E7 repression was mediated by activation of the Rb tumor suppressor pathway and the assembly of repressing...

  1. QIL1-dependent assembly of MICOS complex-lethal mutation in C19ORF70 resulting in liver disease and severe neurological retardation.

    Science.gov (United States)

    Gödiker, J; Grüneberg, M; DuChesne, I; Reunert, J; Rust, S; Westermann, C; Wada, Y; Classen, G; Langhans, C D; Schlingmann, K P; Rodenburg, R J; Pohlmann, R; Marquardt, T

    2018-04-04

    Seven subunits of the mitochondrial contact site and cristae junction (CJ) organizing system (MICOS) in humans have been recently described in function and structure. QIL1 (also named MIC13) is a small complex that is crucial for the maintenance and assembling of MICOS. A novel mutation of an essential splice site in the C19orf70 gene encoding QIL1 induces severe mitochondrial encephalopathy, hepatopathy and lactate acidosis consistent with psychomotor retardation. In addition, bilateral kidney stones were observed. Disassembly of MICOS complex subunits displays lack of MIC10-MIC26-MIC27-QIL1 subcomplex, resulting in aberrant cristae structure and a loss of cristae junctions and contact sites. In liver and muscle tissue, the activity of the respiratory chain complexes (OXPHOS) was severely impaired. Defects in MICOS complex do not only affect mitochondrial architecture, but also mitochondrial fusion, metabolic signalling, lipid trafficking and cellular electric homeostasis.

  2. Layer-by-Layer Assembly of Fluorine-Free Polyelectrolyte-Surfactant Complexes for the Fabrication of Self-Healing Superhydrophobic Films.

    Science.gov (United States)

    Wu, Mengchun; An, Ni; Li, Yang; Sun, Junqi

    2016-11-29

    Fluorine-free self-healing superhydrophobic films are of significance for practical applications because of their extended service life and cost-effective and eco-friendly preparation process. In this study, we report the fabrication of fluorine-free self-healing superhydrophobic films by layer-by-layer (LbL) assembly of poly(sodium 4-styrenesulfonate) (PSS)-1-octadecylamine (ODA) complexes (PSS-ODA) and poly(allylamine hydrochloride) (PAH)-sodium dodecyl sulfonate (SDS) (PAH-SDS) complexes. The wettability of the LbL-assembled PSS-ODA/PAH-SDS films depends on the film structure and can be tailored by changing the NaCl concentration in aqueous dispersions of PSS-ODA complexes and the number of film deposition cycles. The freshly prepared PSS-ODA/PAH-SDS film with micro- and nanoscaled hierarchical structures is hydrophilic and gradually changes to superhydrophobic in air because the polyelectrolyte-complexed ODA and SDS surfactants tend to migrate to the film surface to cover the film with hydrophobic alkyl chains to lower its surface energy. The large amount of ODA and SDS surfactants loaded in the superhydrophobic PSS-ODA/PAH-SDS films and the autonomic migration of these surfactants to the film surface endow the resultant superhydrophobic films with an excellent self-healing ability to restore the damaged superhydrophobicity. The self-healing superhydrophobic PSS-ODA/PAH-SDS films are mechanically robust and can be deposited on various flat and nonflat substrates. The LbL assembly of oppositely charged polyelectrolyte-surfactant complexes provides a new way for the fabrication of fluorine-free self-healing superhydrophobic films with satisfactory mechanical stability, enhanced reliability, and extended service life.

  3. Synthesis, NMR characterization, X-ray crystal structure of Co(II) Ni(II) and Cu(II) complexes of a pyridine containing self-assembling

    International Nuclear Information System (INIS)

    Ranjbar, M.; Taghavipour, M.; Moghimi, A.; Aghabozorg, H.

    2002-01-01

    In the recent years, the self-assembling systems have been attracted chemists. The intermolecular bond in such systems mainly consists of ion pairing and hydrogen bonding [1,2]. The reaction between self-assembling system liquid LH 2 (py dc=2,6-pyridinedicarboxylic acid and py da=2,6- pyridine diamin) with cobalt (II) nitrate, nickel (II) chloride, and copper (II) acetate in water leads to the formation of self- assemble coordination complexes, [py da.H] 2 [M(py dc) 2 ]. H 2 O, M=Co(II),Ni(II), and Cu(II). The characterization was performed using elemental analysis, ESI mass spectroscopy, 1 H and 13 C NMR and X-ray crystallography. The crystal systems are monoclinic with space group P2 1 /n and four molecules per unit cell. These complexes shows 13 C NMR resonances of cationic counter ion [(py dc,H)] + in DMSO- d 6 but no signal corresponding to the two coordinated ligands [py dc] 2- The metal atoms are six-coordinated with a distorted octahedral geometry. The two [py de] 2- units are almost perpendicular to each other

  4. Self-assembly and energy transfer in artificial light-harvesting complexes of bacteriochlorophyll c with astaxanthin

    Czech Academy of Sciences Publication Activity Database

    Alster, J.; Polívka, Tomáš; Arellano, J.B.; Hříbek, P.; Vácha, František; Hala, J.; Pšenčík, J.

    2012-01-01

    Roč. 111, 1-2 (2012), s. 193-204 ISSN 0166-8595 R&D Projects: GA ČR GA206/09/0375 Institutional research plan: CEZ:AV0Z50510513 Keywords : light- harvesting * astaxanthin * self-assembly * bacteriochlorophyll aggregates Subject RIV: BO - Biophysics Impact factor: 3.150, year: 2012

  5. Friedreich's Ataxia Variants I154F and W155R Diminish Frataxin-Based Activation of the Iron-Sulfur Cluster Assembly Complex

    Energy Technology Data Exchange (ETDEWEB)

    Tsai, Chi-Lin; Bridwell-Rabb, Jennifer; Barondeau, David P

    2011-11-07

    Friedreich's ataxia (FRDA) is a progressive neurodegenerative disease that has been linked to defects in the protein frataxin (Fxn). Most FRDA patients have a GAA expansion in the first intron of their Fxn gene that decreases protein expression. Some FRDA patients have a GAA expansion on one allele and a missense mutation on the other allele. Few functional details are known for the ~15 different missense mutations identified in FRDA patients. Here in vitro evidence is presented that indicates the FRDA I154F and W155R variants bind more weakly to the complex of Nfs1, Isd11, and Isu2 and thereby are defective in forming the four-component SDUF complex that constitutes the core of the Fe-S cluster assembly machine. The binding affinities follow the trend Fxn ~ I154F > W155F > W155A ~ W155R. The Fxn variants also have diminished ability to function as part of the SDUF complex to stimulate the cysteine desulfurase reaction and facilitate Fe-S cluster assembly. Four crystal structures, including the first for a FRDA variant, reveal specific rearrangements associated with the loss of function and lead to a model for Fxn-based activation of the Fe-S cluster assembly complex. Importantly, the weaker binding and lower activity for FRDA variants correlate with the severity of disease progression. Together, these results suggest that Fxn facilitates sulfur transfer from Nfs1 to Isu2 and that these in vitro assays are sensitive and appropriate for deciphering functional defects and mechanistic details for human Fe-S cluster biosynthesis.

  6. 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

  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...... 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....

  8. Viral capsid assembly as a model for protein aggregation diseases: Active processes catalyzed by cellular assembly machines comprising novel drug targets.

    Science.gov (United States)

    Marreiros, Rita; Müller-Schiffmann, Andreas; Bader, Verian; Selvarajah, Suganya; Dey, Debendranath; Lingappa, Vishwanath R; Korth, Carsten

    2015-09-02

    Viruses can be conceptualized as self-replicating multiprotein assemblies, containing coding nucleic acids. Viruses have evolved to exploit host cellular components including enzymes to ensure their replicative life cycle. New findings indicate that also viral capsid proteins recruit host factors to accelerate their assembly. These assembly machines are RNA-containing multiprotein complexes whose composition is governed by allosteric sites. In the event of viral infection, the assembly machines are recruited to support the virus over the host and are modified to achieve that goal. Stress granules and processing bodies may represent collections of such assembly machines, readily visible by microscopy but biochemically labile and difficult to isolate by fractionation. We hypothesize that the assembly of protein multimers such as encountered in neurodegenerative or other protein conformational diseases, is also catalyzed by assembly machines. In the case of viral infection, the assembly machines have been modified by the virus to meet the virus' need for rapid capsid assembly rather than host homeostasis. In the case of the neurodegenerative diseases, it is the monomers and/or low n oligomers of the so-called aggregated proteins that are substrates of assembly machines. Examples for substrates are amyloid β peptide (Aβ) and tau in Alzheimer's disease, α-synuclein in Parkinson's disease, prions in the prion diseases, Disrupted-in-schizophrenia 1 (DISC1) in subsets of chronic mental illnesses, and others. A likely continuum between virus capsid assembly and cell-to-cell transmissibility of aggregated proteins is remarkable. Protein aggregation diseases may represent dysfunction and dysregulation of these assembly machines analogous to the aberrations induced by viral infection in which cellular homeostasis is pathologically reprogrammed. In this view, as for viral infection, reset of assembly machines to normal homeostasis should be the goal of protein aggregation

  9. Structure-Function Analysis of Friedreich's Ataxia Mutants Reveals Determinants of Frataxin Binding and Activation of the Fe-S Assembly Complex

    Energy Technology Data Exchange (ETDEWEB)

    Bridwell-Rabb, Jennifer; Winn, Andrew M; Barondeau, David P [TAM

    2012-08-01

    Friedreich's ataxia (FRDA) is a progressive neurodegenerative disease associated with the loss of function of the protein frataxin (FXN) that results from low FXN levels due to a GAA triplet repeat expansion or, occasionally, from missense mutations in the FXN gene. Here biochemical and structural properties of FXN variants, including three FRDA missense mutations (N146K, Q148R, and R165C) and three related mutants (N146A, Q148G, and Q153A), were determined in an effort to understand the structural basis for the loss of function. In vitro assays revealed that although the three FRDA missense mutations exhibited similar losses of cysteine desulfurase and Fe-S cluster assembly activities, the causes for these activation defects were distinct. The R165C variant exhibited a kcat/KM higher than that of native FXN but weak binding to the NFS1, ISD11, and ISCU2 (SDU) complex, whereas the Q148R variant exhibited the lowest kcat/KM of the six tested FXN variants and only a modest binding deficiency. The order of the FXN binding affinities for the SDU Fe-S assembly complex was as follows: FXN > Q148R > N146A > Q148G > N146K > Q153A > R165C. Four different classes of FXN variants were identified on the basis of their biochemical properties. Together, these structure-function studies reveal determinants for the binding and allosteric activation of the Fe-S assembly complex and provide insight into how FRDA missense mutations are functionally compromised.

  10. Rolling cycle amplification based single-color quantum dots–ruthenium complex assembling dyads for homogeneous and highly selective detection of DNA

    Energy Technology Data Exchange (ETDEWEB)

    Su, Chen; Liu, Yufei; Ye, Tai; Xiang, Xia; Ji, Xinghu; He, Zhike, E-mail: zhkhe@whu.edu.cn

    2015-01-01

    Graphical abstract: A universal, label-free, homogeneous, highly sensitive, and selective fluorescent biosensor for DNA detection is developed by using rolling-circle amplification (RCA) based single-color quantum dots–ruthenium complex (QDs–Ru) assembling dyads. - Highlights: • The single-color QDs–Ru assembling dyads were applied in homogeneous DNA assay. • This biosensor exhibited high selectivity against base mismatched sequences. • This biosensor could be severed as universal platform for the detection of ssDNA. • This sensor could be used to detect the target in human serum samples. • This DNA sensor had a good selectivity under the interference of other dsDNA. - Abstract: In this work, a new, label-free, homogeneous, highly sensitive, and selective fluorescent biosensor for DNA detection is developed by using rolling-circle amplification (RCA) based single-color quantum dots–ruthenium complex (QDs–Ru) assembling dyads. This strategy includes three steps: (1) the target DNA initiates RCA reaction and generates linear RCA products; (2) the complementary DNA hybridizes with the RCA products to form long double-strand DNA (dsDNA); (3) [Ru(phen){sub 2}(dppx)]{sup 2+} (dppx = 7,8-dimethyldipyrido [3,2-a:2′,3′-c] phenanthroline) intercalates into the long dsDNA with strong fluorescence emission. Due to its strong binding propensity with the long dsDNA, [Ru(phen){sub 2}(dppx)]{sup 2+} is removed from the surface of the QDs, resulting in restoring the fluorescence of the QDs, which has been quenched by [Ru(phen){sub 2}(dppx)]{sup 2+} through a photoinduced electron transfer process and is overlaid with the fluorescence of dsDNA bonded Ru(II) polypyridyl complex (Ru-dsDNA). Thus, high fluorescence intensity is observed, and is related to the concentration of target. This sensor exhibits not only high sensitivity for hepatitis B virus (HBV) ssDNA with a low detection limit (0.5 pM), but also excellent selectivity in the complex matrix. Moreover

  11. A yeast two-hybrid screen for SYP-3 interactors identifies SYP-4, a component required for synaptonemal complex assembly and chiasma formation in Caenorhabditis elegans meiosis.

    Directory of Open Access Journals (Sweden)

    Sarit Smolikov

    2009-10-01

    Full Text Available The proper assembly of the synaptonemal complex (SC between homologs is critical to ensure accurate meiotic chromosome segregation. The SC is a meiotic tripartite structure present from yeast to humans, comprised of proteins assembled along the axes of the chromosomes and central region (CR proteins that bridge the two chromosome axes. Here we identify SYP-4 as a novel structural component of the SC in Caenorhabditis elegans. SYP-4 interacts in a yeast two-hybrid assay with SYP-3, one of components of the CR of the SC, and is localized at the interface between homologs during meiosis. SYP-4 is essential for the localization of SYP-1, SYP-2, and SYP-3 CR proteins onto chromosomes, thereby playing a crucial role in the stabilization of pairing interactions between homologous chromosomes. In the absence of SYP-4, the levels of recombination intermediates, as indicated by RAD-51 foci, are elevated in mid-prophase nuclei, and crossover recombination events are significantly reduced. The lack of chiasmata observed in syp-4 mutants supports the elevated levels of chromosome nondisjunction manifested in high embryonic lethality. Altogether our findings place SYP-4 as a central player in SC formation and broaden our understanding of the structure of the SC and its assembly.

  12. NSF- and SNARE-mediated membrane fusion is required for nuclear envelope formation and completion of nuclear pore complex assembly in Xenopus laevis egg extracts.

    Science.gov (United States)

    Baur, Tina; Ramadan, Kristijan; Schlundt, Andreas; Kartenbeck, Jürgen; Meyer, Hemmo H

    2007-08-15

    Despite the progress in understanding nuclear envelope (NE) reformation after mitosis, it has remained unclear what drives the required membrane fusion and how exactly this is coordinated with nuclear pore complex (NPC) assembly. Here, we show that, like other intracellular fusion reactions, NE fusion in Xenopus laevis egg extracts is mediated by SNARE proteins that require activation by NSF. Antibodies against Xenopus NSF, depletion of NSF or the dominant-negative NSF(E329Q) variant specifically inhibited NE formation. Staging experiments further revealed that NSF was required until sealing of the envelope was completed. Moreover, excess exogenous alpha-SNAP that blocks SNARE function prevented membrane fusion and caused accumulation of non-flattened vesicles on the chromatin surface. Under these conditions, the nucleoporins Nup107 and gp210 were fully recruited, whereas assembly of FxFG-repeat-containing nucleoporins was blocked. Together, we define NSF- and SNARE-mediated membrane fusion events as essential steps during NE formation downstream of Nup107 recruitment, and upstream of membrane flattening and completion of NPC assembly.

  13. 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.

  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. Dynein light chain 1 induces assembly of large Bim complexes on mitochondria that stabilize Mcl-1 and regulate apoptosis.

    Science.gov (United States)

    Singh, Prafull Kumar; Roukounakis, Aristomenis; Frank, Daniel O; Kirschnek, Susanne; Das, Kushal Kumar; Neumann, Simon; Madl, Josef; Römer, Winfried; Zorzin, Carina; Borner, Christoph; Haimovici, Aladin; Garcia-Saez, Ana; Weber, Arnim; Häcker, Georg

    2017-09-01

    The Bcl-2 family protein Bim triggers mitochondrial apoptosis. Bim is expressed in nonapoptotic cells at the mitochondrial outer membrane, where it is activated by largely unknown mechanisms. We found that Bim is regulated by formation of large protein complexes containing dynein light chain 1 (DLC1). Bim rapidly inserted into cardiolipin-containing membranes in vitro and recruited DLC1 to the membrane. Bim binding to DLC1 induced the formation of large Bim complexes on lipid vesicles, on isolated mitochondria, and in intact cells. Native gel electrophoresis and gel filtration showed Bim-containing mitochondrial complexes of several hundred kilodaltons in all cells tested. Bim unable to form complexes was consistently more active than complexed Bim, which correlated with its substantially reduced binding to anti-apoptotic Bcl-2 proteins. At endogenous levels, Bim surprisingly bound only anti-apoptotic Mcl-1 but not Bcl-2 or Bcl-X L , recruiting only Mcl-1 into large complexes. Targeting of DLC1 by RNAi in human cell lines induced disassembly of Bim-Mcl-1 complexes and the proteasomal degradation of Mcl-1 and sensitized the cells to the Bcl-2/Bcl-X L inhibitor ABT-737. Regulation of apoptosis at mitochondria thus extends beyond the interaction of monomers of proapoptotic and anti-apoptotic Bcl-2 family members but involves more complex structures of proteins at the mitochondrial outer membrane, and targeting complexes may be a novel therapeutic strategy. © 2017 Singh et al.; Published by Cold Spring Harbor Laboratory Press.

  16. Herpes simplex virus 1 DNA is in unstable nucleosomes throughout the lytic infection cycle, and the instability of the nucleosomes is independent of DNA replication.

    Science.gov (United States)

    Lacasse, Jonathan J; Schang, Luis M

    2012-10-01

    Herpes simplex virus 1 (HSV-1) DNA is chromatinized during latency and consequently regularly digested by micrococcal nuclease (MCN) to nucleosome-size fragments. In contrast, MCN digests HSV-1 DNA in lytically infected cells to mostly heterogeneous sizes. Yet HSV-1 DNA coimmunoprecipitates with histones during lytic infections. We have shown that at 5 h postinfection, most nuclear HSV-1 DNA is in particularly unstable nucleoprotein complexes and consequently is more accessible to MCN than DNA in cellular chromatin. HSV-1 DNA was quantitatively recovered at this time in complexes with the biophysical properties of mono- to polynucleosomes following a modified MCN digestion developed to detect potential unstable intermediates. We proposed that most HSV-1 DNA is in unstable nucleosome-like complexes during lytic infections. Physiologically, nucleosome assembly typically associates with DNA replication, although DNA replication transiently disrupts nucleosomes. It therefore remained unclear whether the instability of the HSV-1 nucleoprotein complexes was related to the ongoing viral DNA replication. Here we tested whether HSV-1 DNA is in unstable nucleosome-like complexes before, during, or after the peak of viral DNA replication or when HSV-1 DNA replication is inhibited. HSV-1 DNA was quantitatively recovered in complexes fractionating as mono- to polynucleosomes from nuclei harvested at 2, 5, 7, or 9 h after infection, even if viral DNA replication was inhibited. Therefore, most HSV-1 DNA is in unstable nucleosome-like complexes throughout the lytic replication cycle, and the instability of these complexes is surprisingly independent of HSV-1 DNA replication. The specific accessibility of nuclear HSV-1 DNA, however, varied at different times after infection.

  17. Core-satellites assembly of silver nanoparticles on a single gold nanoparticle via metal ion-mediated complex.

    Science.gov (United States)

    Choi, Inhee; Song, Hyeon Don; Lee, Suseung; Yang, Young In; Kang, Taewook; Yi, Jongheop

    2012-07-25

    We report core-satellites (Au-Ag) coupled plasmonic nanoassemblies based on bottom-up, high-density assembly of molecular-scale silver nanoparticles on a single gold nanoparticle surface, and demonstrate direct observation and quantification of enhanced plasmon coupling (i.e., intensity amplification and apparent spectra shift) in a single particle level. We also explore metal ion sensing capability based on our coupled plasmonic core-satellites, which enabled at least 1000 times better detection limit as compared to that of a single plasmonic nanoparticle. Our results demonstrate and suggest substantial promise for the development of coupled plasmonic nanostructures for ultrasensitive detection of various biological and chemical analytes.

  18. Discovery of a Chllorophyll Binding Protein Complex Involved in the Early Steps of Photosystem II Assembly in Synechocystis

    Czech Academy of Sciences Publication Activity Database

    Knoppová, Jana; Sobotka, Roman; Tichý, Martin; Jianfeng, Yu; Koník, P.; Halada, Petr; Nixon, P. J.; Komenda, Josef

    2014-01-01

    Roč. 26, č. 4 (2014), s. 1200-1212 ISSN 1040-4651 R&D Projects: GA ČR P501/11/0377; GA MŠk ED2.1.00/03.0110 Grant - others:UK Biotechnology and Biological Sciences Research Council(GB) BB/F020554/1; UK Biotechnology and Biological Sciences Research Council(GB) BB/L003260/1; Magistrát hl. m. Prahy(CZ) CZ.2.16/3.1.00/24023 Institutional support: RVO:61388971 Keywords : Synechocystis * photosystem II * assembly * proteins Subject RIV: EE - Microbiology, Virology Impact factor: 9.338, year: 2014

  19. GABenchToB: a genome assembly benchmark tuned on bacteria and benchtop sequencers.

    Directory of Open Access Journals (Sweden)

    Sebastian Jünemann

    Full Text Available De novo genome assembly is the process of reconstructing a complete genomic sequence from countless small sequencing reads. Due to the complexity of this task, numerous genome assemblers have been developed to cope with different requirements and the different kinds of data provided by sequencers within the fast evolving field of next-generation sequencing technologies. In particular, the recently introduced generation of benchtop sequencers, like Illumina's MiSeq and Ion Torrent's Personal Genome Machine (PGM, popularized the easy, fast, and cheap sequencing of bacterial organisms to a broad range of academic and clinical institutions. With a strong pragmatic focus, here, we give a novel insight into the line of assembly evaluation surveys as we benchmark popular de novo genome assemblers based on bacterial data generated by benchtop sequencers. Therefore, single-library assemblies were generated, assembled, and compared to each other by metrics describing assembly contiguity and accuracy, and also by practice-oriented criteria as for instance computing time. In addition, we extensively analyzed the effect of the depth of coverage on the genome assemblies within reasonable ranges and the k-mer optimization problem of de Bruijn Graph assemblers. Our results show that, although both MiSeq and PGM allow for good genome assemblies, they require different approaches. They not only pair with different assembler types, but also affect assemblies differently regarding the depth of coverage where oversampling can become problematic. Assemblies vary greatly with respect to contiguity and accuracy but also by the requirement on the computing power. Consequently, no assembler can be rated best for all preconditions. Instead, the given kind of data, the demands on assembly quality, and the available computing infrastructure determines which assembler suits best. The data sets, scripts and all additional information needed to replicate our results are freely

  20. Functionalisation of bolaamphiphiles with mononuclear bis(2,2 '-bipyridyl)ruthenium(II) complexes for application in self assembled monolayers

    NARCIS (Netherlands)

    Killeen, JS; Browne, WR; Skupin, M; Fuhrhop, JH; Vos, JG

    2003-01-01

    A novel ruthenium(II) polypyridyl complex connected covalently to a bolaamphiphile, containing amide linkages to provide rigidity via hydrogen bonding in the monolayer, has been prepared. The ruthenium( II) complexes of this ligand and of the intermediates in the synthesis were prepared by modi.

  1. Electrochemical and surface plasmon resonance characterization of β-cyclodextrin-based self-assembled monolayers and evaluation of their inclusion complexes with glucocorticoids

    Science.gov (United States)

    Frasconi, Marco; Mazzei, Franco

    2009-07-01

    This paper describes the characterization of a self-assembled β-cyclodextrin (β-CD)-derivative monolayer (β-CD-SAM) on a gold surface and the study of their inclusion complexes with glucocorticoids. To this aim the arrangement of a self-assembled β-cyclodextrin-derivative monolayer on a gold surface was monitored in situ by means of surface plasmon resonance (SPR) spectroscopy and double-layer capacitance measurements. Film thickness and dielectric constant were evaluated for a monolayer of β-CD using one-color-approach SPR. The selectivity of the β-CD host surface was verified by using electroactive species permeable and impermeable in the β-CD cavity. The redox probe was selected according to its capacity to permeate the β-CD monolayer and its electrochemical behavior. In order to evaluate the feasibility of an inclusion complex between β-CD-SAM with some steroids such as cortisol and cortisone, voltammetric experiments in the presence of the redox probes as molecules competitive with the steroids have been performed. The formation constant of the surface host-guest by β-CD-SAM and the steroids under study was calculated.

  2. Role of the ATPase/helicase maleless (MLE in the assembly, targeting, spreading and function of the male-specific lethal (MSL complex of Drosophila

    Directory of Open Access Journals (Sweden)

    Morra Rosa

    2011-04-01

    other MSL proteins in the cytoplasm. These data suggest that the MSL proteins assemble into complexes or subcomplexes before entering the nucleus. Conclusions This study provides insights into the role that MLE plays in the function of the MSL complex through its association with roX RNAs and the other MSL subunits, and suggests a hypothesis to explain the role of MLE in the synthesis of these RNAs.

  3. Unique Roles of the Non-identical MCM Subunits in DNA Replication Licensing.

    Science.gov (United States)

    Zhai, Yuanliang; Li, Ningning; Jiang, Hanlun; Huang, Xuhui; Gao, Ning; Tye, Bik Kwoon

    2017-07-20

    A family of six homologous subunits, Mcm2, -3, -4, -5, -6, and -7, each with its own unique features, forms the catalytic core of the eukaryotic replicative helicase. The necessity of six similar but non-identical subunits has been a mystery since its initial discovery. Recent cryo-EM structures of the Mcm2-7 (MCM) double hexamer, its precursors, and the origin recognition complex (ORC)-Cdc6-Cdt1-Mcm2-7 (OCCM) intermediate showed that each of these subunits plays a distinct role in orchestrating the assembly of the pre-replication complex (pre-RC) by ORC-Cdc6 and Cdt1. Copyright © 2017 Elsevier Inc. All rights reserved.

  4. Delayed vaccine virus replication in chickens vaccinated subcutaneously with an immune complex infectious bursal disease vaccine: Quantification of vaccine virus by real-time polymerase chain reaction

    OpenAIRE

    Iván, Judit; Velhner, Maja; Ursu, Krisztina; Germán, Péter; Mató, Tamás; Drén, Csaba Nick; Mészáros, János

    2005-01-01

    The distribution of the immune complex vaccine virus for infectious bursal disease (IBD) in tissue was examined and the viral loads of the organs were quantitatively compared. One-day-old specific pathogen free (SPF) and maternally immune broiler chickens were injected subcutaneously with the vaccine. Lymphoid and non-lymphoid tissues were collected at various time intervals during the experiment to test for infectious bursal disease virus (IBDV)-RNA by using reverse transcriptase-polymerase ...

  5. Complex assembly, crystallization and preliminary X-ray crystallographic studies of rhesus macaque MHC Mamu-A*01 complexed with an immunodominant SIV-Gag nonapeptide

    International Nuclear Information System (INIS)

    Chu, Fuliang; Lou, Zhiyong; Gao, Bin; Bell, John I.; Rao, Zihe; Gao, George F.

    2005-01-01

    Crystallization of the first rhesus macaque MHC class I complex. Simian immunodeficiency virus (SIV) infection in rhesus macaques has been used as the best model for the study of human immunodeficiency virus (HIV) infection in humans, especially in the cytotoxic T-lymphocyte (CTL) response. However, the structure of rhesus macaque (or any other monkey model) major histocompatibility complex class I (MHC I) presenting a specific peptide (the ligand for CTL) has not yet been elucidated. Here, using in vitro refolding, the preparation of the complex of the rhesus macaque MHC I allele (Mamu-A*01) with human β 2 m and an immunodominant peptide, CTPYDINQM (Gag-CM9), derived from SIV Gag protein is reported. The complex (45 kDa) was crystallized; the crystal belongs to space group I422, with unit-cell parameters a = b = 183.8, c = 155.2 Å. The crystal contains two molecules in the asymmetric unit and diffracts X-rays to 2.8 Å resolution. The structure is being solved by molecular replacement and this is the first attempt to determined the crystal structure of a peptide–nonhuman primate MHC complex

  6. Chromatin challenges during DNA replication and repair

    DEFF Research Database (Denmark)

    Groth, Anja; Rocha, Walter; Verreault, Alain

    2007-01-01

    Inheritance and maintenance of the DNA sequence and its organization into chromatin are central for eukaryotic life. To orchestrate DNA-replication and -repair processes in the context of chromatin is a challenge, both in terms of accessibility and maintenance of chromatin organization. To meet...... the challenge of maintenance, cells have evolved efficient nucleosome-assembly pathways and chromatin-maturation mechanisms that reproduce chromatin organization in the wake of DNA replication and repair. The aim of this Review is to describe how these pathways operate and to highlight how the epigenetic...... landscape may be stably maintained even in the face of dramatic changes in chromatin structure....

  7. β-Cyclodextrin- and adamantyl-substituted poly(acrylate self-assembling aqueous networks designed for controlled complexation and release of small molecules

    Directory of Open Access Journals (Sweden)

    Liang Yan

    2017-09-01

    Full Text Available Three aqueous self-assembling poly(acrylate networks have been designed to gain insight into the factors controlling the complexation and release of small molecules within them. These networks are formed between 8.8% 6A-(2-aminoethylamino-6A-deoxy-6A-β-cyclodextrin, β-CDen, randomly substituted poly(acrylate, PAAβ-CDen, and one of the 3.3% 1-(2-aminoethylamidoadamantyl, ADen, 3.0% 1-(6-aminohexylamidoadamantyl, ADhn, or 2.9% 1-(12-aminododecylamidoadamantyl, ADddn, randomly substituted poly(acrylates, PAAADen, PAAADhn and PAAADddn, respectively, such that the ratio of β-CDen to adamantyl substituents is ca. 3:1. The variation of the characteristics of the complexation of the dyes methyl red, methyl orange and ethyl orange in these three networks and by β-cyclodextrin, β-CD, and PAAβ-CDen alone provides insight into the factors affecting dye complexation. The rates of release of the dyes through a dialysis membrane from the three aqueous networks show a high dependence on host–guest complexation between the β-CDen substituents and the dyes as well as the structure and the viscosity of the network as shown by ITC, 1H NMR and UV–vis spectroscopy, and rheological studies. Such networks potentially form a basis for the design of controlled drug release systems.

  8. Inorganic self-assembly through sequential complexation in the formation of bimetallic and trimetallic architectures from multisite ligands based on 5,5'-disubstituted 2,2'-bipyridines

    Directory of Open Access Journals (Sweden)

    Machado Vanderlei G.

    2003-01-01

    Full Text Available Two multisite 5,5'-disubstituted 2,2'-bipyridine ligands containing N-methyl hydroxamic acids as substituents [5,5'-bis(N-methylhydroxamic-2,2'-bipyridine (4 and 5-methyl-5'-(N-methylhydroxamic-2,2-bipyridine (10] were synthesized. These ligands were used in order to illustrate the strategy of self-assembly through sequential complexation. According to this concept, the first metal added organizes the ligands disposed in 5,5'-positions to accomodate the second metal ion that is sequentially added. Thus, addition of Fe2+ to a solution of 4 led to a Fe2+-tris(bipyridine complex. Addition of Fe3+ to this solution yielded a trimetallic architecture, which was characterized. Ligand 10 yielded a mixture of bimetallic architectures through complexation with Fe2+ followed by Fe3+ ions. However, if the order of metal addition is changed, only one bimetallic complex is obtained. This is due to the fact that the first metal ion added (Fe3+ acts as a template, organizing the bipyridine ligands and preforming an adequate cavity for the Fe2+ ion.

  9. Arp2/3 complex ATP hydrolysis promotes lamellipodial actin network disassembly but is dispensable for assembly

    Science.gov (United States)

    Ingerman, Elena; Hsiao, Jennifer Ying

    2013-01-01

    We examined the role of ATP hydrolysis by the Arp2/3 complex in building the leading edge of a cell by studying the effects of hydrolysis defects on the behavior of the complex in the lamellipodial actin network of Drosophila S2 cells and in a reconstituted, in vitro, actin-based motility system. In S2 cells, nonhydrolyzing Arp2 and Arp3 subunits expanded and delayed disassembly of lamellipodial actin networks and the effect of mutant subunits was additive. Arp2 and Arp3 ATP hydrolysis mutants remained in lamellipodial networks longer and traveled greater distances from the plasma membrane, even in networks still containing wild-type Arp2/3 complex. In vitro, wild-type and ATP hydrolysis mutant Arp2/3 complexes each nucleated actin and built similar dendritic networks. However, networks constructed with Arp2/3 hydrolysis-defective mutants were more resistant to disassembly by cofilin. Our results indicate that ATP hydrolysis on both Arp2 and Arp3 contributes to dissociation of the complex from the actin network but is not strictly necessary for lamellipodial network disassembly. PMID:23439681

  10. MLL/WDR5 Complex Regulates Kif2A Localization to Ensure Chromosome Congression and Proper Spindle Assembly during Mitosis.

    Science.gov (United States)

    Ali, Aamir; Veeranki, Sailaja Naga; Chinchole, Akash; Tyagi, Shweta

    2017-06-19

    Mixed-lineage leukemia (MLL), along with multisubunit (WDR5, RbBP5, ASH2L, and DPY30) complex catalyzes the trimethylation of H3K4, leading to gene activation. Here, we characterize a chromatin-independent role for MLL during mitosis. MLL and WDR5 localize to the mitotic spindle apparatus, and loss of function of MLL complex by RNAi results in defects in chromosome congression and compromised spindle formation. We report interaction of MLL complex with several kinesin and dynein motors. We further show that the MLL complex associates with Kif2A, a member of the Kinesin-13 family of microtubule depolymerase, and regulates the spindle localization of Kif2A during mitosis. We have identified a conserved WDR5 interaction (Win) motif, so far unique to the MLL family, in Kif2A. The Win motif of Kif2A engages in direct interactions with WDR5 for its spindle localization. Our findings highlight a non-canonical mitotic function of MLL complex, which may have a direct impact on chromosomal stability, frequently compromised in cancer. Copyright © 2017 Elsevier Inc. All rights reserved.

  11. Virus-sized self-assembling lamellar complexes between plasmid DNA and cationic micelles promote gene transfer

    Science.gov (United States)

    Pitard, Bruno; Aguerre, Olivier; Airiau, Marc; Lachagès, Anne-Marie; Boukhnikachvili, Tsiala; Byk, Gérardo; Dubertret, Catherine; Herviou, Christian; Scherman, Daniel; Mayaux, Jean-François; Crouzet, Joël

    1997-01-01

    Gene therapy is based on the vectorization of genes to target cells and their subsequent expression. Cationic amphiphile-mediated delivery of plasmid DNA is the nonviral gene transfer method most often used. We examined the supramolecular structure of lipopolyamine/plasmid DNA complexes under various condensing conditions. Plasmid DNA complexation with lipopolyamine micelles whose mean diameter was 5 nm revealed three domains, depending on the lipopolyamine/plasmid DNA ratio. These domains respectively corresponded to negatively, neutrally, and positively charged complexes. Transmission electron microscopy and x-ray scattering experiments on complexes originating from these three domains showed that although their morphology depends on the lipopolyamine/plasmid DNA ratio, their particle structure consists of ordered domains characterized by even spacing of 80 Å, irrespective of the lipid/DNA ratio. The most active lipopolyamine/DNA complexes for gene transfer were positively charged. They were characterized by fully condensed DNA inside spherical particles (diameter: 50 nm) sandwiched between lipid bilayers. These results show that supercoiled plasmid DNA is able to transform lipopolyamine micelles into a supramolecular organization characterized by ordered lamellar domains. PMID:9405626

  12. 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.

  13. 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.

  14. DNA replication in pathogens: Unique properties and possible ...

    Indian Academy of Sciences (India)

    asd

    Eukaryotic DNA Replication Initiation and Elongation. 1. 2. 3 5. 4. 6. ARS. Origin Recognition. Complex (ORC). MCM2-7. CDC6. 1. 2. 3 5. 4. ARS. Replication Initiation. ATP Hydrolysis? Yeast. 6. 2. 2. Replication Elongation. Is ORC function and cell cycle regulation is conserved in. Plasmodium falciparum?

  15. Hepatitis C virus translation preferentially depends on active RNA replication.

    Directory of Open Access Journals (Sweden)

    Helene Minyi Liu

    Full Text Available Hepatitis C virus (HCV RNA initiates its replication on a detergent-resistant membrane structure derived from the endoplasmic reticulum (ER in the HCV replicon cells. By performing a pulse-chase study of BrU-labeled HCV RNA, we found that the newly-synthesized HCV RNA traveled along the anterograde-membrane traffic and moved away from the ER. Presumably, the RNA moved to the site of translation or virion assembly in the later steps of viral life cycle. In this study, we further addressed how HCV RNA translation was regulated by HCV RNA trafficking. When the movement of HCV RNA from the site of RNA synthesis to the Golgi complex was blocked by nocodazole, an inhibitor of ER-Golgi transport, HCV protein translation was surprisingly enhanced, suggesting that the translation of viral proteins occurred near the site of RNA synthesis. We also found that the translation of HCV proteins was dependent on active RNA synthesis: inhibition of viral RNA synthesis by an NS5B inhibitor resulted in decreased HCV viral protein synthesis even when the total amount of intracellular HCV RNA remained unchanged. Furthermore, the translation activity of the replication-defective HCV replicons or viral RNA with an NS5B mutation was greatly reduced as compared to that of the corresponding wildtype RNA. By performing live cell labeling of newly synthesized HCV RNA and proteins, we further showed that the newly synthesized HCV proteins colocalized with the newly synthesized viral RNA, suggesting that HCV RNA replication and protein translation take place at or near the same site. Our findings together indicate that the translation of HCV RNA is coupled to RNA replication and that the both processes may occur at the same subcellular membrane compartments, which we term the replicasome.

  16. Assembly factors as a new class of disease genes for mitochondrial complex I deficiency: cause, pathology and treatment options.

    NARCIS (Netherlands)

    Nouws, J.; Nijtmans, L.G.J.; Smeitink, J.A.M.; Vogel, R.O.

    2012-01-01

    Complex I deficiency is the most frequent cause of oxidative phosphorylation disorders. The disease features a large diversity of clinical symptoms often leading to progressive encephalomyopathies with a fatal outcome. There is currently no cure, and although disease-causing mutations have been

  17. Structure function studies on the lipoate-acetyltransferase--component-X-core assembly of the ox heart pyruvate dehydrogenase complex.

    Science.gov (United States)

    Hodgson, J A; De Marcucci, O G; Lindsay, J G

    1988-02-01

    Component X, the recently recognised subunit of mammalian pyruvate dehydrogenase complex, was shown by immune blotting to be present in all of nine tissues dissected from rat. This finding indicated that component X was not an isoenzyme of the lipoate acetyltransferase (E2) associated with one or a limited number of tissues. Native pyruvate dehydrogenase complex was shown to bind IgG raised to isolated component X, indicating that there were at least some regions of the X subunit exposed at the periphery of the complex. Lipoyl groups of ox heart pyruvate dehydrogenase complex were specifically cross-linked by reaction with phenylene-o-bismaleimide in the presence of pyruvate and the subunits contributing to the products of cross-linking were identified by immune blotting. Species with very high Mr containing both E2 and component X, were formed in high yield, as well as apparent E2/E2 and E2/X dimers and trimers and an X/X dimer. These results showed that acetylated lipoyl groups of different E2 and X subunits were able to interact in all possible combinations. The types of cross-linked E2 products formed suggested that two thiols, reactible with phenylene-o-bismaleimide, were rapidly generated in the presence of pyruvate. The results were most easily explained by the presence of two acetylatable lipoyl groups on each E2 polypeptide.

  18. Assembly and Regulation of the Membrane Attack Complex Based on Structures of C5b6 and sC5b9

    Directory of Open Access Journals (Sweden)

    Michael A. Hadders

    2012-03-01

    Full Text Available Activation of the complement system results in formation of membrane attack complexes (MACs, pores that disrupt lipid bilayers and lyse bacteria and other pathogens. Here, we present the crystal structure of the first assembly intermediate, C5b6, together with a cryo-electron microscopy reconstruction of a soluble, regulated form of the pore, sC5b9. Cleavage of C5 to C5b results in marked conformational changes, distinct from those observed in the homologous C3-to-C3b transition. C6 captures this conformation, which is preserved in the larger sC5b9 assembly. Together with antibody labeling, these structures reveal that complement components associate through sideways alignment of the central MAC-perforin (MACPF domains, resulting in a C5b6-C7-C8β-C8α-C9 arc. Soluble regulatory proteins below the arc indicate a potential dual mechanism in protection from pore formation. These results provide a structural framework for understanding MAC pore formation and regulation, processes important for fighting infections and preventing complement-mediated tissue damage.

  19. CCDC39 is required for assembly of inner dynein arms and the dynein regulatory complex and for normal ciliary motility in humans and dogs.

    Science.gov (United States)

    Merveille, Anne-Christine; Davis, Erica E; Becker-Heck, Anita; Legendre, Marie; Amirav, Israel; Bataille, Géraldine; Belmont, John; Beydon, Nicole; Billen, Frédéric; Clément, Annick; Clercx, Cécile; Coste, André; Crosbie, Rachelle; de Blic, Jacques; Deleuze, Stephane; Duquesnoy, Philippe; Escalier, Denise; Escudier, Estelle; Fliegauf, Manfred; Horvath, Judith; Hill, Kent; Jorissen, Mark; Just, Jocelyne; Kispert, Andreas; Lathrop, Mark; Loges, Niki Tomas; Marthin, June K; Momozawa, Yukihide; Montantin, Guy; Nielsen, Kim G; Olbrich, Heike; Papon, Jean-François; Rayet, Isabelle; Roger, Gilles; Schmidts, Miriam; Tenreiro, Henrique; Towbin, Jeffrey A; Zelenika, Diana; Zentgraf, Hanswalter; Georges, Michel; Lequarré, Anne-Sophie; Katsanis, Nicholas; Omran, Heymut; Amselem, Serge

    2011-01-01

    Primary ciliary dyskinesia (PCD) is an inherited disorder characterized by recurrent infections of the upper and lower respiratory tract, reduced fertility in males and situs inversus in about 50% of affected individuals (Kartagener syndrome). It is caused by motility defects in the respiratory cilia that are responsible for airway clearance, the flagella that propel sperm cells and the nodal monocilia that determine left-right asymmetry. Recessive mutations that cause PCD have been identified in genes encoding components of the outer dynein arms, radial spokes and cytoplasmic pre-assembly factors of axonemal dyneins, but these mutations account for only about 50% of cases of PCD. We exploited the unique properties of dog populations to positionally clone a new PCD gene, CCDC39. We found that loss-of-function mutations in the human ortholog underlie a substantial fraction of PCD cases with axonemal disorganization and abnormal ciliary beating. Functional analyses indicated that CCDC39 localizes to ciliary axonemes and is essential for assembly of inner dynein arms and the dynein regulatory complex.

  20. Regulation of Rvb1/Rvb2 by a Domain within the INO80 Chromatin Remodeling Complex Implicates the Yeast Rvbs as Protein Assembly Chaperones

    Directory of Open Access Journals (Sweden)

    Coral Y. Zhou

    2017-06-01

    Full Text Available The hexameric AAA+ ATPases Rvb1 and Rvb2 (Rvbs are essential for diverse processes ranging from metabolic signaling to chromatin remodeling, but their functions are unknown. While originally thought to act as helicases, recent proposals suggest that Rvbs act as protein assembly chaperones. However, experimental evidence for chaperone-like behavior is lacking. Here, we identify a potent protein activator of the Rvbs, a domain in the Ino80 ATPase subunit of the INO80 chromatin-remodeling complex, termed Ino80INS. Ino80INS stimulates Rvbs’ ATPase activity by 16-fold while concomitantly promoting their dodecamerization. Using mass spectrometry, cryo-EM, and integrative modeling, we find that Ino80INS binds asymmetrically along the dodecamerization interface, resulting in a conformationally flexible dodecamer that collapses into hexamers upon ATP addition. Our results demonstrate the chaperone-like potential of Rvb1/Rvb2 and suggest a model where binding of multiple clients such as Ino80 stimulates ATP-driven cycling between hexamers and dodecamers, providing iterative opportunities for correct subunit assembly.

  1. Assembling Sustainable Territories

    DEFF Research Database (Denmark)

    Vandergeest, Peter; Ponte, Stefano; Bush, Simon

    2015-01-01

    The authors show how certification assembles ‘sustainable’ territories through a complex layering of regulatory authority in which both government and nongovernment entities claim rule-making authority, sometimes working together, sometimes in parallel, sometimes competitively. It is argued...

  2. Transcriptome analysis of complex I-deficient patients reveals distinct expression programs for subunits and assembly factors of the oxidative phosphorylation system.

    Science.gov (United States)

    van der Lee, Robin; Szklarczyk, Radek; Smeitink, Jan; Smeets, Hubert J M; Huynen, Martijn A; Vogel, Rutger

    2015-09-15

    Transcriptional control of mitochondrial metabolism is essential for cellular function. A better understanding of this process will aid the elucidation of mitochondrial disorders, in particular of the many genetically unsolved cases of oxidative phosphorylation (OXPHOS) deficiency. Yet, to date only few studies have investigated nuclear gene regulation in the context of OXPHOS deficiency. In this study we performed RNA sequencing of two control and two complex I-deficient patient cell lines cultured in the presence of compounds that perturb mitochondrial metabolism: chloramphenicol, AICAR, or resveratrol. We combined this with a comprehensive analysis of mitochondrial and nuclear gene expression patterns, co-expression calculations and transcription factor binding sites. Our analyses show that subsets of mitochondrial OXPHOS genes respond opposingly to chloramphenicol and AICAR, whereas the response of nuclear OXPHOS genes is less consistent between cell lines and treatments. Across all samples nuclear OXPHOS genes have a significantly higher co-expression with each other than with other genes, including those encoding mitochondrial proteins. We found no evidence for complex-specific mRNA expression regulation: subunits of different OXPHOS complexes are similarly (co-)expressed and regulated by a common set of transcription factors. However, we did observe significant differences between the expression of nuclear genes for OXPHOS subunits versus assembly factors, suggesting divergent transcription programs. Furthermore, complex I co-expression calculations identified 684 genes with a likely role in OXPHOS biogenesis and function. Analysis of evolutionarily conserved transcription factor binding sites in the promoters of these genes revealed almost all known OXPHOS regulators (including GABP, NRF1/2, SP1, YY1, E-box factors) and a set of novel candidates (ELK1, KLF7, SP4, EHF, ZNF143, and TEL2). OXPHOS genes share an expression program distinct from other genes

  3. Acadian variant of Fanconi syndrome is caused by mitochondrial respiratory chain complex I deficiency due to a non-coding mutation in complex I assembly factor NDUFAF6

    Czech Academy of Sciences Publication Activity Database

    Hartmannová, H.; Piherová, L.; Tauchmannová, Kateřina; Kidd, K.; Acott, P. D.; Crocker, J. F. S.; Oussedik, Y.; Mallet, M.; Hodaňová, K.; Stránecký, V.; Přistoupilová, A.; Barešová, V.; Jedličková, I.; Živná, M.; Sovová, J.; Hůlková, H.; Robins, V.; Vrbacký, Marek; Pecina, Petr; Kaplanová, Vilma; Houštěk, Josef; Mráček, Tomáš; Thibeault, Y.; Bleyer, A. J.; Kmoch, S.

    2016-01-01

    Roč. 25, č. 18 (2016), s. 4062-4079 ISSN 0964-6906 R&D Projects: GA ČR(CZ) GB14-36804G; GA MŠk(CZ) LL1204 Institutional support: RVO:67985823 Keywords : Acadian variant of Fanconi syndrome * mitochondrial complex I deficiency * NDUFAF6 * C8ORF38 * non-coding mutation * alternative splicing variant * protein isoforms Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 5.340, year: 2016

  4. Self-assembly in lanthanum(III) and calcium(II) complexes of salicylaldimines derived from putrescine

    Energy Technology Data Exchange (ETDEWEB)

    Pospieszna-Markiewicz, Izabela; Kaczmarek, Malgorzata T.; Kubicki, Maciej [Department of Bioinorganic Chemistry, Adam Mickiewicz University, 60-780 Poznan (Poland); Radecka-Paryzek, Wanda [Department of Bioinorganic Chemistry, Adam Mickiewicz University, 60-780 Poznan (Poland)], E-mail: wrp@amu.edu.pl

    2008-02-28

    The one-step template reaction of salicylaldehyde with putrescine, a biogenic diamine, in the presence of lanthanum(III) or calcium(II) ions produces salicylaldimine complexes containing the N,N'-bis(salicylidene)-1,4-butanediamine ligand L as a result of the [2 + 1] Schiff base condensation. The X-ray diffraction studies of the lanthanum complex reveals an infinite [La{sub 2}L{sub 4}(NO{sub 3}){sub 6}]{sub {infinity}} polymeric structure based on networks of 10-coordinated La(III) nodes linked by bridging unionized ligands that use exclusively the oxygens as donor atoms with the nitrogen atoms not being involved in coordination.

  5. Rac1 augments Wnt signaling by stimulating β-catenin–lymphoid enhancer factor-1 complex assembly independent of β-catenin nuclear import

    Science.gov (United States)

    Jamieson, Cara; Lui, Christina; Brocardo, Mariana G.; Martino-Echarri, Estefania; Henderson, Beric R.

    2015-01-01

    ABSTRACT β-Catenin transduces the Wnt signaling pathway and its nuclear accumulation leads to gene transactivation and cancer. Rac1 GTPase is known to stimulate β-catenin-dependent transcription of Wnt target genes and we confirmed this activity. Here we tested the recent hypothesis that Rac1 augments Wnt signaling by enhancing β-catenin nuclear import; however, we found that silencing/inhibition or up-regulation of Rac1 had no influence on nuclear accumulation of β-catenin. To better define the role of Rac1, we employed proximity ligation assays (PLA) and discovered that a significant pool of Rac1–β-catenin protein complexes redistribute from the plasma membrane to the nucleus upon Wnt or Rac1 activation. More importantly, active Rac1 was shown to stimulate the formation of nuclear β-catenin–lymphoid enhancer factor 1 (LEF-1) complexes. This regulation required Rac1-dependent phosphorylation of β-catenin at specific serines, which when mutated (S191A and S605A) reduced β-catenin binding to LEF-1 by up to 50%, as revealed by PLA and immunoprecipitation experiments. We propose that Rac1-mediated phosphorylation of β-catenin stimulates Wnt-dependent gene transactivation by enhancing β-catenin–LEF-1 complex assembly, providing new insight into the mechanism of cross-talk between Rac1 and canonical Wnt/β-catenin signaling. PMID:26403202

  6. The rice DUF1620-containing and WD40-like repeat protein is required for the assembly of the restoration of fertility complex.

    Science.gov (United States)

    Qin, Xiaojian; Huang, Qi; Xiao, Haijun; Zhang, Qiannan; Ni, Chenzi; Xu, Yanghong; Liu, Gai; Yang, Daichang; Zhu, Yingguo; Hu, Jun

    2016-05-01

    Cytoplasmic male sterility (CMS) and restoration of fertility (Rf) are widely distributed in plant species utilized by humans. RF5 and GRP162 are subunits of the restoration of fertility complex (RFC) in Hong-Lian rice. Despite the fact that the RFC is 400-500 kDa in size, the other proteins or factors in the complex still remain unknown. Here, we identified RFC subunit 3, which encodes a DUF1620-containing and WD40-like repeat protein (RFC3) that is present in all tissues but highly expressed in leaves. We established that RFC3 interacts with both RF5 and GRP162 in vitro and in vivo, and is transported into the mitochondria as a membrane protein. Furthermore, CMS RNA (atp6-orfH79) and CMS cytotoxic protein (ORFH79) accumulate when RFC3 is silenced in restorer lines. We presented the analysis with blue-native polyacrylamide gel electrophoresis, indicating that RFC is disrupted in the RNAi line. We concluded that RCF3 is indispensable as a scaffold protein for the assembly of the RFC complex. We unveil a new molecular player of the RFC in the Rf pathway in rice and propose the model of RFC based on these data. © 2016 The Authors. New Phytologist © 2016 New Phytologist Trust.

  7. Cas5d Protein Processes Pre-crRNA and Assembles into a Cascade-like Interference Complex in Subtype I-C/Dvulg CRISPR-Cas System

    Energy Technology Data Exchange (ETDEWEB)

    Nam, Ki Hyun; Haitjema, Charles; Liu, Xueqi; Ding, Fran; Wang, Hongwei; DeLisa, Matthew P.; Ke, Ailong (Yale); (Cornell); (Tsinghua)

    2012-10-10

    Clustered regularly interspaced short palindromic repeats (CRISPRs), together with an operon of CRISPR-associated (Cas) proteins, form an RNA-based prokaryotic immune system against exogenous genetic elements. Cas5 family proteins are found in several type I CRISPR-Cas systems. Here, we report the molecular function of subtype I-C/Dvulg Cas5d from Bacillus halodurans. We show that Cas5d cleaves pre-crRNA into unit length by recognizing both the hairpin structure and the 3 single stranded sequence in the CRISPR repeat region. Cas5d structure reveals a ferredoxin domain-based architecture and a catalytic triad formed by Y46, K116, and H117 residues. We further show that after pre-crRNA processing, Cas5d assembles with crRNA, Csd1, and Csd2 proteins to form a multi-sub-unit interference complex similar to Escherichia coli Cascade (CRISPR-associated complex for antiviral defense) in architecture. Our results suggest that formation of a crRNA-presenting Cascade-like complex is likely a common theme among type I CRISPR subtypes.

  8. Assembly of Photosynthetic Antenna Protein Complexes from Algae for Development of Nano-biodevice and Its Fuelization

    Science.gov (United States)

    2013-05-20

    device for solar fuel production from CO2 and water. On going: Development of artificial leaf for solar fuel hydrogen or methanol production from CO2...e- e- PSII Mn Hydrogen Evolution or Carbon Dioxide Fixation Water Photolysis HCHO CH3OH Fuel Material H2 2H+ Hydrogenase(Hase) e- Water Photolysis...and the antenna complexes by modifying the pigment Cars and BChls or chlorophylls as well as the supporting peptides. These modified photosynthetic

  9. To Be or Not To Be T4: Evidence of a Complex Evolutionary Pathway of Head Structure and Assembly in Giant Salmonella Virus SPN3US

    Directory of Open Access Journals (Sweden)

    Bazla Ali

    2017-11-01

    Full Text Available Giant Salmonella phage SPN3US has a 240-kb dsDNA genome and a large complex virion composed of many proteins for which the functions of most are undefined. We recently determined that SPN3US shares a core set of genes with related giant phages and sequenced and characterized 18 amber mutants to facilitate its use as a genetic model system. Notably, SPN3US and related giant phages contain a bolus of ejection proteins within their heads, including a multi-subunit virion RNA polymerase (vRNAP, that enter the host cell with the DNA during infection. In this study, we characterized the SPN3US virion using mass spectrometry to gain insight into its head composition and the features that its head shares with those of related giant phages and with T4 phage. SPN3US has only homologs to the T4 proteins critical for prohead shell formation, the portal and major capsid proteins, as well as to the major enzymes essential for head maturation, the prohead protease and large terminase subunit. Eight of ~50 SPN3US head proteins were found to undergo proteolytic processing at a cleavage motif by the prohead protease gp245. Gp245 undergoes auto-cleavage of its C-terminus, suggesting this is a conserved activation and/or maturation feature of related phage proteases. Analyses of essential head gene mutants showed that the five subunits of the vRNAP must be assembled for any subunit to be incorporated into the prohead, although the assembled vRNAP must then undergo subsequent major conformational rearrangements in the DNA packed capsid to allow ejection through the ~30 Å diameter tail tube for transcription from the injected DNA. In addition, ejection protein candidate gp243 was found to play a critical role in head assembly. Our analyses of the vRNAP and gp243 mutants highlighted an unexpected dichotomy in giant phage head maturation: while all analyzed giant phages have a homologous protease that processes major capsid and portal proteins, processing of ejection

  10. Respective Functions of Two Distinct Siwi Complexes Assembled during PIWI-Interacting RNA Biogenesis in Bombyx Germ Cells

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    Kazumichi M. Nishida

    2015-01-01

    Full Text Available PIWI-interacting RNA (piRNA biogenesis consists of two sequential steps: primary piRNA processing and the ping-pong cycle that depends on reciprocal Slicer-mediated RNA cleavage by PIWI proteins. However, the molecular functions of the factors involved remain elusive. Here, we show that RNAs cleaved by a Bombyx mori PIWI, Siwi, remain bound to the protein upon cleavage but are released by a DEAD box protein BmVasa. BmVasa copurifies with Siwi but not another PIWI BmAgo3. A lack of BmVasa does not affect primary piRNA processing but abolishes the ping-pong cycle. Siwi also forms a complex with BmSpn-E and BmQin. This complex is physically separable from the Siwi/BmVasa complex. BmSpn-E, unlike BmVasa, is necessary for primary piRNA production. We propose a model for piRNA biogenesis, where the BmSpn-E/BmQin dimer binds Siwi to function in primary piRNA processing, whereas BmVasa, by associating with Siwi, ensures target RNA release upon cleavage to facilitate the ping-pong cycle.

  11. Mutation of the zebrafish nucleoporin elys sensitizes tissue progenitors to replication stress.

    Directory of Open Access Journals (Sweden)

    Gangarao Davuluri

    2008-10-01

    Full Text Available The recessive lethal mutation flotte lotte (flo disrupts development of the zebrafish digestive system and other tissues. We show that flo encodes the ortholog of Mel-28/Elys, a highly conserved gene that has been shown to be required for nuclear integrity in worms and nuclear pore complex (NPC assembly in amphibian and mammalian cells. Maternal elys expression sustains zebrafish flo mutants to larval stages when cells in proliferative tissues that lack nuclear pores undergo cell cycle arrest and apoptosis. p53 mutation rescues apoptosis in the flo retina and optic tectum, but not in the intestine, where the checkpoint kinase Chk2 is activated. Chk2 inhibition and replication stress induced by DNA synthesis inhibitors were lethal to flo larvae. By contrast, flo mutants were not sensitized to agents that cause DNA double strand breaks, thus showing that loss of Elys disrupts responses to selected replication inhibitors. Elys binds Mcm2-7 complexes derived from Xenopus egg extracts. Mutation of elys reduced chromatin binding of Mcm2, but not binding of Mcm3 or Mcm4 in the flo intestine. These in vivo data indicate a role for Elys in Mcm2-chromatin interactions. Furthermore, they support a recently proposed model in which replication origins licensed by excess Mcm2-7 are required for the survival of human cells exposed to replication stress.

  12. Cell-Free and Cell-Based Approaches to Explore the Roles of Host Membranes and Lipids in the Formation of Viral Replication Compartment Induced by Tombusviruses.

    Science.gov (United States)

    Nagy, Peter D; Pogany, Judit; Xu, Kai

    2016-03-03

    Plant positive strand RNA viruses are intracellular infectious agents that take advantage of cellular lipids and membranes to support replication and protect viral RNA from degradation by host antiviral responses. In this review, we discuss how Tomato bushy stunt virus (TBSV) co-opts lipid transfer proteins and modulates lipid metabolism and transport to facilitate the assembly of the membrane-bound viral replicase complexes within intricate replication compartments. Identification and characterization of the proviral roles of specific lipids and proteins involved in lipid metabolism based on results from yeast (Saccharomyces cerevisiae) model host and cell-free approaches are discussed. The review also highlights the advantage of using liposomes with chemically defined composition to identify specific lipids required for TBSV replication. Remarkably, all the known steps in TBSV replication are dependent on cellular lipids and co-opted membranes.

  13. Adenovirus DNA Replication

    Science.gov (United States)

    Hoeben, Rob C.; Uil, Taco G.

    2013-01-01

    Adenoviruses have attracted much attention as probes to study biological processes such as DNA replication, transcription, splicing, and cellular transformation. More recently these viruses have been used as gene-transfer vectors and oncolytic agents. On the other hand, adenoviruses are notorious pathogens in people with compromised immune functions. This article will briefly summarize the basic replication strategy of adenoviruses and the key proteins involved and will deal with the new developments since 2006. In addition, we will cover the development of antivirals that interfere with human adenovirus (HAdV) replication and the impact of HAdV on human disease. PMID:23388625

  14. Gene 5.5 protein of bacteriophage T7 in complex with Escherichia coli nucleoid protein H-NS and transfer RNA masks transfer RNA priming in T7 DNA replication.

    Science.gov (United States)

    Zhu, Bin; Lee, Seung-Joo; Tan, Min; Wang, En-Duo; Richardson, Charles C

    2012-05-22

    DNA primases provide oligoribonucleotides for DNA polymerase to initiate lagging strand synthesis. A deficiency in the primase of bacteriophage T7 to synthesize primers can be overcome by genetic alterations that decrease the expression of T7 gene 5.5, suggesting an alternative mechanism to prime DNA synthesis. The product of gene 5.5 (gp5.5) forms a stable complex with the Escherichia coli histone-like protein H-NS and transfer RNAs (tRNAs). The 3'-terminal sequence (5'-ACCA-3') of tRNAs is identical to that of a functional primer synthesized by T7 primase. Mutations in T7 that suppress the inability of primase reduce the amount of gp5.5 and thus increase the pool of tRNA to serve as primers. Alterations in T7 gene 3 facilitate tRNA priming by reducing its endonuclease activity that cleaves at the tRNA-DNA junction. The tRNA bound to gp5.5 recruits H-NS. H-NS alone inhibits reactions involved in DNA replication, but the binding to gp5.5-tRNA complex abolishes this inhibition.

  15. The Type VI Secretion TssEFGK-VgrG Phage-Like Baseplate Is Recruited to the TssJLM Membrane Complex via Multiple Contacts and Serves As Assembly Platform for Tail Tube/Sheath Polymerization.

    Directory of Open Access Journals (Sweden)

    Yannick R Brunet

    2015-10-01

    Full Text Available The Type VI secretion system (T6SS is a widespread weapon dedicated to the delivery of toxin proteins into eukaryotic and prokaryotic cells. The 13 T6SS subunits assemble a cytoplasmic contractile structure anchored to the cell envelope by a membrane-spanning complex. This structure is evolutionarily, structurally and functionally related to the tail of contractile bacteriophages. In bacteriophages, the tail assembles onto a protein complex, referred to as the baseplate, that not only serves as a platform during assembly of the tube and sheath, but also triggers the contraction of the sheath. Although progress has been made in understanding T6SS assembly and function, the composition of the T6SS baseplate remains mostly unknown. Here, we report that six T6SS proteins-TssA, TssE, TssF, TssG, TssK and VgrG-are required for proper assembly of the T6SS tail tube, and a complex between VgrG, TssE,-F and-G could be isolated. In addition, we demonstrate that TssF and TssG share limited sequence homologies with known phage components, and we report the interaction network between these subunits and other baseplate and tail components. In agreement with the baseplate being the assembly platform for the tail, fluorescence microscopy analyses of functional GFP-TssF and TssK-GFP fusion proteins show that these proteins assemble stable and static clusters on which the sheath polymerizes. Finally, we show that recruitment of the baseplate to the apparatus requires initial positioning of the membrane complex and contacts between TssG and the inner membrane TssM protein.

  16. Photosensitized electron transfer within a self-assembled norharmane-2'-deoxyadenosine 5'-monophosphate (dAMP) complex.

    Science.gov (United States)

    Gonzalez, M Micaela; Rasse-Suriani, Federico A O; Franca, Carlos A; Diez, Reinaldo Pis; Gholipour, Yousef; Nonami, Hiroshi; Erra-Balsells, Rosa; Cabrerizo, Franco M

    2012-12-21

    Norharmane is a compound that belongs to a family of alkaloids called β-carbolines (βCs). These alkaloids are present in a wide range of biological systems, playing a variety of significant photo-dependent roles. Upon UV-A irradiation, βCs are able to act as efficient photosensitizers. In this work, we have investigated the photosensitized oxidation of 2'-deoxyadenosine 5'-monophosphate (dAMP) by norharmane in an aqueous phase, upon UV-A (350 nm) irradiation. The effect of the pH was evaluated on both the interactions between norharmane and dAMP in the ground and electronic excited states, and on the dAMP photosensitized oxidation. A quite strong static interaction between norharmane and dAMP was observed, especially under those pH conditions where the protonated form of the alkaloid is present (pH dAMP complex is the operative mechanism in the dAMP photosensitization.

  17. 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.

  18. Interaction between Nbp35 and Cfd1 proteins of cytosolic Fe-S cluster assembly reveals a stable complex formation in Entamoeba histolytica.

    Directory of Open Access Journals (Sweden)

    Shadab Anwar

    Full Text Available Iron-Sulfur (Fe-S proteins are involved in many biological functions such as electron transport, photosynthesis, regulation of gene expression and enzymatic activities. Biosynthesis and transfer of Fe-S clusters depend on Fe-S clusters assembly processes such as ISC, SUF, NIF, and CIA systems. Unlike other eukaryotes which possess ISC and CIA systems, amitochondriate Entamoeba histolytica has retained NIF & CIA systems for Fe-S cluster assembly in the cytosol. In the present study, we have elucidated interaction between two proteins of E. histolytica CIA system, Cytosolic Fe-S cluster deficient 1 (Cfd1 protein and Nucleotide binding protein 35 (Nbp35. In-silico analysis showed that structural regions ranging from amino acid residues (P33-K35, G131-V135 and I147-E151 of Nbp35 and (G5-V6, M34-D39 and G46-A52 of Cfd1 are involved in the formation of protein-protein complex. Furthermore, Molecular dynamic (MD simulations study suggested that hydrophobic forces surpass over hydrophilic forces between Nbp35 and Cfd1 and Van-der-Waal interaction plays crucial role in the formation of stable complex. Both proteins were separately cloned, expressed as recombinant fusion proteins in E. coli and purified to homogeneity by affinity column chromatography. Physical interaction between Nbp35 and Cfd1 proteins was confirmed in vitro by co-purification of recombinant Nbp35 with thrombin digested Cfd1 and in vivo by pull down assay and immunoprecipitation. The insilico, in vitro as well as in vivo results prove a stable interaction between these two proteins, supporting the possibility of its involvement in Fe-S cluster transfer to target apo-proteins through CIA machinery in E. histolytica. Our study indicates that initial synthesis of a Fe-S precursor in mitochondria is not necessary for the formation of Cfd1-Nbp35 complex. Thus, Cfd1 and Nbp35 with the help of cytosolic NifS and NifU proteins can participate in the maturation of non-mitosomal Fe-S proteins

  19. A new member of a family of ATPases is essential for assembly of mitochondrial respiratory chain and ATP synthetase complexes in Saccharomyces cerevisiae.

    Science.gov (United States)

    Tzagoloff, A; Yue, J; Jang, J; Paul, M F

    1994-10-21

    Respiration-defective pet mutants of Saccharomyces cerevisiae, assigned to complementation group G25, are grossly deficient in mitochondrial respiratory and ATPase complexes. This phenotype is usually found in strains impaired in mitochondrial protein synthesis. The G25 mutants, however, synthesize all of the proteins encoded by mitochondrial DNA. The mutants are also able to import and process cytoplasmically derived subunits of these enzymes. These results are most compatible with the idea that the gene defined by G25 mutants (RCA1) codes for a protein essential for the assembly of functional respiratory and ATPase complexes. The RCA1 gene has been cloned by complementation of an rca1 mutant with a yeast genomic library. The sequence of the encoded product shows Rca1 protein to be a new member of a recently described family of ATPases. The Rca1 protein is a mitochondrial membrane protein and is the third known member of this family implicated to function in the biogenesis of mitochondria. The primary structure of Rca1 protein indicates several distinct domains in addition to the common purine nucleotide binding region shared by all members of this protein family. One, located in the amino-terminal half, contains two hydrophobic stretches of sufficient length to span a membrane lipid bilayer.

  20. Discovery and investigation of anticancer ruthenium-arene Schiff-base complexes via water-promoted combinatorial three-component assembly.

    Science.gov (United States)

    Chow, Mun Juinn; Licona, Cynthia; Yuan Qiang Wong, Daniel; Pastorin, Giorgia; Gaiddon, Christian; Ang, Wee Han

    2014-07-24

    The structural diversity of metal scaffolds makes them a viable alternative to traditional organic scaffolds for drug design. Combinatorial chemistry and multicomponent reactions, coupled with high-throughput screening, are useful techniques in drug discovery, but they are rarely used in metal-based drug design. We report the optimization and validation of a new combinatorial, metal-based, three-component assembly reaction for the synthesis of a library of 442 Ru-arene Schiff-base (RAS) complexes. These RAS complexes were synthesized in a one-pot, on-a-plate format using commercially available starting materials under aqueous conditions. The library was screened for their anticancer activity, and several cytotoxic lead compounds were identified. In particular, [(η6-1,3,5-triisopropylbenzene)RuCl(4-methoxy-N-(2-quinolinylmethylene)aniline)]Cl (4) displayed low micromolar IC50 values in ovarian cancers (A2780, A2780cisR), breast cancer (MCF7), and colorectal cancer (HCT116, SW480). The absence of p53 activation or changes in IC50 value between p53+/+ and p53-/- cells suggests that 4 and possibly the other lead compounds may act independently of the p53 tumor suppressor gene frequently mutated in cancer.

  1. Structural basis for DNA strand separation by a hexameric replicative helicase

    Science.gov (United States)

    Chaban, Yuriy; Stead, Jonathan A.; Ryzhenkova, Ksenia; Whelan, Fiona; Lamber, Ekaterina P.; Antson, Alfred; Sanders, Cyril M.; Orlova, Elena V.

    2015-01-01

    Hexameric helicases are processive DNA unwinding machines but how they engage with a replication fork during unwinding is unknown. Using electron microscopy and single particle analysis we determined structures of the intact hexameric helicase E1 from papillomavirus and two complexes of E1 bound to a DNA replication fork end-labelled with protein tags. By labelling a DNA replication fork with streptavidin (dsDNA end) and Fab (5′ ssDNA) we located the positions of these labels on the helicase surface, showing that at least 10 bp of dsDNA enter the E1 helicase via a side tunnel. In the currently accepted ‘steric exclusion’ model for dsDNA unwinding, the active 3′ ssDNA strand is pulled through a central tunnel of the helicase motor domain as the dsDNA strands are wedged apart outside the protein assembly. Our structural observations together with nuclease footprinting assays indicate otherwise: strand separation is taking place inside E1 in a chamber above the helicase domain and the 5′ passive ssDNA strands exits the assembly through a separate tunnel opposite to the dsDNA entry point. Our data therefore suggest an alternative to the current general model for DNA unwinding by hexameric helicases. PMID:26240379

  2. Giant panda BAC library construction and assembly of a 650-kb contig spanning major histocompatibility complex class II region

    Directory of Open Access Journals (Sweden)

    Pan Hui-Juan

    2007-09-01

    Full Text Available Abstract Background Giant panda is rare and endangered species endemic to China. The low rates of reproductive success and infectious disease resistance have severely hampered the development of captive and wild populations of the giant panda. The major histocompatibility complex (MHC plays important roles in immune response and reproductive system such as mate choice and mother-fetus bio-compatibility. It is thus essential to understand genetic details of the giant panda MHC. Construction of a bacterial artificial chromosome (BAC library will provide a new tool for panda genome physical mapping and thus facilitate understanding of panda MHC genes. Results A giant panda BAC library consisting of 205,800 clones has been constructed. The average insert size was calculated to be 97 kb based on the examination of 174 randomly selected clones, indicating that the giant panda library contained 6.8-fold genome equivalents. Screening of the library with 16 giant panda PCR primer pairs revealed 6.4 positive clones per locus, in good agreement with an expected 6.8-fold genomic coverage of the library. Based on this BAC library, we constructed a contig map of the giant panda MHC class II region from BTNL2 to DAXX spanning about 650 kb by a three-step method: (1 PCR-based screening of the BAC library with primers from homologous MHC class II gene loci, end sequences and BAC clone shotgun sequences, (2 DNA sequencing validation of positive clones, and (3 restriction digest fingerprinting verification of inter-clone overlapping. Conclusion The identifications of genes and genomic regions of interest are greatly favored by the availability of this giant panda BAC library. The giant panda BAC library thus provides a useful platform for physical mapping, genome sequencing or complex analysis of targeted genomic regions. The 650 kb sequence-ready BAC contig map of the giant panda MHC class II region from BTNL2 to DAXX, verified by the three-step method, offers a

  3. Determination of optimal assemblies of software-based Ortho-SUV frame for correction of complex midfoot and hindfoot deformities

    Directory of Open Access Journals (Sweden)

    L. N. Solomin

    2014-01-01

    Full Text Available Aim: to find the optimal configuration of passive computer-assisted ORTO-SUV device for the correction of complicated midfoot and hindfoot deformities. Material and methods: The study was carried out using plastic “shin-foot”complexes, on which different versions of Orto-SUV device layout: five versions for midfoot deformities and three versions for hindfoot deformities. A model providing maximum amplitude of the movement was considered optimal. Results: Optimal layout for midfoot provides dorsiflexion of 41,1±1,7°; plantar flexion - 32,1±1,4°; supination - 53,3±1,8°; pronation - 35±1,6°; abduction - 44,2±2,1°; adduction - 43,2±1,7°. Optimal layout for the correction of hindfoot deformities provides rotation in sagittal plane upwards - 96,5±2,4°;rotation in sagittal plane downwards - 36,2±1,4°;“varusation” (angulationmedially in the frontal plane - 31±1,3°; “valgusation” (in the frontal plane outwards - 44,5±1,9°; movement upwards and backwards, angle of 45° to the horizontal plane - 86,4±1,2 mm; movement downwards and anteriorly, angle of 45° to the horizontal plane - 81,5±1,1 mm.

  4. Complexation of uranyl ion with sulfonates. One- to three-dimensional assemblies with 1,5- and 2,7-naphthalenedisulfonates

    Energy Technology Data Exchange (ETDEWEB)

    Thuery, Pierre [NIMBE, CEA, CNRS, Universite Paris-Saclay, CEA Saclay, Gif-sur-Yvette (France); Harrowfield, Jack [ISIS, Universite de Strasbourg (France)

    2017-02-03

    Uranyl nitrate was treated with the sodium salt of either 1,5- or 2,7-naphthalenedisulfonate (1,5-ndsNa{sub 2} or 2,7-ndsNa{sub 2}, respectively) under (solvo)-hydrothermal conditions, in the presence of additional coligands and/or metal cations, to give six new complexes, which were characterized by determination of their crystal structures. Complex [UO{sub 2}(1,5-nds)(H{sub 2}O)] (1) crystallizes as a three-dimensional (3D) framework, with both sulfonate groups coordinated in the O,O{sup '}-bridging mode. In the presence of the N-chelating species 2,2{sup '}-bipyridine (bipy) or 1,10-phenanthroline (phen), the three complexes [(UO{sub 2}){sub 2}(1,5-nds)(OH){sub 2}(bipy){sub 2}].H{sub 2}O (2), [(UO{sub 2}){sub 2}(1,5-nds)(OH){sub 2}(bipy){sub 2}].bipy (3) and [(UO{sub 2}){sub 2}(1,5-nds)(OH){sub 2}(phen){sub 2}] (4) were obtained, in which doubly hydroxide-bridged uranyl dimers are assembled into one-dimensional (1D) chains by bis(unidentate) disulfonate ligands. Complex [Cu(bipy){sub 2}Cl][UO{sub 2}(2,7-nds)(OH)].H{sub 2}O (5) displays anionic, two-dimensional (2D) sheets in which unidentate/O,O{sup '}-bridging disulfonate ligands link hydroxide-bridged uranyl dimers. In the additional presence of cucurbit[6]uril (CB6), complex [(UO{sub 2}){sub 4}Na{sub 4}(2,7-nds){sub 2}(CB6)Cl{sub 4}O{sub 2}(H{sub 2}O){sub 10}].5H{sub 2}O (6) crystallizes as a 3D framework of intricate architecture, with bis(μ{sub 3}-oxo)-bridged uranyl tetranuclear moieties connected to CB6-bound sodium cations by doubly O,O{sup '}-bridging disulfonates. Complexes 2 and 4 display intense and well-resolved uranyl emission in the solid state, while nearly complete quenching is observed in 3 and 5. (copyright 2017 WILEY-VCH Verlag GmbH and Co. KGaA, Weinheim)

  5. Transformation from kinetically into thermodynamically controlled self-organization of complex helical columns with 3D periodicity assembled from dendronized perylene bisimides.

    Science.gov (United States)

    Percec, Virgil; Sun, Hao-Jan; Leowanawat, Pawaret; Peterca, Mihai; Graf, Robert; Spiess, Hans W; Zeng, Xiangbing; Ungar, Goran; Heiney, Paul A

    2013-03-13

    The dendronized perylene 3,4:9,10-tetracarboxylic acid bisimide (PBI), (3,4,5)12G1-1-PBI, was reported by our laboratory to self-assemble into complex helical columns containing dimers of dendronized PBI with one molecule in each stratum, with different intra- and interdimer rotation angles but identical intra- and interdimer distance of 3.5 Å, exhibiting a four-strata 2(1) helical repeat. A thermodynamically controlled 2D columnar hexagonal phase with short-range intracolumnar order represents the thermodynamic product at high temperature, while a kinetically controlled monoclinic columnar array with 3D periodicity is the thermodynamic product at low temperature. With heating and cooling rates higher than 10 °C/min to 1 °C/min, at low temperature the 2D columnar periodic array is the kinetic product for this dendronized PBI. Here the synthesis and structural analysis of a library of (3,4,5)nG1-m-PBI with n = 12 to 6 and m = 1 are reported. A combination of differential scanning calorimetry, X-ray diffraction on powder and orientated fibers, including pattern simulation and electron density map reconstruction, and solid-state NMR, all as a function of temperature and heating and cooling rate, was employed for their structural analysis. It was discovered that at low temperature the as-prepared n = 12 to 10 exhibit a 3D layered array that transforms irreversibly into columnar periodicities during heating and cooling. Also the kinetically controlled 3D columnar phase of n = 12 becomes thermodynamically controlled for n = 10, 9, 8, 7, and 6. This unprecedented transformation is expected to facilitate the design of functions from dendronized PBI and other self-assembling building blocks.

  6. Animal Mitochondrial DNA Replication

    Science.gov (United States)

    Ciesielski, Grzegorz L.; Oliveira, Marcos T.; Kaguni, Laurie S.

    2016-01-01

    Recent advances in the field of mitochondrial DNA (mtDNA) replication highlight the diversity of both the mechanisms utilized and the structural and functional organization of the proteins at mtDNA replication fork, despite the simplicity of the animal mtDNA genome. DNA polymerase γ, mtDNA helicase and mitochondrial single-stranded DNA-binding protein- the key replisome proteins, have evolved distinct structural features and biochemical properties. These appear to be correlated with mtDNA genomic features in different metazoan taxa and with their modes of DNA replication, although a substantial integrative research is warranted to establish firmly these links. To date, several modes of mtDNA replication have been described for animals: rolling circle, theta, strand-displacement, and RITOLS/bootlace. Resolution of a continuing controversy relevant to mtDNA replication in mammals/vertebrates will have a direct impact on the mechanistic interpretation of mtDNA-related human diseases. Here we review these subjects, integrating earlier and recent data to provide a perspective on the major challenges for future research. PMID:27241933

  7. New histone supply regulates replication fork speed and PCNA unloading

    DEFF Research Database (Denmark)

    Mejlvang, Jakob; Feng, Yunpeng; Alabert, Constance

    2014-01-01

    Correct duplication of DNA sequence and its organization into chromatin is central to genome function and stability. However, it remains unclear how cells coordinate DNA synthesis with provision of new histones for chromatin assembly to ensure chromosomal stability. In this paper, we show...... that replication fork speed is dependent on new histone supply and efficient nucleosome assembly. Inhibition of canonical histone biosynthesis impaired replication fork progression and reduced nucleosome occupancy on newly synthesized DNA. Replication forks initially remained stable without activation...... of conventional checkpoints, although prolonged histone deficiency generated DNA damage. PCNA accumulated on newly synthesized DNA in cells lacking new histones, possibly to maintain opportunity for CAF-1 recruitment and nucleosome assembly. Consistent with this, in vitro and in vivo analysis showed that PCNA...

  8. 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......In recent years polymer components with surface microstructures have been in rising demand for applications such as lab-on-a-chip and optical components. Injection moulding has proven to be a feasible and efficient way to manufacture such components. In injection moulding the mould surface...... 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...

  9. Psychology, replication & beyond.

    Science.gov (United States)

    Laws, Keith R

    2016-06-01

    Modern psychology is apparently in crisis and the prevailing view is that this partly reflects an inability to replicate past findings. If a crisis does exists, then it is some kind of 'chronic' crisis, as psychologists have been censuring themselves over replicability for decades. While the debate in psychology is not new, the lack of progress across the decades is disappointing. Recently though, we have seen a veritable surfeit of debate alongside multiple orchestrated and well-publicised replication initiatives. The spotlight is being shone on certain areas and although not everyone agrees on how we should interpret the outcomes, the debate is happening and impassioned. The issue of reproducibility occupies a central place in our whig history of psychology.

  10. Registered Replication Report

    DEFF Research Database (Denmark)

    Bouwmeester, S.; Verkoeijen, P. P.J.L.; Aczel, B.

    2017-01-01

    In an anonymous 4-person economic game, participants contributed more money to a common project (i.e., cooperated) when required to decide quickly than when forced to delay their decision (Rand, Greene & Nowak, 2012), a pattern consistent with the social heuristics hypothesis proposed by Rand...... 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...

  11. 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...

  12. Paleoproterozoic (ca. 1.8 Ga) arc magmatism in the Lützow-Holm Complex, East Antarctica: Implications for crustal growth and terrane assembly in erstwhile Gondwana fragments

    Science.gov (United States)

    Takahashi, Kazuki; Tsunogae, Toshiaki; Santosh, M.; Takamura, Yusuke; Tsutsumi, Yukiyasu

    2018-05-01

    lithological data from the region, suggest that the LHC can be divided into three units: Neoarchean (ca. 2.5 Ga) unit in the southern LHC (Shirase Orthogneiss or "Shirase microcontinent"), Neoproterozoic (ca. 1.0 Ga) unit in the northern LHC, and supracrustal unit in the central LHC with fragments of Paleoproterozoic (ca. 1.8 Ga) and minor Neoarchean (ca. 2.5 Ga) and Neoproterozoic (ca. 1.0 Ga) magmatic arcs. The 1.8 Ga arc magmatism inferred in this study has also been reported from adjacent Gondwana fragments such as the Highland Complex in Sri Lanka, and the Trivandrum and Nagercoil Blocks in southern India. Although the ca. 1.8 Ga arc-magmatic event is coeval in these regions, the Paleoproterozoic supracrustal unit in the central LHC may not be contiguous with those in the Highland Complex of Sri Lanka because recent studies have shown that the Vijayan Complex in Sri Lanka and the ca. 1.0 Ga northern LHC possibly were part of a single crustal unit (northern Lützow-Holm-Vijayan Complex) within the Kalahari Block. The supracrustal unit possibly marks part of a discrete suture formed by the collision of the ca. 2.5 Ga southern LHC (Shirase microcontinent) and the ca. 1.0 Ga northern Lützow-Holm-Vijayan Complex during the latest Neoproterozoic-Cambrian Gondwana amalgamation, which might be coeval with the collision of the Vijayan and Wanni Complexes and the formation of the Highland Complex in Sri Lanka. Our study provides new insights on crustal growth and terrane assembly in the ancient continental blocks of Gondwana.

  13. DDX3 directly regulates TRAF3 ubiquitination and acts as a scaffold to co-ordinate assembly of signalling complexes downstream from MAVS.

    Science.gov (United States)

    Gu, Lili; Fullam, Anthony; McCormack, Niamh; Höhn, Yvette; Schröder, Martina

    2017-02-15

    The human DEAD-box helicase 3 (DDX3) has been shown to contribute to type I interferon (IFN) induction downstream from antiviral pattern recognition receptors. It binds to TANK-binding kinase 1 and IκB-kinase-ε (IKKε), the two key kinases mediating activation of IFN regulatory factor (IRF) 3 and IRF7. We previously demonstrated that DDX3 facilitates IKKε activation downstream from RIG-I and then links the activated kinase to IRF3. In the present study, we probed the interactions between DDX3 and other key signalling molecules in the RIG-I pathway and identified a novel direct interaction between DDX3 and TNF receptor-associated factor 3 (TRAF3) mediated by a TRAF-interaction motif in the N-terminus of DDX3, which was required for TRAF3 ubiquitination. Interestingly, we observed two waves of K63-linked TRAF3 ubiquitination following RIG-I activation by Sendai virus (SeV) infection, both of which were suppressed by DDX3 knockdown. We also investigated the spatiotemporal formation of endogenous downstream signalling complexes containing the mitochondrial antiviral signalling (MAVS) adaptor, DDX3, IκB-kinase-ε (IKKε), TRAF3 and IRF3. DDX3 was recruited to MAVS early after SeV infection, suggesting that it might mediate subsequent recruitment of other molecules. Indeed, knockdown of DDX3 prevented the formation of TRAF3-MAVS and TRAF3-IKKε complexes. Based on our data, we propose that early TRAF3 ubiquitination is required for the formation of a stable MAVS-TRAF3 complex, while the second wave of TRAF3 ubiquitination mediates IRF3 recruitment and activation. Our study characterises DDX3 as a multifunctional adaptor molecule that co-ordinates assembly of different TRAF3, IKKε and IRF3-containing signalling complexes downstream from MAVS. Additionally, it provides novel insights into the role of TRAF3 in RIG-I signalling. © 2017 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.

  14. Assembly of the Lactuca sativa, L. cv. Tizian draft genome sequence reveals differences within major resistance complex 1 as compared to the cv. Salinas reference genome.

    Science.gov (United States)

    Verwaaijen, Bart; Wibberg, Daniel; Nelkner, Johanna; Gordin, Miriam; Rupp, Oliver; Winkler, Anika; Bremges, Andreas; Blom, Jochen; Grosch, Rita; Pühler, Alfred; Schlüter, Andreas

    2018-02-10

    Lettuce (Lactuca sativa, L.) is an important annual plant of the family Asteraceae (Compositae). The commercial lettuce cultivar Tizian has been used in various scientific studies investigating the interaction of the plant with phytopathogens or biological control agents. Here, we present the de novo draft genome sequencing and gene prediction for this specific cultivar derived from transcriptome sequence data. The assembled scaffolds amount to a size of 2.22 Gb. Based on RNAseq data, 31,112 transcript isoforms were identified. Functional predictions for these transcripts were determined within the GenDBE annotation platform. Comparison with the cv. Salinas reference genome revealed a high degree of sequence similarity on genome and transcriptome levels, with an average amino acid identity of 99%. Furthermore, it was observed that two large regions are either missing or are highly divergent within the cv. Tizian genome compared to cv. Salinas. One of these regions covers the major resistance complex 1 region of cv. Salinas. The cv. Tizian draft genome sequence provides a valuable resource for future functional and transcriptome analyses focused on this lettuce cultivar. Copyright © 2017 Elsevier B.V. All rights reserved.

  15. Study of the synthesis and self-assembly of CO2-philic copolymers with complexing groups: application to decontamination in supercritical CO2 medium

    International Nuclear Information System (INIS)

    Ribaut, T.

    2009-10-01

    In the frame of sustainable development, a priority is to decrease the volume of nuclear wastes. The use of supercritical carbon dioxide (scCO 2 ) could allow to solve this problem. The aim of this study is to extract an ionic or particle cobalt contamination deposited on textile lab coats. The strategy uses CO 2 -philic/CO 2 -phobic copolymers soluble in scCO 2 and containing complexing groups. This approach combines the use of amphiphilic copolymers for steric stabilization of particles, of surfactants able to self-assemble to promote extraction and of ligands. Controlled radical polymerization is used to synthesize fluorinated gradient or block copolymers. Cloud point curves of the copolymers are determined experimentally in scCO 2 . Prediction of polymer/scCO 2 phase diagrams was assessed by Perturbed-Chain Statistical Associating Fluid Theory (PC-SAFT) modeling. Gradient copolymers appear more advantageous than block copolymers due to their solubility in much milder conditions of pressure and temperature. Small-angle neutron scattering (SANS) allowed us to evidence the pressure-induced aggregation of the gradient copolymers in scCO 2 . Their interface properties were demonstrated: they allow to form water-in-CO 2 microemulsions and to stabilize cobalt hydroxide dispersions in scCO 2 . Lastly, in presence of a very low quantity of water, Co 2+ ions were removed with a rate of 37 % from a cotton/polyester matrix by a gradient copolymer. (author)

  16. Synthesis of an S T = 7 [Mn 3 ] Mixed-Valence Complex Based on 1,3-Propanediol Ligand Derivatives and Its One-Dimensional Assemblies

    KAUST Repository

    Huang, Jian

    2013-10-07

    Controlled organization of high-spin complexes and single-molecule magnets is a great challenge in molecular magnetism in order to study the effect of the intercomplex magnetic interactions on the intrinsic properties of a given magnetic object. In this work, a new ST = 7 trinuclear mixed-valence Mn complex, [MnIIIMnII 2(LA) 2(Br)4(CH3OH)6] ·Br· (CH3OH)1.5·(H2O)0.5 (1), is reported using a pyridinium-functionalized 1,3-propanediol ligand (H 2LABr = 1-(3-bromo-2,2-bis(hydroxymethyl)propyl)pyridinium bromide). Using azido anions as bridging ligands and different pyridinium-functionalized 1,3-propanediol ligands (H2LBBr = 1-(3-bromo-2,2-bis(hydroxymethyl)propyl)-4-picolinium bromide; H 2LCBr = 1-(3-bromo-2,2-bis(hydroxymethyl)propyl)-3,5- lutidinium bromide), the linear [MnIIIMnII 2L2X4]+ building block has been assembled into one-dimensional coordination networks: [MnIIIMn II 2(LA)2(Br)4(CH 3OH)4(N3)]·((C2H 5)2O)1.25 (2∞), [MnIIIMn II 2(LB)2(Br)4(C 2H5OH)(CH3OH)(H2O) 2(N3)]·(H2O)0.25 (3∞), and [MnIIIMnII 2(LC) 2(Cl)3.8(Br)0.2(C2H 5OH)3(CH3OH)(N3)] (4∞). The syntheses, characterization, crystal structures, and magnetic properties of these new [Mn3]-based materials are reported. © 2013 American Chemical Society.

  17. 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

  18. Replication studies in longevity

    DEFF Research Database (Denmark)

    Varcasia, O; Garasto, S; Rizza, T

    2001-01-01

    In Danes we replicated the 3'APOB-VNTR gene/longevity association study previously carried out in Italians, by which the Small alleles (less than 35 repeats) had been identified as frailty alleles for longevity. In Danes, neither genotype nor allele frequencies differed between centenarians and 20...

  19. Porcine Reproductive and Respiratory Syndrome Virus nsp1α Inhibits NF-κB Activation by Targeting the Linear Ubiquitin Chain Assembly Complex.

    Science.gov (United States)

    Jing, Huiyuan; Fang, Liurong; Ding, Zhen; Wang, Dang; Hao, Wenqi; Gao, Li; Ke, Wenting; Chen, Huanchun; Xiao, Shaobo

    2017-02-01

    Linear ubiquitination, a newly discovered posttranslational modification, is catalyzed by the linear ubiquitin chain assembly complex (LUBAC), which is composed of three subunits: one catalytic subunit HOIP and two accessory molecules, HOIL-1L and SHARPIN. Accumulating evidence suggests that linear ubiquitination plays a crucial role in innate immune signaling and especially in the activation of the NF-κB pathway by conjugating linear polyubiquitin chains to NF-κB essential modulator (NEMO, also called IKKγ), the regulatory subunit of the IKK complex. Porcine reproductive and respiratory syndrome virus (PRRSV), an Arterivirus that has devastated the swine industry worldwide, is an ideal model to study the host's disordered inflammatory responses after viral infection. Here, we found that LUBAC-induced NF-κB and proinflammatory cytokine expression can be inhibited in the early phase of PRRSV infection. Screening the PRRSV-encoded proteins showed that nonstructural protein 1α (nsp1α) suppresses LUBAC-mediated NF-κB activation and its CTE domain is required for the inhibition. Mechanistically, nsp1α binds to HOIP/HOIL-1L and impairs the interaction between HOIP and SHARPIN, thus reducing the LUBAC-dependent linear ubiquitination of NEMO. Moreover, PRRSV infection also blocks LUBAC complex formation and NEMO linear-ubiquitination, the important step for transducing NF-κB signaling. This unexpected finding demonstrates a previously unrecognized role of PRRSV nsp1α in modulating LUBAC signaling and explains an additional mechanism of immune modulation by PRRSV. Porcine reproductive and respiratory syndrome (PRRS) is one of the most important veterinary infectious diseases in countries with intensive swine industries. PRRS virus (PRRSV) infection usually suppresses proinflammatory cytokine expression in the early stage of infection, whereas it induces an inflammatory storm in the late stage. However, precisely how the virus is capable of doing so remains obscure

  20. Collective helicity switching of a DNA-coat assembly

    Science.gov (United States)

    Kim, Yongju; Li, Huichang; He, Ying; Chen, Xi; Ma, Xiaoteng; Lee, Myongsoo

    2017-07-01

    Hierarchical assemblies of biomolecular subunits can carry out versatile tasks at the cellular level with remarkable spatial and temporal precision. As an example, the collective motion and mutual cooperation between complex protein machines mediate essential functions for life, such as replication, synthesis, degradation, repair and transport. Nucleic acid molecules are far less dynamic than proteins and need to bind to specific proteins to form hierarchical structures. The simplest example of these nucleic acid-based structures is provided by a rod-shaped tobacco mosaic virus, which consists of genetic material surrounded by coat proteins. Inspired by the complexity and hierarchical assembly of viruses, a great deal of effort has been devoted to design similarly constructed artificial viruses. However, such a wrapping approach makes nucleic acid dynamics insensitive to environmental changes. This limitation generally restricts, for example, the amplification of the conformational dynamics between the right-handed B form to the left-handed Z form of double-stranded deoxyribonucleic acid (DNA). Here we report a virus-like hierarchical assembly in which the native DNA and a synthetic coat undergo repeated collective helicity switching triggered by pH change under physiological conditions. We also show that this collective helicity inversion occurs during translocation of the DNA-coat assembly into intracellular compartments. Translating DNA conformational dynamics into a higher level of hierarchical dynamics may provide an approach to create DNA-based nanomachines.

  1. Coordination of Genomic RNA Packaging with Viral Assembly in HIV-1

    Directory of Open Access Journals (Sweden)

    Chris Hellmund

    2016-07-01

    Full Text Available The tremendous progress made in unraveling the complexities of human immunodeficiency virus (HIV replication has resulted in a library of drugs to target key aspects of the replication cycle of the virus. Yet, despite this accumulated wealth of knowledge, we still have much to learn about certain viral processes. One of these is virus assembly, where the viral genome and proteins come together to form infectious progeny. Here we review this topic from the perspective of how the route to production of an infectious virion is orchestrated by the viral genome, and we compare and contrast aspects of the assembly mechanisms employed by HIV-1 with those of other RNA viruses.

  2. Bacteriophage SPP1 DNA replication strategies promote viral and disable host replication in vitro.

    Science.gov (United States)

    Seco, Elena M; Zinder, John C; Manhart, Carol M; Lo Piano, Ambra; McHenry, Charles S; Ayora, Silvia

    2013-02-01

    Complex viruses that encode their own initiation proteins and subvert the host's elongation apparatus have provided valuable insights into DNA replication. Using purified bacteriophage SPP1 and Bacillus subtilis proteins, we have reconstituted a rolling circle replication system that recapitulates genetically defined protein requirements. Eleven proteins are required: phage-encoded helicase (G40P), helicase loader (G39P), origin binding protein (G38P) and G36P single-stranded DNA-binding protein (SSB); and host-encoded PolC and DnaE polymerases, processivity factor (β(2)), clamp loader (τ-δ-δ') and primase (DnaG). This study revealed a new role for the SPP1 origin binding protein. In the presence of SSB, it is required for initiation on replication forks that lack origin sequences, mimicking the activity of the PriA replication restart protein in bacteria. The SPP1 replisome is supported by both host and viral SSBs, but phage SSB is unable to support B. subtilis replication, likely owing to its inability to stimulate the PolC holoenzyme in the B. subtilis context. Moreover, phage SSB inhibits host replication, defining a new mechanism by which bacterial replication could be regulated by a viral factor.

  3. Cellular Ubc2/Rad6 E2 ubiquitin-conjugating enzyme facilitates tombusvirus replication in yeast and plants

    Energy Technology Data Exchange (ETDEWEB)

    Imura, Yoshiyuki, E-mail: imura@brs.nihon-u.ac.jp; Molho, Melissa; Chuang, Chingkai; Nagy, Peter D., E-mail: pdnagy2@uky.edu

    2015-10-15

    Mono- and multi-ubiquitination alters the functions and subcellular localization of many cellular and viral proteins. Viruses can co-opt or actively manipulate the ubiquitin network to support viral processes or suppress innate immunity. Using yeast (Saccharomyces cerevisiae) model host, we show that the yeast Rad6p (radiation sensitive 6) E2 ubiquitin-conjugating enzyme and its plant ortholog, AtUbc2, interact with two tombusviral replication proteins and these E2 ubiquitin-conjugating enzymes could be co-purified with the tombusvirus replicase. We demonstrate that TBSV RNA replication and the mono- and bi-ubiquitination level of p33 is decreased in rad6Δ yeast. However, plasmid-based expression of AtUbc2p could complement both defects in rad6Δ yeast. Knockdown of UBC2 expression in plants also decreases tombusvirus accumulation and reduces symptom severity, suggesting that Ubc2p is critical for virus replication in plants. We provide evidence that Rad6p is involved in promoting the subversion of Vps23p and Vps4p ESCRT proteins for viral replicase complex assembly. - Highlights: • Tombusvirus p33 replication protein interacts with cellular RAD6/Ubc2 E2 enzymes. • Deletion of RAD6 reduces tombusvirus replication in yeast. • Silencing of UBC2 in plants inhibits tombusvirus replication. • Mono- and bi-ubiquitination of p33 replication protein in yeast and in vitro. • Rad6p promotes the recruitment of cellular ESCRT proteins into the tombusvirus replicase.

  4. Nidovirus replication structures : hijacking membranes to support viral RNA synthesis

    NARCIS (Netherlands)

    Knoops, Kèvin

    2011-01-01

    Positive-stranded RNA viruses replicate in the cytoplasm of host cells and their replication complexes are associated with modified cell membranes. We investigated the structure of the nidovirus-induced membrane modifications and found that nidoviruses transform the endoplasmic reticulum into a

  5. 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…

  6. Self-assembly of self-assembled molecular triangles

    Indian Academy of Sciences (India)

    While the solution state structure of 1 can be best described as a trinuclear complex, in the solidstate well-fashioned intermolecular - and CH- interactions are observed. Thus, in the solid-state further self-assembly of already self-assembled molecular triangle is witnessed. The triangular panels are arranged in a linear ...

  7. Zika virus inhibits eIF2α-dependent stress granule assembly.

    Directory of Open Access Journals (Sweden)

    Raquel Amorim

    2017-07-01

    Full Text Available Zika virus (ZIKV, a member of the Flaviviridae family, is the most recent emerging arbovirus with pandemic potential. During infection, viruses trigger the host cell stress response, leading to changes in RNA translation and the assembly of large aggregates of stalled translation preinitiation complexes, termed stress granules (SGs. Several reports demonstrate that flaviviruses modulate the assembly of stress granules (SG. As an emerging pathogen, little is known however about how ZIKV modulates the host cell stress response. In this work, we investigate how ZIKV modulates SG assembly. We demonstrate that ZIKV negatively impacts SG assembly under oxidative stress conditions induced by sodium arsenite (Ars, a treatment that leads to the phosphorylation of eIF2α. By contrast, no measurable difference in SG assembly was observed between mock and ZIKV-infected cells treated with sodium selenite (Se or Pateamine A (PatA, compounds that trigger eIF2α-independent SG assembly. Interestingly, ZIKV infection markedly impaired the phosphorylation of eIF2α triggered in Ars-treated infected cells, and the abrogation of SG assembly in ZIKV-infected cells is, at least in part, dependent on eIF2α dephosphorylation. These data demonstrate that ZIKV elicits mechanisms to counteract host anti-viral stress responses to promote a cellular environment propitious for viral replication.

  8. Zika virus inhibits eIF2α-dependent stress granule assembly.

    Science.gov (United States)

    Amorim, Raquel; Temzi, Abdelkrim; Griffin, Bryan D; Mouland, Andrew J

    2017-07-01

    Zika virus (ZIKV), a member of the Flaviviridae family, is the most recent emerging arbovirus with pandemic potential. During infection, viruses trigger the host cell stress response, leading to changes in RNA translation and the assembly of large aggregates of stalled translation preinitiation complexes, termed stress granules (SGs). Several reports demonstrate that flaviviruses modulate the assembly of stress granules (SG). As an emerging pathogen, little is known however about how ZIKV modulates the host cell stress response. In this work, we investigate how ZIKV modulates SG assembly. We demonstrate that ZIKV negatively impacts SG assembly under oxidative stress conditions induced by sodium arsenite (Ars), a treatment that leads to the phosphorylation of eIF2α. By contrast, no measurable difference in SG assembly was observed between mock and ZIKV-infected cells treated with sodium selenite (Se) or Pateamine A (PatA), compounds that trigger eIF2α-independent SG assembly. Interestingly, ZIKV infection markedly impaired the phosphorylation of eIF2α triggered in Ars-treated infected cells, and the abrogation of SG assembly in ZIKV-infected cells is, at least in part, dependent on eIF2α dephosphorylation. These data demonstrate that ZIKV elicits mechanisms to counteract host anti-viral stress responses to promote a cellular environment propitious for viral replication.

  9. 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.

  10. 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.

  11. Nanoparticle assemblies and superstructures

    National Research Council Canada - National Science Library

    Kotov, Nicholas A

    2006-01-01

    ... building blocks of larger and more complex systems. Therefore, the present challenge of nanoscale science is to shift from making certain building blocks to organizing them in one-, two-, and three-dimensional structures. Such assemblies and superstructures are the next logical step in the development of nanoscience and nanotechnology. In this re...

  12. Assembling sustainable territories

    NARCIS (Netherlands)

    Vandergeest, Peter; Ponte, Stefano; Bush, Simon

    2015-01-01

    The authors show how certification assembles ‘sustainable’ territories through a complex layering of regulatory authority in which both government and nongovernment entities claim rule-making authority, sometimes working together, sometimes in parallel, sometimes competitively. It is argued that

  13. 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

  14. DNA replication origins in archaea

    Directory of Open Access Journals (Sweden)

    Zhenfang eWu

    2014-04-01

    Full Text Available DNA replication initiation, which starts at specific chromosomal site (known as replication origins, is the key regulatory stage of chromosome replication. Archaea, the third domain of life, use a single or multiple origin(s to initiate replication of their circular chromosomes. The basic structure of replication origins is conserved among archaea, typically including an AT-rich unwinding region flanked by several conserved repeats (origin recognition box, ORB that are located adjacent to a replication initiator gene. Both the ORB sequence and the adjacent initiator gene are considerably diverse among different replication origins, while in silico and genetic analyses have indicated the specificity between the initiator genes and their cognate origins. These replicator-initiator pairings are reminiscent of the oriC-dnaA system in bacteria, and a model for the negative regulation of origin activity by a downstream cluster of ORB elements has been recently proposed in haloarchaea. Moreover, comparative genomic analyses have revealed that the mosaics of replicator-initiator pairings in archaeal chromosomes originated from the integration of extrachromosomal elements. This review summarizes the research progress in understanding of archaeal replication origins with particular focus on the utilization, control and evolution of multiple replication origins in haloarchaea.

  15. Molecular Biology of Rotavirus Entry and Replication

    Science.gov (United States)

    Ruiz, Marie Christine; Leon, Theresa; Diaz, Yuleima; Michelangeli, Fabian

    2009-01-01

    Rotavirus is a nonenveloped, double-stranded, RNA virus belonging to the Reoviridae family and is the major etiological agent of viral gastroenteritis in young children and young animals. Remarkable progress in the understanding of the rotavirus cycle has been made in the last 10 years. The knowledge of viral replication thus far acquired is based on structural studies, the expression and coexpression of individual viral proteins, silencing of individual genes by siRNAs, and the effects that these manipulations have on the physiology of the infected cell. The functions of the individual rotavirus proteins have been largely dissected; however, the interactions between them and with cell proteins, and the molecular mechanisms of virus replication, are just beginning to be understood. These advancements represent the basis for the development of effective vaccination and rational therapeutic strategies to combat rotavirus infection and diarrhea syndromes. In this paper, we review and try to integrate the new knowledge about rotavirus entry, replication, and assembly, and pose some of the questions that remain to be solved. PMID:20024520

  16. Optical tweezers reveal how proteins alter replication

    Science.gov (United States)

    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.

  17. On Constraints in Assembly Planning

    Energy Technology Data Exchange (ETDEWEB)

    Calton, T.L.; Jones, R.E.; Wilson, R.H.

    1998-12-17

    Constraints on assembly plans vary depending on product, assembly facility, assembly volume, and many other factors. Assembly costs and other measures to optimize vary just as widely. To be effective, computer-aided assembly planning systems must allow users to express the plan selection criteria that appIy to their products and production environments. We begin this article by surveying the types of user criteria, both constraints and quality measures, that have been accepted by assembly planning systems to date. The survey is organized along several dimensions, including strategic vs. tactical criteria; manufacturing requirements VS. requirements of the automated planning process itself and the information needed to assess compliance with each criterion. The latter strongly influences the efficiency of planning. We then focus on constraints. We describe a framework to support a wide variety of user constraints for intuitive and efficient assembly planning. Our framework expresses all constraints on a sequencing level, specifying orders and conditions on part mating operations in a number of ways. Constraints are implemented as simple procedures that either accept or reject assembly operations proposed by the planner. For efficiency, some constraints are supplemented with special-purpose modifications to the planner's algorithms. Fast replanning enables an interactive plan-view-constrain-replan cycle that aids in constraint discovery and documentation. We describe an implementation of the framework in a computer-aided assembly planning system and experiments applying the system to a number of complex assemblies, including one with 472 parts.

  18. 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...

  19. Conserved Features of the PB2 627 Domain Impact Influenza Virus Polymerase Function and Replication

    Science.gov (United States)

    Kirui, James; Bucci, Michael D.; Poole, Daniel S.

    2014-01-01

    ABSTRACT Successful replication of influenza virus requires the coordinated expression of viral genes and replication of the genome by the viral polymerase, composed of the subunits PA, PB1, and PB2. Polymerase activity is regulated by both viral and host factors, yet the mechanisms of regulation and how they contribute to viral pathogenicity and tropism are poorly understood. To characterize these processes, we created a series of mutants in the 627 domain of the PB2 subunit. This domain contains a conserved “P[F/P]AAAPP” sequence motif and the well-described amino acid 627, whose identity regulates host range. A lysine present at position 627 in most mammalian viral isolates creates a basic face on the domain surface and confers high-level activity in humans compared to the glutamic acid found at this position in avian isolates. Mutation of the basic face or the P[F/P]AAAPP motif impaired polymerase activity, assembly of replication complexes, and viral replication. Most of these residues are required for general polymerase activity, whereas PB2 K586 and R589 were preferentially required for function in human versus avian cells. Thus, these data identify residues in the 627 domain and other viral proteins that regulate polymerase activity, highlighting the importance of the surface charge and structure of this domain for virus replication and host adaptation. IMPORTANCE Influenza virus faces barriers to transmission across species as it emerges from its natural reservoir in birds to infect mammals. The viral polymerase is an important regulator of this process and undergoes discrete changes to adapt to replication in mammals. Many of these changes occur in the polymerase subunit PB2. Here we describe the systematic analysis of a key region in PB2 that controls species-specific polymerase activity. We report the importance of conserved residues that contribute to the overall charge of the protein as well as those that likely affect protein structure. These

  20. Coordination of Hepatitis C Virus Assembly by Distinct Regulatory Regions in Nonstructural Protein 5A.

    Directory of Open Access Journals (Sweden)

    Margarita Zayas

    2016-01-01

    Full Text Available Hepatitis C virus (HCV nonstructural protein (NS5A is a RNA-binding protein composed of a N-terminal membrane anchor, a structured domain I (DI and two intrinsically disordered domains (DII and DIII interacting with viral and cellular proteins. While DI and DII are essential for RNA replication, DIII is required for assembly. How these processes are orchestrated by NS5A is poorly understood. In this study, we identified a highly conserved basic cluster (BC at the N-terminus of DIII that is critical for particle assembly. We generated BC mutants and compared them with mutants that are blocked at different stages of the assembly process: a NS5A serine cluster (SC mutant blocked in NS5A-core interaction and a mutant lacking the envelope glycoproteins (ΔE1E2. We found that BC mutations did not affect core-NS5A interaction, but strongly impaired core-RNA association as well as virus particle envelopment. Moreover, BC mutations impaired RNA-NS5A interaction arguing that the BC might be required for loading of core protein with viral RNA. Interestingly, RNA-core interaction was also reduced with the ΔE1E2 mutant, suggesting that nucleocapsid formation and envelopment are coupled. These findings argue for two NS5A DIII determinants regulating assembly at distinct, but closely linked steps: (i SC-dependent recruitment of replication complexes to core protein and (ii BC-dependent RNA genome delivery to core protein, triggering encapsidation that is tightly coupled to particle envelopment. These results provide a striking example how a single viral protein exerts multiple functions to coordinate the steps from RNA replication to the assembly of infectious virus particles.

  1. Fuel assembly

    International Nuclear Information System (INIS)

    Abe, Hideaki; Sakai, Takao; Ishida, Tomio; Yokota, Norikatsu.

    1992-01-01

    The lower ends of a plurality of plate-like shape memory alloys are secured at the periphery of the upper inside of the handling head of a fuel assembly. As the shape memory alloy, a Cu-Zn alloy, a Ti-Pd alloy or a Fe-Ni alloy is used. When high temperature coolants flow out to the handling head, the shape memory alloy deforms by warping to the outer side more greatly toward the upper portion thereof with the temperature increase of the coolants. As the result, the shape of the flow channel of the coolants is changed so as to enlarge at the exit of the upper end of the fuel assembly. Then, the pressure loss of the coolants in the fuel assembly is decreased by the enlargement. Accordingly, the flow rate of the coolants in the fuel assembly is increased to lower the temperature of the coolants. Further, high temperature coolants and low temperature coolants are mixed sufficiently just above the fuel assembly. This can suppress the temperature fluctuation of the mixed coolants in the upper portion of the reactor core, thereby enabling to decrease a fatigue and failures of the structural components in the upper portion of the reactor core. (I.N.)

  2. Redefining bacterial origins of replication as centralized information processors.

    Science.gov (United States)

    Marczynski, Gregory T; Rolain, Thomas; Taylor, James A

    2015-01-01

    In this review we stress the differences between eukaryotes and bacteria with respect to their different cell cycles, replication mechanisms and genome organizations. One of the most basic and underappreciated differences is that a bacterial chromosome uses only one ori while eukaryotic chromosome uses multiple oris. Consequently, eukaryotic oris work redundantly in a cell cycle divided into separate phases: First inactive replication proteins assemble on eukaryotic oris, and then they await conditions (in the separate "S-phase") that activate only the ori-bound and pre-assembled replication proteins. S-phase activation (without re-assembly) ensures that a eukaryotic ori "fires" (starts replication) only once and that each chromosome consistently duplicates only once per cell cycle. This precise chromosome duplication does not require precise multiple ori firing in S-phase. A eukaryotic ori can fire early, late or not at all. The single bacterial ori has no such margin for error and a comparable imprecision is lethal. Single ori usage is not more primitive; it is a totally different strategy that distinguishes bacteria. We further argue that strong evolutionary pressures created more sophisticated single ori systems because bacteria experience extreme and rapidly changing conditions. A bacterial ori must rapidly receive and process much information in "real-time" and not just in "cell cycle time." This redefinition of bacterial oris as centralized information processors makes at least two important predictions: First that bacterial oris use many and yet to be discovered control mechanisms and second that evolutionarily distinct bacteria will use many very distinct control mechanisms. We review recent literature that supports both predictions. We will highlight three key examples and describe how negative-feedback, phospho-relay, and chromosome-partitioning systems act to regulate chromosome replication. We also suggest future studies and discuss using replication

  3. Fuel assembly

    International Nuclear Information System (INIS)

    Nakatsuka, Masafumi; Matsuzuka, Ryuji.

    1976-01-01

    Object: To provide a fuel assembly which can decrease pressure loss of coolant to uniform temperature. Structure: A sectional area of a flow passage in the vicinity of an inner peripheral surface of a wrapper tube is limited over the entire length to prevent the temperature of a fuel element in the outermost peripheral portion from being excessively decreased to thereby flatten temperature distribution. To this end, a plurality of pincture-frame-like sheet metals constituting a spacer for supporting a fuel assembly, which has a plurality of fuel elements planted lengthwise and in given spaced relation within the wrapper tube, is disposed in longitudinal grooves and in stacked fashion to form a substantially honeycomb-like space in cross section. The fuel elements are inserted and supported in the space to form a fuel assembly. (Kamimura, M.)

  4. Polyelectrolyte-surfactant complexes of poly[3,5-bis(dimethylaminomethyl)-4-hydroxystyrene]-block-poly(ethylene oxide) and sodium dodecyl sulfate: anomalous self-assembly behavior

    Czech Academy of Sciences Publication Activity Database

    Hajduová, J.; Procházka, K.; Šlouf, Miroslav; Angelov, Borislav; Mountrichas, G.; Pispas, S.; Štěpánek, M.

    2013-01-01

    Roč. 29, č. 18 (2013), s. 5443-5449 ISSN 0743-7463 R&D Projects: GA TA ČR TE01020118; GA ČR(CZ) GA13-02938S Institutional support: RVO:61389013 Keywords : polymer self-assemblies * light and X-ray scattering * electron microsocopy Subject RIV: JA - Electronics ; Optoelectronics, Electrical Engineering Impact factor: 4.384, year: 2013

  5. Novel theranostic zinc phthalocyanine-phospholipid complex self-assembled nanoparticles for imaging-guided targeted photodynamic treatment with controllable ROS production and shape-assisted enhanced cellular uptake.

    Science.gov (United States)

    Ma, Jinyuan; Li, Yang; Liu, Guihua; Li, Ai; Chen, Yilin; Zhou, Xinyi; Chen, Dengyue; Hou, Zhenqing; Zhu, Xuan

    2018-02-01

    The novel drug delivery system based on self-assembly of zinc phthalocyanine-soybean phosphatidylcholine (ZnPc-SPC) complex was developed by a co-solvent method followed by a nanoprecipitaion technique. DSPE-PEG-methotrexate (DSPE-PEG-MTX) was introduced on the surface of ZnPc-SPC self-assembled nanoparticles (ZS) to endow them with folate receptor-targeting property. NMR, XRD, FTIR, and UV-vis-NIR analysis demonstrated the weak molecular interaction between ZnPc and SPC. The ZS functionalized with DSPE-PEG-MTX (ZSPM) was successfully constructed with an average particle size of ∼170nm, a narrow size distribution, and could remain physiologically stable for at least 7days. In vitro cellular uptake and cytotoxicity studies demonstrated that ZSPM exhibited stronger cellular uptake efficacy and photodynamic cytotoxicity against HeLa and MCF-7 cells than ZS functionalized with DSPE-mPEG (ZSP) and free ZnPc. More importantly, ZSPM showed the enhanced accumulation effect at the tumor region compared with ZSP by the active-plus-passive targeting via enhanced permeability and retention (EPR) effect and folate receptor-mediated endocytosis. Furthermore, in vivo antitumor effect and histological analysis demonstrated the superior tumor growth inhibition effect of ZSPM. In addition, the needle-shape ZSP (ZSPN) exhibited better in vitro cellular uptake and in vivo tumor accumulation compared with ZSP due to the shape-assisted effect. Moreover, the interesting off-on switch effect of reactive oxygen species (ROS) production of ZnPc-SPC complex-based nanoparticles was discovered to achieve photodynamic treatment in a controllable way. These findings suggested that the ZnPc-SPC complex-based self-assembled nanoparticles could serve as a promising and effective formulation to achieve tumor-targeting fluorescence imaging and enhanced photodynamic treatment. Copyright © 2017. Published by Elsevier B.V.

  6. Replication of bacteriophage lambda DNA

    International Nuclear Information System (INIS)

    Tsurimoto, T.; Matsubara, K.

    1983-01-01

    In this paper results of studies on the mechanism of bacteriophage lambda replication using molecular biological and biochemical approaches are reported. The purification of the initiator proteins, O and P, and the role of the O and P proteins in the initiation of lambda DNA replication through interactions with specific DNA sequences are described. 47 references, 15 figures

  7. Fuel assemblies

    International Nuclear Information System (INIS)

    Nakamura, Mitsuya; Yamashita, Jun-ichi; Mochida, Takaaki.

    1986-01-01

    Purpose: To improve the fuel economy by increasing the reactivity at the latter burning stage of fuel assemblies and thereby increasing the burn-up degree. Constitution: At the later stage of the burning where the infinite multiplication factor of a fuel assembly is lowered, fuel rods are partially discharged to increase the fuel-moderator volume ratio in the fuel assembly. Then, plutonium is positively burnt by bringing the ratio near to an optimum point where the infinite multiplication factor becomes maximum and the reactivity of the fuel assembly is increased by utilizing the spectral shift effect. The number of the fuel rods to be removed is selected so as to approach the fuel-moderator atom number ratio where the infinite multiplication factor is maximum. Further, the positions where the thermal neutron fluxes are low are most effective for removing the rods and those positions between which no fuel rods are present and which are adjacent with neither the channel box nor the water rods are preferred. The rods should be removed at the time when the burning is proceeded at lest for one cycle. The reactivity is thus increased and the burn-up degree of fuels upon taking-out can be improved. (Kamimura, M.)

  8. Valve assembly

    International Nuclear Information System (INIS)

    Sandling, M.

    1981-01-01

    An improved valve assembly, used for controlling the flow of radioactive slurry, is described. Radioactive contamination of the air during removal or replacement of the valve is prevented by sucking air from the atmosphere through a portion of the structure above the valve housing. (U.K.)

  9. 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.

  10. 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.

  11. Geometric reasoning about assembly tools

    Energy Technology Data Exchange (ETDEWEB)

    Wilson, R.H.

    1997-01-01

    Planning for assembly requires reasoning about various tools used by humans, robots, or other automation to manipulate, attach, and test parts and subassemblies. This paper presents a general framework to represent and reason about geometric accessibility issues for a wide variety of such assembly tools. Central to the framework is a use volume encoding a minimum space that must be free in an assembly state to apply a given tool, and placement constraints on where that volume must be placed relative to the parts on which the tool acts. Determining whether a tool can be applied in a given assembly state is then reduced to an instance of the FINDPLACE problem. In addition, the author presents more efficient methods to integrate the framework into assembly planning. For tools that are applied either before or after their target parts are mated, one method pre-processes a single tool application for all possible states of assembly of a product in polynomial time, reducing all later state-tool queries to evaluations of a simple expression. For tools applied after their target parts are mated, a complementary method guarantees polynomial-time assembly planning. The author presents a wide variety of tools that can be described adequately using the approach, and surveys tool catalogs to determine coverage of standard tools. Finally, the author describes an implementation of the approach in an assembly planning system and experiments with a library of over one hundred manual and robotic tools and several complex assemblies.

  12. Stability versus exchange : A paradox in DNA replication

    NARCIS (Netherlands)

    Åberg, Christoffer; Duderstadt, Karl E.; van Oijen, Antoine M.

    2016-01-01

    Multi-component biological machines, comprising individual proteins with specialized functions, perform a variety of essential processes in cells. Once assembled, most such complexes are considered very stable, retaining individual constituents as long as required. However, rapid and frequent

  13. 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....

  14. 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 in the si......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...

  15. The Causes and Consequences of Topological Stress during DNA Replication.

    Science.gov (United States)

    Keszthelyi, Andrea; Minchell, Nicola E; Baxter, Jonathan

    2016-12-21

    The faithful replication of sister chromatids is essential for genomic integrity in every cell division. The replication machinery must overcome numerous difficulties in every round of replication, including DNA topological stress. Topological stress arises due to the double-stranded helical nature of DNA. When the strands are pulled apart for replication to occur, the intertwining of the double helix must also be resolved or topological stress will arise. This intrinsic problem is exacerbated by specific chromosomal contexts encountered during DNA replication. The convergence of two replicons during termination, the presence of stable protein-DNA complexes and active transcription can all lead to topological stresses being imposed upon DNA replication. Here we describe how replication forks respond to topological stress by replication fork rotation and fork reversal. We also discuss the genomic contexts where topological stress is likely to occur in eukaryotes, focusing on the contribution of transcription. Finally, we describe how topological stress, and the ways forks respond to it, may contribute to genomic instability in cells.

  16. Geminin: a major DNA replication safeguard in higher eukaryotes

    DEFF Research Database (Denmark)

    Melixetian, Marina; Helin, Kristian

    2004-01-01

    Eukaryotes have evolved multiple mechanisms to restrict DNA replication to once per cell cycle. These mechanisms prevent relicensing of origins of replication after initiation of DNA replication in S phase until the end of mitosis. Most of our knowledge of mechanisms controlling prereplication...... complex (preRC) formation are based on studies from yeast and Xenopus, while much less is known for mammalian cells. Here we discuss our recent data demonstrating that Geminin is required for preventing rereplication in human normal and cancer cells....

  17. Replication origin-flanking roadblocks reveal origin-licensing dynamics and altered sequence dependence.

    Science.gov (United States)

    Warner, Megan D; Azmi, Ishara F; Kang, Sukhyun; Zhao, Yanding; Bell, Stephen P

    2017-12-29

    In eukaryotes, DNA replication initiates from multiple origins of replication for timely genome duplication. These sites are selected by origin licensing, during which the core enzyme of the eukaryotic DNA replicative helicase, the Mcm2-7 (minichromosome maintenance) complex, is loaded at each origin. This origin licensing requires loading two Mcm2-7 helicases around origin DNA in a head-to-head orientation. Current models suggest that the origin-recognition complex (ORC) and cell-division cycle 6 (Cdc6) proteins recognize and encircle origin DNA and assemble an Mcm2-7 double-hexamer around adjacent double-stranded DNA. To test this model and assess the location of Mcm2-7 initial loading, we placed DNA-protein roadblocks at defined positions adjacent to the essential ORC-binding site within Saccharomyces cerevisiae origin DNA. Roadblocks were made either by covalent cross-linking of the HpaII methyltransferase to DNA or through binding of a transcription activator-like effector (TALE) protein. Contrary to the sites of Mcm2-7 recruitment being precisely defined, only single roadblocks that inhibited ORC-DNA binding showed helicase loading defects. We observed inhibition of helicase loading without inhibition of ORC-DNA binding only when roadblocks were placed on both sides of the origin to restrict sliding of a helicase-loading intermediate. Consistent with a sliding helicase-loading intermediate, when either one of the flanking roadblocks was eliminated, the remaining roadblock had no effect on helicase loading. Interestingly, either origin-flanking nucleosomes or roadblocks resulted in helicase loading being dependent on an additional origin sequence known to be a weaker ORC-DNA-binding site. Together, our findings support a model in which sliding helicase-loading intermediates increase the flexibility of the DNA sequence requirements for origin licensing. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  18. Kaposi's Sarcoma-Associated Herpesvirus K8 Is an RNA Binding Protein That Regulates Viral DNA Replication in Coordination with a Noncoding RNA.

    Science.gov (United States)

    Liu, Dongcheng; Wang, Yan; Yuan, Yan

    2018-01-10

    KSHV lytic replication and constant primary infection of fresh cells are crucial for viral tumorigenicity. Virus-encoded b-Zip family protein K8 plays an important role in viral DNA replication in both viral reactivation and de novo infection. The mechanism underlying the functional role of K8 in the viral life cycle is elusive. Here we report that K8 is a RNA binding protein, which also associates with many proteins including other RNA binding proteins. Many K8-involved protein-protein interactions are mediated by RNA. Using a c ross l inking and i mmuno p recipitation (CLIP) procedure combined with high-throughput sequencing, RNAs that are associated with K8 in BCBL-1 cells were identified, that include both viral (PAN, T1.4, T0.7 and etc.) and cellular (MALAT-1, MRP, 7SK and etc.) RNAs. An RNA-binding motif in K8 was defined, and mutation of the motif abolished the ability of K8 binding to many noncoding RNAs as well as viral DNA replication during de novo infection, suggesting that the K8 functions in viral replication are carried out through RNA association. The function of K8 and associated T1.4 RNA was investigated in details and results showed that T1.4 mediates the binding of K8 with ori-Lyt DNA. T1.4-K8 complex physically bound to KSHV ori-Lyt DNA and recruited other proteins and cofactors to assemble replication complex. Depletion of T1.4 abolished the DNA replication in primary infection. These findings provide mechanistic insights into the role of K8 in coordination with T1.4 RNA in regulating KSHV DNA replication during de novo infection. Importance Genome wide analyses of the mammalian transcriptome revealed that a large proportion of sequence previously annotated as noncoding region are actually transcribed and give rise to stable RNAs. Emergence of a large number of noncoding RNAs suggests that functional RNA-protein complexes exampled by ribosome or spliceosome are not ancient relics of the last riboorganism but would be well adapted for

  19. Fuel assemblies

    International Nuclear Information System (INIS)

    Echigoya, Hironori; Nomata, Terumitsu.

    1983-01-01

    Purpose: To render the axial distribution relatively flat. Constitution: First nuclear element comprises a fuel can made of zircalloy i.e., the metal with less neutron absorption, which is filled with a plurality of UO 2 pellets and sealed by using a lower end plug, a plenum spring and an upper end plug by means of welding. Second fuel element is formed by substituting a part of the UO 2 pellets with a water tube which is sealed with water and has a space for allowing the heat expansion. The nuclear fuel assembly is constituted by using the first and second fuel elements together. In such a structure, since water reflects neutrons and decrease their leakage to increase the temperature, reactivity is added at the upper portion of the fuel assembly to thereby flatten the axial power distribution. Accordingly, stable operation is possible only by means of deep control rods while requiring no shallow control rods. (Sekiya, K.)

  20. Replication-uncoupled histone deposition during adenovirus DNA replication.

    Science.gov (United States)

    Komatsu, Tetsuro; Nagata, Kyosuke

    2012-06-01

    In infected cells, the chromatin structure of the adenovirus genome DNA plays critical roles in its genome functions. Previously, we reported that in early phases of infection, incoming viral DNA is associated with both viral core protein VII and cellular histones. Here we show that in late phases of infection, newly synthesized viral DNA is also associated with histones. We also found that the knockdown of CAF-1, a histone chaperone that functions in the replication-coupled deposition of histones, does not affect the level of histone H3 bound on viral chromatin, although CAF-1 is accumulated at viral DNA replication foci together with PCNA. Chromatin immunoprecipitation assays using epitope-tagged histone H3 demonstrated that histone variant H3.3, which is deposited onto the cellular genome in a replication-independent manner, is selectively associated with both incoming and newly synthesized viral DNAs. Microscopic analyses indicated that histones but not USF1, a transcription factor that regulates viral late gene expression, are excluded from viral DNA replication foci and that this is achieved by the oligomerization of the DNA binding protein (DBP). Taken together, these results suggest that histone deposition onto newly synthesized viral DNA is most likely uncoupled with viral DNA replication, and a possible role of DBP oligomerization in this replication-uncoupled histone deposition is discussed.

  1. Assembling consumption

    DEFF Research Database (Denmark)

    Assembling Consumption marks a definitive step in the institutionalisation of qualitative business research. By gathering leading scholars and educators who study markets, marketing and consumption through the lenses of philosophy, sociology and anthropology, this book clarifies and applies...... the investigative tools offered by assemblage theory, actor-network theory and non-representational theory. Clear theoretical explanation and methodological innovation, alongside empirical applications of these emerging frameworks will offer readers new and refreshing perspectives on consumer culture and market...

  2. Influenza a virus assembly intermediates fuse in the cytoplasm.

    Directory of Open Access Journals (Sweden)

    Seema S Lakdawala

    2014-03-01

    Full Text Available Reassortment of influenza viral RNA (vRNA segments in co-infected cells can lead to the emergence of viruses with pandemic potential. Replication of influenza vRNA occurs in the nucleus of infected cells, while progeny virions bud from the plasma membrane. However, the intracellular mechanics of vRNA assembly into progeny virions is not well understood. Here we used recent advances in microscopy to explore vRNA assembly and transport during a productive infection. We visualized four distinct vRNA segments within a single cell using fluorescent in situ hybridization (FISH and observed that foci containing more than one vRNA segment were found at the external nuclear periphery, suggesting that vRNA segments are not exported to the cytoplasm individually. Although many cytoplasmic foci contain multiple vRNA segments, not all vRNA species are present in every focus, indicating that assembly of all eight vRNA segments does not occur prior to export from the nucleus. To extend the observations made in fixed cells, we used a virus that encodes GFP fused to the viral polymerase acidic (PA protein (WSN PA-GFP to explore the dynamics of vRNA assembly in live cells during a productive infection. Since WSN PA-GFP colocalizes with viral nucleoprotein and influenza vRNA segments, we used it as a surrogate for visualizing vRNA transport in 3D and at high speed by inverted selective-plane illumination microscopy. We observed cytoplasmic PA-GFP foci colocalizing and traveling together en route to the plasma membrane. Our data strongly support a model in which vRNA segments are exported from the nucleus as complexes that assemble en route to the plasma membrane through dynamic colocalization events in the cytoplasm.

  3. General Assembly

    CERN Multimedia

    Staff Association

    2016-01-01

    5th April, 2016 – Ordinary General Assembly of the Staff Association! In the first semester of each year, the Staff Association (SA) invites its members to attend and participate in the Ordinary General Assembly (OGA). This year the OGA will be held on Tuesday, April 5th 2016 from 11:00 to 12:00 in BE Auditorium, Meyrin (6-2-024). During the Ordinary General Assembly, the activity and financial reports of the SA are presented and submitted for approval to the members. This is the occasion to get a global view on the activities of the SA, its financial management, and an opportunity to express one’s opinion, including taking part in the votes. Other points are listed on the agenda, as proposed by the Staff Council. Who can vote? Only “ordinary” members (MPE) of the SA can vote. Associated members (MPA) of the SA and/or affiliated pensioners have a right to vote on those topics that are of direct interest to them. Who can give his/her opinion? The Ordinary General Asse...

  4. Fuel assembly

    International Nuclear Information System (INIS)

    Ueda, Sei; Ando, Ryohei; Mitsutake, Toru.

    1995-01-01

    The present invention concerns a fuel assembly suitable to a BWR-type reactor and improved especially with the nuclear characteristic, heat performance, hydraulic performance, dismantling or assembling performance and economical property. A part of poison rods are formed as a large-diameter/multi-region poison rods having a larger diameter than a fuel rod. A large number of fuel rods are disposed surrounding a large diameter water rod and a group of the large-diameter/multi-region poison rods in adjacent with the water rod. The large-diameter water rod has a burnable poison at the tube wall portion. At least a portion of the large-diameter poison rods has a coolant circulation portion allowing coolants to circulate therethrough. Since the large-diameter poison rods are disposed at a position of high neutron fluxes, a large neutron multiplication factor suppression effect can be provided, thereby enabling to reduce the number of burnable poison rods relative to fuels. As a result, power peaking in the fuel assembly is moderated and a greater amount of plutonium can be loaded. In addition the flow of cooling water which tends to gather around the large diameter water rod can be controlled to improve cooling performance of fuels. (N.H.)

  5. Illustrating how mechanical assemblies work

    KAUST Repository

    Mitra, Niloy J.

    2010-07-26

    How things work visualizations use a variety of visual techniques to depict the operation of complex mechanical assemblies. We present an automated approach for generating such visualizations. Starting with a 3D CAD model of an assembly, we first infer the motions of individual parts and the interactions between parts based on their geometry and a few user specified constraints. We then use this information to generate visualizations that incorporate motion arrows, frame sequences and animation to convey the causal chain of motions and mechanical interactions between parts. We present results for a wide variety of assemblies. © 2010 ACM.

  6. Illustrating how mechanical assemblies work

    KAUST Repository

    Mitra, Niloy J.

    2013-01-01

    How-things-work visualizations use a variety of visual techniques to depict the operation of complex mechanical assemblies. We present an automated approach for generating such visualizations. Starting with a 3D CAD model of an assembly, we first infer the motions of the individual parts and the interactions across the parts based on their geometry and a few user-specified constraints. We then use this information to generate visualizations that incorporate motion arrows, frame sequences, and animation to convey the causal chain of motions and mechanical interactions across parts. We demonstrate our system on a wide variety of assemblies. © 2013 ACM 0001-0782/13/01.

  7. Synchronous replication of remote storage

    OpenAIRE

    Mirzoev, Dr. Timur

    2014-01-01

    Storage replication is one of the essential requirements for network environments. While many forms of Network Attached Storage (NAS), Storage Area Networks (SAN) and other forms of network storage exist, there is a need for a reliable synchronous storage replication technique between distant sites (less than 1 mile). Such technology allows setting new standards for network failover and failback systems for virtual servers; specifically, addressing the growing need for effective disaster reco...

  8. Formation, surface characterization, and electrocatalytic application of self-assembled monolayer films of tetra-substituted manganese, iron, and cobalt benzylthio phthalocyanine complexes

    CSIR Research Space (South Africa)

    Akinbulu, IA

    2011-10-01

    Full Text Available Molecular thin films of manganese (SAM-2), iron (SAM-3), and cobalt (SAM-4) phthalocyanine complexes, non-peripherally tetra-substituted with benzylmercapto, were formed on polycrystalline gold disc electrode by selfassembly technique. Surface...

  9. Influence of electric field on fine structure of exciton complexes in CdTe/ZnTe self-assembled quantum dot

    International Nuclear Information System (INIS)

    Kowalik, K.; Kudelski, A.; Krebs, O.; Voisin, P.; Golnik, A.; Suffczynski, J.; Gaj, J.A.; Karczewski, G.; Kossut, J.

    2006-01-01

    We report on micro-photoluminescence (μ-PL) measurements of CdTe/ZnTe self-assembled quantum dots. Jumps in the line positions are observed and attributed to fluctuations of local electrostatic environment. We find that fine structures of exciton, biexciton and charged exciton originating from the same QD are correlated with line positions. We observe stronger influence of the field on the excitonic states of lower binding energy. The local field likely originates from charges localized at defects in the close vicinity of the QD, and correlations indicate that its direction is not completely random. (copyright 2006 WILEY-VCH Verlag GmbH and Co. KGaA, Weinheim) (orig.)

  10. CCDC39 is required for assembly of inner dynein arms and the dynein regulatory complex and for normal ciliary motility in humans and dogs

    DEFF Research Database (Denmark)

    Merveille, Anne-Christine; Davis, Erica E; Becker-Heck, Anita

    2011-01-01

    cilia that are responsible for airway clearance, the flagella that propel sperm cells and the nodal monocilia that determine left-right asymmetry. Recessive mutations that cause PCD have been identified in genes encoding components of the outer dynein arms, radial spokes and cytoplasmic pre......-assembly factors of axonemal dyneins, but these mutations account for only about 50% of cases of PCD. We exploited the unique properties of dog populations to positionally clone a new PCD gene, CCDC39. We found that loss-of-function mutations in the human ortholog underlie a substantial fraction of PCD cases......Primary ciliary dyskinesia (PCD) is an inherited disorder characterized by recurrent infections of the upper and lower respiratory tract, reduced fertility in males and situs inversus in about 50% of affected individuals (Kartagener syndrome). It is caused by motility defects in the respiratory...

  11. Static Electricity-Responsive Supramolecular Assembly.

    Science.gov (United States)

    Jintoku, Hirokuni; Ihara, Hirotaka; Matsuzawa, Yoko; Kihara, Hideyuki

    2017-12-01

    Stimuli-responsive materials can convert between molecular scale and macroscopic scale phenomena. Two macroscopic static electricity-responsive phenomena based on nanoscale supramolecular assemblies of a zinc porphyrin derivative are presented. One example involves the movement of supramolecular assemblies in response to static electricity. The assembly of a pyridine (Py) complex of the above-mentioned derivative in cyclohexane is drawn to a positively charged material, whereas the assembly of a 3,5-dimethylpyridine complex is drawn to a negatively charged material. The second phenomenon involves the movement of a non-polar solvent in response to static electrical stimulation. A cyclohexane solution containing a small quantity of the Py-complexed assembly exhibited a strong movement response towards negatively charged materials. Based on spectroscopic measurements and electron microscope observations, it was revealed that the assembled formation generates the observed response to static electricity. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  12. Shingle assembly

    Science.gov (United States)

    Dinwoodie, Thomas L.

    2007-02-20

    A barrier, such as a PV module, is secured to a base by a support to create a shingle assembly with a venting region defined between the barrier and base for temperature regulation. The first edge of one base may be interengageable with the second edge of an adjacent base to be capable of resisting first and second disengaging forces oriented perpendicular to the edges and along planes oriented parallel to and perpendicular to the base. A deflector may be used to help reduce wind uplift forces.

  13. Heater assembly

    International Nuclear Information System (INIS)

    Lee, K.; Ueng, Tzoushin.

    1991-01-01

    An electrical resistance heater, installed in the H1 borehole, is used to thermally perturb the rock mass through a controlled heating and cooling cycle. Heater power levels are controlled by a Variac power transformer and are measured by wattmeters. Temperatures are measured by thermocouples on the borehole wall and on the heater assembly. Power and temperature values are recorded by the DAS described in Chapter 12. The heater assembly consists of a 3.55-m (11.6-ft) long by 20.3-cm (8-in.) O.D., Type 304 stainless steel pipe, containing a tubular hairpin heating element. The element has a heated length of 3 m (9.84 ft). The power rating of the element is 10 kW; however, we plan to operate the unit at a maximum power of only 3 kW. The heater is positioned with its midpoint directly below the axis of the P2 borehole, as shown in the borehole configuration diagram. This heater midpoint position corresponds to a distance of approximately 8.5 m (27.9 ft) from the H1 borehole collar. A schematic of the heater assembly in the borehole is shown. The distance from the borehole collar to the closest point on the assembly (the front end) is 6.5 m (21.3 ft). A high-temperature inflatable packer, used to seal the borehole for moisture collection, is positioned 50 cm (19.7 in.) ahead of the heater front end. The heater is supported and centralized within the borehole by two skids, fabricated from 25-mm (1-in.) O.D. stainless steel pipe. Thermocouples are installed at a number of locations in the H1 borehole. Four thermocouples that are attached to the heater skin monitor temperatures on the outer surface of the can, while three thermocouples that are held in place by rock sections monitor borehole wall temperatures beneath the heater. Temperatures are also monitored at the heater terminal and on the packer hardware

  14. Assembling consumption

    DEFF Research Database (Denmark)

    Assembling Consumption marks a definitive step in the institutionalisation of qualitative business research. By gathering leading scholars and educators who study markets, marketing and consumption through the lenses of philosophy, sociology and anthropology, this book clarifies and applies the i...... societies. This is an essential reading for both seasoned scholars and advanced students of markets, economies and social forms of consumption....... the investigative tools offered by assemblage theory, actor-network theory and non-representational theory. Clear theoretical explanation and methodological innovation, alongside empirical applications of these emerging frameworks will offer readers new and refreshing perspectives on consumer culture and market...

  15. Template role of double-stranded RNA in tombusvirus replication.

    Science.gov (United States)

    Kovalev, Nikolay; Pogany, Judit; Nagy, Peter D

    2014-05-01

    Replication of plus-strand RNA [(+)RNA] viruses of plants is a relatively simple process that involves complementary minus-strand RNA [(-)RNA] synthesis and subsequent (+)RNA synthesis. However, the actual replicative form of the (-)RNA template in the case of plant (+)RNA viruses is not yet established unambiguously. In this paper, using a cell-free replication assay supporting a full cycle of viral replication, we show that replication of Tomato bushy stunt virus (TBSV) leads to the formation of double-stranded RNA (dsRNA). Using RNase digestion, DNAzyme, and RNA mobility shift assays, we demonstrate the absence of naked (-)RNA templates during replication. Time course experiments showed the rapid appearance of dsRNA earlier than the bulk production of new (+)RNAs, suggesting an active role for dsRNA in replication. Radioactive nucleotide chase experiments showed that the mechanism of TBSV replication involves the use of dsRNA templates in strand displacement reactions, where the newly synthesized plus strand replaces the original (+)RNA in the dsRNA. We propose that the use of dsRNA as a template for (+)RNA synthesis by the viral replicase is facilitated by recruited host DEAD box helicases and the viral p33 RNA chaperone protein. Altogether, this replication strategy allows TBSV to separate minus- and plus-strand syntheses in time and regulate asymmetrical RNA replication that leads to abundant (+)RNA progeny. Positive-stranded RNA viruses of plants use their RNAs as the templates for replication. First, the minus strand is synthesized by the viral replicase complex (VRC), which then serves as a template for new plus-strand synthesis. To characterize the nature of the (-)RNA in the membrane-bound viral replicase, we performed complete RNA replication of Tomato bushy stunt virus (TBSV) in yeast cell-free extracts and in plant extracts. The experiments demonstrated that the TBSV (-)RNA is present as a double-stranded RNA that serves as the template for TBSV

  16. Architecture of the Yeast Mitochondrial Iron-Sulfur Cluster Assembly Machinery: THE SUB-COMPLEX FORMED BY THE IRON DONOR, Yfh1 PROTEIN, AND THE SCAFFOLD, Isu1 PROTEIN.

    Science.gov (United States)

    Ranatunga, Wasantha; Gakh, Oleksandr; Galeano, Belinda K; Smith, Douglas Y; Söderberg, Christopher A G; Al-Karadaghi, Salam; Thompson, James R; Isaya, Grazia

    2016-05-06

    The biosynthesis of Fe-S clusters is a vital process involving the delivery of elemental iron and sulfur to scaffold proteins via molecular interactions that are still poorly defined. We reconstituted a stable, functional complex consisting of the iron donor, Yfh1 (yeast frataxin homologue 1), and the Fe-S cluster scaffold, Isu1, with 1:1 stoichiometry, [Yfh1]24·[Isu1]24 Using negative staining transmission EM and single particle analysis, we obtained a three-dimensional reconstruction of this complex at a resolution of ∼17 Å. In addition, via chemical cross-linking, limited proteolysis, and mass spectrometry, we identified protein-protein interaction surfaces within the complex. The data together reveal that [Yfh1]24·[Isu1]24 is a roughly cubic macromolecule consisting of one symmetric Isu1 trimer binding on top of one symmetric Yfh1 trimer at each of its eight vertices. Furthermore, molecular modeling suggests that two subunits of the cysteine desulfurase, Nfs1, may bind symmetrically on top of two adjacent Isu1 trimers in a manner that creates two putative [2Fe-2S] cluster assembly centers. In each center, conserved amino acids known to be involved in sulfur and iron donation by Nfs1 and Yfh1, respectively, are in close proximity to the Fe-S cluster-coordinating residues of Isu1. We suggest that this architecture is suitable to ensure concerted and protected transfer of potentially toxic iron and sulfur atoms to Isu1 during Fe-S cluster assembly. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  17. A human Polycomb isoform lacking the Pc box does not participate to PRC1 complexes but forms protein assemblies and represses transcription

    DEFF Research Database (Denmark)

    Völkel, Pamela; Le Faou, Perrine; Vandamme, Julien

    2012-01-01

    and alternative splicing events, the human CBX2 locus produces two transcripts: a 5-exon transcript that encodes the 532-amino acid CBX2-1 isoform that contains the conserved chromodomain and Pc box and a 4-exon transcript encoding a shorter isoform, CBX2-2, lacking the Pc box but still possessing a chromodomain....... Using biochemical approaches and a novel in vivo imaging assay, we show that the short CBX2-2 isoform lacking the Pc box, does not participate in PRC1 protein complexes, but self-associates in vivo and forms complexes of high molecular weight. Furthermore, the CBX2 short isoform is still able to repress...

  18. Fuel assembly

    International Nuclear Information System (INIS)

    Chaki, Masao; Nishida, Koji; Karasawa, Hidetoshi; Kanazawa, Toru; Orii, Akihito; Nagayoshi, Takuji; Kashiwai, Shin-ichi; Masuhara, Yasuhiro

    1998-01-01

    The present invention concerns a fuel assembly, for a BWR type nuclear reactor, comprising fuel rods in 9 x 9 matrix. The inner width of the channel box is about 132mm and the length of the fuel rods which are not short fuel rods is about 4m. Two water rods having a circular cross section are arranged on a diagonal line in a portion of 3 x 3 matrix at the center of the fuel assembly, and two fuel rods are disposed at vacant spaces, and the number of fuel rods is 74. Eight fuel rods are determined as short fuel rods among 74 fuel rods. Assuming the fuel inventory in the short fuel rod as X(kg), and the fuel inventory in the fuel rods other than the short fuel rods as Y(kg), X and Y satisfy the relation: X + Y ≥ 173m, Y ≤ - 9.7X + 292, Y ≤ - 0.3X + 203 and X > 0. Then, even when the short fuel rods are used, the fuel inventory is increased and fuel economy can be improved. (I.N.)

  19. General Assembly

    CERN Multimedia

    Staff Association

    2016-01-01

    Mardi 5 avril à 11 h 00 BE Auditorium Meyrin (6-2-024) Conformément aux statuts de l’Association du personnel, une Assemblée générale ordinaire est organisée une fois par année (article IV.2.1). Projet d’ordre du jour : Adoption de l’ordre du jour. Approbation du procès-verbal de l’Assemblée générale ordinaire du 5 mai 2015. Présentation et approbation du rapport d’activités 2015. Présentation et approbation du rapport financier 2015. Présentation et approbation du rapport des vérificateurs aux comptes pour 2015. Programme de travail 2016. Présentation et approbation du projet de budget 2016 Approbation du taux de cotisation pour 2017. Modifications aux Statuts de l'Association du personnel proposée. Élections des membres de la Commissio...

  20. General Assembly

    CERN Multimedia

    Staff Association

    2017-01-01

    Conformément aux statuts de l’Association du personnel, une Assemblée générale ordinaire est organisée une fois par année (article IV.2.1). Projet d’ordre du jour : Adoption de l’ordre du jour. Approbation du procès-verbal de l’Assemblée générale ordinaire du 5 avril 2016. Présentation et approbation du rapport d’activités 2016. Présentation et approbation du rapport financier 2016. Présentation et approbation du rapport des vérificateurs aux comptes pour 2016. Programme de travail 2017. Présentation et approbation du projet de budget 2017 Approbation du taux de cotisation pour 2018. Modifications aux Statuts de l'Association du personnel proposées. Élections des membres de la Commission électorale. Élections des vérifica...

  1. General Assembly

    CERN Multimedia

    Staff Association

    2015-01-01

    Mardi 5 mai à 11 h 00 Salle 13-2-005 Conformément aux statuts de l’Association du personnel, une Assemblée générale ordinaire est organisée une fois par année (article IV.2.1). Projet d’ordre du jour : 1- Adoption de l’ordre du jour. 2- Approbation du procès-verbal de l’Assemblée générale ordinaire du 22 mai 2014. 3- Présentation et approbation du rapport d’activités 2014. 4- Présentation et approbation du rapport financier 2014. 5- Présentation et approbation du rapport des vérificateurs aux comptes pour 2014. 6- Programme 2015. 7- Présentation et approbation du projet de budget 2015 et taux de cotisation pour 2015. 8- Pas de modifications aux Statuts de l'Association du personnel proposée. 9- Élections des membres de la Commission é...

  2. General assembly

    CERN Multimedia

    Staff Association

    2015-01-01

    Mardi 5 mai à 11 h 00 Salle 13-2-005 Conformément aux statuts de l’Association du personnel, une Assemblée générale ordinaire est organisée une fois par année (article IV.2.1). Projet d’ordre du jour : Adoption de l’ordre du jour. Approbation du procès-verbal de l’Assemblée générale ordinaire du 22 mai 2014. Présentation et approbation du rapport d’activités 2014. Présentation et approbation du rapport financier 2014. Présentation et approbation du rapport des vérificateurs aux comptes pour 2014. Programme 2015. Présentation et approbation du projet de budget 2015 et taux de cotisation pour 2015. Pas de modifications aux Statuts de l'Association du personnel proposée. Élections des membres de la Commission électorale. &am...

  3. Fuel assembly

    International Nuclear Information System (INIS)

    Nomata, Terumitsu.

    1993-01-01

    Among fuel pellets to be loaded to fuel cans of a fuel assembly, fuel pellets having a small thermal power are charged in a region from the end of each of spacers up to about 50mm on the upstream of coolants that flow vertically at the periphery of fuel rods. Coolants at the periphery of fuel rods are heated by the heat generation, to result in voids. However, since cooling effect on the upstream of the spacers is low due to influences of the spacers. Further, since the fuel pellets disposed in the upstream region have small thermal power, a void coefficient is not increased. Even if a thermal power exceeding cooling performance should be generated, there is no worry of causing burnout in the upstream region. Even if burnout should be caused, safety margin and reliability relative to burnout are improved, to increase an allowable thermal power, thereby enabling to improve integrity and reliability of fuel rods and fuel assemblies. (N.H.)

  4. Several tetratricopeptide repeat (TPR) motifs of FANCG are required for assembly of the BRCA2/D1-D2-G-X3 complex, FANCD2 monoubiquitylation and phleomycin resistance

    Energy Technology Data Exchange (ETDEWEB)

    Wilson, James B. [Molecular Oncology and Stem Cell Research Group, School of Biological Sciences, University of Liverpool, Biosciences Building, Crown Street, Liverpool L69 7ZB (United Kingdom); Blom, Eric [Department of Clinical Genetics and Human Genetics, VU University Medical Center, Van der Boechorststraat 7, NL-1081 BT Amsterdam (Netherlands); Cunningham, Ryan; Xiao, Yuxuan [Molecular Oncology and Stem Cell Research Group, School of Biological Sciences, University of Liverpool, Biosciences Building, Crown Street, Liverpool L69 7ZB (United Kingdom); Kupfer, Gary M. [Departments of Pediatrics and Pathology, Yale University School of Medicine, Section of Hematology/Oncology, 333 Cedar Street, New Haven, CT 0652 (United States); Jones, Nigel J., E-mail: njjones@liv.ac.uk [Molecular Oncology and Stem Cell Research Group, School of Biological Sciences, University of Liverpool, Biosciences Building, Crown Street, Liverpool L69 7ZB (United Kingdom)

    2010-07-07

    The Fanconi anaemia (FA) FANCG protein is an integral component of the FA nuclear core complex that is required for monoubiquitylation of FANCD2. FANCG is also part of another protein complex termed D1-D2-G-X3 that contains FANCD2 and the homologous recombination repair proteins BRCA2 (FANCD1) and XRCC3. Formation of the D1-D2-G-X3 complex is mediated by serine-7 phosphorylation of FANCG and occurs independently of the FA core complex and FANCD2 monoubiquitylation. FANCG contains seven tetratricopeptide repeat (TPR) motifs that mediate protein-protein interactions and here we show that mutation of several of the TPR motifs at a conserved consensus residue ablates the in vivo binding activity of FANCG. Expression of mutated TPR1, TPR2, TPR5 and TPR6 in Chinese hamster fancg mutant NM3 fails to functionally complement its hypersensitivities to mitomycin C (MMC) and phleomycin and fails to restore FANCD2 monoubiquitylation. Using co-immunoprecipitation analysis, we demonstrate that these TPR-mutated FANCG proteins fail to interact with BRCA2, XRCC3, FANCA or FANCF. The interactions of other proteins in the D1-D2-G-X3 complex are also absent, including the interaction of BRCA2 with both the monoubiquitylated (FANCD2-L) and non-ubiquitylated (FANCD2-S) isoforms of FANCD2. Interestingly, a mutation of TPR7 (R563E), that complements the MMC and phleomycin hypersensitivity of human FA-G EUFA316 cells, fails to complement NM3, despite the mutated FANCG protein co-precipitating with FANCA, BRCA2 and XRCC3. Whilst interaction of TPR7-mutated FANCG with FANCF does appear to be reduced in NM3, FANCD2 is monoubiquitylated suggesting that sub-optimal interactions of FANCG in the core complex and the D1-D2-G-X3 complex are responsible for the observed MMC- and phleomycin-hypersensitivity, rather than a defect in FANCD2 monoubiquitylation. Our data demonstrate that FANCG functions as a mediator of protein-protein interactions and is vital for the assembly of multi-protein complexes

  5. 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.

  6. Electrical-field-induced structural change and charge transfer of lanthanide-salophen complexes assembled on carbon nanotube field effect transistor devices.

    OpenAIRE

    Magadur , Gurvan; Bouanis , Fatima Zara; Norman , Evgeny; Guillot , Regis; Lauret , Jean Sebastien; Huc , Vincent; Cojocaru , Costel Sorin; Mallah , Talal

    2012-01-01

    International audience; The application of a negative gate voltage on a carbon nanotube field effect transistor decorated by a binuclear Tb(III) complex leads to the generation of a negatively charged mononuclear one, presenting an electron density transfer to the nanotube and ambipolar behaviour.

  7. Investigating the early stages of Photosystem II assembly in Synechocystis sp. PCC 6803: isolation of CP47 and CP43 complexe

    Czech Academy of Sciences Publication Activity Database

    Boehm, M.; Romero, E.; Reisinger, V.; Yu, J.; Komenda, Josef; Eichacker, L. A.; Dekker, J. P.; Nixon, P. J.

    2011-01-01

    Roč. 286, č. 17 (2011), 14812-14819 ISSN 0021-9258 R&D Projects: GA AV ČR IAA400200801 Institutional research plan: CEZ:AV0Z50200510 Keywords : ENERGY CHLOROPHYLL STATES * ANTENNA PROTEIN COMPLEX * OXYGEN-EVOLVING CENTER Subject RIV: EE - Microbiology, Virology Impact factor: 4.773, year: 2011

  8. Singlet and triplet energy transfer dynamics in self-assembled axial porphyrin-anthracene complexes: towards supra-molecular structures for photon upconversion.

    Science.gov (United States)

    Gray, Victor; Küçüköz, Betül; Edhborg, Fredrik; Abrahamsson, Maria; Moth-Poulsen, Kasper; Albinsson, Bo

    2018-03-14

    Energy and electron transfer reactions are central to many different processes and research fields, from photosynthesis and solar energy harvesting to biological and medical applications. Herein we report a comprehensive study of the singlet and triplet energy transfer dynamics in porphyrin-anthracene coordination complexes. Seven newly synthesized pyridine functionalized anthracene ligands, five with various bridge lengths and two dendrimer structures containing three and seven anthracene units, were prepared. We found that triplet energy transfer from ruthenium octaethylporphyrin to an axially coordinated anthracene is possible, and is in some cases followed by back triplet energy transfer to the porphyrin. The triplet energy transfer follows an exponential distance dependence with an attenuation factor, β, of 0.64 Å -1 . Further, singlet energy transfer from anthracene to the ruthenium porphyrin appears to follow a R 6 Förster distance dependence. Porphyrin-anthracene complexes are also used as triplet sensitizers for triplet-triplet annihilation (TTA) based photon upconversion, demonstrating their potential for photophysical and photochemical applications. The triplet lifetime of the complex is extended by the anthracene ligands, resulting in a threefold increase in the upconversion efficiency, Φ UC to 4.5%, compared to the corresponding ruthenium porphyrin-pyridine complex. Based on the results herein we discuss the future design of supra-molecular structures for TTA upconversion.

  9. The effects of linear assembly of two carbazole groups on acid-base and DNA-binding properties of a ruthenium(II) complex

    Science.gov (United States)

    Chen, Xi; Xue, Long-Xin; Ju, Chun-Chuan; Wang, Ke-Zhi

    2013-07-01

    A novel Ru(II) complex of [Ru(bpy)2(Hbcpip)](ClO4)2 {where bpy = 2,2-bipyridine, Hbcpip = 2-(4-(9H-3,9'-bicarbazol-9-yl)phenyl)-1H-imidazo[4,5-f][1,10]phenanthroline} is synthesized and characterized. Calf-thymus DNA-binding properties of the complex were studied by UV-vis absorption and luminescence titrations, steady-state emission quenching by [Fe(CN)6]4-, DNA competitive binding with ethidium bromide, thermal denaturation and DNA viscosity measurements. The results indicate that the complex partially intercalated into the DNA with a binding constant of (5.5 ± 1.4) × 105 M-1 in buffered 50 mM NaCl. The acid-base properties of the complex were also studied by UV-visible and luminescence spectrophotometric pH titrations, and ground- and excited-state acidity ionization constant values were derived.

  10. Break induced replication in eukaryotes: mechanisms, functions, and consequences.

    Science.gov (United States)

    Sakofsky, Cynthia J; Malkova, Anna

    2017-08-01

    Break-induced replication (BIR) is an important pathway specializing in repair of one-ended double-strand DNA breaks (DSBs). This type of DSB break typically arises at collapsed replication forks or at eroded telomeres. BIR initiates by invasion of a broken DNA end into a homologous template followed by initiation of DNA synthesis that can proceed for hundreds of kilobases. This synthesis is drastically different from S-phase replication in that instead of a replication fork, BIR proceeds via a migrating bubble and is associated with conservative inheritance of newly synthesized DNA. This unusual mode of DNA replication is responsible for frequent genetic instabilities associated with BIR, including hyper-mutagenesis, which can lead to the formation of mutation clusters, extensive loss of heterozygosity, chromosomal translocations, copy-number variations and complex genomic rearrangements. In addition to budding yeast experimental systems that were initially employed to investigate eukaryotic BIR, recent studies in different organisms including humans, have provided multiple examples of BIR initiated within different cellular contexts, including collapsed replication fork and telomere maintenance in the absence of telomerase. In addition, significant progress has been made towards understanding microhomology-mediated BIR (MMBIR) that can promote complex chromosomal rearrangements, including those associated with cancer and those leading to a number of neurological disorders in humans.

  11. An experimental and theoretical magneto-structural study of polynuclear Ni(II) complexes assembled from a versatile bis(salicylaldehyde)diamine polytopic ligand.

    Science.gov (United States)

    Oyarzabal, Itziar; Ruiz, José; Mota, Antonio J; Rodríguez-Diéguez, Antonio; Seco, José M; Colacio, Enrique

    2015-04-21

    Six novel Ni(II) complexes, ranging from mononuclear to tetranuclear, have been prepared from the polytopic symmetrical Mannich base ligand N,N'-dimethyl-N,N'-bis(2-hidroxy-3-formyl-5-bromo-benzyl)ethylenediamine (H2L) and different anionic coligands: [Ni(H2L)(NO3)(H2O)]NO3·H2O (), [Ni2(μ-L)(acac)2(H2O)]·CH3CN (), [Ni2(μ-L)(μ-OAc)(NCS)] (), [Ni3(μ-L)2(μ-OH2)2(H2O)(CH3CN)](NO3)2·4CH3CN (), [Ni4(μ-L)2(μ-OAc)2(μ-OCH3)2]·6H2O·2CH3OH () and [Ni4(μ-L)2(μ-OAc)2(μ-N3)2]·2H2O·CH3OH (). These complexes have been characterized by single crystal X-ray diffraction, magnetic measurements and DFT theoretical calculations. The structural analysis of these complexes reveals that the anionic coligand and reaction conditions play a fundamental role in determining their final structures and magnetic properties. Compound contains a monomeric cationic unit with the nickel ion coordinated in the external O4 site of the compartmental ligand H2L, which acts in a neutral zwitterionic form. Complexes and are dinuclear Ni2 neutral entities, in which the Ni(II) ions are connected through two μ-phenoxido bridging groups. The Ni(O)2Ni bridging fragment in is almost planar, whereas in is bent due to the additional presence of a syn-syn acetate bridge connecting the Ni(II) atoms. Complex has a bent trinuclear structure with double μ-phenoxido/μ-water bridges between the central and terminal nickel atoms. Complexes and are Ni4 complexes with defective dicubane structures, in which triple μ-phenoxido/μ1,1,1-X/syn-syn acetate and double μ-phenoxido/μ1,1,1-X mixed bridges connect central and terminal Ni(II) atoms, whereas double μ1,1,1-X bridging ligands link the central Ni(II) ions (X = methoxido and azido groups for and , respectively). Magnetic susceptibility measurements reveal that complex shows a moderate antiferromagnetic interaction between the Ni(II) ions through the double di-μ-phenoxido bridge, leading to a S = 0 ground state. Compared to , complex shows a much

  12. The Chromatin Assembly Factor Complex 1 (CAF1) and 5-Azacytidine (5-AzaC) Affect Cell Motility in Src-transformed Human Epithelial Cells.

    Science.gov (United States)

    Endo, Akinori; Ly, Tony; Pippa, Raffaella; Bensaddek, Dalila; Nicolas, Armel; Lamond, Angus I

    2017-01-06

    Tumor invasion into surrounding stromal tissue is a hallmark of high grade, metastatic cancers. Oncogenic transformation of human epithelial cells in culture can be triggered by activation of v-Src kinase, resulting in increased cell motility, invasiveness, and tumorigenicity and provides a valuable model for studying how changes in gene expression cause cancer phenotypes. Here, we show that epithelial cells transformed by activated Src show increased levels of DNA methylation and that the methylation inhibitor 5-azacytidine (5-AzaC) potently blocks the increased cell motility and invasiveness induced by Src activation. A proteomic screen for chromatin regulators acting downstream of activated Src identified the replication-dependent histone chaperone CAF1 as an important factor for Src-mediated increased cell motility and invasion. We show that Src causes a 5-AzaC-sensitive decrease in both mRNA and protein levels of the p150 (CHAF1A) and p60 (CHAF1B), subunits of CAF1. Depletion of CAF1 in untransformed epithelial cells using siRNA was sufficient to recapitulate the increased motility and invasive phenotypes characteristic of transformed cells without activation of Src. Maintaining high levels of CAF1 by exogenous expression suppressed the increased cell motility and invasiveness phenotypes when Src was activated. These data identify a critical role of CAF1 in the dysregulation of cell invasion and motility phenotypes seen in transformed cells and also highlight an important role for epigenetic remodeling through DNA methylation for Src-mediated induction of cancer phenotypes. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  13. The Chromatin Assembly Factor Complex 1 (CAF1) and 5-Azacytidine (5-AzaC) Affect Cell Motility in Src-transformed Human Epithelial Cells*

    Science.gov (United States)

    Endo, Akinori; Ly, Tony; Pippa, Raffaella; Bensaddek, Dalila; Nicolas, Armel; Lamond, Angus I.

    2017-01-01

    Tumor invasion into surrounding stromal tissue is a hallmark of high grade, metastatic cancers. Oncogenic transformation of human epithelial cells in culture can be triggered by activation of v-Src kinase, resulting in increased cell motility, invasiveness, and tumorigenicity and provides a valuable model for studying how changes in gene expression cause cancer phenotypes. Here, we show that epithelial cells transformed by activated Src show increased levels of DNA methylation and that the methylation inhibitor 5-azacytidine (5-AzaC) potently blocks the increased cell motility and invasiveness induced by Src activation. A proteomic screen for chromatin regulators acting downstream of activated Src identified the replication-dependent histone chaperone CAF1 as an important factor for Src-mediated increased cell motility and invasion. We show that Src causes a 5-AzaC-sensitive decrease in both mRNA and protein levels of the p150 (CHAF1A) and p60 (CHAF1B), subunits of CAF1. Depletion of CAF1 in untransformed epithelial cells using siRNA was sufficient to recapitulate the increased motility and invasive phenotypes characteristic of transformed cells without activation of Src. Maintaining high levels of CAF1 by exogenous expression suppressed the increased cell motility and invasiveness phenotypes when Src was activated. These data identify a critical role of CAF1 in the dysregulation of cell invasion and motility phenotypes seen in transformed cells and also highlight an important role for epigenetic remodeling through DNA methylation for Src-mediated induction of cancer phenotypes. PMID:27872192

  14. Phosphorylation of the Ndc80 complex protein, HEC1, by Nek2 kinase modulates chromosome alignment and signaling of the spindle assembly checkpoint.

    Science.gov (United States)

    Wei, Randy; Ngo, Bryan; Wu, Guikai; Lee, Wen-Hwa

    2011-10-01

    The spindle assemble checkpoint (SAC) is critical for accurate chromosome segregation. Hec1 contributes to chromosome segregation in part by mediating SAC signaling and chromosome alignment. However, the molecular mechanism by which Hec1 modulates checkpoint signaling and alignment remains poorly understood. We found that Hec1 serine 165 (S165) is preferentially phosphorylated at kinetochores. Phosphorylated Hec1 serine 165 (pS165) specifically localized to kinetochores of misaligned chromosomes, showing a spatiotemporal distribution characteristic of SAC molecules. Expressing an RNA interference (RNAi)-resistant S165A mutant in Hec1-depleted cells permitted normal progression to metaphase, but accelerated the metaphase-to-anaphase transition. The S165A cells were defective in Mad1 and Mad2 localization to kinetochores, regardless of attachment status. These cells often entered anaphase with lagging chromosomes and elicited increased segregation errors and cell death. In contrast, expressing S165E mutant in Hec1-depleted cells triggered defective chromosome alignment and severe mitotic arrest associated with increased Mad1/Mad2 signals at prometaphase kinetochores. A small portion of S165E cells eventually bypassed the SAC but showed severe segregation errors. Nek2 is the primary kinase responsible for kinetochore pS165, while PP1 phosphatase may dephosphorylate pS165 during SAC silencing. Taken together, these results suggest that modifications of Hec1 S165 serve as an important mechanism in modulating SAC signaling and chromosome alignment.

  15. Viral and Cellular Components of AAV2 Replication Compartments

    Science.gov (United States)

    Vogel, Rebecca; Seyffert, Michael; Pereira, Bruna de Andrade; Fraefel, Cornel

    2013-01-01

    Adeno-associated virus 2 (AAV2) is a helpervirus-dependent parvovirus with a bi-phasic life cycle comprising latency in absence and lytic replication in presence of a helpervirus, such as adenovirus (Ad) or herpes simplex virus type 1 (HSV-1). Helpervirus-supported AAV2 replication takes place in replication compartments (RCs) in the cell nucleus where virus DNA replication and transcription occur. RCs consist of a defined set of helper virus-, AAV2-, and cellular proteins. Here we compare the profile of cellular proteins recruited into AAV2 RCs or identified in Rep78-associated complexes when either Ad or HSV-1 is the helpervirus, and we discuss the potential roles of some of these proteins in AAV2 and helpervirus infection. PMID:24222808

  16. Regulation of beta cell replication

    DEFF Research Database (Denmark)

    Lee, Ying C; Nielsen, Jens Høiriis

    2008-01-01

    Beta cell mass, at any given time, is governed by cell differentiation, neogenesis, increased or decreased cell size (cell hypertrophy or atrophy), cell death (apoptosis), and beta cell proliferation. Nutrients, hormones and growth factors coupled with their signalling intermediates have been...... suggested to play a role in beta cell mass regulation. In addition, genetic mouse model studies have indicated that cyclins and cyclin-dependent kinases that determine cell cycle progression are involved in beta cell replication, and more recently, menin in association with cyclin-dependent kinase...... inhibitors has been demonstrated to be important in beta cell growth. In this review, we consider and highlight some aspects of cell cycle regulation in relation to beta cell replication. The role of cell cycle regulation in beta cell replication is mostly from studies in rodent models, but whether...

  17. Defects of mitochondrial DNA replication.

    Science.gov (United States)

    Copeland, William C

    2014-09-01

    Mitochondrial DNA is replicated by DNA polymerase γ in concert with accessory proteins such as the mitochondrial DNA helicase, single-stranded DNA binding protein, topoisomerase, and initiating factors. Defects in mitochondrial DNA replication or nucleotide metabolism can cause mitochondrial genetic diseases due to mitochondrial DNA deletions, point mutations, or depletion, which ultimately cause loss of oxidative phosphorylation. These genetic diseases include mitochondrial DNA depletion syndromes such as Alpers or early infantile hepatocerebral syndromes, and mitochondrial DNA deletion disorders, such as progressive external ophthalmoplegia, ataxia-neuropathy, or mitochondrial neurogastrointestinal encephalomyopathy. This review focuses on our current knowledge of genetic defects of mitochondrial DNA replication (POLG, POLG2, C10orf2, and MGME1) that cause instability of mitochondrial DNA and mitochondrial disease. © The Author(s) 2014.

  18. Biomarkers of replicative senescence revisited

    DEFF Research Database (Denmark)

    Nehlin, Jan

    2016-01-01

    Biomarkers of replicative senescence can be defined as those ultrastructural and physiological variations as well as molecules whose changes in expression, activity or function correlate with aging, as a result of the gradual exhaustion of replicative potential and a state of permanent cell cycle...... arrest. The biomarkers that characterize the path to an irreversible state of cell cycle arrest due to proliferative exhaustion may also be shared by other forms of senescence-inducing mechanisms. Validation of senescence markers is crucial in circumstances where quiescence or temporary growth arrest may...... be triggered or is thought to be induced. Pre-senescence biomarkers are also important to consider as their presence indicate that induction of aging processes is taking place. The bona fide pathway leading to replicative senescence that has been extensively characterized is a consequence of gradual reduction...

  19. Personality and Academic Motivation: Replication, Extension, and Replication

    Science.gov (United States)

    Jones, Martin H.; McMichael, Stephanie N.

    2015-01-01

    Previous work examines the relationships between personality traits and intrinsic/extrinsic motivation. We replicate and extend previous work to examine how personality may relate to achievement goals, efficacious beliefs, and mindset about intelligence. Approximately 200 undergraduates responded to the survey with a 150 participants replicating…

  20. The Psb27 Assembly Factor Binds to the CP43 Complex of Photosystem II in the Cyanobacterium Synechocystis sp PCC 6803

    Czech Academy of Sciences Publication Activity Database

    Komenda, Josef; Knoppová, Jana; Kopečná, Jana; Sobotka, Roman; Halada, Petr; Yu, J.; Nickelsen, J.; Boehm, M.; Nixon, P. J.

    2012-01-01

    Roč. 158, č. 1 (2012), s. 476-486 ISSN 0032-0889 R&D Projects: GA ČR(CZ) GAP501/11/0377; GA ČR GAP501/10/1000; GA MŠk LC07017 Institutional research plan: CEZ:AV0Z50200510 Keywords : MEMBRANE-PROTEIN COMPLEXES * SP PCC-6803 * D1 PROTEIN Subject RIV: EE - Microbiology, Virology Impact factor: 6.555, year: 2012

  1. The Assembly Factor SDHAF2 Is Dispensable for Flavination of the Catalytic Subunit of Mitochondrial Complex II in Breast Cancer Cells

    Czech Academy of Sciences Publication Activity Database

    Bezawork-Geleta, A.; Dong, L.; Rohlena, Jakub; Neužil, Jiří

    2016-01-01

    Roč. 291, č. 41 (2016), s. 21414-21420 ISSN 0021-9258 R&D Projects: GA ČR GA15-02203S; GA ČR(CZ) GA16-22823S; GA MŠk(CZ) ED1.1.00/02.0109 Institutional support: RVO:86652036 Keywords : cancer biology * mitochondrial respiratory chain complex * SUCCINATE-UBIQUINONE OXIDOREDUCTASE Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 4.125, year: 2016

  2. Formation of assemblies comprising Ru-polypyridine complexes and CdSe nanocrystals studied by ATR-FTIR spectroscopy and DFT modeling.

    Science.gov (United States)

    Koposov, Alexey Y; Cardolaccia, Thomas; Albert, Victor; Badaeva, Ekaterina; Kilina, Svetlana; Meyer, Thomas J; Tretiak, Sergei; Sykora, Milan

    2011-07-05

    The interaction between CdSe nanocrystals (NCs) passivated with trioctylphosphine oxide (TOPO) ligands and a series of Ru-polypyridine complexes-[Ru(bpy)(3)](PF(6))(2) (1), [Ru(bpy)(2)(mcb)](PF(6))(2) (2), [Ru(bpy)(mcb)(2)](BarF)(2) (3), and [Ru(tpby)(2)(dcb)](PF(6))(2) (4) (where bpy = 2,2'-bipyridine, mcb = 4-carboxy-4'-methyl-2,2'-bipyridine, tbpy = 4,4'-di-tert-butyl-2,2'-bipyridine; dcb = 4,4'-dicarboxy-2,2'-bipyridine, and BarF = tetrakis[3,5-bis(trifluoromethyl)phenyl]borate)-was studied by attenuated total reflectance FTIR (ATR-FTIR) and modeled using density functional theory (DFT). ATR-FTIR studies reveal that when the solid film of NCs is exposed to an acetonitrile solution of 2, 3, or 4, the complexes chemically bind to the NC surface through their carboxylic acid groups, replacing TOPO ligands. The corresponding spectral changes are observed on a time scale of minutes. In the case of 2, the FTIR spectral changes clearly show that the complex adsorption is associated with a loss of proton from the carboxylic acid group. In the case of 3 and 4, deprotonation of the anchoring group is also detected, while the second, "spectrator" carboxylic acid group remains protonated. The observed energy difference between the symmetric, ν(s), and asymmetric, ν(as), stretch of the deprotonated carboxylic acid group suggests that the complexes are bound to the NC surface via a bridging mode. The results of DFT modeling are consistent with the experiment, showing that for the deprotonated carboxylic acid group the coupling to two Cd atoms via a bridging mode is the energetically most favorable mode of attachment for all nonequivalent NC surface sites and that the attachment of the protonated carboxylic acid is thermodynamically significantly less favorable. © 2011 American Chemical Society

  3. International Expansion through Flexible Replication

    DEFF Research Database (Denmark)

    Jonsson, Anna; Foss, Nicolai Juul

    2011-01-01

    to local environments and under the impact of new learning. To illuminate these issues, we draw on a longitudinal in-depth study of Swedish home furnishing giant IKEA, involving more than 70 interviews. We find that IKEA has developed organizational mechanisms that support an ongoing learning process aimed...... at frequent modification of the format for replication. Another finding is that IKEA treats replication as hierarchical: lower-level features (marketing efforts, pricing, etc.) are allowed to vary across IKEA stores in response to market-based learning, while higher-level features (fundamental values, vision...

  4. Spacer-Controlled Supramolecular Assemblies of Cu(II with Bis(2-Hydroxyphenylimine Ligands. from Monoligand Complexes to Double-Stranded Helicates and Metallomacrocycles

    Directory of Open Access Journals (Sweden)

    Norman Kelly

    2016-09-01

    Full Text Available Reaction of Cu(NO32·3H2O or Cu(CH3COO2·H2O with the bis(2-hydroxyphenylimine ligands H2L1-H2L4 gave four Cu(II complexes of composition [Cu2(L1(NO32(H2O]·MeOH, [Cu2(L22], [Cu2(L32] and [Cu2(L42]·2MeOH. Depending on the spacer unit, the structures are characterized by a dinuclear arrangement of Cu(II within one ligand (H2L1, by a double-stranded [2+2] helical binding mode (H2L2 and H2L3 and a [2 + 2] metallomacrocycle formation (H2L4. In these complexes, the Cu(II coordination geometries are quite different, varying between common square planar or square pyramidal arrangements, and rather rare pentagonal bipyramidal and tetrahedral geometries. In addition, solution studies of the complex formation using UV/Vis and ESI-MS as well as solvent extraction are reported.

  5. Synthetic assembly of novel avidin-biotin-GlcNAc (ABG) complex as an attractive bio-probe and its interaction with wheat germ agglutinin (WGA).

    Science.gov (United States)

    Kumari, Amrita; Koyama, Tetsuo; Hatano, Ken; Matsuoka, Koji

    2016-10-01

    A tetravalent GlcNAc pendant glycocluster was constructed with terminal biotin through C6 linker. To acquire the multivalent carbohydrate-protein interactions, we synthesized a glycopolymer of tetrameric structure using N-acetyl-d-glucosamine (GlcNAc) as the target carbohydrate by the use of 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) as coupling reagent, followed by biotin-avidin complexation leading to the formation of glycocluster of avidin-biotin-GlcNAc conjugate (ABG complex). The dynamic light scattering (DLS) system was implied for size detection and to check the binding affinity of GlcNAc conjugate with a WGA lectin we use fluorometric assay by means of specific excitation of tryptophan at λex 295nm and it was found to be very high Ka∼1.39×10(7) M(-1) in case of ABG complex as compared to GlcNAc only Ka∼1.01×10(4) M(-1) with the phenomenon proven to be due to glycocluster effect. Copyright © 2016 Elsevier Inc. All rights reserved.

  6. Cytoplasmic translocation of polypyrimidine tract-binding protein and its binding to viral RNA during Japanese encephalitis virus infection inhibits virus replication.

    Directory of Open Access Journals (Sweden)

    Deepika Bhullar

    Full Text Available Japanese encephalitis virus (JEV has a single-stranded, positive-sense RNA genome containing a single open reading frame flanked by the 5'- and 3'-non-coding regions (NCRs. The virus genome replicates via a negative-sense RNA intermediate. The NCRs and their complementary sequences in the negative-sense RNA are the sites for assembly of the RNA replicase complex thereby regulating the RNA synthesis and virus replication. In this study, we show that the 55-kDa polypyrimidine tract-binding protein (PTB interacts in vitro with both the 5'-NCR of the positive-sense genomic RNA--5NCR(+, and its complementary sequence in the negative-sense replication intermediate RNA--3NCR(-. The interaction of viral RNA with PTB was validated in infected cells by JEV RNA co-immunoprecipitation and JEV RNA-PTB colocalization experiments. Interestingly, we observed phosphorylation-coupled translocation of nuclear PTB to cytoplasmic foci that co-localized with JEV RNA early during JEV infection. Our studies employing the PTB silencing and over-expression in cultured cells established an inhibitory role of PTB in JEV replication. Using RNA-protein binding assay we show that PTB competitively inhibits association of JEV 3NCR(- RNA with viral RNA-dependent RNA polymerase (NS5 protein, an event required for the synthesis of the plus-sense genomic RNA. cAMP is known to promote the Protein kinase A (PKA-mediated PTB phosphorylation. We show that cells treated with a cAMP analogue had an enhanced level of phosphorylated PTB in the cytoplasm and a significantly suppressed JEV replication. Data presented here show a novel, cAMP-induced, PTB-mediated, innate host response that could effectively suppress JEV replication in mammalian cells.

  7. Breaking the cycle: impact of sterically-tailored tetra(pyrazolyl)lutidines on the self-assembly of silver(I) complexes.

    Science.gov (United States)

    Morin, Tyler J; Merkel, Andrew; Lindeman, Sergey V; Gardinier, James R

    2010-09-06

    A improved preparation of the pentadentate ligand alpha,alpha,alpha',alpha'-tetra(pyrazolyl)lutidine, pz(4)lut, and the syntheses of three new alkyl-substituted pyrazolyl derivatives pz(4')(4)lut (pz(4') = 4-methylpyrazolyl), pz*(4)lut (pz* = 3,5-dimethylpyrazolyl), and pz(DIP)(4)lut (pz(DIP) = 3,5-diisopropylpyrazolyl) are described. The silver(I) complexes of these ligands were studied to ascertain the impact of pyrazolyl substitution, if any, on their binding modes and on solubility issues. In the solid state, [Ag(pz(4)lut)](BF(4)) (1), [Ag(pz(4')(4)lut)](BF(4)) (2), and [Ag(pz*(4)lut)](BF(4)) (3) give cyclic dications as a result of two ligands sandwiching two silver centers where each ligand binds the metals through only pyrazolyl nitrogen donors. This cyclic motif is similar to those observed in the silver complexes of tetra(pyridyl)lutidine PY5-R derivatives (where the central pyridyl does not bind) and in related tetra(pyrazolyl)-m-xylene complexes. While suitable single crystals of [Ag(pz(DIP)(4)lut)](BF(4)) (4) could not be obtained, those of [Ag(pz(DIP)(4)lut)](OTf) (5) showed infinite polymeric chains secured via silver-bound mu-kappa(2)N(pz),kappa(2)N(pz)- ligands. The different binding mode of the latter ligand versus the former three is likely due to unfavorable steric interactions between the bulky iso-propyl (pyrazolyl) substituents and the central pyridyl rings of hypothetical cyclic architectures. The combined electrospray ionization mass spectrometry (ESI(+)-MS), variable-temperature NMR (VT NMR), and diffusion pulsed field-gradient spin-echo (PFGSE) NMR data indicate that the solid state structures of each 1-5 are neither retained nor static in CD(3)CN, rather the cations are monomeric in solution.

  8. Self assembly of dialkoxo bridged dinuclear Fe(III) complex of pyridoxal Schiff base with C-C bond formation - structure, spectral and magnetic properties

    Czech Academy of Sciences Publication Activity Database

    Murašková, V.; Szabó, N.; Pižl, M.; Hoskovcová, I.; Dušek, Michal; Huber, Š.; Sedmidubský, D.

    2017-01-01

    Roč. 461, May (2017), s. 111-119 ISSN 0020-1693 R&D Projects: GA ČR(CZ) GA15-12653S; GA MŠk LO1603 EU Projects: European Commission(XE) CZ.2.16/3.1.00/24510 Institutional support: RVO:68378271 Keywords : iron (III) dinuclear complex * dialkoxo bridged pyridoxal Schiff base * C-C bond * crystal structure * magnetic properties Subject RIV: BM - Solid Matter Physics ; Magnetism OBOR OECD: Condensed matter physics (including formerly solid state physics, supercond.) Impact factor: 2.002, year: 2016

  9. 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 conserve