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Sample records for active dna replication

  1. Mrc1 is a replication fork component whose phosphorylation in response to DNA replication stress activates Rad53

    Osborn, Alexander J.; Elledge, Stephen J.

    2003-01-01

    When DNA replication is stalled, a signal transduction pathway is activated that promotes the stability of stalled forks and resumption of DNA synthesis. In budding yeast, this pathway includes the kinases Mec1 and Rad53. Here we report that the Mediator protein Mrc1, which is required for normal DNA replication and for activation of Rad53, is present at replication forks. Mrc1 initially binds early-replicating sequences and moves along chromatin with the replication f...

  2. Replication stress activates DNA repair synthesis in mitosis

    Minocherhomji, Sheroy; Ying, Songmin; Bjerregaard, Victoria A;

    2015-01-01

    mitosis serves as the trigger for completion of DNA replication at CFS loci in human cells. Given that this POLD3-dependent mitotic DNA synthesis is enhanced in aneuploid cancer cells that exhibit intrinsically high levels of chromosomal instability (CIN(+)) and replicative stress, we suggest that...

  3. Checkpoint Activation of an Unconventional DNA Replication Program in Tetrahymena.

    Sandoval, Pamela Y; Lee, Po-Hsuen; Meng, Xiangzhou; Kapler, Geoffrey M

    2015-07-01

    The intra-S phase checkpoint kinase of metazoa and yeast, ATR/MEC1, protects chromosomes from DNA damage and replication stress by phosphorylating subunits of the replicative helicase, MCM2-7. Here we describe an unprecedented ATR-dependent pathway in Tetrahymena thermophila in which the essential pre-replicative complex proteins, Orc1p, Orc2p and Mcm6p are degraded in hydroxyurea-treated S phase cells. Chromosomes undergo global changes during HU-arrest, including phosphorylation of histone H2A.X, deacetylation of histone H3, and an apparent diminution in DNA content that can be blocked by the deacetylase inhibitor sodium butyrate. Most remarkably, the cell cycle rapidly resumes upon hydroxyurea removal, and the entire genome is replicated prior to replenishment of ORC and MCMs. While stalled replication forks are elongated under these conditions, DNA fiber imaging revealed that most replicating molecules are produced by new initiation events. Furthermore, the sole origin in the ribosomal DNA minichromosome is inactive and replication appears to initiate near the rRNA promoter. The collective data raise the possibility that replication initiation occurs by an ORC-independent mechanism during the recovery from HU-induced replication stress. PMID:26218270

  4. Damage-induced DNA replication stalling relies on MAPK-activated protein kinase 2 activity

    Kopper, F.; Bierwirth, C.; Schon, M.;

    2013-01-01

    DNA damage can obstruct replication forks, resulting in replicative stress. By siRNA screening, we identified kinases involved in the accumulation of phosphohistone 2AX (gamma H2AX) upon UV irradiation-induced replication stress. Surprisingly, the strongest reduction of phosphohistone 2AX followed...... replication impaired by gemcitabine or by Chk1 inhibition. This rescue strictly depended on transiesion DNA polymerases. In conclusion, instead of being an unavoidable consequence of DNA damage, alterations of replication speed and origin firing depend on MK2-mediated signaling....... knockdown of the MAP kinase-activated protein kinase 2 (MK2), a kinase currently implicated in p38 stress signaling and G2 arrest. Depletion or inhibition of MK2 also protected cells from DNA damage-induced cell death, and mice deficient for MK2 displayed decreased apoptosis in the skin upon UV irradiation...

  5. Escherichia coli DnaE Polymerase Couples Pyrophosphatase Activity to DNA Replication.

    Fabio Lapenta

    Full Text Available DNA Polymerases generate pyrophosphate every time they catalyze a step of DNA elongation. This elongation reaction is generally believed as thermodynamically favoured by the hydrolysis of pyrophosphate, catalyzed by inorganic pyrophosphatases. However, the specific action of inorganic pyrophosphatases coupled to DNA replication in vivo was never demonstrated. Here we show that the Polymerase-Histidinol-Phosphatase (PHP domain of Escherichia coli DNA Polymerase III α subunit features pyrophosphatase activity. We also show that this activity is inhibited by fluoride, as commonly observed for inorganic pyrophosphatases, and we identified 3 amino acids of the PHP active site. Remarkably, E. coli cells expressing variants of these catalytic residues of α subunit feature aberrant phenotypes, poor viability, and are subject to high mutation frequencies. Our findings indicate that DNA Polymerases can couple DNA elongation and pyrophosphate hydrolysis, providing a mechanism for the control of DNA extension rate, and suggest a promising target for novel antibiotics.

  6. Viral DNA Replication-Dependent DNA Damage Response Activation during BK Polyomavirus Infection

    Verhalen, Brandy; Justice, Joshua L.; Imperiale, Michael J; Jiang, Mengxi

    2015-01-01

    BK polyomavirus (BKPyV) reactivation is associated with severe human disease in kidney and bone marrow transplant patients. The interplay between viral and host factors that regulates the productive infection process remains poorly understood. We have previously reported that the cellular DNA damage response (DDR) is activated upon lytic BKPyV infection and that its activation is required for optimal viral replication in primary kidney epithelial cells. In this report, we set out to determine...

  7. ATPase activity measurement of DNA replicative helicase from Bacillus stearothermophilus by malachite green method.

    Yang, Mu; Wang, Ganggang

    2016-09-15

    The DnaB helicase from Bacillus stearothermophilus (DnaBBst) was a model protein for studying the bacterial DNA replication. In this work, a non-radioactive method for measuring ATPase activity of DnaBBst helicase was described. The working parameters and conditions were optimized. Furthermore, this method was applied to investigate effects of DnaG primase, ssDNA and helicase loader protein (DnaI) on ATPase activity of DnaBBst. Our results showed this method was sensitive and efficient. Moreover, it is suitable for the investigation of functional interaction between DnaB and related factors. PMID:27372608

  8. Sulfolobus Replication Factor C stimulates the activity of DNA Polymerase B1

    Xing, Xuanxuan; Zhang, Likui; Guo, Li;

    2014-01-01

    Replication factor C (RFC) is known to function in loading proliferating cell nuclear antigen (PCNA) onto primed DNA, allowing PCNA to tether DNA polymerase for highly processive DNA synthesis in eukaryotic and archaeal replication. In this report, we show that an RFC complex from the...... with the ability of RFC to facilitate DNA binding by PolB1 through protein-protein interaction. These results suggest that Sulfolobus RFC may play a role in recruiting DNA polymerase for efficient primer extension, in addition to clamp loading, during DNA replication....... hyperthermophilic archaea of the genus Sulfolobus physically interacts with DNA polymerase B1 (PolB1) and enhances both the polymerase and 3'-5' exonuclease activities of PolB1 in an ATP-independent manner. Stimulation of the PolB1 activity by RFC is independent of the ability of RFC to bind DNA but is consistent...

  9. Archaeal DNA replication.

    Kelman, Lori M; Kelman, Zvi

    2014-01-01

    DNA replication is essential for all life forms. Although the process is fundamentally conserved in the three domains of life, bioinformatic, biochemical, structural, and genetic studies have demonstrated that the process and the proteins involved in archaeal DNA replication are more similar to those in eukaryal DNA replication than in bacterial DNA replication, but have some archaeal-specific features. The archaeal replication system, however, is not monolithic, and there are some differences in the replication process between different species. In this review, the current knowledge of the mechanisms governing DNA replication in Archaea is summarized. The general features of the replication process as well as some of the differences are discussed. PMID:25421597

  10. Replicating animal mitochondrial DNA

    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.

  11. Modeling DNA Replication.

    Bennett, Joan

    1998-01-01

    Recommends the use of a model of DNA made out of Velcro to help students visualize the steps of DNA replication. Includes a materials list, construction directions, and details of the demonstration using the model parts. (DDR)

  12. DNA Replication Origins

    Leonard, Alan C.; Méchali, Marcel

    2013-01-01

    The onset of genomic DNA synthesis requires precise interactions of specialized initiator proteins with DNA at sites where the replication machinery can be loaded. These sites, defined as replication origins, are found at a few unique locations in all of the prokaryotic chromosomes examined so far. However, replication origins are dispersed among tens of thousands of loci in metazoan chromosomes, thereby raising questions regarding the role of specific nucleotide sequences and chromatin envir...

  13. Modeling inhomogeneous DNA replication kinetics.

    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.

  14. cis-active elements from mouse chromosomal DNA suppress simian virus 40 DNA replication.

    Hartl, M.; Willnow, T; Fanning, E

    1990-01-01

    Simian virus 40 (SV40)-containing DNA was rescued after the fusion of SV40-transformed VLM cells with permissive COS1 monkey cells and cloned, and prototype plasmid clones were characterized. A 2-kilobase mouse DNA fragment fused with the rescued SV40 DNA, and derived from mouse DNA flanking the single insert of SV40 DNA in VLM cells, was sequenced. Insertion of the intact rescued mouse sequence, or two nonoverlapping fragments of it, into wild-type SV40 plasmid DNA suppressed replication of ...

  15. Protein Phosphatase 1 Recruitment by Rif1 Regulates DNA Replication Origin Firing by Counteracting DDK Activity

    Anoushka Davé; Carol Cooley; Mansi Garg; Alessandro Bianchi

    2014-01-01

    Summary The firing of eukaryotic origins of DNA replication requires CDK and DDK kinase activities. DDK, in particular, is involved in setting the temporal program of origin activation, a conserved feature of eukaryotes. Rif1, originally identified as a telomeric protein, was recently implicated in specifying replication timing in yeast and mammals. We show that this function of Rif1 depends on its interaction with PP1 phosphatases. Mutations of two PP1 docking motifs in Rif1 lead to early re...

  16. Chromium reduces the in vitro activity and fidelity of DNA replication mediated by the human cell DNA synthesome

    Hexavalent chromium Cr(VI) is known to be a carcinogenic metal ion, with a complicated mechanism of action. It can be found within our environment in soil and water contaminated by manufacturing processes. Cr(VI) ion is readily taken up by cells, and is recognized to be both genotoxic and cytotoxic; following its reduction to the stable trivalent form of the ion, chromium(Cr(III)), within cells. This form of the ion is known to impede the activity of cellular DNA polymerase and polymerase-mediated DNA replication. Here, we report the effects of chromium on the activity and fidelity of the DNA replication process mediated by the human cell DNA synthesome. The DNA synthesome is a functional multiprotein complex that is fully competent to carry-out each phase of the DNA replication process. The IC50 of Cr(III) toward the activity of DNA synthesome-associated DNA polymerases α, δ and ε is 15, 45 and 125 μM, respectively. Cr(III) inhibits synthesome-mediated DNA synthesis (IC50 = 88 μM), and significantly reduces the fidelity of synthesome-mediated DNA replication. The mutation frequency induced by the different concentrations of Cr(III) ion used in our assays ranges from 2-13 fold higher than that which occurs spontaneously, and the types of mutations include single nucleotide substitutions, insertions, and deletions. Single nucleotide substitutions are the predominant type of mutation, and they occur primarily at GC base-pairs. Cr(III) ion produces a lower number of transition and a higher number of transversion mutations than occur spontaneously. Unlike Cr(III), Cr(VI) ion has little effect on the in vitro DNA synthetic activity and fidelity of the DNA synthesome, but does significantly inhibit DNA synthesis in intact cells. Cell growth and proliferation is also arrested by increasing concentrations of Cr(VI) ion. Our studies provide evidence indicating that the chromium ion induced decrease in the fidelity and activity of synthesome mediated DNA replication

  17. Multiple Rad5 activities mediate sister chromatid recombination to bypass DNA damage at stalled replication forks.

    Minca, Eugen C; Kowalski, David

    2010-06-11

    DNA damage that blocks replication is bypassed in order to complete chromosome duplication and preserve cell viability and genome stability. Rad5, a PCNA polyubiquitin ligase and DNA-dependent ATPase in yeast, is orthologous to putative tumor suppressors and controls error-free damage bypass by an unknown mechanism. To identify the mechanism in vivo, we investigated the roles of Rad5 and analyzed the DNA structures that form during damage bypass at site-specific stalled forks present at replication origins. Rad5 mediated the formation of recombination-dependent, X-shaped DNA structures containing Holliday junctions between sister chromatids. Mutants lacking these damage-induced chromatid junctions were defective in resolving stalled forks, restarting replication, and completing chromosome duplication. Rad5 polyubiquitin ligase and ATPase domains both contributed to replication fork recombination. Our results indicate that multiple activities of Rad5 function coordinately with homologous recombination factors to enable replication template switch events that join sister chromatids at stalled forks and bypass DNA damage. PMID:20541998

  18. Origin DNA Melting and Unwinding in DNA Replication

    Gai, Dahai; Chang, Y Paul; Chen, Xiaojiang S.

    2010-01-01

    Genomic DNA replication is a necessary step in the life cycles of all organisms. To initiate DNA replication, the double-stranded DNA (dsDNA) at the origin of replication must be separated or melted; this melted region is propagated and a mature replication fork is formed. To accomplish origin recognition, initial DNA melting, and the eventual formation of a replication fork, coordinated activity of initiators, helicases, and other cellular factors are required. In this review, we focus on re...

  19. Replication stress and oxidative damage contribute to aberrant constitutive activation of DNA damage signalling in human gliomas

    Bartkova, J; Hamerlik, P; Stockhausen, Marie;

    2010-01-01

    brain and grade II astrocytomas, despite the degree of DDR activation was higher in grade II tumors. Markers indicative of ongoing DNA replication stress (Chk1 activation, Rad17 phosphorylation, replication protein A foci and single-stranded DNA) were present in GBM cells under high- or low...... and indicate that replication stress, rather than oxidative stress, fuels the DNA damage signalling in early stages of astrocytoma development.......Malignant gliomas, the deadliest of brain neoplasms, show rampant genetic instability and resistance to genotoxic therapies, implicating potentially aberrant DNA damage response (DDR) in glioma pathogenesis and treatment failure. Here, we report on gross, aberrant constitutive activation of DNA...

  20. Differential activation of intra-S-phase checkpoint in response to tripchlorolide and its effects on DNA replication

    Yan REN; Jia Rui WU

    2004-01-01

    DNA replication is tightly regulated during the S phase of the cell cycle, and the activation of the intra-S-phase checkpoint due to DNA damage usually results in arrest of DNA synthesis. However, the molecular details about the correlation between the checkpoint and regulation of DNA replication are still unclear. To investigate the connections between DNA replication and DNA damage checkpoint, a DNA-damage reagent, tripchlorolide, was applied to CHO (Chinese ovary hamster) cells at early- or middle-stages of the S phase. The early-S-phase treatment with TC significantly delayed the progression of the S phase and caused the phosphorylation of the Chk1 checkpoint protein, whereas the middle-S-phase treatment only slightly slowed down the progression of the S phase. Furthermore, the analysis of DNA replication patterns revealed that replication pattern Ⅱ was greatly prolonged in the cells treated with the drug during the early-S phase, whereas the late-replication patterns of these cells were hardly detected, suggesting that the activation of the intra-S-phase checkpoint inhibits the late-origin firing of DNA replication. We conclude that cells at different stages of the S phase are differentially sensitive to the DNA-damage reagent, and the activation of the intra-Sphase checkpoint blocks the DNA replication progression in the late stage of S phase.

  1. Protein Phosphatase 1 Recruitment by Rif1 Regulates DNA Replication Origin Firing by Counteracting DDK Activity

    Anoushka Davé

    2014-04-01

    Full Text Available The firing of eukaryotic origins of DNA replication requires CDK and DDK kinase activities. DDK, in particular, is involved in setting the temporal program of origin activation, a conserved feature of eukaryotes. Rif1, originally identified as a telomeric protein, was recently implicated in specifying replication timing in yeast and mammals. We show that this function of Rif1 depends on its interaction with PP1 phosphatases. Mutations of two PP1 docking motifs in Rif1 lead to early replication of telomeres in budding yeast and misregulation of origin firing in fission yeast. Several lines of evidence indicate that Rif1/PP1 counteract DDK activity on the replicative MCM helicase. Our data suggest that the PP1/Rif1 interaction is downregulated by the phosphorylation of Rif1, most likely by CDK/DDK. These findings elucidate the mechanism of action of Rif1 in the control of DNA replication and demonstrate a role of PP1 phosphatases in the regulation of origin firing.

  2. Optimal Placement of Origins for DNA Replication

    Karschau, Jens; Blow, J. Julian; de Moura, Alessandro P. S.

    2012-01-01

    DNA replication is an essential process in biology and its timing must be robust so that cells can divide properly. Random fluctuations in the formation of replication starting points, called origins, and the subsequent activation of proteins lead to variations in the replication time. We analyse these stochastic properties of DNA and derive the positions of origins corresponding to the minimum replication time. We show that under some conditions the minimization of replication time leads to ...

  3. HBO1 histone acetylase activity is essential for DNA replication licensing and inhibited by geminin

    Miotto, Benoit; Struhl, Kevin

    2010-01-01

    HBO1, an H4-specific histone acetylase, is a co-activator of the DNA replication licensing factor Cdt1. HBO1 acetylase activity is required for licensing, because a HAT-defective mutant of HBO1 bound at origins is unable to load the MCM complex. H4 acetylation at origins is cell-cycle regulated, with maximal activity at the G1/S transition, and co-expression of HBO1 and Jade1 increases histone acetylation and MCM complex loading. Overexpression of the Set8 histone H4 tail-binding domain speci...

  4. Genome-wide localization of Rrm3 and Pif1 DNA helicases at stalled active and inactive DNA replication forks of Saccharomyces cerevisiae.

    Rossi, Silvia Emma; Carotenuto, Walter; Giannattasio, Michele

    2016-03-01

    The genome of the budding yeast Saccharomyces cerevisiae is sequenced and the location and dynamic of activation of DNA replication origins are known. G1 synchronized yeast cells can be released into S-phase in the presence of hydroxyurea (HU) (1), which slows down DNA replication and retains replication forks in proximity of DNA replication origins. In this condition, the Chromatin Immuno-Precipitation on chip (ChIP on chip) (2-4) of replisome components allows the precise localization of all active DNA replication forks. This analysis can be coupled with the ssDNA-BromodeoxyUridine (ssDNA-BrdU) Immuno-Precipitation on chip (ssDNA-BrdU IP on chip) technique (5-7), which detects the location of newly synthesized DNA. Comparison of binding and BrdU incorporation profiles allows to locate a factor of interest at DNA replication forks genome wide. We present datasets deposited in the gene expression omnibus (GEO) database under accession number GSE68214, which show how the DNA helicases Rrm3 and Pif1 (8) associate to active and inactive DNA replication forks. PMID:26981397

  5. Genome-wide localization of Rrm3 and Pif1 DNA helicases at stalled active and inactive DNA replication forks of Saccharomyces cerevisiae

    Silvia Emma Rossi

    2016-03-01

    Full Text Available The genome of the budding yeast Saccharomyces cerevisiae is sequenced and the location and dynamic of activation of DNA replication origins are known. G1 synchronized yeast cells can be released into S-phase in the presence of hydroxyurea (HU (1, which slows down DNA replication and retains replication forks in proximity of DNA replication origins. In this condition, the Chromatin Immuno-Precipitation on chip (ChIP on chip (2–4 of replisome components allows the precise localization of all active DNA replication forks. This analysis can be coupled with the ssDNA-BromodeoxyUridine (ssDNA-BrdU Immuno-Precipitation on chip (ssDNA-BrdU IP on chip technique (5–7, which detects the location of newly synthesized DNA. Comparison of binding and BrdU incorporation profiles allows to locate a factor of interest at DNA replication forks genome wide. We present datasets deposited in the gene expression omnibus (GEO database under accession number GSE68214, which show how the DNA helicases Rrm3 and Pif1 (8 associate to active and inactive DNA replication forks.

  6. STRUCTURE-ACTIVITY STUDY OF PARACETAMOL ANALOGUES: INHIBITION OF REPLICATIVE DNA SYNTHESIS IN V79 CHINESE HAMSTER CELLS

    Experimental and theoretical evidence pertaining to cytotoxic and genotoxic activity of paracetamol in biological systems was used to formulate a simple mechanistic hypothesis to explain the relative inhibition of replicative DNA synthesis by a series of 19 structurally similar p...

  7. Stable DNA replication: interplay between DNA replication, homologous recombination, and transcription.

    Kogoma, T

    1997-01-01

    Chromosome replication in Escherichia coli is normally initiated at oriC, the origin of chromosome replication. E. coli cells possess at least three additional initiation systems for chromosome replication that are normally repressed but can be activated under certain specific conditions. These are termed the stable DNA replication systems. Inducible stable DNA replication (iSDR), which is activated by SOS induction, is proposed to be initiated from a D-loop, an early intermediate in homologo...

  8. Controlled rereplication at DNA replication origins

    Gómez, María

    2008-01-01

    No-more-than-once per cell cycle initiation of DNA replication is a golden rule to maintain genome stability and guarantee cell survival. In our recent work we discovered that small genome fragments of about 200 bp are repeatedly synthesised at human DNA replication origins at the time of origin firing during S phase in normal cells. Rereplicated DNA fragments coincide physical and temporarily with replication origin activity, implying that their generation is intimately associated with the i...

  9. Targeted mutagenesis of the human papillomavirus type 16 E2 transactivation domain reveals separable transcriptional activation and DNA replication functions.

    Sakai, H; Yasugi, T; Benson, J D; Dowhanick, J J; Howley, P M

    1996-03-01

    The E2 gene products of papillomavirus play key roles in viral replication, both as regulators of viral transcription and as auxiliary factors that act with E1 in viral DNA replication. We have carried out a detailed structure-function analysis of conserved amino acids within the N-terminal domain of the human papillomavirus type 16 (HPV16) E2 protein. These mutants were tested for their transcriptional activation activities as well as transient DNA replication and E1 binding activities. Analysis of the stably expressed mutants revealed that the transcriptional activation and replication activities of HPV16 E2 could be dissociated. The 173A mutant was defective for the transcriptional activation function but retained wild-type DNA replication activity, whereas the E39A mutant wild-type transcriptional activation function but was defective in transient DNA replication assays. The E39A mutant was also defective for HPV16 E1 binding in vitro, suggesting that the ability of E2 protein to form a complex with E1 appears to be essential for its function as an auxiliary replication factor. PMID:8627680

  10. Constitutive stable DNA replication in Escherichia coli cells lacking type 1A topoisomerase activity.

    Martel, Makisha; Balleydier, Aurélien; Sauriol, Alexandre; Drolet, Marc

    2015-11-01

    Type 1A topoisomerases (topos) are ubiquitous enzymes involved in supercoiling regulation and in the maintenance of genome stability. Escherichia coli possesses two type 1A enzymes, topo I (topA) and topo III (topB). Cells lacking both enzymes form very long filaments and have severe chromosome segregation and growth defects. We previously found that RNase HI overproduction or a dnaT::aph mutation could significantly correct these phenotypes. This leads us to hypothesize that they were related to unregulated replication originating from R-loops, i.e. constitutive stable DNA replication (cSDR). cSDR, first observed in rnhA (RNase HI) mutants, is characterized by its persistence for several hours following protein synthesis inhibition and by its requirement for primosome components, including DnaT. Here, to visualize and measure cSDR, the incorporation of the nucleotide analog ethynyl deoxyuridine (EdU) during replication in E. coli cells pre-treated with protein synthesis inhibitors, was revealed by "click" labeling with Alexa Fluor(®) 488 in fixed cells, and flow cytometry analysis. cSDR was detected in rnhA mutants, but not in wild-type strains, and the number of cells undergoing cSDR was significantly reduced by the introduction of the dnaT::aph mutation. cSDR was also found in topA, double topA topB but not in topB null cells. This result is consistent with the established function of topo I in the inhibition of R-loop formation. Moreover, our finding that topB rnhA mutants are perfectly viable demonstrates that topo III is not uniquely required during cSDR. Thus, either topo I or III can provide the type 1A topo activity that is specifically required during cSDR to allow chromosome segregation. PMID:26444226

  11. Genome-wide localization of Rrm3 and Pif1 DNA helicases at stalled active and inactive DNA replication forks of Saccharomyces cerevisiae

    Silvia Emma Rossi; Walter Carotenuto; Michele Giannattasio

    2016-01-01

    The genome of the budding yeast Saccharomyces cerevisiae is sequenced and the location and dynamic of activation of DNA replication origins are known. G1 synchronized yeast cells can be released into S-phase in the presence of hydroxyurea (HU) (1), which slows down DNA replication and retains replication forks in proximity of DNA replication origins. In this condition, the Chromatin Immuno-Precipitation on chip (ChIP on chip) (2–4) of replisome components allows the precise localization of al...

  12. Genome-wide localization of Rrm3 and Pif1 DNA helicases at stalled active and inactive DNA replication forks of Saccharomyces cerevisiae

    Rossi, Silvia Emma; Carotenuto, Walter; Giannattasio, Michele

    2015-01-01

    The genome of the budding yeast Saccharomyces cerevisiae is sequenced and the location and dynamic of activation of DNA replication origins are known. G1 synchronized yeast cells can be released into S-phase in the presence of hydroxyurea (HU) (1), which slows down DNA replication and retains replication forks in proximity of DNA replication origins. In this condition, the Chromatin Immuno-Precipitation on chip (ChIP on chip) (2–4) of replisome components allows the precise localization of al...

  13. Replication Protein A (RPA) deficiency activates the Fanconi anemia DNA repair pathway.

    Jang, Seok-Won; Jung, Jin Ki; Kim, Jung Min

    2016-09-01

    The Fanconi anemia (FA) pathway regulates DNA inter-strand crosslink (ICL) repair. Despite our greater understanding of the role of FA in ICL repair, its function in the preventing spontaneous genome instability is not well understood. Here, we show that depletion of replication protein A (RPA) activates the FA pathway. RPA1 deficiency increases chromatin recruitment of FA core complex, leading to FANCD2 monoubiquitination (FANCD2-Ub) and foci formation in the absence of DNA damaging agents. Importantly, ATR depletion, but not ATM, abolished RPA1 depletion-induced FANCD2-Ub, suggesting that ATR activation mediated FANCD2-Ub. Interestingly, we found that depletion of hSSB1/2-INTS3, a single-stranded DNA-binding protein complex, induces FANCD2-Ub, like RPA1 depletion. More interestingly, depletion of either RPA1 or INTS3 caused increased accumulation of DNA damage in FA pathway deficient cell lines. Taken together, these results indicate that RPA deficiency induces activation of the FA pathway in an ATR-dependent manner, which may play a role in the genome maintenance. PMID:27398742

  14. Synchronization of DNA array replication kinetics

    Manturov, Alexey O.; Grigoryev, Anton V.

    2016-04-01

    In the present work we discuss the features of the DNA replication kinetics at the case of multiplicity of simultaneously elongated DNA fragments. The interaction between replicated DNA fragments is carried out by free protons that appears at the every nucleotide attachment at the free end of elongated DNA fragment. So there is feedback between free protons concentration and DNA-polymerase activity that appears as elongation rate dependence. We develop the numerical model based on a cellular automaton, which can simulate the elongation stage (growth of DNA strands) for DNA elongation process with conditions pointed above and we study the possibility of the DNA polymerases movement synchronization. The results obtained numerically can be useful for DNA polymerase movement detection and visualization of the elongation process in the case of massive DNA replication, eg, under PCR condition or for DNA "sequencing by synthesis" sequencing devices evaluation.

  15. Replication of bacteriophage lambda DNA

    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

  16. Multiple Rad5 activities mediate sister chromatid recombination to bypass DNA damage at stalled replication forks

    Minca, Eugen C; Kowalski, David

    2010-01-01

    DNA damage that blocks replication is bypassed in order to complete chromosome duplication and preserve cell viability and genome stability. Rad5, a PCNA polyubiquitin ligase and DNA-dependent ATPase in yeast, is orthologous to putative tumor suppressors and controls error-free damage bypass by an unknown mechanism. To identify the mechanism in vivo, we investigated the roles of Rad5 and analyzed the DNA structures that form during damage bypass at site-specific stalled forks present at repli...

  17. Pyrimidine Pool Disequilibrium Induced by a Cytidine Deaminase Deficiency Inhibits PARP-1 Activity, Leading to the Under Replication of DNA.

    Simon Gemble

    2015-07-01

    Full Text Available Genome stability is jeopardized by imbalances of the dNTP pool; such imbalances affect the rate of fork progression. For example, cytidine deaminase (CDA deficiency leads to an excess of dCTP, slowing the replication fork. We describe here a novel mechanism by which pyrimidine pool disequilibrium compromises the completion of replication and chromosome segregation: the intracellular accumulation of dCTP inhibits PARP-1 activity. CDA deficiency results in incomplete DNA replication when cells enter mitosis, leading to the formation of ultrafine anaphase bridges between sister-chromatids at "difficult-to-replicate" sites such as centromeres and fragile sites. Using molecular combing, electron microscopy and a sensitive assay involving cell imaging to quantify steady-state PAR levels, we found that DNA replication was unsuccessful due to the partial inhibition of basal PARP-1 activity, rather than slower fork speed. The stimulation of PARP-1 activity in CDA-deficient cells restores replication and, thus, chromosome segregation. Moreover, increasing intracellular dCTP levels generates under-replication-induced sister-chromatid bridges as efficiently as PARP-1 knockdown. These results have direct implications for Bloom syndrome (BS, a rare genetic disease combining susceptibility to cancer and genomic instability. BS results from mutation of the BLM gene, encoding BLM, a RecQ 3'-5' DNA helicase, a deficiency of which leads to CDA downregulation. BS cells thus have a CDA defect, resulting in a high frequency of ultrafine anaphase bridges due entirely to dCTP-dependent PARP-1 inhibition and independent of BLM status. Our study describes previously unknown pathological consequences of the distortion of dNTP pools and reveals an unexpected role for PARP-1 in preventing DNA under-replication and chromosome segregation defects.

  18. Transcriptional activation function is not required for stimulation of DNA replication by bovine papillomavirus type 1 E2.

    Grossel, M J; Sverdrup, F; Breiding, D E; Androphy, E J

    1996-01-01

    Bovine papillomavirus type 1 replication was previously shown to require both the E1 initiator protein and the E2 transactivator protein. We show here that E1, in the absence of E2, is sufficient for low-level bovine papillomavirus type 1 DNA replication in C-33A cells. In addition, studies of genetically isolated E2 point mutants demonstrate that enhancement of replication by E2 does not require its transcriptional activation function. The uncoupling of the E2 functions suggests that stimula...

  19. Targeting DNA Replication Stress for Cancer Therapy

    Jun Zhang

    2016-08-01

    Full Text Available The human cellular genome is under constant stress from extrinsic and intrinsic factors, which can lead to DNA damage and defective replication. In normal cells, DNA damage response (DDR mediated by various checkpoints will either activate the DNA repair system or induce cellular apoptosis/senescence, therefore maintaining overall genomic integrity. Cancer cells, however, due to constitutive growth signaling and defective DDR, may exhibit “replication stress” —a phenomenon unique to cancer cells that is described as the perturbation of error-free DNA replication and slow-down of DNA synthesis. Although replication stress has been proven to induce genomic instability and tumorigenesis, recent studies have counterintuitively shown that enhancing replicative stress through further loosening of the remaining checkpoints in cancer cells to induce their catastrophic failure of proliferation may provide an alternative therapeutic approach. In this review, we discuss the rationale to enhance replicative stress in cancer cells, past approaches using traditional radiation and chemotherapy, and emerging approaches targeting the signaling cascades induced by DNA damage. We also summarize current clinical trials exploring these strategies and propose future research directions including the use of combination therapies, and the identification of potential new targets and biomarkers to track and predict treatment responses to targeting DNA replication stress.

  20. Identification of Peptides That Inhibit the DNA Binding, trans-Activator, and DNA Replication Functions of the Human Papillomavirus Type 11 E2 Protein

    Deng, Su-Jun; Pearce, Kenneth H.; Dixon, Eric P.; Hartley, Kelly A.; Stanley, Thomas B.; Lobe, David C.; Garvey, Edward P.; Kost, Thomas A.; Petty, Regina L.; Rocque, Warren J.; Alexander, Kenneth A.; Underwood, Mark R.

    2004-01-01

    Peptide antagonists of the human papillomavirus type 11 (HPV-11) E2-DNA association were identified using a filamentous bacteriophage random peptide library. Synthetic peptides antagonized the E2-DNA interaction, effectively blocked E2-mediated transcriptional activation of a reporter gene in cell culture, and inhibited E1-E2-mediated HPV-11 DNA replication in vitro. These peptides may prove to be useful tools for characterizing E2 function and for exploring the effectiveness of E2-inhibitor-...

  1. Where does DNA replication start in archaea?

    Vas, Amit; Leatherwood, Janet

    2000-01-01

    Genome-wide measures of DNA strand composition have been used to find archaeal DNA replication origins. Archaea seem to replicate using a single origin (as do eubacteria) even though archaeal replication factors are more like those of eukaryotes.

  2. Replication stress and oxidative damage contribute to aberrant constitutive activation of DNA damage signalling in human gliomas

    Bartkova, J; Hamerlik, P; Stockhausen, Marie;

    2010-01-01

    damage signalling in low- and high-grade human gliomas, and analyze the sources of such endogenous genotoxic stress. Based on analyses of human glioblastoma multiforme (GBM) cell lines, normal astrocytes and clinical specimens from grade II astrocytomas (n=41) and grade IV GBM (n=60), we conclude that...... brain and grade II astrocytomas, despite the degree of DDR activation was higher in grade II tumors. Markers indicative of ongoing DNA replication stress (Chk1 activation, Rad17 phosphorylation, replication protein A foci and single-stranded DNA) were present in GBM cells under high- or low......-oxygen culture conditions and in clinical specimens of both low- and high-grade tumors. The observed global checkpoint signaling, in contrast to only focal areas of overabundant p53 (indicative of p53 mutation) in grade II astrocytomas, are consistent with DDR activation being an early event in gliomagenesis...

  3. Mutator phenotypes due to DNA replication infidelity

    Arana, Mercedes E.; Kunkel, Thomas A.

    2010-01-01

    This article considers the fidelity of DNA replication performed by eukaryotic DNA polymerases involved in replicating the nuclear genome. DNA replication fidelity can vary widely depending on the DNA polymerase, the composition of the error, the flanking sequence, the presence of DNA damage and the ability to correct errors. As a consequence, defects in processes that determine DNA replication fidelity can confer strong mutator phenotypes whose specificity can help determine the molecular na...

  4. Rolling-circle amplification for the detection of active porcine circovirus type 2 DNA replication in vitro.

    Navidad, Paolo Dominic; Li, Hao; Mankertz, Annette; Meehan, Brian

    2008-09-01

    Porcine circovirus type 2 (PCV2) infections in pigs have diverse clinical presentations and are considered economically important diseases worldwide. However, despite intensive research, the early pathogenesis of PCV2 and the primary target cells for PCV2 infections and replication are still unknown. Rolling-circle amplification (RCA) is an amplification technique for small, circular DNA templates that essentially mimics rolling-circle replication in vitro. In this study, the amplification of PCV2-specific DNAs using randomly primed RCA has been demonstrated. This novel approach has circumvented the normal requirement for conventional virus isolation procedures for the characterization of PCV2 DNAs from clinical samples. In addition, the potential utility of a strand-specific derivative of RCA was further investigated. Specifically, strand-specific RCA for the detection of active virus replication following the amplification of complementary sense PCV2 DNAs, which occur as double-stranded replicative intermediates that are present only during de novo viral DNA replication both in vitro and in vivo has been demonstrated. PMID:18606463

  5. Long Terminal Repeat Circular DNA as Markers of Active Viral Replication of Human T Lymphotropic Virus-1 in Vivo.

    Fox, James M; Hilburn, Silva; Demontis, Maria-Antonietta; Brighty, David W; Rios Grassi, Maria Fernanda; Galvão-Castro, Bernardo; Taylor, Graham P; Martin, Fabiola

    2016-03-01

    Clonal expansion of human T-lymphotropic virus type-1 (HTLV-1) infected cells in vivo is well documented. Unlike human immunodeficiency virus type 1 (HIV-1), HTLV-1 plasma RNA is sparse. The contribution of the "mitotic" spread of HTLV-1 compared with infectious spread of the virus to HTLV-1 viral burden in established infection is uncertain. Since extrachromosomal long terminal repeat (LTR) DNA circles are indicators of viral replication in HIV-1 carriers with undetectable plasma HIV RNA, we hypothesised that HTLV-1 LTR circles could indicate reverse transcriptase (RT) usage and infectious activity. 1LTR and 2LTR DNA circles were measured in HTLV-1 cell lines and peripheral blood mononuclear cells (PBMC) of asymptomatic carriers (ACs) and patients with HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) or adult T cell leukaemia/lymphoma (ATLL). 1LTR DNA circles were detected in 14/20 patients at a mean of 1.38/100 PBMC but did not differentiate disease status nor correlate with HTLV-1 DNA copies. 2LTR DNA circles were detected in 30/31 patients and at higher concentrations in patients with HTLV-1-associated diseases, independent of HTLV-1 DNA load. In an incident case the 2LTR DNA circle concentration increased 2.1 fold at the onset of HAM/TSP compared to baseline. Detectable and fluctuating levels of HTLV-1 DNA circles in patients indicate viral RT usage and virus replication. Our results indicate HTLV-1 viral replication capacity is maintained in chronic infection and may be associated with disease onset. PMID:26985903

  6. Cellular factors required for papillomavirus DNA replication.

    Melendy, T; Sedman, J; Stenlund, A

    1995-01-01

    In vitro replication of papillomavirus DNA has been carried out with a combination of purified proteins and partially purified extracts made from human cells. DNA synthesis requires the viral E1 protein and the papillomavirus origin of replication. The E2 protein stimulates DNA synthesis in a binding site-independent manner. Papillomavirus DNA replication is also dependent on the cellular factors replication protein A, replication factor C, and proliferating-cell nuclear antigen as well as a ...

  7. Uncoupling of Sister Replisomes during Eukaryotic DNA Replication

    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. So

  8. DNA Replication Reaches the Breaking Point

    Petrini, John H.J.

    2009-01-01

    DNA strand breaks that result in stalled or damaged replication forks can be detrimental to the DNA replication process. In this issue, Doksani et al. (2009) examine the impact of a single double-stranded DNA break on replication in the budding yeast, Saccharomyces cerevisiae.

  9. Yeast Rad5 Protein Required for Postreplication Repair Has a DNA Helicase Activity Specific for Replication Fork Regression

    Blastyák, András; Pintér, Lajos; Unk, Ildiko; Prakash, Louise; Prakash, Satya; Haracska, Lajos

    2007-01-01

    Summary Lesions in the template DNA strand block the progression of the replication fork. In the yeast Saccharomyces cerevisiae, replication through DNA lesions is mediated by different Rad6-Rad18-dependent means, which include translesion synthesis and a Rad5-dependent postreplicational repair pathway that repairs the discontinuities that form in the DNA synthesized from damaged templates. Although translesion synthesis is well characterized, little is known about the mechanisms that modulat...

  10. Prevention of DNA re-replication in eukaryotic cells

    Lan N. Truong; Xiaohua Wu

    2011-01-01

    DNA replication is a highly regulated process involving a number of licensing and replication factors that function in a carefully orchestrated manner to faithfully replicate DNA during every cell cycle. Loss of proper licensing control leads to deregulated DNA replication including DNA re-replication, which can cause genome instability and tumorigenesis. Eukaryotic organisms have established several conserved mechanisms to prevent DNA re-replication and to counteract its potentially harmful effects. These mechanisms include tightly controlled regulation of licensing factors and activation of cell cycle and DNA damage checkpoints.Deregulated licensing control and its associated compromised checkpoints have both been observed in tumor cells, indicating that proper functioning of these pathways is essential for maintaining genome stability. In this review, we discuss the regulatory mechanisms of licensing control, the deleterious consequences when both licensing and checkpoints are compromised, and present possible mechanisms to prevent re-replication in order to maintain genome stability.

  11. DNA polymerase I modulates inducible stable DNA replication in Escherichia coli.

    Ruscitti, T; Linn, S.(Florida International University, Miami, USA)

    1992-01-01

    Mutants of Escherichia coli lacking RNase HI activity and cells induced for the SOS response express modes of DNA replication independent of protein synthesis, called constitutive and induced stable DNA replication, respectively. We report here that mutants deleted for the polA gene express induced stable DNA replication at approximately 25-fold the rate of wild-type cells, whereas constitutive stable DNA replication is not enhanced.

  12. Transcription regulatory elements are punctuation marks for DNA replication.

    Mirkin, Ekaterina V; Castro Roa, Daniel; Nudler, Evgeny; Mirkin, Sergei M

    2006-05-01

    Collisions between DNA replication and transcription significantly affect genome organization, regulation, and stability. Previous studies have described collisions between replication forks and elongating RNA polymerases. Although replication collisions with the transcription-initiation or -termination complexes are potentially even more important because most genes are not actively transcribed during DNA replication, their existence and mechanisms remained unproven. To address this matter, we have designed a bacterial promoter that binds RNA polymerase and maintains it in the initiating mode by precluding the transition into the elongation mode. By using electrophoretic analysis of replication intermediates, we have found that this steadfast transcription-initiation complex inhibits replication fork progression in an orientation-dependent manner during head-on collisions. Transcription terminators also appeared to attenuate DNA replication, but in the opposite, codirectional orientation. Thus, transcription regulatory signals may serve as "punctuation marks" for DNA replication in vivo. PMID:16670199

  13. Do transcriptional enhancers also augment DNA replication?

    O'Connor, D T; Subramani, S

    1988-01-01

    Enhancers are DNA elements that augment transcription in cis, independent of distance and orientation. Evidence such as hormone dependent neoplastic cell growth and the stimulation of viral replication by sequences present in enhancers suggests that enhancers may also directly affect DNA replication. We tested this hypothesis in recombinant plasmids by asking whether sequences that stimulated DNA replication shared the properties of transcriptional enhancers. The homologous simian virus 40 (S...

  14. Replication licensing and the DNA damage checkpoint

    Cook, Jeanette Gowen

    2009-01-01

    Accurate and timely duplication of chromosomal DNA requires that replication be coordinated with processes that ensure genome integrity. Significant advances in determining how the earliest steps in DNA replication are affected by DNA damage have highlighted some of the mechanisms to establish that coordination. Recent insights have expanded the relationship between the ATM and ATR-dependent checkpoint pathways and the proteins that bind and function at replication origins. These findings sug...

  15. DNA Replication via Entanglement Swapping

    Pusuluk, Onur

    2010-01-01

    Quantum effects are mainly used for the determination of molecular shapes in molecular biology, but quantum information theory may be a more useful tool to understand the physics of life. Molecular biology assumes that function is explained by structure, the complementary geometries of molecules and weak intermolecular hydrogen bonds. However, both this assumption and its converse are possible if organic molecules and quantum circuits/protocols are considered as hardware and software of living systems that are co-optimized during evolution. In this paper, we try to model DNA replication as a multiparticle entanglement swapping with a reliable qubit representation of nucleotides. In the model, molecular recognition of a nucleotide triggers an intrabase entanglement corresponding to a superposition state of different tautomer forms. Then, base pairing occurs by swapping intrabase entanglements with interbase entanglements.

  16. Cryptic single-stranded-DNA binding activities of the phage λ P and Escherichia coli DnaC replication initiation proteins facilitate the transfer of E. coli DnaB helicase onto DNA

    Learn, Brian A.; Um, Soo-Jong; Huang, Li; McMacken, Roger

    1997-01-01

    The bacteriophage λ P and Escherichia coli DnaC proteins are known to recruit the bacterial DnaB replicative helicase to initiator complexes assembled at the phage and bacterial origins, respectively. These specialized nucleoprotein assemblies facilitate the transfer of one or more molecules of DnaB helicase onto the chromosome; the transferred DnaB, in turn, promotes establishment of a processive replication fork apparatus. To learn more about the mechanism of the DnaB transfer reaction, we ...

  17. Cisplatin-Induced DNA Damage Activates Replication Checkpoint Signaling Components that Differentially Affect Tumor Cell SurvivalS⃞

    Wagner, Jill M.; Karnitz, Larry M.

    2009-01-01

    Cisplatin and other platinating agents are some of the most widely used chemotherapy agents. These drugs exert their antiproliferative effects by creating intrastrand and interstrand DNA cross-links, which block DNA replication. The cross-links mobilize signaling and repair pathways, including the Rad9-Hus1-Rad1-ATR-Chk1 pathway, a pathway that helps tumor cells survive the DNA damage inflicted by many chemotherapy agents. Here we show that Rad9 and ATR play critical r...

  18. Single molecule analysis of Trypanosoma brucei DNA replication dynamics.

    Calderano, Simone Guedes; Drosopoulos, William C; Quaresma, Marina Mônaco; Marques, Catarina A; Kosiyatrakul, Settapong; McCulloch, Richard; Schildkraut, Carl L; Elias, Maria Carolina

    2015-03-11

    Eukaryotic genome duplication relies on origins of replication, distributed over multiple chromosomes, to initiate DNA replication. A recent genome-wide analysis of Trypanosoma brucei, the etiological agent of sleeping sickness, localized its replication origins to the boundaries of multigenic transcription units. To better understand genomic replication in this organism, we examined replication by single molecule analysis of replicated DNA. We determined the average speed of replication forks of procyclic and bloodstream form cells and we found that T. brucei DNA replication rate is similar to rates seen in other eukaryotes. We also analyzed the replication dynamics of a central region of chromosome 1 in procyclic forms. We present evidence for replication terminating within the central part of the chromosome and thus emanating from both sides, suggesting a previously unmapped origin toward the 5' extremity of chromosome 1. Also, termination is not at a fixed location in chromosome 1, but is rather variable. Importantly, we found a replication origin located near an ORC1/CDC6 binding site that is detected after replicative stress induced by hydroxyurea treatment, suggesting it may be a dormant origin activated in response to replicative stress. Collectively, our findings support the existence of more replication origins in T. brucei than previously appreciated. PMID:25690894

  19. DNA replication: polymerase epsilon as a non-catalytic converter of the helicase.

    Zegerman, Philip

    2013-04-01

    In eukaryotes DNA polymerase epsilon (ε) synthesises the leading DNA strand during replication. A new study provides insight into how this polymerase also functions independently of its enzyme activity to assemble and activate the replicative helicase. PMID:23578873

  20. Genome instability induced by structured DNA and replication fork restart

    Schalbetter, Stephanie

    2012-01-01

    DNA replication is a central mechanism to all forms of life. Errors occurring during DNA replication can result in mutagenesis and genome rearrangements, which can cause various diseases. In this work I have investigated the stability of direct tandem repeats (TRs) in the context of replication and replication-associated repair mechanisms. During DNA replication the replication fork encounters many obstacles, such as DNA-protein barriers, secondary DNA structures and DNA lesions. How and if r...

  1. The Many Roles of PCNA in Eukaryotic DNA Replication.

    Boehm, E M; Gildenberg, M S; Washington, M T

    2016-01-01

    Proliferating cell nuclear antigen (PCNA) plays critical roles in many aspects of DNA replication and replication-associated processes, including translesion synthesis, error-free damage bypass, break-induced replication, mismatch repair, and chromatin assembly. Since its discovery, our view of PCNA has evolved from a replication accessory factor to the hub protein in a large protein-protein interaction network that organizes and orchestrates many of the key events at the replication fork. We begin this review article with an overview of the structure and function of PCNA. We discuss the ways its many interacting partners bind and how these interactions are regulated by posttranslational modifications such as ubiquitylation and sumoylation. We then explore the many roles of PCNA in normal DNA replication and in replication-coupled DNA damage tolerance and repair processes. We conclude by considering how PCNA can interact physically with so many binding partners to carry out its numerous roles. We propose that there is a large, dynamic network of linked PCNA molecules at and around the replication fork. This network would serve to increase the local concentration of all the proteins necessary for DNA replication and replication-associated processes and to regulate their various activities. PMID:27241932

  2. Self-replication of DNA rings

    Kim, Junghoon; Lee, Junwye; Hamada, Shogo; Murata, Satoshi; Ha Park, Sung

    2015-06-01

    Biology provides numerous examples of self-replicating machines, but artificially engineering such complex systems remains a formidable challenge. In particular, although simple artificial self-replicating systems including wooden blocks, magnetic systems, modular robots and synthetic molecular systems have been devised, such kinematic self-replicators are rare compared with examples of theoretical cellular self-replication. One of the principal reasons for this is the amount of complexity that arises when you try to incorporate self-replication into a physical medium. In this regard, DNA is a prime candidate material for constructing self-replicating systems due to its ability to self-assemble through molecular recognition. Here, we show that DNA T-motifs, which self-assemble into ring structures, can be designed to self-replicate through toehold-mediated strand displacement reactions. The inherent design of these rings allows the population dynamics of the systems to be controlled. We also analyse the replication scheme within a universal framework of self-replication and derive a quantitative metric of the self-replicability of the rings.

  3. The Cell Cycle Timing of Human Papillomavirus DNA Replication.

    Reinson, Tormi; Henno, Liisi; Toots, Mart; Ustav, Mart; Ustav, Mart

    2015-01-01

    Viruses manipulate the cell cycle of the host cell to optimize conditions for more efficient viral genome replication. One strategy utilized by DNA viruses is to replicate their genomes non-concurrently with the host genome; in this case, the viral genome is amplified outside S phase. This phenomenon has also been described for human papillomavirus (HPV) vegetative genome replication, which occurs in G2-arrested cells; however, the precise timing of viral DNA replication during initial and stable replication phases has not been studied. We developed a new method to quantitate newly synthesized DNA levels and used this method in combination with cell cycle synchronization to show that viral DNA replication is initiated during S phase and is extended to G2 during initial amplification but follows the replication pattern of cellular DNA during S phase in the stable maintenance phase. E1 and E2 protein overexpression changes the replication time from S only to both the S and G2 phases in cells that stably maintain viral episomes. These data demonstrate that the active synthesis and replication of the HPV genome are extended into the G2 phase to amplify its copy number and the duration of HPV genome replication is controlled by the level of the viral replication proteins E1 and E2. Using the G2 phase for genome amplification may be an important adaptation that allows exploitation of changing cellular conditions during cell cycle progression. We also describe a new method to quantify newly synthesized viral DNA levels and discuss its benefits for HPV research. PMID:26132923

  4. The Cell Cycle Timing of Human Papillomavirus DNA Replication.

    Tormi Reinson

    Full Text Available Viruses manipulate the cell cycle of the host cell to optimize conditions for more efficient viral genome replication. One strategy utilized by DNA viruses is to replicate their genomes non-concurrently with the host genome; in this case, the viral genome is amplified outside S phase. This phenomenon has also been described for human papillomavirus (HPV vegetative genome replication, which occurs in G2-arrested cells; however, the precise timing of viral DNA replication during initial and stable replication phases has not been studied. We developed a new method to quantitate newly synthesized DNA levels and used this method in combination with cell cycle synchronization to show that viral DNA replication is initiated during S phase and is extended to G2 during initial amplification but follows the replication pattern of cellular DNA during S phase in the stable maintenance phase. E1 and E2 protein overexpression changes the replication time from S only to both the S and G2 phases in cells that stably maintain viral episomes. These data demonstrate that the active synthesis and replication of the HPV genome are extended into the G2 phase to amplify its copy number and the duration of HPV genome replication is controlled by the level of the viral replication proteins E1 and E2. Using the G2 phase for genome amplification may be an important adaptation that allows exploitation of changing cellular conditions during cell cycle progression. We also describe a new method to quantify newly synthesized viral DNA levels and discuss its benefits for HPV research.

  5. DNA ligase I, the replicative DNA ligase

    Howes, Timothy R.L.; Tomkinson, Alan E.

    2012-01-01

    Multiple DNA ligation events are required to join the Okazaki fragments generated during lagging strand DNA synthesis. In eukaryotes, this is primarily carried out by members of the DNA ligase I family. The C-terminal catalytic region of these enzymes is composed of three domains: a DNA binding domain, an adenylation domain and an OB-fold domain. In the absence of DNA, these domains adopt an extended structure but transition into a compact ring structure when they engage a DNA nick, with each...

  6. Chromatin challenges during DNA replication and repair

    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...

  7. Inhibition of helicase activity by a small molecule impairs Werner syndrome helicase (WRN) function in the cellular response to DNA damage or replication stress.

    Aggarwal, Monika; Sommers, Joshua A; Shoemaker, Robert H; Brosh, Robert M

    2011-01-25

    Modulation of DNA repair proteins by small molecules has attracted great interest. An in vitro helicase activity screen was used to identify molecules that modulate DNA unwinding by Werner syndrome helicase (WRN), mutated in the premature aging disorder Werner syndrome. A small molecule from the National Cancer Institute Diversity Set designated NSC 19630 [1-(propoxymethyl)-maleimide] was identified that inhibited WRN helicase activity but did not affect other DNA helicases [Bloom syndrome (BLM), Fanconi anemia group J (FANCJ), RECQ1, RecQ, UvrD, or DnaB). Exposure of human cells to NSC 19630 dramatically impaired growth and proliferation, induced apoptosis in a WRN-dependent manner, and resulted in elevated γ-H2AX and proliferating cell nuclear antigen (PCNA) foci. NSC 19630 exposure led to delayed S-phase progression, consistent with the accumulation of stalled replication forks, and to DNA damage in a WRN-dependent manner. Exposure to NSC 19630 sensitized cancer cells to the G-quadruplex-binding compound telomestatin or a poly(ADP ribose) polymerase (PARP) inhibitor. Sublethal dosage of NSC 19630 and the chemotherapy drug topotecan acted synergistically to inhibit cell proliferation and induce DNA damage. The use of this WRN helicase inhibitor molecule may provide insight into the importance of WRN-mediated pathway(s) important for DNA repair and the replicational stress response. PMID:21220316

  8. Reconsidering DNA Polymerases at the Replication Fork in Eukaryotes

    Stillman, Bruce

    2015-01-01

    The distribution of DNA polymerase activities at the eukaryotic DNA replication fork was “established,” but recent genetic studies in this issue of Molecular Cell raise questions about which polymerases are copying the leading and lagging strand templates (Johnson et al, 2015).

  9. Inhomogeneous DNA replication kinetics is associated with immune system response

    Bechhoefer, John; Gauthier, Michel G.; Norio, Paolo

    2013-03-01

    In eukaryotic organisms, DNA replication is initiated at ``origins,'' launching ``forks'' that spread bidirectionally to replicate the genome. The distribution and firing rate of these origins and the fork progression velocity form the ``replication program.'' Previous models of DNA replication in eukaryotes have assumed firing rates and replication fork velocities to be homogeneous across the genome. But large variations in origin activity and fork velocity do occur. Here, we generalize our replication model to allow for arbitrary spatial variation of initiation rates and fork velocities in a given region of the genome. We derive and solve rate equations for the forks and replication probability, to obtain the mean-field replication program. After testing the model on simulations, we analyze the changes in replication program that occur during B cell development in the mouse. B cells play a major role in the adaptive immune system by producing the antibodies. We show that the process of cell differentiation is associated with a change in replication program, where the zones of high origin initiation rates located in the immunoglobulin heavy-chain locus shift their position as the locus prepares to undergo the recombination events responsible for generating antibody specificity. This work was funded by HSFP and NSERC-Canada (MGG and JB) and by NIH-NIGMS grant R01GM080606 (PN).

  10. RNase H is not involved in the induction of stable DNA replication in Escherichia coli.

    Bialy, H; Kogoma, T

    1986-01-01

    rnh mutations of Escherichia coli inactivating RNase H activity allow the initiation of rounds of DNA replication in the absence of protein synthesis (stable DNA replication). However, levels of RNase H did not change during or after the induction of stable DNA replication in rnh+ strains by incubation with nalidixic acid or UV irradiation.

  11. Spatial regulation and organization of DNA replication within the nucleus

    Natsume, Toyoaki; Tanaka, Tomoyuki U.

    2009-01-01

    Duplication of chromosomal DNA is a temporally and spatially regulated process. The timing of DNA replication initiation at various origins is highly coordinated; some origins fire early and others late during S phase. Moreover, inside the nuclei, the bulk of DNA replication is physically organized in replication factories, consisting of DNA polymerases and other replication proteins. In this review article, we discuss how DNA replication is organized and regulated spatially within the nucleu...

  12. Assembling semiconductor nanocomposites using DNA replication technologies.

    Heimer, Brandon W.; Crown, Kevin K.; Bachand, George David

    2005-11-01

    Deoxyribonucleic acid (DNA) molecules represent Nature's genetic database, encoding the information necessary for all cellular processes. From a materials engineering perspective, DNA represents a nanoscale scaffold with highly refined structure, stability across a wide range of environmental conditions, and the ability to interact with a range of biomolecules. The ability to mass-manufacture functionalized DNA strands with Angstrom-level resolution through DNA replication technology, however, has not been explored. The long-term goal of the work presented in this report is focused on exploiting DNA and in vitro DNA replication processes to mass-manufacture nanocomposite materials. The specific objectives of this project were to: (1) develop methods for replicating DNA strands that incorporate nucleotides with ''chemical handles'', and (2) demonstrate attachment of nanocrystal quantum dots (nQDs) to functionalized DNA strands. Polymerase chain reaction (PCR) and primer extension methodologies were used to successfully synthesize amine-, thiol-, and biotin-functionalized DNA molecules. Significant variability in the efficiency of modified nucleotide incorporation was observed, and attributed to the intrinsic properties of the modified nucleotides. Noncovalent attachment of streptavidin-coated nQDs to biotin-modified DNA synthesized using the primer extension method was observed by epifluorescence microscopy. Data regarding covalent attachment of nQDs to amine- and thiol-functionalized DNA was generally inconclusive; alternative characterization tools are necessary to fully evaluate these attachment methods. Full realization of this technology may facilitate new approaches to manufacturing materials at the nanoscale. In addition, composite nQD-DNA materials may serve as novel recognition elements in sensor devices, or be used as diagnostic tools for forensic analyses. This report summarizes the results obtained over the course of this 1-year

  13. Mutations reducing replication from R-loops suppress the defects of growth, chromosome segregation and DNA supercoiling in cells lacking topoisomerase I and RNase HI activity.

    Usongo, Valentine; Martel, Makisha; Balleydier, Aurélien; Drolet, Marc

    2016-04-01

    R-loop formation occurs when the nascent RNA hybridizes with the template DNA strand behind the RNA polymerase. R-loops affect a wide range of cellular processes and their use as origins of replication was the first function attributed to them. In Escherichia coli, R-loop formation is promoted by the ATP-dependent negative supercoiling activity of gyrase (gyrA and gyrB) and is inhibited by topoisomerase (topo) I (topA) relaxing transcription-induced negative supercoiling. RNase HI (rnhA) degrades the RNA moiety of R-loops. The depletion of RNase HI activity in topA null mutants was previously shown to lead to extensive DNA relaxation, due to DNA gyrase inhibition, and to severe growth and chromosome segregation defects that were partially corrected by overproducing topo III (topB). Here, DNA gyrase assays in crude cell extracts showed that the ATP-dependent activity (supercoiling) of gyrase but not its ATP-independent activity (relaxation) was inhibited in topA null cells lacking RNase HI. To characterize the cellular event(s) triggered by the absence of RNase HI, we performed a genetic screen for suppressors of the growth defect of topA rnhA null cells. Suppressors affecting genes in replication (holC2::aph and dnaT18::aph) nucleotide metabolism (dcd49::aph), RNA degradation (rne59::aph) and fimbriae synthesis (fimD22::aph) were found to reduce replication from R-loops and to restore supercoiling, thus pointing to a correlation between R-loop-dependent replication in topA rnhA mutants and the inhibition of gyrase activity and growth. Interestingly, the position of fimD on the E. coli chromosome corresponds to the site of one of the five main putative origins of replication from R-loops in rnhA null cells recently identified by next-generation sequencing, thus suggesting that the fimD22::aph mutation inactivated one of these origins. Furthermore, we show that topo III overproduction is unable to complement the growth defect of topA rnhA null mutants at low

  14. Specificity and function of Archaeal DNA replication initiator proteins

    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...... investigate the role of ATP binding and hydrolysis in initiator function in vivo and in vitro. We find that the ATP-bound form of Orc1-1 is proficient for replication and implicates hydrolysis of ATP in downregulation of origin activity. Finally, we reveal that ATP and DNA binding by Orc1-1 remodels the...... protein's structure rather than that of the DNA template....

  15. Kinetic model of DNA replication in eukaryotic organisms

    Herrick, J; Bensimon, A; Herrick, John; Bechhoefer, John; Bensimon, Aaron

    2001-01-01

    We formulate a kinetic model of DNA replication that quantitatively describes recent results on DNA replication in the in vitro system of Xenopus laevis prior to the mid-blastula transition. The model describes well a large amount of different data within a simple theoretical framework. This allows one, for the first time, to determine the parameters governing the DNA replication program in a eukaryote on a genome-wide basis. In particular, we have determined the frequency of origin activation in time and space during the cell cycle. Although we focus on a specific stage of development, this model can easily be adapted to describe replication in many other organisms, including budding yeast.

  16. Completion of DNA replication in Escherichia coli

    Wendel, Brian M.; Courcelle, Charmain T.; Courcelle, Justin

    2014-01-01

    The mechanism by which cells recognize and complete replicated regions at their precise doubling point must be remarkably efficient, occurring thousands of times per cell division along the chromosomes of humans. However, this process remains poorly understood. Here we show that, in Escherichia coli, the completion of replication involves an enzymatic system that effectively counts pairs and limits cellular replication to its doubling point by allowing converging replication forks to transiently continue through the doubling point before the excess, over-replicated regions are incised, resected, and joined. Completion requires RecBCD and involves several proteins associated with repairing double-strand breaks including, ExoI, SbcDC, and RecG. However, unlike double-strand break repair, completion occurs independently of homologous recombination and RecA. In some bacterial viruses, the completion mechanism is specifically targeted for inactivation to allow over-replication to occur during lytic replication. The results suggest that a primary cause of genomic instabilities in many double-strand-break-repair mutants arises from an impaired ability to complete replication, independent from DNA damage. PMID:25368150

  17. SMARCAL1 maintains telomere integrity during DNA replication.

    Poole, Lisa A; Zhao, Runxiang; Glick, Gloria G; Lovejoy, Courtney A; Eischen, Christine M; Cortez, David

    2015-12-01

    The SMARCAL1 (SWI/SNF related, matrix-associated, actin-dependent, regulator of chromatin, subfamily A-like 1) DNA translocase is one of several related enzymes, including ZRANB3 (zinc finger, RAN-binding domain containing 3) and HLTF (helicase-like transcription factor), that are recruited to stalled replication forks to promote repair and restart replication. These enzymes can perform similar biochemical reactions such as fork reversal; however, genetic studies indicate they must have unique cellular activities. Here, we present data showing that SMARCAL1 has an important function at telomeres, which present an endogenous source of replication stress. SMARCAL1-deficient cells accumulate telomere-associated DNA damage and have greatly elevated levels of extrachromosomal telomere DNA (C-circles). Although these telomere phenotypes are often found in tumor cells using the alternative lengthening of telomeres (ALT) pathway for telomere elongation, SMARCAL1 deficiency does not yield other ALT phenotypes such as elevated telomere recombination. The activity of SMARCAL1 at telomeres can be separated from its genome-maintenance activity in bulk chromosomal replication because it does not require interaction with replication protein A. Finally, this telomere-maintenance function is not shared by ZRANB3 or HLTF. Our results provide the first identification, to our knowledge, of an endogenous source of replication stress that requires SMARCAL1 for resolution and define differences between members of this class of replication fork-repair enzymes. PMID:26578802

  18. Replication of DNA during barley endosperm development

    Giese, H.

    1992-01-01

    The incorporation of [6-H-3]-thymidine into DNA of developing barley end sperm was examined by autoradiography of cross sections of seeds and DNA analysis. The majority of nuclear divisions took place in the very young endosperm, but as late as 25 days after anthesis there was evidence for DNA re...... replication. The DNA content of the endosperm increases during development and in response to nitrogen application in parallel to the storage protein synthesis profile. The hordein genes were hypersensitive to DNase I treatment throughout development.......The incorporation of [6-H-3]-thymidine into DNA of developing barley end sperm was examined by autoradiography of cross sections of seeds and DNA analysis. The majority of nuclear divisions took place in the very young endosperm, but as late as 25 days after anthesis there was evidence for DNA...

  19. Design and characterization of N2-arylaminopurines which selectively inhibit replicative DNA synthesis and replication-specific DNA polymerases: guanine derivatives active on mammalian DNA polymerase alpha and bacterial DNA polymerase III.

    Wright, G E; Baril, E F; Brown, V M; Brown, N C

    1982-01-01

    The 2-amino substituted derivatives of guanine, N2-(p-n-butylphenyl)guanine (BuPG) and N2-(3',4'-trimethylenephenyl) guanine (TMPG), were synthesized and found to selectively inhibit, respectively, HeLa cell DNA polymerase alpha (po1 alpha) and B. subtilis DNA polymerase III (po1 III). Both purines, like their corresponding uracil analogs, BuAu and TMAU (2,9), were specifically competitive with dGTP in their inhibitory action on their target polymerases. BuPG, the pol alpha-specific purine, w...

  20. The BPV-1 E2 DNA-contact helix cysteine is required for transcriptional activation but not replication in mammalian cells.

    Grossel, M J; Barsoum, J; Prakash, S S; Androphy, E J

    1996-03-01

    The papillomavirus E2 protein contains an amino-terminal region thought necessary and sufficient to support transcriptional activation and a carboxy-terminal region shown to direct sequence-specific DNA binding and dimerization. A cysteine residue in the center of the E2 DNA recognition helix is highly conserved among papillomavirus E2 proteins. Mutations of this cysteine in bovine papillomavirus type 1 E2 to serine and glycine resulted in proteins which failed to activate E2-dependent promoters in mammalian cells. These E2 mutants were DNA-binding competent, dimeric, and nuclear. When fused to the VP16 transactivation domain, C-terminal regions of E2 containing the mutations at 340 supported transcriptional activation, indicating that the heterologous trans-activation domain did not require cysteine in the DNA-binding helix as did the full-length E2 transactivating protein. Although cysteine-340 was required for transcriptional activation it was not required for DNA replication in vivo. Together, these results suggest that the E2 DNA-binding domain may directly contribute to functions of transcriptional activation previously thought limited to the N-terminal domain. PMID:8599215

  1. PTEN Controls the DNA Replication Process through MCM2 in Response to Replicative Stress

    Jiawen Feng

    2015-11-01

    Full Text Available PTEN is a tumor suppressor frequently mutated in human cancers. PTEN inhibits the phosphatidylinositol 3-kinase (PI3K-AKT cascade, and nuclear PTEN guards the genome by multiple mechanisms. Here, we report that PTEN physically associates with the minichromosome maintenance complex component 2 (MCM2, which is essential for DNA replication. Specifically, PTEN dephosphorylates MCM2 at serine 41 (S41 and restricts replication fork progression under replicative stress. PTEN disruption results in unrestrained fork progression upon replication stalling, which is similar to the phenotype of cells expressing the phosphomimic MCM2 mutant S41D. Moreover, PTEN is necessary for prevention of chromosomal aberrations under replication stress. This study demonstrates that PTEN regulates DNA replication through MCM2 and loss of PTEN function leads to replication defects and genomic instability. We propose that PTEN plays a critical role in maintaining genetic stability through a replication-specific mechanism, and this is a crucial facet of PTEN tumor suppressor activity.

  2. DNA replication of mitotic chromatin in Xenopus egg extracts

    Prokhorova, Tatyana A.; Mowrer, Karen; Gilbert, Catherine H.; Walter, Johannes C.

    2003-01-01

    Prereplication complexes are assembled at eukaryotic origins of DNA replication in the G1 phase of the cell cycle, and they are activated in S phase by cyclin-dependent kinase (Cdk)2/cyclin E and Cdk2/cyclin A. Previous experiments using Xenopus nuclear assembly egg extracts suggested that Cdk1/cyclin A, which is normally active in early mitosis, can replace the function of Cdk2 in driving DNA replication, whereas Cdk1/cyclin B, which functions later in mitosis, cannot. Here, we use a complet...

  3. Entropy involved in fidelity of DNA replication

    Arias-Gonzalez, J Ricardo

    2012-01-01

    Information has an entropic character which can be analyzed within the Statistical Theory in molecular systems. R. Landauer and C.H. Bennett showed that a logical copy can be carried out in the limit of no dissipation if the computation is performed sufficiently slowly. Structural and recent single-molecule assays have provided dynamic details of polymerase machinery with insight into information processing. We introduce a rigorous characterization of Shannon Information in biomolecular systems and apply it to DNA replication in the limit of no dissipation. Specifically, we devise an equilibrium pathway in DNA replication to determine the entropy generated in copying the information from a DNA template in the absence of friction. Both the initial state, the free nucleotides randomly distributed in certain concentrations, and the final state, a polymerized strand, are mesoscopic equilibrium states for the nucleotide distribution. We use empirical stacking free energies to calculate the probabilities of incorpo...

  4. DNA primase acts as a molecular brake in DNA replication

    Lee, Jong-Bong; Hite, Richard K.; Hamdan, Samir M.; Xie, X. Sunney; Richardson, Charles C.; Oijen, Antoine M. van

    2006-01-01

    A hallmark feature of DNA replication is the coordination between the continuous polymerization of nucleotides on the leading strand and the discontinuous synthesis of DNA on the lagging strand. This synchronization requires a precisely timed series of enzymatic steps that control the synthesis of a

  5. dnaC-dependent reconstitution of replication forks in Escherichia coli lysates.

    Nüsslein-Crystalla, V; Niedenhof, I; Rein, R.

    1982-01-01

    Lysates of Escherichia coli exhibit a DNA-synthesizing activity that depends on the presence of replication forks and of replication proteins. Replicative activity was reconstituted in vitro by mixing lysates prepared from temperature-sensitive dnaB mutants with wild-type dnaB protein. Lysates of double mutants deficient in both dnaB and dnaC genes could only be complemented by the addition of both dnaB and dnaC proteins, whereas lysates deficient in dnaC protein did not require the addition ...

  6. Evidence for interplay among yeast replicative DNA polymerases alpha, delta and epsilon from studies of exonuclease and polymerase active site mutations

    Pavlov Youri I

    2004-05-01

    Full Text Available Abstract Background DNA polymerase ε (Pol ε is essential for S-phase replication, DNA damage repair and checkpoint control in yeast. A pol2-Y831A mutation leading to a tyrosine to alanine change in the Pol ε active site does not cause growth defects and confers a mutator phenotype that is normally subtle but strong in a mismatch repair-deficient strain. Here we investigate the mechanism responsible for the mutator effect. Results Purified four-subunit Y831A Pol ε turns over more deoxynucleoside triphosphates to deoxynucleoside monophosphates than does wild-type Pol ε, suggesting altered coordination between the polymerase and exonuclease active sites. The pol2-Y831A mutation suppresses the mutator effect of the pol2-4 mutation in the exonuclease active site that abolishes proofreading by Pol ε, as measured in haploid strain with the pol2-Y831A,4 double mutation. Analysis of mutation rates in diploid strains reveals that the pol2-Y831A allele is recessive to pol2-4. In addition, the mutation rates of strains with the pol2-4 mutation in combination with active site mutator mutations in Pol δ and Pol α suggest that Pol ε may proofread certain errors made by Pol α and Pol δ during replication in vivo. Conclusions Our data suggest that Y831A replacement in Pol ε reduces replication fidelity and its participation in chromosomal replication, but without eliminating an additional function that is essential for viability. This suggests that other polymerases can substitute for certain functions of polymerase ε.

  7. Regulation of eukaryotic DNA replication and nuclear structure

    WUJIARUI

    1999-01-01

    In eukaryote,nuclear structure is a key component for the functions of eukaryotic cells.More and more evidences show that the nuclear structure plays important role in regulating DNA replication.The nuclear structure provides a physical barrier for the replication licensing,participates in the decision where DNA replication initiates,and organizes replication proteins as replication factory for DNA replication.Through these works,new concepts on the regulation of DNA replication have emerged,which will be discussed in this minireview.

  8. A moving DNA replication factory in Caulobacter crescentus

    Jensen, Rasmus B.; Wang, Sherry C.; Shapiro, Lucy

    2001-01-01

    The in vivo intracellular location of components of the Caulobacter replication apparatus was visualized during the cell cycle. Replisome assembly occurs at the chromosomal origin located at the stalked cell pole, coincident with the initiation of DNA replication. The replisome gradually moves to midcell as DNA replication proceeds and disassembles upon completion of DNA replication. Although the newly replicated origin regions of the chromosome are rapidly moved to opposite cell poles by an ...

  9. The DNA damage and the DNA replication checkpoints converge at the MBF transcription factor

    Ivanova, Tsvetomira Georgieva, 1978-; Alves-Rodrigues, Isabel; G??mez Escoda, Blanca; Dutta, Chaitali; DeCaprio, James A.; Rhind, Nick; Hidalgo Hernando, Elena; Ayt?? del Olmo, Jos??

    2013-01-01

    In fission yeast cells, Cds1 is the effector kinase of the DNA replication checkpoint. We previously showed that when the DNA replication checkpoint is activated, the repressor Yox1 is phosphorylated and inactivated by Cds1, resulting in activation of MluI-binding factor (MBF)-dependent transcription. This is essential to reinitiate DNA synthesis and for correct G1-to-S transition. Here we show that Cdc10, which is an essential part of the MBF core, is the target of the DNA damage checkpoint....

  10. Saying a firm ‘no’ to DNA re-replication

    Blow, J. Julian; Prokhorova, Tatyana A.

    1999-01-01

    Yeast cells exclude the DNA-replication-initiation factor Mcm4 from the nucleus during the S and G2 phases of the cell cycle, when active replication ‘origins’ must not reassemble. This exclusion is dependent on cyclin-dependent kinases and provides a general mechanism for preventing re-replication of DNA in a single cell cycle.

  11. Human cytomegalovirus induces JC virus DNA replication in human fibroblasts.

    Heilbronn, R; Albrecht, I; Stephan, S; Bürkle, A; zur Hausen, H

    1993-01-01

    JC virus, a human papovavirus, is the causative agent of the demyelinating brain disease progressive multifocal leucoencephalopathy (PML). PML is a rare but fatal disease which develops as a complication of severe immunosuppression. Latent JC virus is harbored by many asymptomatic carriers and is transiently reactivated from the latent state upon immunosuppression. JC virus has a very restricted host range, with human glial cells being the only tissue in which it can replicate at reasonable efficiency. Evidence that latent human cytomegalovirus is harbored in the kidney similar to latent JC virus led to the speculation that during episodes of impaired immunocompetence, cytomegalovirus might serve as helper virus for JC virus replication in otherwise nonpermissive cells. We show here that cytomegalovirus infection indeed leads to considerable JC virus DNA replication in cultured human fibroblasts that are nonpermissive for the replication of JC virus alone. Cytomegalovirus-mediated JC virus replication is dependent on the JC virus origin of replication and T antigen. Ganciclovir-induced inhibition of cytomegalovirus replication is associated with a concomitant inhibition of JC virus replication. These results suggest that reactivation of cytomegalovirus during episodes of immunosuppression might lead to activation of latent JC virus, which would enhance the probability of subsequent PML development. Ganciclovir-induced repression of both cytomegalovirus and JC virus replication may form the rational basis for the development of an approach toward treatment or prevention of PML. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:8248262

  12. Origin of replication of the DNA of a herpesvirus (pseudorabies).

    Ben-Porat, T; Veach, R A

    1980-01-01

    During the first round of the replication of pseudorabies virus DNA, replicating DNA is mainly in the form of circles. The main origin of replication is located inthe region of the molecule bearing the inverted repeat. Replication proceeds unidirectionally from the origin.

  13. Flavin-dependent thymidylate synthase X limits chromosomal DNA replication

    Escartin, Frédéric; Skouloubris, Stéphane; Liebl, Ursula; Myllykallio, Hannu

    2008-01-01

    We have investigated the hitherto unexplored possibility that differences in the catalytic efficiencies of thymidylate synthases ThyX and ThyA, enzymes that produce the essential DNA precursor dTMP, have influenced prokaryotic genome evolution. We demonstrate that DNA replication speed in bacteria and archaea that contain the low-activity ThyX enzyme is up to 10-fold decreased compared with species that contain the catalytically more efficient ThyA. Our statistical studies of >400 genomes ind...

  14. Preventing DNA over-replication: a Cdk perspective

    Porter Andrew CG

    2008-01-01

    Full Text Available Abstract The cell cycle is tightly controlled to ensure that replication origins fire only once per cycle and that consecutive S-phases are separated by mitosis. When controls fail, DNA over-replication ensues: individual origins fire more than once per S-phase (re-replication or consecutive S-phases occur without intervening mitoses (endoreduplication. In yeast the cell cycle is controlled by a single cyclin dependent kinase (Cdk that prevents origin licensing at times when it promotes origin firing, and that is inactivated, via proteolysis of its partner cyclin, as cells undergo mitosis. A quantitative model describes three levels of Cdk activity: low activity allows licensing, intermediate activity allows firing but prevents licensing, and high activity promotes mitosis. In higher eukaryotes the situation is complicated by the existence of additional proteins (geminin, Cul4-Ddb1Cdt2, and Emi1 that control licensing. A current challenge is to understand how these various control mechanisms are co-ordinated and why the degree of redundancy between them is so variable. Here the experimental induction of DNA over-replication is reviewed in the context of the quantitative model of Cdk action. Endoreduplication is viewed as a consequence of procedures that cause Cdk activity to fall below the threshold required to prevent licensing, and re-replication as the result of procedures that increase that threshold value. This may help to explain why over-replication does not necessarily require reduced Cdk activity and how different mechanisms conspire to prevent over-replication. Further work is nevertheless required to determine exactly how losing just one licensing control mechanism often causes over-replication, and why this varies between cell systems.

  15. Human papillomavirus 16 L2 inhibits the transcriptional activation function, but not the DNA replication function, of HPV-16 E2

    Okoye, A.; Cordano, P; Taylor, E.R.; Morgan, I.M.; Everett, R.; Campo, M. S.

    2005-01-01

    In this study we analysed the outcome of the interaction between HPV-16 L2 and E2 on the transactivation and DNA replication functions of E2. When E2 was expressed on its own, it transactivated a number of E2-responsive promoters but co-expression of L2 led to the down-regulation of the transcription transactivation activity of the E2 protein. This repression is not mediated by an increased degradation of the E2 protein. In contrast, the expression of L2 had no effect on the ability of E2 to ...

  16. Emi1 is needed to couple DNA replication with mitosis but does not regulate activation of the mitotic APC/C

    Di Fiore, Barbara; Pines, Jonathon

    2007-01-01

    Ubiquitin-mediated proteolysis is critical for the alternation between DNA replication and mitosis and for the key regulatory events in mitosis. The anaphase-promoting complex/cyclosome (APC/C) is a conserved ubiquitin ligase that has a fundamental role in regulating mitosis and the cell cycle in all eukaryotes. In vertebrate cells, early mitotic inhibitor 1 (Emi1) has been proposed as an important APC/C inhibitor whose destruction may trigger activation of the APC/C at mitosis. However, in t...

  17. DNA polymerase beta can substitute for DNA polymerase I in the initiation of plasmid DNA replication.

    Sweasy, J B; Chen, M.; Loeb, L A

    1995-01-01

    We previously demonstrated that mammalian DNA polymerase beta can substitute for DNA polymerase I of Escherichia coli in DNA replication and in base excision repair. We have now obtained genetic evidence suggesting that DNA polymerase beta can substitute for E. coli DNA polymerase I in the initiation of replication of a plasmid containing a pMB1 origin of DNA replication. Specifically, we demonstrate that a plasmid with a pMB1 origin of replication can be maintained in an E. coli polA mutant ...

  18. Depletion of Cellular Pre-Replication Complex Factors Results in Increased Human Cytomegalovirus DNA Replication

    Tamara Evans Braun; Emma Poole; John Sinclair

    2012-01-01

    Although HCMV encodes many genes required for the replication of its DNA genome, no HCMV-encoded orthologue of the origin binding protein, which has been identified in other herpesviruses, has been identified. This has led to speculation that HCMV may use other viral proteins or possibly cellular factors for the initiation of DNA synthesis. It is also unclear whether cellular replication factors are required for efficient replication of viral DNA during or after viral replication origin recog...

  19. On the scattering of DNA replication completion times

    Meilikhov, E. Z.; Farzetdinova, R. M.

    2015-07-01

    Stochasticity of Eukaryotes' DNA replication should not lead to large fluctuations of replication times, which could result in mitotic catastrophes. Fundamental problem that cells face is how to be ensured that entire genome is replicated on time. We develop analytic approach of calculating DNA replication times, that being simplified and approximate, leads, nevertheless, to results practically coincident with those that were obtained by some sophisticated methods. In the framework of that model we consider replication times' scattering and discuss the influence of repair stopping on kinetics of DNA replication. Our main explicit formulae for DNA replication time t r ∝ ( N is the total number of DNA base pairs) is of general character and explains basic features of DNA replication kinetics.

  20. Enzymes involved in organellar DNA replication in photosynthetic eukaryotes

    Moriyama, Takashi; Sato, Naoki

    2014-01-01

    Plastids and mitochondria possess their own genomes. Although the replication mechanisms of these organellar genomes remain unclear in photosynthetic eukaryotes, several organelle-localized enzymes related to genome replication, including DNA polymerase, DNA primase, DNA helicase, DNA topoisomerase, single-stranded DNA maintenance protein, DNA ligase, primer removal enzyme, and several DNA recombination-related enzymes, have been identified. In the reference Eudicot plant Arabidopsis thaliana...

  1. Timing, coordination, and rhythm: Acrobatics at the DNA replication fork

    Hamdan, Samir

    2010-04-09

    In DNA replication, the antiparallel nature of the parental duplex imposes certain constraints on the activity of the DNA polymerases that synthesize new DNA. The leading-strand polymerase advances in a continuous fashion, but the lagging-strand polymerase is forced to restart at short intervals. In several prokaryotic systems studied so far, this problem is solved by the formation of a loop in the lagging strand of the replication fork to reorient the lagging-strand DNA polymerase so that it advances in parallel with the leading-strand polymerase. The replication loop grows and shrinks during each cycle of Okazaki fragment synthesis. The timing of Okazaki fragment synthesis and loop formation is determined by a subtle interplay of enzymatic activities at the fork. Recent developments in single-molecule techniques have enabled the direct observation of these processes and have greatly contributed to a better understanding of the dynamic nature of the replication fork. Here, we will review recent experimental advances, present the current models, and discuss some of the exciting developments in the field. 2010 by The American Society for Biochemistry and Molecular Biology, Inc.

  2. Drosophila Ctf4 is essential for efficient DNA replication and normal cell cycle progression

    Christensen Tim W; Gosnell Justin A

    2011-01-01

    Abstract Background Proper coordination of the functions at the DNA replication fork is vital to the normal functioning of a cell. Specifically the precise coordination of helicase and polymerase activity is crucial for efficient passage though S phase. The Ctf4 protein has been shown to be a central member of the replication fork and links the replicative MCM helicase and DNA polymerase α primase. In addition, it has been implicated as a member of a complex that promotes replication fork sta...

  3. Specific binding of a cellular DNA replication protein to the origin of replication of adenovirus DNA

    1984-01-01

    Nuclear factor I, a 47-kilodalton protein, purified from nuclear extracts of uninfected HeLa cells, is involved in the initiation and possibly the elongation of replicating adenovirus (Ad) DNA in vitro. The binding of nuclear factor I to DNA has been monitored by a filter binding assay of nuclear factor I to DNA has been monitored by a filter binding assay using plasmid pLA1 DNA, which contains a 3,290 base-pair fragment derived from the left-hand terminus (coordinates, 0-9.4 map units) of Ad...

  4. Role of Polymer Loops in DNA Replication

    Jun, S; Jun, Suckjoon; Bechhoefer, John

    2003-01-01

    Loop formation in long molecules occurs many places in nature, from solutions of carbon nanotubes to polymers inside a cell. In this article, we review theoretical studies of the static and dynamic properties of polymer loops. For example, long polymers must search many configurations to find a "target" binding site, while short polymers are stiff and resist bending. In between, there is an optimal loop size, which balances the entropy of long loops against the energetic cost of short loops. We show that such simple pictures of loop formation can explain several long-standing observations in DNA replication, quantitatively.

  5. FBH1 promotes DNA double-strand breakage and apoptosis in response to DNA replication stress.

    Jeong, Yeon-Tae; Rossi, Mario; Cermak, Lukas; Saraf, Anita; Florens, Laurence; Washburn, Michael P; Sung, Patrick; Schildkraut, Carl L; Schildkraut, Carl; Pagano, Michele

    2013-01-21

    Proper resolution of stalled replication forks is essential for genome stability. Purification of FBH1, a UvrD DNA helicase, identified a physical interaction with replication protein A (RPA), the major cellular single-stranded DNA (ssDNA)-binding protein complex. Compared with control cells, FBH1-depleted cells responded to replication stress with considerably fewer double-strand breaks (DSBs), a dramatic reduction in the activation of ATM and DNA-PK and phosphorylation of RPA2 and p53, and a significantly increased rate of survival. A minor decrease in ssDNA levels was also observed. All these phenotypes were rescued by wild-type FBH1, but not a FBH1 mutant lacking helicase activity. FBH1 depletion had no effect on other forms of genotoxic stress in which DSBs form by means that do not require ssDNA intermediates. In response to catastrophic genotoxic stress, apoptosis prevents the persistence and propagation of DNA lesions. Our findings show that FBH1 helicase activity is required for the efficient induction of DSBs and apoptosis specifically in response to DNA replication stress. PMID:23319600

  6. Replication stalling by catalytically impaired Twinkle induces mitochondrial DNA rearrangements in cultured cells

    Pohjoismaki, J.L.; Goffart, S.; Spelbrink, J.N.

    2011-01-01

    Pathological mitochondrial DNA (mtDNA) rearrangements have been proposed to result from repair of double-strand breaks caused by blockage of mitochondrial DNA (mtDNA) replication. As mtDNA deletions are seen only in post-mitotic tissues, it has been suggested that they are selected out in actively d

  7. Bloom’s syndrome helicase and Mus81 are required to induce transient double-strand DNA breaks in response to DNA replication stress

    Shimura, Tsutomu; Torres, Michael J.; Melvenia M Martin; Rao, V. Ashutosh; Pommier, Yves; Katsura, Mari; Miyagawa, Kiyoshi; Aladjem, Mirit I

    2007-01-01

    Perturbed DNA replication either activates a cell cycle checkpoint, which halts DNA replication, or decreases the rate of DNA synthesis without activating a checkpoint. Here we report that at low doses, replication inhibitors did not activate a cell cycle checkpoint, but they did activate a process that required functional Bloom’s syndrome-associated (BLM) helicase, Mus81 nuclease and ATR kinase to induce transient double stranded DNA breaks. The induction of transient DNA breaks was accompan...

  8. Geminin: a major DNA replication safeguard in higher eukaryotes

    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...

  9. Visualizing Single-molecule DNA Replication with Fluorescence Microscopy

    Tanner, Nathan A.; Loparo, Joseph J.; Oijen, Antoine M. van

    2009-01-01

    We describe a simple fluorescence microscopy-based real-time method for observing DNA replication at the single-molecule level. A circular, forked DNA template is attached to a functionalized glass coverslip and replicated extensively after introduction of replication proteins and nucleotides. The g

  10. Initiation of bacteriophage lambda DNA replication in vitro with purified lambda replication proteins.

    Wold, M S; Mallory, J B; Roberts, J D; LeBowitz, J H; McMacken, R

    1982-01-01

    We have developed a soluble enzyme system that replicates exogenously added plasmid DNA (lambda dv) bearing the replication origin of the bacteriophage lambda chromosome. The system contains pure phage lambda O and P replication proteins and a partially purified mixture of Escherichia coli replication proteins [the enzyme system of Fuller, R.S., Kaguni, J.M. & Kornberg, A. (1981) Proc. Natl. Acad. Sci. USA 78, 7370-7374). The features of lambda dv replication in this system closely resemble t...

  11. Mouse zygotes respond to severe sperm DNA damage by delaying paternal DNA replication and embryonic development.

    Joanna E Gawecka

    Full Text Available Mouse zygotes do not activate apoptosis in response to DNA damage. We previously reported a unique form of inducible sperm DNA damage termed sperm chromatin fragmentation (SCF. SCF mirrors some aspects of somatic cell apoptosis in that the DNA degradation is mediated by reversible double strand breaks caused by topoisomerase 2B (TOP2B followed by irreversible DNA degradation by a nuclease(s. Here, we created zygotes using spermatozoa induced to undergo SCF (SCF zygotes and tested how they responded to moderate and severe paternal DNA damage during the first cell cycle. We found that the TUNEL assay was not sensitive enough to identify the breaks caused by SCF in zygotes in either case. However, paternal pronuclei in both groups stained positively for γH2AX, a marker for DNA damage, at 5 hrs after fertilization, just before DNA synthesis, while the maternal pronuclei were negative. We also found that both pronuclei in SCF zygotes with moderate DNA damage replicated normally, but paternal pronuclei in the SCF zygotes with severe DNA damage delayed the initiation of DNA replication by up to 12 hrs even though the maternal pronuclei had no discernable delay. Chromosomal analysis of both groups confirmed that the paternal DNA was degraded after S-phase while the maternal pronuclei formed normal chromosomes. The DNA replication delay caused a marked retardation in progression to the 2-cell stage, and a large portion of the embryos arrested at the G2/M border, suggesting that this is an important checkpoint in zygotic development. Those embryos that progressed through the G2/M border died at later stages and none developed to the blastocyst stage. Our data demonstrate that the zygote responds to sperm DNA damage through a non-apoptotic mechanism that acts by slowing paternal DNA replication and ultimately leads to arrest in embryonic development.

  12. Reactivation of DNA replication of the parvovirus MVM in UV preirradiated mouse cells

    The parvovirus Minute-Virus-of-Mice (MVM) was used to probe the DNA replication activities expressed by mouse fibroblasts. This system allowed us to study quantitatively the effect of UV-induced DNA lesions on the progression of DNA replication in vivo. MVM was UV-irradiated prior to infection. Pyrimidine dimers induced in the viral genome account for the reduced level of intracellular viral DNA synthesis, assuming that most of these lesions block viral DNA replication in unirradiated cells. The inhibition of damaged MVM DNA synthesis is less severe if the host cells themselves are irradiated prior to virus infection. This stimulation of viral DNA replication in pretreated cells might account for the UV-enhanced viral reactivation phenomenon, i.e. the increased survival of nuclear-replicating viruses propagated in cells preexposed to various genotoxic agents

  13. Break-Induced Replication Requires DNA Damage-Induced Phosphorylation of Pif1 and Leads to Telomere Lengthening

    Yulia Vasianovich; Harrington, Lea A.; Svetlana Makovets

    2014-01-01

    Broken replication forks result in DNA breaks that are normally repaired via homologous recombination or break induced replication (BIR). Mild insufficiency in the replicative ligase Cdc9 in budding yeast Saccharomyces cerevisiae resulted in a population of cells with persistent DNA damage, most likely due to broken replication forks, constitutive activation of the DNA damage checkpoint and longer telomeres. This telomere lengthening required functional telomerase, the core DNA damage signali...

  14. Managing Single-Stranded DNA during Replication Stress in Fission Yeast

    Sarah A. Sabatinos

    2015-09-01

    Full Text Available Replication fork stalling generates a variety of responses, most of which cause an increase in single-stranded DNA. ssDNA is a primary signal of replication distress that activates cellular checkpoints. It is also a potential source of genome instability and a substrate for mutation and recombination. Therefore, managing ssDNA levels is crucial to chromosome integrity. Limited ssDNA accumulation occurs in wild-type cells under stress. In contrast, cells lacking the replication checkpoint cannot arrest forks properly and accumulate large amounts of ssDNA. This likely occurs when the replication fork polymerase and helicase units are uncoupled. Some cells with mutations in the replication helicase (mcm-ts mimic checkpoint-deficient cells, and accumulate extensive areas of ssDNA to trigger the G2-checkpoint. Another category of helicase mutant (mcm4-degron causes fork stalling in early S-phase due to immediate loss of helicase function. Intriguingly, cells realize that ssDNA is present, but fail to detect that they accumulate ssDNA, and continue to divide. Thus, the cellular response to replication stalling depends on checkpoint activity and the time that replication stress occurs in S-phase. In this review we describe the signs, signals, and symptoms of replication arrest from an ssDNA perspective. We explore the possible mechanisms for these effects. We also advise the need for caution when detecting and interpreting data related to the accumulation of ssDNA.

  15. Entanglement Swapping Model of DNA Replication

    Pusuluk, Onur

    2011-01-01

    Molecular biology explains function of molecules by their geometric and electronic structures which are mainly determined by utilization of quantum effects in chemistry. However, further quantum effects are not thought to play any significant role in the essential processes of life. On the contrary, consideration of quantum circuits/protocols and organic molecules as software and hardware of living systems that are co-optimized during evolution, may be useful to pass over the difficulties raised by biochemical complexity and to understand the physics of life. In this sense, we model DNA replication with a reliable qubit representation of the nucleotides: 1) molecular recognition of a nucleotide is assumed to trigger an intrabase entanglement corresponding to a superposition of different tautomer forms and 2) pairing of complementary nucleotides is described by swapping intrabase entanglements with interbase entanglements. We examine possible realizations of quantum circuits/protocols to be used to obtain intr...

  16. A quantitative and high-throughput assay of human papillomavirus DNA replication.

    Gagnon, David; Fradet-Turcotte, Amélie; Archambault, Jacques

    2015-01-01

    Replication of the human papillomavirus (HPV) double-stranded DNA genome is accomplished by the two viral proteins E1 and E2 in concert with host DNA replication factors. HPV DNA replication is an established model of eukaryotic DNA replication and a potential target for antiviral therapy. Assays to measure the transient replication of HPV DNA in transfected cells have been developed, which rely on a plasmid carrying the viral origin of DNA replication (ori) together with expression vectors for E1 and E2. Replication of the ori-plasmid is typically measured by Southern blotting or PCR analysis of newly replicated DNA (i.e., DpnI digested DNA) several days post-transfection. Although extremely valuable, these assays have been difficult to perform in a high-throughput and quantitative manner. Here, we describe a modified version of the transient DNA replication assay that circumvents these limitations by incorporating a firefly luciferase expression cassette in cis of the ori. Replication of this ori-plasmid by E1 and E2 results in increased levels of firefly luciferase activity that can be accurately quantified and normalized to those of Renilla luciferase expressed from a control plasmid, thus obviating the need for DNA extraction, digestion, and analysis. We provide a detailed protocol for performing the HPV type 31 DNA replication assay in a 96-well plate format suitable for small-molecule screening and EC50 determinations. The quantitative and high-throughput nature of the assay should greatly facilitate the study of HPV DNA replication and the identification of inhibitors thereof. PMID:25348316

  17. Direct interaction between cohesin complex and DNA replication machinery

    Structural maintenance of chromosome 1 (Smc1) is a multifunctional protein, which has been implicated in sister chromatid cohesion, DNA recombination and repair, and the activation of cell cycle checkpoints by ionizing radiation, ultraviolet light, and other genotoxic agents. In order to identify the proteins that interact with Smc1, we conducted the Tandem affinity purification (TAP) technique and analyzed the Smc1-interacting proteins via MALDI-TOF mass spectrometry. We identified minichromosome maintenance 7 (Mcm7), an essential component of the pre-replication complex, as a novel Smc1-interacting protein. Co-immunoprecipitation revealed an interaction occurring between Smc1 and Mcm7, both in vitro and in vivo. Using a GST pull-down assay, we determined that Smc1 interacts physically with Mcm7 via its N-terminal and hinge regions, and Mcm7 interacts with Smc1 via its middle region. Interestingly, we also discovered that Smc1 interacts with other DNA replication proteins, including Mcm6, RFC1, and DNA polymerase α. These results suggest that a functional link exists between the cohesin complex and DNA replication proteins

  18. The DNA damage and the DNA replication checkpoints converge at the MBF transcription factor

    Ivanova, Tsvetomira; Alves-Rodrigues, Isabel; Gómez-Escoda, Blanca; Dutta, Chaitali; DeCaprio, James A.; Rhind, Nick; Hidalgo, Elena; Ayté, José

    2013-01-01

    In fission yeast cells, Cds1 is the effector kinase of the DNA replication checkpoint. We previously showed that when the DNA replication checkpoint is activated, the repressor Yox1 is phosphorylated and inactivated by Cds1, resulting in activation of MluI-binding factor (MBF)–dependent transcription. This is essential to reinitiate DNA synthesis and for correct G1-to-S transition. Here we show that Cdc10, which is an essential part of the MBF core, is the target of the DNA damage checkpoint. When fission yeast cells are treated with DNA-damaging agents, Chk1 is activated and phosphorylates Cdc10 at its carboxy-terminal domain. This modification is responsible for the repression of MBF-dependent transcription through induced release of MBF from chromatin. This inactivation of MBF is important for survival of cells challenged with DNA-damaging agents. Thus Yox1 and Cdc10 couple normal cell cycle regulation in unperturbed conditions and the DNA replication and DNA damage checkpoints into a single transcriptional complex. PMID:24006488

  19. The DNA damage and the DNA replication checkpoints converge at the MBF transcription factor.

    Ivanova, Tsvetomira; Alves-Rodrigues, Isabel; Gómez-Escoda, Blanca; Dutta, Chaitali; DeCaprio, James A; Rhind, Nick; Hidalgo, Elena; Ayté, José

    2013-11-01

    In fission yeast cells, Cds1 is the effector kinase of the DNA replication checkpoint. We previously showed that when the DNA replication checkpoint is activated, the repressor Yox1 is phosphorylated and inactivated by Cds1, resulting in activation of MluI-binding factor (MBF)-dependent transcription. This is essential to reinitiate DNA synthesis and for correct G1-to-S transition. Here we show that Cdc10, which is an essential part of the MBF core, is the target of the DNA damage checkpoint. When fission yeast cells are treated with DNA-damaging agents, Chk1 is activated and phosphorylates Cdc10 at its carboxy-terminal domain. This modification is responsible for the repression of MBF-dependent transcription through induced release of MBF from chromatin. This inactivation of MBF is important for survival of cells challenged with DNA-damaging agents. Thus Yox1 and Cdc10 couple normal cell cycle regulation in unperturbed conditions and the DNA replication and DNA damage checkpoints into a single transcriptional complex. PMID:24006488

  20. Universal Temporal Profile of Replication Origin Activation in Eukaryotes

    Goldar, Arach

    2011-03-01

    The complete and faithful transmission of eukaryotic genome to daughter cells involves the timely duplication of mother cell's DNA. DNA replication starts at multiple chromosomal positions called replication origin. From each activated replication origin two replication forks progress in opposite direction and duplicate the mother cell's DNA. While it is widely accepted that in eukaryotic organisms replication origins are activated in a stochastic manner, little is known on the sources of the observed stochasticity. It is often associated to the population variability to enter S phase. We extract from a growing Saccharomyces cerevisiae population the average rate of origin activation in a single cell by combining single molecule measurements and a numerical deconvolution technique. We show that the temporal profile of the rate of origin activation in a single cell is similar to the one extracted from a replicating cell population. Taking into account this observation we exclude the population variability as the origin of observed stochasticity in origin activation. We confirm that the rate of origin activation increases in the early stage of S phase and decreases at the latter stage. The population average activation rate extracted from single molecule analysis is in prefect accordance with the activation rate extracted from published micro-array data, confirming therefore the homogeneity and genome scale invariance of dynamic of replication process. All these observations point toward a possible role of replication fork to control the rate of origin activation.

  1. Patterning quantum dot arrays using DNA replication principles.

    Crown, Kevin K.; Bachand, George David

    2004-11-01

    The convergence of nanoscience and biotechnology has opened the door to the integration of a wide range of biological molecules and processes with synthetic materials and devices. A primary biomolecule of interest has been DNA based upon its role as information storage in living systems, as well as its ability to withstand a wide range of environmental conditions. DNA also offers unique chemistries and interacts with a range of biomolecules, making it an ideal component in biological sensor applications. The primary goal of this project was to develop methods that utilize in vitro DNA synthesis to provide spatial localization of nanocrystal quantum dots (nQDs). To accomplish this goal, three specific technical objectives were addressed: (1) attachment of nQDs to DNA nucleotides, (2) demonstrating the synthesis of nQD-DNA strands in bulk solution, and (3) optimizing the ratio of unlabeled to nQD-labeled nucleotides. DNA nucleotides were successfully attached to nQDs using the biotin-streptavidin linkage. Synthesis of 450-nm long, nQD-coated DNA strands was demonstrated using a DNA template and the polymerase chain reaction (PCR)-based method of DNA amplification. Modifications in the synthesis process and conditions were subsequently used to synthesize 2-{micro}m long linear nQD-DNA assemblies. In the case of the 2-{micro}m structures, both the ratio of streptavidin-coated nQDs to biotinylated dCTP, and streptavidin-coated nQD-dCTPs to unlabeled dCTPs affected the ability to synthesize the nQD-DNA assemblies. Overall, these proof-of-principles experiments demonstrated the successful synthesis of nQD-DNA using DNA templates and in vitro replication technologies. Continued development of this technology may enable rapid, spatial patterning of semiconductor nanoparticles with Angstrom-level resolution, as well as optically active probes for DNA and other biomolecular analyses.

  2. Identification of replication factor C from Saccharomyces cerevisiae: a component of the leading-strand DNA replication complex.

    Fien, K; Stillman, B

    1992-01-01

    A number of proteins have been isolated from human cells on the basis of their ability to support DNA replication in vitro of the simian virus 40 (SV40) origin of DNA replication. One such protein, replication factor C (RFC), functions with the proliferating cell nuclear antigen (PCNA), replication protein A (RPA), and DNA polymerase delta to synthesize the leading strand at a replication fork. To determine whether these proteins perform similar roles during replication of DNA from origins in...

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

    Jared T. Nordman

    2014-11-01

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

  4. Multiple regulatory systems coordinate DNA replication with cell growth in Bacillus subtilis.

    Heath Murray

    2014-10-01

    Full Text Available In many bacteria the rate of DNA replication is linked with cellular physiology to ensure that genome duplication is coordinated with growth. Nutrient-mediated growth rate control of DNA replication initiation has been appreciated for decades, however the mechanism(s that connects these cell cycle activities has eluded understanding. In order to help address this fundamental question we have investigated regulation of DNA replication in the model organism Bacillus subtilis. Contrary to the prevailing view we find that changes in DnaA protein level are not sufficient to account for nutrient-mediated growth rate control of DNA replication initiation, although this regulation does require both DnaA and the endogenous replication origin. We go on to report connections between DNA replication and several essential cellular activities required for rapid bacterial growth, including respiration, central carbon metabolism, fatty acid synthesis, phospholipid synthesis, and protein synthesis. Unexpectedly, the results indicate that multiple regulatory systems are involved in coordinating DNA replication with cell physiology, with some of the regulatory systems targeting oriC while others act in a oriC-independent manner. We propose that distinct regulatory systems are utilized to control DNA replication in response to diverse physiological and chemical changes.

  5. Inhibition of DNA replication, DNA repair synthesis, and DNA polymerases α and δ by butylphenyl deoxyguanosine triphosphate

    Semiconservative DNA replication in growing mammalian cells and ultraviolet (UV)-induced DNA repair synthesis in nongrowing mammalian cells are mediated by one or both of the aphidicolin-sensitive DNA polymerases, α and/or δ. They have studied the inhibition of replication and repair synthesis in permeable human cells by N2 (p-n-butylphenyl)-2'-deoxyguanosine-5'-triphosphate (BuPh dGTP), an agent which inhibits polymerase α strongly and polymerase δ weakly. Both processes are inhibited by BuPh-dGTP in competition with dGTP. The K/sub i/'s are, for replication, 2-3 μM and, for repair synthesis, 3-4 μM, consistent with the involvement of the same DNA polymerase in both processes. Inhibition of isolated human polymerase α by BuPh-dGTP is also competitive with dGTP, but the K/sub i/ is approximately 10 nM, several hundred-fold lower than the K/sub i/'s of replication and repair synthesis. Isolated polymerase δ is inhibited by BuPh-dGTP at doses similar to those which inhibit replication and repair synthesis, however, attempts to determine the K/sub i/ of polymerase δ were hampered by the finding that the dependence of δ activity on deoxyribunucleotide concentration is parabolic at low doses. This behavior differs from the behavior of polymerase α and of cellular DNA replication and repair synthesis, all of which show a simple, hyperbolic relationship between activity and deoxyribonucleotide concentration. Thus, inhibition of DNA replication and UV induced DNA repair synthesis by BuPh dGTP is quantitatively similar to DNA polymerase δ, but some other characteristics of the cellular processes are more similar to those of polymerase α

  6. Replication factors transiently associate with mtDNA at the mitochondrial inner membrane to facilitate replication

    Rajala, N.; Gerhold, J.M.; Martinsson, P.; Klymov, A.; Spelbrink, H.

    2014-01-01

    Mitochondrial DNA (mtDNA) is organized in discrete protein-DNA complexes, nucleoids, that are usually considered to be mitochondrial-inner-membrane associated. Here we addressed the association of replication factors with nucleoids and show that endogenous mtDNA helicase Twinkle and single-stranded

  7. Anaphase onset before complete DNA replication with intact checkpoint responses

    Torres-Rosell, Jordi; De Piccoli, Giacomo; Cordon-Preciado, Violeta;

    2007-01-01

    Cellular checkpoints prevent mitosis in the presence of stalled replication forks. Whether checkpoints also ensure the completion of DNA replication before mitosis is unknown. Here, we show that in yeast smc5-smc6 mutants, which are related to cohesin and condensin, replication is delayed, most...

  8. A novel cell permeable DNA replication and repair marker.

    Herce, Henry D; Rajan, Malini; Lättig-Tünnemann, Gisela; Fillies, Marion; Cardoso, M Cristina

    2014-01-01

    Proliferating Cell Nuclear Antigen (PCNA) is a key protein in DNA replication and repair. The dynamics of replication and repair in live cells is usually studied introducing translational fusions of PCNA. To obviate the need for transfection and bypass the problem of difficult to transfect and/or short lived cells, we have now developed a cell permeable replication and/or repair marker. The design of this marker has three essential molecular components: (1) an optimized artificial PCNA binding peptide; (2) a cell-penetrating peptide, derived from the HIV-1 Trans Activator of Transcription (TAT); (3) an in vivo cleavable linker, linking the two peptides. The resulting construct was taken up by human, hamster and mouse cells within minutes of addition to the media. Inside the cells, the cargo separated from the vector peptide and bound PCNA effectively. Both replication and repair sites could be directly labeled in live cells making it the first in vivo cell permeable peptide marker for these two fundamental cellular processes. Concurrently, we also introduced a quick peptide based PCNA staining method as an alternative to PCNA antibodies for immunofluorescence applications. In summary, we present here a versatile tool to instantaneously label repair and replication processes in fixed and live cells. PMID:25484186

  9. Chromatin Assembly at Kinetochores Is Uncoupled from DNA Replication

    Shelby, Richard D.; Monier, Karine; Sullivan, Kevin F.

    2000-01-01

    The specification of metazoan centromeres does not depend strictly on centromeric DNA sequences, but also requires epigenetic factors. The mechanistic basis for establishing a centromeric “state” on the DNA remains unclear. In this work, we have directly examined replication timing of the prekinetochore domain of human chromosomes. Kinetochores were labeled by expression of epitope-tagged CENP-A, which stably marks prekinetochore domains in human cells. By immunoprecipitating CENP-A mononucleosomes from synchronized cells pulsed with [3H]thymidine we demonstrate that CENP-A–associated DNA is replicated in mid-to-late S phase. Cytological analysis of DNA replication further demonstrated that centromeres replicate asynchronously in parallel with numerous other genomic regions. In contrast, quantitative Western blot analysis demonstrates that CENP-A protein synthesis occurs later, in G2. Quantitative fluorescence microscopy and transient transfection in the presence of aphidicolin, an inhibitor of DNA replication, show that CENP-A can assemble into centromeres in the absence of DNA replication. Thus, unlike most genomic chromatin, histone synthesis and assembly are uncoupled from DNA replication at the kinetochore. Uncoupling DNA replication from CENP-A synthesis suggests that regulated chromatin assembly or remodeling could play a role in epigenetic centromere propagation. PMID:11086012

  10. Roles of replication protein-A subunits 2 and 3 in DNA replication fork movement in Saccharomyces cerevisiae

    Replication Protein-A, the eukaryotic SSB, consists of a large subunit (RPA1) with strong ssDNA binding activity and two smaller subunits (RPA2 and 3) that may cooperate with RPA1 to bind ssDNA in a higher-order mode. To determine the in vivo function of the two smaller subunits and the potential role of higher-order ssDNA binding, we isolated an assortment of heat-lethal mutations in the genes encoding RPA2 and RPA3. At the permissive temperature, the mutants show a range of effects on DNA replication fidelity and sensitivities to UV and MMS. At the nonpermissive temperature, four out of five RPA2 mutants show a fast-stop DNA synthesis phenotype typical of a replication fork block. In contrast, the fifth RPA2 mutant and all RPA3 mutants are able to complete at least one round of DNA replication at the nonpermissive temperature. The effect of these mutations on the stability of the RPA complex was tested using a coprecipitation assay. At the nonpermissive temperature, we find that RPA1 and RPA2 are dissociated in the fast-stop mutants, but not in the slow-stop mutants. Thus, replication fork movement in vivo requires the association of at least two subunits of RPA. This result is consistent with the hypothesis that RPA functions in vivo by binding ssDNA in a higher-order mode

  11. Polyomavirus origin for DNA replication comprises multiple genetic elements.

    Muller, W J; Mueller, C R; Mes, A M; Hassell, J A

    1983-01-01

    To define the minimal cis-acting sequences required for polyomavirus DNA replication (ori), we constructed a number of polyomavirus-plasmid recombinants and measured their replicative capacity after transfection of a permissive mouse cell line capable of providing polyomavirus large T antigen in trans (MOP cells). Recombinant plasmids containing a 251-base-pair fragment of noncoding viral DNA replicate efficiently in MOP cells. Mutational analyses of these viral sequences revealed that they c...

  12. Replication of cloned DNA containing the Alu family sequence during cell extract-promoting simian virus 40 DNA synthesis.

    Ariga, H

    1984-01-01

    The replicating activity of several cloned DNAs containing putative origin sequences was examined in a cell-free extract that absolutely depends on simian virus 40 (SV40) T antigen promoting initiation of SV40 DNA replication in vitro. Of the three DNAs containing the human Alu family sequence (BLUR8), the origin of (Saccharomyces cerevisiae plasmid 2 micron DNA (pJD29), and the yeast autonomous replicating sequence (YRp7), only BLUR8 was active as a template. Replication in a reaction mixtur...

  13. Enzymes involved in organellar DNA replication in photosynthetic eukaryotes

    Takashi eMoriyama

    2014-09-01

    Full Text Available Plastids and mitochondria possess their own genomes. Although the replication mechanisms of these organellar genomes remain unclear in photosynthetic eukaryotes, several organelle-localized enzymes related to genome replication, including DNA polymerase, DNA primase, DNA helicase, DNA topoisomerase, single-stranded DNA maintenance protein, DNA ligase, primer removal enzyme, and several DNA recombination-related enzymes, have been identified. In the reference Eudicot plant Arabidopsis thaliana, the replication-related enzymes of plastids and mitochondria are similar because many of them are dual targeted to both organelles, whereas in the red alga Cyanidioschyzon merolae, plastids and mitochondria contain different replication machinery components. The enzymes involved in organellar genome replication in green plants and red algae were derived from different origins, including proteobacterial, cyanobacterial, and eukaryotic lineages. In the present review, we summarize the available data for enzymes related to organellar genome replication in green plants and red algae. In addition, based on the type and distribution of replication enzymes in photosynthetic eukaryotes, we discuss the transitional history of replication enzymes in the organelles of plants.

  14. YabA of Bacillus subtilis controls DnaA-mediated replication initiation but not the transcriptional response to replication stress

    Goranov, Alexi I.; Breier, Adam M.; Merrikh, Houra; Grossman, Alan D.

    2009-01-01

    yabA encodes a negative regulator of replication initiation in Bacillus subtilis and homologues are found in many other Gram-positive species. YabA interacts with the β-processivity clamp (DnaN) of DNA polymerase and with the replication initiator and transcription factor DnaA. Because of these interactions, YabA has been proposed to modulate the activity of DnaA. We investigated the role of YabA in regulating replication initiation and the activity of DnaA as a transcription factor. We foun...

  15. DNA Damage Reduces the Quality, but Not the Quantity of Human Papillomavirus 16 E1 and E2 DNA Replication.

    Bristol, Molly L; Wang, Xu; Smith, Nathan W; Son, Minkyeong P; Evans, Michael R; Morgan, Iain M

    2016-01-01

    Human papillomaviruses (HPVs) are causative agents in almost all cervical carcinomas. HPVs are also causative agents in head and neck cancer, the cases of which are increasing rapidly. Viral replication activates the DNA damage response (DDR) pathway; associated proteins are recruited to replication foci, and this pathway may serve to allow for viral genome amplification. Likewise, HPV genome double-strand breaks (DSBs) could be produced during replication and could lead to linearization and viral integration. Many studies have shown that viral integration into the host genome results in unregulated expression of the viral oncogenes, E6 and E7, promoting HPV-induced carcinogenesis. Previously, we have demonstrated that DNA-damaging agents, such as etoposide, or knocking down viral replication partner proteins, such as topoisomerase II β binding protein I (TopBP1), does not reduce the level of DNA replication. Here, we investigated whether these treatments alter the quality of DNA replication by HPV16 E1 and E2. We confirm that knockdown of TopBP1 or treatment with etoposide does not reduce total levels of E1/E2-mediated DNA replication; however, the quality of replication is significantly reduced. The results demonstrate that E1 and E2 continue to replicate under genomically-stressed conditions and that this replication is mutagenic. This mutagenesis would promote the formation of substrates for integration of the viral genome into that of the host, a hallmark of cervical cancer. PMID:27338449

  16. DNA Damage Reduces the Quality, but Not the Quantity of Human Papillomavirus 16 E1 and E2 DNA Replication

    Molly L. Bristol

    2016-06-01

    Full Text Available Human papillomaviruses (HPVs are causative agents in almost all cervical carcinomas. HPVs are also causative agents in head and neck cancer, the cases of which are increasing rapidly. Viral replication activates the DNA damage response (DDR pathway; associated proteins are recruited to replication foci, and this pathway may serve to allow for viral genome amplification. Likewise, HPV genome double-strand breaks (DSBs could be produced during replication and could lead to linearization and viral integration. Many studies have shown that viral integration into the host genome results in unregulated expression of the viral oncogenes, E6 and E7, promoting HPV-induced carcinogenesis. Previously, we have demonstrated that DNA-damaging agents, such as etoposide, or knocking down viral replication partner proteins, such as topoisomerase II β binding protein I (TopBP1, does not reduce the level of DNA replication. Here, we investigated whether these treatments alter the quality of DNA replication by HPV16 E1 and E2. We confirm that knockdown of TopBP1 or treatment with etoposide does not reduce total levels of E1/E2-mediated DNA replication; however, the quality of replication is significantly reduced. The results demonstrate that E1 and E2 continue to replicate under genomically-stressed conditions and that this replication is mutagenic. This mutagenesis would promote the formation of substrates for integration of the viral genome into that of the host, a hallmark of cervical cancer.

  17. Involvement of proliferating cell nuclear antigen (cyclin) in DNA replication in living cells.

    Zuber, M; Tan, E M; Ryoji, M

    1989-01-01

    Proliferating cell nuclear antigen (PCNA) (also called cyclin) is known to stimulate the activity of DNA polymerase delta but not the other DNA polymerases in vitro. We injected a human autoimmune antibody against PCNA into unfertilized eggs of Xenopus laevis and examined the effects of this antibody on the replication of injected plasmid DNA as well as egg chromosomes. The anti-PCNA antibody inhibited plasmid replication by up to 67%, demonstrating that PCNA is involved in plasmid replicatio...

  18. USP7 is a SUMO deubiquitinase essential for DNA replication

    Lecona, Emilio; Rodriguez-Acebes, Sara; Specks, Julia;

    2016-01-01

    Post-translational modification of proteins by ubiquitin (Ub) and Ub-like modifiers regulates DNA replication. We have previously shown that chromatin around replisomes is rich in SUMO and poor in Ub, whereas mature chromatin exhibits an opposite pattern. How this SUMO-rich, Ub-poor environment is...... maintained at sites of DNA replication in mammalian cells remains unexplored. Here we identify USP7 as a replisome-enriched SUMO deubiquitinase that is essential for DNA replication. By acting on SUMO and SUMOylated proteins, USP7 counteracts their ubiquitination. Inhibition or genetic deletion of USP7 leads...... to the accumulation of Ub on SUMOylated proteins, which are displaced away from replisomes. Our findings provide a model explaining the differential accumulation of SUMO and Ub at replication forks and identify an essential role of USP7 in DNA replication that should be considered in the development...

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

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

    2016-08-01

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

  20. Mitochondria DNA Replication and DNA Methylation in the Metabolic Memory Associated with Continued Progression of Diabetic Retinopathy

    Tewari, Shikha; Zhong, Qing; Santos, Julia M.; Kowluru, Renu A.

    2012-01-01

    Diabetes-induced damage to the retinal mtDNA replication system compromises the transcriptional activity and the mitochondria remains dysfunctional even after hyperglycemic insult is terminated, resulting in the continued progression of diabetic retinopathy.

  1. Roles of DNA polymerase epsilon and TopBP1 in DNA replication and damage response

    Hillukkala, T.

    2006-01-01

    Abstract During DNA replication cells accurately copy their DNA to transfer the genetic information to daughter cells. DNA polymerases synthesise the new DNA strand using the old strand as a template. Other functions of DNA polymerases are recombination linked and DNA iamage repair linked DNA synthesis, regulation of replication complex formation and regulation of transcription – a process in which the genetic information is transformed into an RNA sequence needed to guide protein synthesi...

  2. DNA intercalator stimulates influenza transcription and virus replication

    Poon Leo LM

    2011-03-01

    Full Text Available Abstract Influenza A virus uses its host transcription machinery to facilitate viral RNA synthesis, an event that is associated with cellular RNA polymerase II (RNAPII. In this study, various RNAPII transcription inhibitors were used to investigate the effect of RNAPII phosphorylation status on viral RNA transcription. A low concentration of DNA intercalators, such as actinomycin D (ActD, was found to stimulate viral polymerase activity and virus replication. This effect was not observed in cells treated with RNAPII kinase inhibitors. In addition, the loss of RNAPIIa in infected cells was due to the shift of nonphosphorylated RNAPII (RNAPIIa to hyperphosphorylated RNAPII (RNAPIIo.

  3. Acute MUS81 depletion leads to replication fork slowing and a constitutive DNA damage response

    Xing, Meichun; Wang, Xiaohui; Palmai-Pallag, Timea;

    2015-01-01

    The MUS81 protein belongs to a conserved family of DNA structure-specific nucleases that play important roles in DNA replication and repair. Inactivation of the Mus81 gene in mice has no major deleterious consequences for embryonic development, although cancer susceptibility has been reported. We...... have investigated the role of MUS81 in human cells by acutely depleting the protein using shRNAs. We found that MUS81 depletion from human fibroblasts leads to accumulation of ssDNA and a constitutive DNA damage response that ultimately activates cellular senescence. Moreover, we show that MUS81 is...... required for efficient replication fork progression during an unperturbed S-phase, and for recovery of productive replication following replication stalling. These results demonstrate essential roles for the MUS81 nuclease in maintenance of replication fork integrity....

  4. A novel cell permeable DNA replication and repair marker

    Herce, Henry D.; Rajan, Malini; Lättig-Tünnemann, Gisela; Fillies, Marion; Cardoso, M. Cristina

    2014-01-01

    Proliferating Cell Nuclear Antigen (PCNA) is a key protein in DNA replication and repair. The dynamics of replication and repair in live cells is usually studied introducing translational fusions of PCNA. To obviate the need for transfection and bypass the problem of difficult to transfect and/or short lived cells, we have now developed a cell permeable replication and/or repair marker. The design of this marker has three essential molecular components: (1) an optimized artificial PCNA bindin...

  5. Structural properties of replication origins in yeast DNA sequences

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

  6. Nanoscale topographical replication of graphene architecture by artificial DNA nanostructures

    Moon, Y.; Shin, J.; Seo, S.; Park, J.; Dugasani, S. R.; Woo, S. H.; Park, T.; Park, S. H.; Ahn, J. R.

    2014-06-01

    Despite many studies on how geometry can be used to control the electronic properties of graphene, certain limitations to fabrication of designed graphene nanostructures exist. Here, we demonstrate controlled topographical replication of graphene by artificial deoxyribonucleic acid (DNA) nanostructures. Owing to the high degree of geometrical freedom of DNA nanostructures, we controlled the nanoscale topography of graphene. The topography of graphene replicated from DNA nanostructures showed enhanced thermal stability and revealed an interesting negative temperature coefficient of sheet resistivity when underlying DNA nanostructures were denatured at high temperatures.

  7. Nanoscale topographical replication of graphene architecture by manufactured DNA nanostructures

    Moon, Youngkwon; Shin, Jihoon; Seo, Soonbeom; Park, Sung Ha; Ahn, Joung Real

    2015-03-01

    Despite many studies on how geometry can be used to control the electronic properties of graphene, certain limitations to fabrication of designed graphene nanostructures exist. Here, we demonstrate controlled topographical replication of graphene by artificial deoxyribonucleic acid (DNA) nanostructures. Owing to the high degree of geometrical freedom of DNA nanostructures, we controlled the nanoscale topography of graphene. The topography of graphene replicated from DNA nanostructures showed enhanced thermal stability and revealed an interesting negative temperature coefficient of sheet resistivity when underlying DNA nanostructures were denatured at high temperatures.

  8. Nanoscale topographical replication of graphene architecture by artificial DNA nanostructures

    Moon, Y.; Seo, S.; Park, J.; Park, T.; Ahn, J. R., E-mail: jrahn@skku.edu [Department of Physics, Sungkyunkwan University, Suwon 440-746 (Korea, Republic of); Shin, J.; Dugasani, S. R. [Sungkyunkwan Advanced Institute of Nanotechnology (SAINT), Sungkyunkwan University, Suwon 440-746 (Korea, Republic of); Woo, S. H. [College of Pharmacy, Chungnam National University, Daejeon 305-764 (Korea, Republic of); Park, S. H., E-mail: sunghapark@skku.edu [Department of Physics, Sungkyunkwan University, Suwon 440-746 (Korea, Republic of); Sungkyunkwan Advanced Institute of Nanotechnology (SAINT), Sungkyunkwan University, Suwon 440-746 (Korea, Republic of)

    2014-06-09

    Despite many studies on how geometry can be used to control the electronic properties of graphene, certain limitations to fabrication of designed graphene nanostructures exist. Here, we demonstrate controlled topographical replication of graphene by artificial deoxyribonucleic acid (DNA) nanostructures. Owing to the high degree of geometrical freedom of DNA nanostructures, we controlled the nanoscale topography of graphene. The topography of graphene replicated from DNA nanostructures showed enhanced thermal stability and revealed an interesting negative temperature coefficient of sheet resistivity when underlying DNA nanostructures were denatured at high temperatures.

  9. Diversification of DnaA dependency for DNA replication in cyanobacterial evolution.

    Ohbayashi, Ryudo; Watanabe, Satoru; Ehira, Shigeki; Kanesaki, Yu; Chibazakura, Taku; Yoshikawa, Hirofumi

    2016-05-01

    Regulating DNA replication is essential for all living cells. The DNA replication initiation factor DnaA is highly conserved in prokaryotes and is required for accurate initiation of chromosomal replication at oriC. DnaA-independent free-living bacteria have not been identified. The dnaA gene is absent in plastids and some symbiotic bacteria, although it is not known when or how DnaA-independent mechanisms were acquired. Here, we show that the degree of dependency of DNA replication on DnaA varies among cyanobacterial species. Deletion of the dnaA gene in Synechococcus elongatus PCC 7942 shifted DNA replication from oriC to a different site as a result of the integration of an episomal plasmid. Moreover, viability during the stationary phase was higher in dnaA disruptants than in wild-type cells. Deletion of dnaA did not affect DNA replication or cell growth in Synechocystis sp. PCC 6803 or Anabaena sp. PCC 7120, indicating that functional dependency on DnaA was already lost in some nonsymbiotic cyanobacterial lineages during diversification. Therefore, we proposed that cyanobacteria acquired DnaA-independent replication mechanisms before symbiosis and such an ancestral cyanobacterium was the sole primary endosymbiont to form a plastid precursor. PMID:26517699

  10. Functional amyloids as inhibitors of plasmid DNA replication.

    Molina-García, Laura; Gasset-Rosa, Fátima; Moreno-Del Álamo, María; Fernández-Tresguerres, M Elena; Moreno-Díaz de la Espina, Susana; Lurz, Rudi; Giraldo, Rafael

    2016-01-01

    DNA replication is tightly regulated to constrain the genetic material within strict spatiotemporal boundaries and copy numbers. Bacterial plasmids are autonomously replicating DNA molecules of much clinical, environmental and biotechnological interest. A mechanism used by plasmids to prevent over-replication is 'handcuffing', i.e. inactivating the replication origins in two DNA molecules by holding them together through a bridge built by a plasmid-encoded initiator protein (Rep). Besides being involved in handcuffing, the WH1 domain in the RepA protein assembles as amyloid fibres upon binding to DNA in vitro. The amyloid state in proteins is linked to specific human diseases, but determines selectable and epigenetically transmissible phenotypes in microorganisms. Here we have explored the connection between handcuffing and amyloidogenesis of full-length RepA. Using a monoclonal antibody specific for an amyloidogenic conformation of RepA-WH1, we have found that the handcuffed RepA assemblies, either reconstructed in vitro or in plasmids clustering at the bacterial nucleoid, are amyloidogenic. The replication-inhibitory RepA handcuff assembly is, to our knowledge, the first protein amyloid directly dealing with DNA. Built on an amyloid scaffold, bacterial plasmid handcuffs can bring a novel molecular solution to the universal problem of keeping control on DNA replication initiation. PMID:27147472

  11. A phage-encoded inhibitor of Escherichia coli DNA replication targets the DNA polymerase clamp loader.

    Yano, Sho T; Rothman-Denes, Lucia B

    2011-03-01

    Coliphage N4 infection leads to shut-off of host DNA replication without inhibition of host transcription or translation. We report the identification and characterization of gp8, the N4 gene product responsible for this phenotype. N4 gp8 is an Escherichia coli bacteriostatic inhibitor that colocalizes with the E. coli replisome in a replication-dependent manner. Gp8 was purified and observed to cross-link to complexes containing the replicative DNA polymerase, DNAP III, in vivo. Purified gp8 inhibits DNA polymerization by DNA polymerase III holoenzyme in vitro by interfering with polymerase processivity. Gp8 specifically inhibits the clamp-loading activity of DNAP III by targeting the delta subunit of the DNAP III clamp loader; E. coli mutations conferring gp8 resistance were identified in the holA gene, encoding delta. Delta and gp8 interact in vitro; no interaction was detected between gp8 inactive mutants and wild-type delta or between delta gp8-resistant mutants and wild-type gp8. Therefore, this work identifies the DNAP III clamp loader as a new target for inhibition of bacterial growth. Finally, we show that gp8 is not essential in N4 development under laboratory conditions, but its activity contributes to phage yield. PMID:21205014

  12. Priming DNA Replication from Triple Helix Oligonucleotides: Possible Threestranded DNA in DNA Polymerases

    Lestienne, Patrick P.

    2011-01-01

    Triplex associate with a duplex DNA presenting the same polypurine or polypyrimidine-rich sequence in an antiparallel orientation. So far, triplex forming oligonucleotides (TFOs) are known to inhibit transcription, replication, and to induce mutations. A new property of TFO is reviewed here upon analysis of DNA breakpoint yielding DNA rearrangements; the synthesized sequence of the first direct repeat displays a skewed polypurine- rich sequence. This synthesized sequence can bind the second h...

  13. Impaired DNA replication within progenitor cell pools promotes leukemogenesis.

    Ganna Bilousova

    2005-12-01

    Full Text Available Impaired cell cycle progression can be paradoxically associated with increased rates of malignancies. Using retroviral transduction of bone marrow progenitors followed by transplantation into mice, we demonstrate that inhibition of hematopoietic progenitor cell proliferation impairs competition, promoting the expansion of progenitors that acquire oncogenic mutations which restore cell cycle progression. Conditions that impair DNA replication dramatically enhance the proliferative advantage provided by the expression of Bcr-Abl or mutant p53, which provide no apparent competitive advantage under conditions of healthy replication. Furthermore, for the Bcr-Abl oncogene the competitive advantage in contexts of impaired DNA replication dramatically increases leukemogenesis. Impaired replication within hematopoietic progenitor cell pools can select for oncogenic events and thereby promote leukemia, demonstrating the importance of replicative competence in the prevention of tumorigenesis. The demonstration that replication-impaired, poorly competitive progenitor cell pools can promote tumorigenesis provides a new rationale for links between tumorigenesis and common human conditions of impaired DNA replication such as dietary folate deficiency, chemotherapeutics targeting dNTP synthesis, and polymorphisms in genes important for DNA metabolism.

  14. The DNA Replication Stress Hypothesis of Alzheimer’s Disease

    Yuri B. Yurov

    2011-01-01

    Full Text Available A well-recognized theory of Alzheimer’s disease (AD pathogenesis suggests ectopic cell cycle events to mediate neurodegeneration. Vulnerable neurons of the AD brain exhibit biomarkers of cell cycle progression and DNA replication suggesting a reentry into the cell cycle. Chromosome reduplication without proper cell cycle completion and mitotic division probably causes neuronal cell dysfunction and death. However, this theory seems to require some inputs in accordance with the generally recognized amyloid cascade theory as well as to explain causes and consequences of genomic instability (aneuploidy in the AD brain. We propose that unscheduled and incomplete DNA replication (replication stress destabilizes (epigenomic landscape in the brain and leads to DNA replication “catastrophe” causing cell death during the S phase (replicative cell death. DNA replication stress can be a key element of the pathogenetic cascade explaining the interplay between ectopic cell cycle events and genetic instabilities in the AD brain. Abnormal cell cycle reentry and somatic genome variations can be used for updating the cell cycle theory introducing replication stress as a missing link between cell genetics and neurobiology of AD.

  15. Ubiquitin-dependent DNA damage bypass is separable from genome replication

    Daigaku, Yasukazu; Davies, Adelina A.; Ulrich, Helle D.

    2010-01-01

    Postreplication repair (PRR) is a pathway that allows cells to bypass or overcome lesions during DNA replication1. In eukaryotes, damage bypass is activated by ubiquitylation of the replication clamp PCNA through components of the RAD6 pathway2. Whereas monoubiquitylation of PCNA allows mutagenic translesion synthesis by damage-tolerant DNA polymerases3-5, polyubiquitylation is required for an error-free pathway that likely involves a template switch to the undamaged sister chromatid6. Both t...

  16. Nucleotide sequence analysis of regions of adenovirus 5 DNA containing the origins of DNA replication

    The purpose of the investigations described is the determination of nucleotide sequences at the molecular ends of the linear adenovirus type 5 DNA. Knowledge of the primary structure at the termini of this DNA molecule is of particular interest in the study of the mechanism of replication of adenovirus DNA. The initiation- and termination sites of adenovirus DNA replication are located at the ends of the DNA molecule. (Auth.)

  17. p53 Protein Is a Suppressor of Papillomavirus DNA Amplificational Replication

    Lepik, Dina; Ilves, Ivar; Kristjuhan, Arnold; Maimets, Toivo; Ustav, Mart

    1998-01-01

    p53 protein was able to block human and bovine papillomavirus DNA amplificational replication while not interfering with Epstein-Barr virus oriP once-per-cell cycle replication. Oligomerization, intact DNA-binding, replication protein A-binding, and proline-rich domains of the p53 protein were essential for efficient inhibition, while the N-terminal transcriptional activation and C-terminal regulatory domains were dispensable for the suppressor activity of the p53 protein. The inhibition of r...

  18. The Bacillus subtilis DnaD and DnaB Proteins Exhibit Different DNA Remodelling Activities

    Zhang, Wenke; Carneiro, Maria J. V. M.; Turner, Ian J.; ALLEN, Stephanie; Roberts, Clive J.; Soultanas, Panos

    2005-01-01

    Primosomal protein cascades load the replicative helicase onto DNA. In Bacillus subtilis a putative primosomal cascade involving the DnaD-DnaB-DnaI proteins has been suggested to participate in both the DnaA and PriA-dependent loading of the replicative helicase DnaC onto the DNA. Recently we discovered that DnaD has a global remodelling DNA activity suggesting a more widespread role in bacterial nucleoid architecture. Here, we show that DnaB forms a “square-like” tetramer with a hole in the ...

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

    Amnon Koren

    2010-08-01

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

  20. Error correction during DNA replication: DNAP as Dr. Jekyll-and-Mr. Hyde

    Sharma, Ajeet K

    2012-01-01

    DNA polymerase (DNAP) is a dual-purpose enzyme that plays two opposite roles in two different situations during DNA replication. It plays its normal role as a {\\it polymerase} catalyzing the elongation of a new DNA molecule by adding a monomer. However, it can switch to the role of an {\\it exonuclease} and shorten the same DNA by cleavage of the last incorporated monomer from the nascent DNA. Just as misincorporated nucleotides can escape exonuclease causing replication error, correct nucleotide may get sacrificed unnecessarily by erroneous cleavage. The interplay of polymerase and exonuclease activities of a DNAP is explored here by developing a minimal stochastic kinetic model of DNA replication. Exact analytical expressions are derived for a few key statistical distributions; these characterize the temporal patterns in the mechanical stepping and the chemical (cleavage) reaction. The Michaelis-Menten-like analytical expression derived for the average rates of these two processes not only demonstrate the ef...

  1. Porcine circovirus: transcription and rolling-circle DNA replication

    This review summarizes the molecular studies pertaining to porcine circovirus (PCV) transcription and DNA replication. The genome of PCV is circular, single-stranded DNA and contains 1759-1768 nucleotides. Both the genome-strand (packaged in the virus particle) and the complementary-strand (synthesi...

  2. A Paper Model of DNA Structure and Replication.

    Sigismondi, Linda A.

    1989-01-01

    A paper model which is designed to give students a hands-on experience during lecture and blackboard instruction on DNA structure is provided. A list of materials, paper patterns, and procedures for using the models to teach DNA structure and replication are given. (CW)

  3. The hunt for origins of DNA replication in multicellular eukaryotes

    Urban, J. M.; Foulk, M. S.; Casella, Cinzia;

    2015-01-01

    Origins of DNA replication (ORIs) occur at defined regions in the genome. Although DNA sequence defines the position of ORIs in budding yeast, the factors for ORI specification remain elusive in metazoa. Several methods have been used recently to map ORIs in metazoan genomes with the hope that fe...

  4. Dynamics of plant DNA replication based on PCNA visualization.

    Yokoyama, Ryohei; Hirakawa, Takeshi; Hayashi, Seri; Sakamoto, Takuya; Matsunaga, Sachihiro

    2016-01-01

    DNA replication is an essential process for the copying of genomic information in living organisms. Imaging of DNA replication in tissues and organs is mainly performed using fixed cells after incorporation of thymidine analogs. To establish a useful marker line to measure the duration of DNA replication and analyze the dynamics of DNA replication, we focused on the proliferating cell nuclear antigen (PCNA), which functions as a DNA sliding clamp for replicative DNA polymerases and is an essential component of replisomes. In this study we produced an Arabidopsis thaliana line expressing PCNA1 fused with the green fluorescent protein under the control of its own promoter (pAtPCNA1::AtPCNA1-EGFP). The duration of the S phase measured using the expression line was consistent with that measured after incorporation of a thymidine analog. Live cell imaging revealed that three distinct nuclear localization patterns (whole, dotted, and speckled) were sequentially observable. These whole, dotted, and speckled patterns of subnuclear AtPCNA1 signals were indicative of the G1 or G2 phase, early S phase and late S phase, respectively. The results indicate that the pAtPCNA1::AtPCNA1-EGFP line is a useful marker line for visualization of S-phase progression in live plant organs. PMID:27417498

  5. Analysis of the cruciform binding activity of recombinant 14-3-3zeta-MBP fusion protein, its heterodimerization profile with endogenous 14-3-3 isoforms, and effect on mammalian DNA replication in vitro.

    Alvarez, David; Callejo, Mario; Shoucri, Rami; Boyer, Lee; Price, Gerald B; Zannis-Hadjopoulos, Maria

    2003-06-17

    The human cruciform binding protein (CBP), a member of the 14-3-3 protein family, has been recently identified as an origin of DNA replication binding protein and involved in DNA replication. Here, pure recombinant 14-3-3zeta tagged with maltose binding protein (r14-3-3zeta-MBP) at its N-terminus was tested for binding to cruciform DNA either in the absence or presence of F(TH), a CBP-enriched fraction, by electromobility shift assay (EMSA), followed by Western blot analysis of the electroeluted CBP-cruciform DNA complex. The r14-3-3zeta-MBP was found to have cruciform binding activity only after preincubation with F(TH). Anti-MBP antibody immunoprecipitation of F(TH) preincubated with r14-3-3zeta-MBP, followed by Western blot analysis with antibodies specific to the beta, gamma, epsilon, zeta, and sigma 14-3-3 isoforms showed that r14-3-3zeta-MBP heterodimerized with the endogenous beta, epsilon, and zeta isoforms present in the F(TH) but not with the gamma or sigma isoforms. Immunoprecipitation of endogenous 14-3-3zeta from nuclear extracts (NE) of HeLa cells that were either serum-starved (s-s) or blocked at the G(1)/S or G(2)/M phases of the cell cycle revealed that at G(1)/S and G(2)/M, the zeta isoform heterodimerized only with the beta and epsilon isoforms, while in s-s extracts, the 14-3-3zeta/epsilon heterodimer was never detected, and the 14-3-3zeta/beta heterodimer was seldom detected. Furthermore, addition of r14-3-3zeta-MBP to HeLa cell extracts used in a mammalian in vitro replication system increased the replication level of p186, a plasmid bearing the minimal 186-bp origin of the monkey origin of DNA replication ors8, by approximately 3.5-fold. The data suggest that specific dimeric combinations of the 14-3-3 isoforms have CBP activity and that upregulation of this activity leads to an increase in DNA replication. PMID:12795617

  6. Inhibition and recovery of the replication of depurinated parvovirus DNA in mouse fibroblasts

    Vos, J.M.; Avalosse, B.; Su, Z.Z.; Rommelaere, J.

    1984-01-01

    Apurinic sites were introduced in the single-stranded DNA of parvovirus minute-virus-of-mice (MVM) and their effect on viral DNA synthesis was measured in mouse fibroblasts. Approximately one apurinic site per viral genome, is sufficient to block its replication in untreated cells. The exposure of host cells to a sublethal dose of UV-light 15 hours prior to virus infection, enhances their ability to support the replication of depurinated MVM. Cell preirradiation induces the apparent overcome of 10-15% of viral DNA replication blocks. These results indicate that apurinic sites prevent mammalian cells from replicating single-stranded DNA unless a recovery process is activated by cell UV-irradiation.

  7. DNA is a co-factor for its own replication in Xenopus egg extracts

    Lebofsky, Ronald; van Oijen, Antoine M.; Walter, Johannes C.

    2011-01-01

    Soluble Xenopus egg extracts efficiently replicate added plasmids using a physiological mechanism, and thus represent a powerful system to understand vertebrate DNA replication. Surprisingly, DNA replication in this system is highly sensitive to plasmid concentration, being undetectable below simila

  8. Assays Used to Study the DNA Replication Checkpoint in Fission Yeast

    Noguchi, Eishi; Ansbach, Alison B.; Noguchi, Chiaki; Russell, Paul

    2009-01-01

    The DNA replication checkpoint, also known as the intra-S or S-phase checkpoint, plays a central role in ensuring the accuracy of DNA replication. When replication is impeded by DNA damage or other conditions, this checkpoint delays cell cycle progression and coordinates resumption of replication with DNA repair pathways. One of its critical functions is to stabilize stalled replication forks in a replication-competent state, presumably by maintaining proper assembly of replisome components a...

  9. DNA-PKcs and PARP1 Bind to Unresected Stalled DNA Replication Forks Where They Recruit XRCC1 to Mediate Repair.

    Ying, Songmin; Chen, Zhihui; Medhurst, Annette L; Neal, Jessica A; Bao, Zhengqiang; Mortusewicz, Oliver; McGouran, Joanna; Song, Xinming; Shen, Huahao; Hamdy, Freddie C; Kessler, Benedikt M; Meek, Katheryn; Helleday, Thomas

    2016-03-01

    A series of critical pathways are responsible for the detection, signaling, and restart of replication forks that encounter blocks during S-phase progression. Small base lesions may obstruct replication fork progression and processing, but the link between repair of small lesions and replication forks is unclear. In this study, we investigated a hypothesized role for DNA-PK, an important enzyme in DNA repair, in cellular responses to DNA replication stress. The enzyme catalytic subunit DNA-PKcs was phosphorylated on S2056 at sites of stalled replication forks in response to short hydroxyurea treatment. Using DNA fiber experiments, we found that catalytically active DNA-PK was required for efficient replication restart of stalled forks. Furthermore, enzymatically active DNA-PK was also required for PARP-dependent recruitment of XRCC1 to stalled replication forks. This activity was enhanced by preventing Mre11-dependent DNA end resection, suggesting that XRCC1 must be recruited early to an unresected stalled fork. We also found that XRCC1 was required for effective restart of a subset of stalled replication forks. Overall, our work suggested that DNA-PK and PARP-dependent recruitment of XRCC1 is necessary to effectively protect, repair, and restart stalled replication forks, providing new insight into how genomic stability is preserved. Cancer Res; 76(5); 1078-88. ©2015 AACR. PMID:26603896

  10. Nuclear organization of DNA replication in primary mammalian cells

    Kennedy, Brian K.; Barbie, David A; Classon, Marie; Dyson, Nicholas; Harlow, Ed

    2000-01-01

    Using methods that conserve nuclear architecture, we have reanalyzed the spatial organization of the initiation of mammalian DNA synthesis. Contrary to the commonly held view that replication begins at hundreds of dispersed nuclear sites, primary fibroblasts initiate synthesis in a limited number of foci that contain replication proteins, surround the nucleolus, and overlap with previously identified internal lamin A/C structures. These foci are established in early G1-phase and also contain ...

  11. DNA Polymerases Divide the Labor of Genome Replication.

    Lujan, Scott A; Williams, Jessica S; Kunkel, Thomas A

    2016-09-01

    DNA polymerases synthesize DNA in only one direction, but large genomes require RNA priming and bidirectional replication from internal origins. We review here the physical, chemical, and evolutionary constraints underlying these requirements. We then consider the roles of the major eukaryotic replicases, DNA polymerases α, δ, and ɛ, in replicating the nuclear genome. Pol α has long been known to extend RNA primers at origins and on Okazaki fragments that give rise to the nascent lagging strand. Taken together, more recent results of mutation and ribonucleotide incorporation mapping, electron microscopy, and immunoprecipitation of nascent DNA now lead to a model wherein Pol ɛ and Pol δ, respectively, synthesize the majority of the nascent leading and lagging strands of undamaged DNA. PMID:27262731

  12. Priming DNA Replication from Triple Helix Oligonucleotides: Possible Threestranded DNA in DNA Polymerases

    Patrick P. Lestienne

    2011-01-01

    Full Text Available Triplex associate with a duplex DNA presenting the same polypurine or polypyrimidine-rich sequence in an antiparallel orientation. So far, triplex forming oligonucleotides (TFOs are known to inhibit transcription, replication, and to induce mutations. A new property of TFO is reviewed here upon analysis of DNA breakpoint yielding DNA rearrangements; the synthesized sequence of the first direct repeat displays a skewed polypurine- rich sequence. This synthesized sequence can bind the second homologous duplex sequence through the formation of a triple helix, which is able to prime further DNA replication. In these case, the d(G-rich Triple Helix Primers (THP bind the homologous strand in a parallel manner, possibly via a RecA-like mechanism. This novel property is shared by all tested DNA polymerases: phage, retrovirus, bacteria, and human. These features may account for illegitimate initiation of replication upon single-strand breakage and annealing to a homologous sequence where priming may occur. Our experiments suggest that DNA polymerases can bind three instead of two polynucleotide strands in their catalytic centre.

  13. Priming DNA replication from triple helix oligonucleotides: possible threestranded DNA in DNA polymerases.

    Lestienne, Patrick P

    2011-01-01

    Triplex associate with a duplex DNA presenting the same polypurine or polypyrimidine-rich sequence in an antiparallel orientation. So far, triplex forming oligonucleotides (TFOs) are known to inhibit transcription, replication, and to induce mutations. A new property of TFO is reviewed here upon analysis of DNA breakpoint yielding DNA rearrangements; the synthesized sequence of the first direct repeat displays a skewed polypurine- rich sequence. This synthesized sequence can bind the second homologous duplex sequence through the formation of a triple helix, which is able to prime further DNA replication. In these case, the d(G)-rich Triple Helix Primers (THP) bind the homologous strand in a parallel manner, possibly via a RecA-like mechanism. This novel property is shared by all tested DNA polymerases: phage, retrovirus, bacteria, and human. These features may account for illegitimate initiation of replication upon single-strand breakage and annealing to a homologous sequence where priming may occur. Our experiments suggest that DNA polymerases can bind three instead of two polynucleotide strands in their catalytic centre. PMID:22229092

  14. Nonenzymatic Role for WRN in Preserving Nascent DNA Strands after Replication Stress

    Fengtao Su

    2014-11-01

    Full Text Available WRN, the protein defective in Werner syndrome (WS, is a multifunctional nuclease involved in DNA damage repair, replication, and genome stability maintenance. It was assumed that the nuclease activities of WRN were critical for these functions. Here, we report a nonenzymatic role for WRN in preserving nascent DNA strands following replication stress. We found that lack of WRN led to shortening of nascent DNA strands after replication stress. Furthermore, we discovered that the exonuclease activity of MRE11 was responsible for the shortening of newly replicated DNA in the absence of WRN. Mechanistically, the N-terminal FHA domain of NBS1 recruits WRN to replication-associated DNA double-stranded breaks to stabilize Rad51 and to limit the nuclease activity of its C-terminal binding partner MRE11. Thus, this previously unrecognized nonenzymatic function of WRN in the stabilization of nascent DNA strands sheds light on the molecular reason for the origin of genome instability in WS individuals.

  15. Direct observation of enzymes replicating DNA using a single-molecule DNA stretching assay

    Kulczyk, A.W.; Tanner, N.A.; Loparo, J.J.; Richardson, C.C.; Oijen, A.M. van

    2010-01-01

    We describe a method for observing real time replication of individual DNA molecules mediated by proteins of the bacteriophage replication system. Linearized lambda DNA is modified to have a biotin on the end of one strand, and a digoxigenin moiety on the other end of the same strand. The biotinylat

  16. USP7 is a SUMO deubiquitinase essential for DNA replication

    Lecona, Emilio; Rodriguez-Acebes, Sara; Specks, Julia; Lopez-Contreras, Andres J; Ruppen, Isabel; Murga, Matilde; Muñoz, Javier; Mendez, Juan; Fernandez-Capetillo, Oscar

    2016-01-01

    Post-translational modification of proteins by ubiquitin (Ub) and Ub-like modifiers regulates various aspects of DNA replication. We previously showed that the chromatin around replisomes is rich in SUMO and depleted in Ub, whereas an opposite pattern is observed in mature chromatin. How this SUMO-rich/Ub-low environment is maintained at sites of DNA replication is not known. Here we identify USP7 as a replisome-enriched SUMO deubiquitinase that is essential for DNA replication. By acting on SUMO and SUMOylated proteins, USP7 counteracts their ubiquitination. Chemical inhibition or genetic deletion of USP7 leads to the accumulation of Ub on SUMOylated proteins, which are displaced to chromatin away from replisomes. Our findings provide a model to explain the differential accumulation of SUMO and Ub at replication forks, and identify an essential role of USP7 in DNA replication that should be taken into account for the use of USP7 inhibitors as anticancer agents. PMID:26950370

  17. Homologous recombination-dependent initiation of DNA replication from DNA damage-inducible origins in Escherichia coli.

    Asai, T; Sommer, S; Bailone, A; Kogoma, T

    1993-01-01

    Escherichia coli cells induced for the SOS response express inducible stable DNA replication (iSDR) as an SOS function. Initiation of iSDR is independent of transcription, translation and DnaA protein, which are essential for initiation of DNA replication from oriC. We found that a recA mutant that is defective in recombination but proficient in SOS induction could not elicit iSDR. In contrast, iSDR was enhanced by recD and recJ mutations that inactivate the exonuclease V activity of the RecB...

  18. Gastric cancer associated variant of DNA polymerase beta (Leu22Pro) promotes DNA replication associated double strand breaks.

    Rozacky, Jenna; Nemec, Antoni A; Sweasy, Joann B; Kidane, Dawit

    2015-09-15

    DNA polymerase beta (Pol β) is a key enzyme for the protection against oxidative DNA lesions via its role in base excision repair (BER). Approximately 1/3 of tumors studied to date express Pol β variant proteins, and several tumors overexpress Pol β. Pol β possesses DNA polymerase and dRP lyase activities, both of which are known to be important for efficient BER. The dRP lyase activity resides within the 8kDa amino terminal domain of Pol β, is responsible for removal of the 5' phosphate group (5'-dRP). The DNA polymerase subsequently fills the gaps. Previously, we demonstrated that the human gastric cancer-associated variant of Pol β (Leu22Pro (L22P)) lacks dRP lyase function in vitro. Here, we report that L22P-expressing cells harbor significantly increased replication associated DNA double strand breaks (DSBs) and defective maintenance of the nascent DNA strand (NDS) during replication stress. Moreover, L22P-expressing cells are sensitive to PARP1 inhibitors, which suggests trapped PARP1 binds to the 5'-dRP group and blocks replications forks, resulting in fork collapse and DSBs. Our data suggest that the normal function of the dRP lyase is critical to maintain replication fork integrity and prevent replication fork collapse to DSBs and cellular transformation. PMID:26090616

  19. A novel endoribonuclease cleaves at a priming site of mouse mitochondrial DNA replication.

    Chang, D D; Clayton, D A

    1987-01-01

    Priming at the mouse mitochondrial origin of heavy-strand DNA replication is effected by transcripts from the light-strand promoter. The transition from RNA synthesis to DNA synthesis occurs at specific locations between 75 and 165 nucleotides downstream from the transcriptional initiation site. We have identified and partially purified an endonucleolytic activity that cleaves RNA accurately near one of these transition sites; this finding implies a role of specific RNA processing in DNA repl...

  20. Functional redundancy between DNA ligases I and III in DNA replication in vertebrate cells

    Arakawa, Hiroshi; Bednar, Theresa; Wang, Minli; Paul, Katja; Mladenov, Emil; Bencsik-Theilen, Alena A.; Iliakis, George

    2012-01-01

    In eukaryotes, the three families of ATP-dependent DNA ligases are associated with specific functions in DNA metabolism. DNA ligase I (LigI) catalyzes Okazaki-fragment ligation at the replication fork and nucleotide excision repair (NER). DNA ligase IV (LigIV) mediates repair of DNA double strand breaks (DSB) via the canonical non-homologous end-joining (NHEJ) pathway. The evolutionary younger DNA ligase III (LigIII) is restricted to higher eukaryotes and has been associated with base excision (BER) and single strand break repair (SSBR). Here, using conditional knockout strategies for LIG3 and concomitant inactivation of the LIG1 and LIG4 genes, we show that in DT40 cells LigIII efficiently supports semi-conservative DNA replication. Our observations demonstrate a high functional versatility for the evolutionary new LigIII in DNA replication and mitochondrial metabolism, and suggest the presence of an alternative pathway for Okazaki fragment ligation. PMID:22127868

  1. Proteotoxic stress induces a cell-cycle arrest by stimulating Lon to degrade the replication initiator DnaA.

    Jonas, Kristina; Liu, Jing; Chien, Peter; Laub, Michael T

    2013-08-01

    The decision to initiate DNA replication is a critical step in the cell cycle of all organisms. Cells often delay replication in the face of stressful conditions, but the underlying mechanisms remain incompletely defined. Here, we demonstrate in Caulobacter crescentus that proteotoxic stress induces a cell-cycle arrest by triggering the degradation of DnaA, the conserved replication initiator. A depletion of available Hsp70 chaperone, DnaK, either through genetic manipulation or heat shock, induces synthesis of the Lon protease, which can directly degrade DnaA. Unexpectedly, we find that unfolded proteins, which accumulate following a loss of DnaK, also allosterically activate Lon to degrade DnaA, thereby ensuring a cell-cycle arrest. Our work reveals a mechanism for regulating DNA replication under adverse growth conditions. Additionally, our data indicate that unfolded proteins can actively and directly alter substrate recognition by cellular proteases. PMID:23911325

  2. p53 represses human papillomavirus type 16 DNA replication via the viral E2 protein

    Morgan Iain M

    2008-01-01

    Full Text Available Abstract Background Human papillomavirus (HPV DNA replication can be inhibited by the cellular tumour suppressor protein p53. However, the mechanism through which p53 inhibits viral replication and the role that this might play in the HPV life cycle are not known. The papillomavirus E2 protein is required for efficient HPV DNA replication and also regulates viral gene expression. E2 represses transcription of the HPV E6 and E7 oncogenes and can thereby modulate indirectly host cell proliferation and survival. In addition, the E2 protein from HPV 16 has been shown to bind p53 and to be capable of inducing apoptosis independently of E6 and E7. Results Here we use a panel of E2 mutants to confirm that mutations which block the induction of apoptosis via this E6/E7-independent pathway, have little or no effect on the induction of apoptosis by the E6/E7-dependent pathway. Although these mutations in E2 do not affect the ability of the protein to mediate HPV DNA replication, they do abrogate the repressive effects of p53 on the transcriptional activity of E2 and prevent the inhibition of E2-dependent HPV DNA replication by p53. Conclusion These data suggest that p53 down-regulates HPV 16 DNA replication via the E2 protein.

  3. DNA ligase I selectively affects DNA synthesis by DNA polymerases delta and epsilon suggesting differential functions in DNA replication and repair.

    Mossi, R; Ferrari, E.; Hübscher, U

    1998-01-01

    The joining of single-stranded breaks in double-stranded DNA is an essential step in many important processes such as DNA replication, DNA repair, and genetic recombination. Several data implicate a role for DNA ligase I in DNA replication, probably coordinated by the action of other enzymes and proteins. Since both DNA polymerases delta and epsilon show multiple functions in different DNA transactions, we investigated the effect of DNA ligase I on various DNA synthesis events catalyzed by th...

  4. DNA breaks early in replication in B cell cancers

    Research by scientists at the NCI has identified a new class of DNA sites in cells that break early in the replication process. They found that these break sites correlate with damage often seen in B cell cancers, such as diffuse large B cell lymphoma.

  5. The absence of a DNA replication checkpoint in porcine zygotes

    Vacková, I.; Křen, Radomír; Loi, P.; Krylov, V.; Fulka Jr., J.

    2006-01-01

    Roč. 14, 1 (2006), s. 33-37. ISSN 0967-1994 Institutional research plan: CEZ:AV0Z50450515 Keywords : checkpoint * DNA replication * fertilization Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 0.782, year: 2006

  6. DNA replication at the single-molecule level

    Stratmann, S A; van Oijen, A M

    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 i

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

    Egel, Richard

    2004-01-01

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

  8. Quantitative live imaging of endogenous DNA replication in mammalian cells.

    Andrew Burgess

    Full Text Available Historically, the analysis of DNA replication in mammalian tissue culture cells has been limited to static time points, and the use of nucleoside analogues to pulse-label replicating DNA. Here we characterize for the first time a novel Chromobody cell line that specifically labels endogenous PCNA. By combining this with high-resolution confocal time-lapse microscopy, and with a simplified analysis workflow, we were able to produce highly detailed, reproducible, quantitative 4D data on endogenous DNA replication. The increased resolution allowed accurate classification and segregation of S phase into early-, mid-, and late-stages based on the unique subcellular localization of endogenous PCNA. Surprisingly, this localization was slightly but significantly different from previous studies, which utilized over-expressed GFP tagged forms of PCNA. Finally, low dose exposure to Hydroxyurea caused the loss of mid- and late-S phase localization patterns of endogenous PCNA, despite cells eventually completing S phase. Taken together, these results indicate that this simplified method can be used to accurately identify and quantify DNA replication under multiple and various experimental conditions.

  9. Localization of adenovirus DNA replication in KB cells

    Vlak, J.M.; Rozijn, Th.H.; Spies, F.

    1975-01-01

    The localization of adenovirus type 5 DNA replication has been investigated by both fractionation of isolated nuclei and electron-microscope autoradiography. Nuclear fractionation by means of the M-band-technique of Tremblay et al. (Tremblay, G. Y., Daniels, M. J., and Schaechter, M. (1969). J. Mol.

  10. Inferring the spatiotemporal DNA replication program from noisy data

    Baker, A.; Bechhoefer, J.

    2014-03-01

    We generalize a stochastic model of DNA replication to the case where replication-origin-initiation rates vary locally along the genome and with time. Using this generalized model, we address the inverse problem of inferring initiation rates from experimental data concerning replication in cell populations. Previous work based on curve fitting depended on arbitrarily chosen functional forms for the initiation rate, with free parameters that were constrained by the data. We introduce a nonparametric method of inference that is based on Gaussian process regression. The method replaces specific assumptions about the functional form of the initiation rate with more general prior expectations about the smoothness of variation of this rate, along the genome and in time. Using this inference method, we recover, with high precision, simulated replication schemes from noisy data that are typical of current experiments.

  11. Proteasome-dependent degradation of replisome components regulates faithful DNA replication

    Roseaulin, Laura C; Noguchi, Chiaki; Noguchi, Eishi

    2013-01-01

    The replication machinery, or the replisome, collides with a variety of obstacles during the normal process of DNA replication. In addition to damaged template DNA, numerous chromosome regions are considered to be difficult to replicate owing to the presence of DNA secondary structures and DNA-binding proteins. Under these conditions, the replication fork stalls, generating replication stress. Stalled forks are prone to collapse, posing serious threats to genomic integrity. It is generally th...

  12. Properties of SV40 large T antigen involved in the replication of SV40 DNA

    The role of phosphorylation in regulating the biochemical properties of SV40 large T antigen (Tag) was analyzed. Treatment of purified Tag with calf intestinal alkaline phosphatase (CIAP) removed 80% of the 32P label. CIAP treated Tag displayed an increased ability to support SV40 DNA replication in vitro. This paralleled an activation of specific DNA binding to site II, an essential element within the SV40 origin of replication (ori). The ATPase activity of dephosphorylated Tag remained unchanged. To evaluate the properties of Tag devoid of mammalian post translational modifications, a bacterial expression system was used. Purified E. coli Tag did not bind to site II, and therefore could not unwind ori containing plasmids or efficiently replicate SV40 DNA in vitro. However, E. coli Tag functioned as a helicase, and bound to DNA fragments containing either site I or the wild type (wt) ori in a manner identical to mammalian Tag. A single DNA binding domain is responsible for specific DNA binding to either of two different sites embedded in the SV40 ori, and the intrinsic preference of full length Tag for either site I, or site II appears to be modulated by phosphorylation. Several Tag point mutants were examined in a series of assays which reflect partial biochemical reactions required for the in vitro replication of SV40 DNA

  13. Properties of SV40 large T antigen involved in the replication of SV40 DNA

    Mohr, I.J.

    1989-01-01

    The role of phosphorylation in regulating the biochemical properties of SV40 large T antigen (Tag) was analyzed. Treatment of purified Tag with calf intestinal alkaline phosphatase (CIAP) removed 80% of the {sup 32}P label. CIAP treated Tag displayed an increased ability to support SV40 DNA replication in vitro. This paralleled an activation of specific DNA binding to site II, an essential element within the SV40 origin of replication (ori). The ATPase activity of dephosphorylated Tag remained unchanged. To evaluate the properties of Tag devoid of mammalian post translational modifications, a bacterial expression system was used. Purified E. coli Tag did not bind to site II, and therefore could not unwind ori containing plasmids or efficiently replicate SV40 DNA in vitro. However, E. coli Tag functioned as a helicase, and bound to DNA fragments containing either site I or the wild type (wt) ori in a manner identical to mammalian Tag. A single DNA binding domain is responsible for specific DNA binding to either of two different sites embedded in the SV40 ori, and the intrinsic preference of full length Tag for either site I, or site II appears to be modulated by phosphorylation. Several Tag point mutants were examined in a series of assays which reflect partial biochemical reactions required for the in vitro replication of SV40 DNA.

  14. The logic of DNA replication in double-stranded DNA viruses: insights from global analysis of viral genomes

    Kazlauskas, Darius; Krupovic, Mart; Venclovas, Česlovas

    2016-01-01

    Genomic DNA replication is a complex process that involves multiple proteins. Cellular DNA replication systems are broadly classified into only two types, bacterial and archaeo-eukaryotic. In contrast, double-stranded (ds) DNA viruses feature a much broader diversity of DNA replication machineries. Viruses differ greatly in both completeness and composition of their sets of DNA replication proteins. In this study, we explored whether there are common patterns underlying this extreme diversity...

  15. Cloning of an origin of DNA replication of Xenopus laevis

    DNA fragments of Xenopus laevis, the African frog, were cloned in the EcoRI site of the Eschrichia coli plasmid pACYC189 and tested for ability to initiate and complete replication of the recombinant plasmid when injected into unfertilized eggs of X. laevis. After measurement of the [3H]-thymidine incorporation per egg for a number of recombinant plasmids, pSW14 and pSW9, which respectively contain a small segment (550 base pairs) and several kilobases of frog DNA, were selected for more extensive analysis. In spite of the small size of th segment in pSW14, it incorporates in 2 hr at least 3 times as much labeled thymidine as either pSW9 or the vector alone. To determine the number of replications of pSW14, a novel method was employed. The results showed that about 50% of the labeled, supercoiled DNA recovered from eggs after 4 hr was sensitive to EcoRI digestion, which indicates that most of the DNA that incorporated [3H]thymidine had replicated twice during the 4 hr in the unfertilized eggs of X. laevis. We conclude the pSW14 has a functional origin in the Xenopus DNA segment

  16. Epigallocatechin gallate inhibits HBV DNA synthesis in a viral replication - inducible cell line

    Wei He; Li-Xia Li; Qing-Jiao Liao; Chun-Lan Liu; Xu-Lin Chen

    2011-01-01

    AIM: To analyze the antiviral mechanism of Epigallocatechin gallate (EGCG) against hepatitis B virus (HBV) replication. METHODS: In this research, the HBV-replicating cell line HepG2.117 was used to investigate the antiviral mechanism of EGCG. Cytotoxicity of EGCG was analyzed by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Hepatitis B virus e antigen (HBeAg) and hepatitis B virus surface antigen (HBsAg) in the supernatant were detected by enzyme-linked immunosorbent assay. Precore mRNA and pregenomic RNA (pgRNA) levels were determined by semi-quantitative reverse transcription polymerase chain reaction (PCR) assay. The effect of EGCG on HBV core promoter activity was measured by dual luciferase reporter assay. HBV covalently closed circular DNA and replicative intermediates of DNA were quantified by real-time PCR assay. RESULTS: When HepG2.117 cells were grown in the presence of EGCG, the expression of HBeAg was suppressed, however, the expression of HBsAg was not affected. HBV precore mRNA level was also downregulated by EGCG, while the transcription of precore mRNA was not impaired. The synthesis of both HBV covalently closed circular DNA and replicative intermediates of DNA were reduced by EGCG treatment to a similar extent, however, HBV pgRNA transcripted from chromosome-integrated HBV genome was not affected by EGCG treatment, indicating that EGCG targets only replicative intermediates of DNA synthesis. CONCLUSION: In HepG2.117 cells, EGCG inhibits HBV replication by impairing HBV replicative intermediates of DNA synthesis and such inhibition results in reduced production of HBV covalently closed circular DNA.

  17. Linking DNA replication to heterochromatin silencing and epigenetic inheritance

    Qing Li; Zhiguo Zhang

    2012-01-01

    Chromatin is organized into distinct functional domains.During mitotic cell division,both genetic information encoded in DNA sequence and epigenetic information embedded in chromatin structure must be faithfully duplicated.The inheritance of epigenetic states is critical in maintaining the genome integrity and gene expression state.In this review,we will discuss recent progress on how proteins known to be involved in DNA replication and DNA replication-coupled nucleosome assembly impact on the inheritance and maintenance of heterochromatin,a tightly compact chromatin structure that silences gene transcription.As heterochromatin is important in regulating gene expression and maintaining genome stability,understanding how heterochromatin states are inherited during S phase of the cell cycle is of fundamental importance.

  18. Drosophila Ctf4 is essential for efficient DNA replication and normal cell cycle progression

    Christensen Tim W

    2011-04-01

    Full Text Available Abstract Background Proper coordination of the functions at the DNA replication fork is vital to the normal functioning of a cell. Specifically the precise coordination of helicase and polymerase activity is crucial for efficient passage though S phase. The Ctf4 protein has been shown to be a central member of the replication fork and links the replicative MCM helicase and DNA polymerase α primase. In addition, it has been implicated as a member of a complex that promotes replication fork stability, the Fork Protection Complex (FPC, and as being important for sister chromatid cohesion. As such, understanding the role of Ctf4 within the context of a multicellular organism will be integral to our understanding of its potential role in developmental and disease processes. Results We find that Drosophila Ctf4 is a conserved protein that interacts with members of the GINS complex, Mcm2, and Polymerase α primase. Using in vivo RNAi knockdown of CTF4 in Drosophila we show that Ctf4 is required for viability, S phase progression, sister chromatid cohesion, endoreplication, and coping with replication stress. Conclusions Ctf4 remains a central player in DNA replication. Our findings are consistent with what has been previously reported for CTF4 function in yeast, Xenopus extracts, and human tissue culture. We show that Ctf4 function is conserved and that Drosophila can be effectively used as a model to further probe the precise function of Ctf4 as a member of the replication fork and possible roles in development.

  19. Plasmid P1 replication: negative control by repeated DNA sequences.

    Chattoraj, D; Cordes, K.; Abeles, A

    1984-01-01

    The incompatibility locus, incA, of the unit-copy plasmid P1 is contained within a fragment that is essentially a set of nine 19-base-pair repeats. One or more copies of the fragment destabilizes the plasmid when present in trans. Here we show that extra copies of incA interfere with plasmid DNA replication and that a deletion of most of incA increases plasmid copy number. Thus, incA is not essential for replication but is required for its control. When cloned in a high-copy-number vector, pi...

  20. The DNA repair helicase UvrD is essential for replication fork reversal in replication mutants

    Flores, Maria Jose; Bidnenko, Vladimir; Michel, Bénédicte

    2004-01-01

    Replication forks arrested by inactivation of the main Escherichia coli DNA polymerase (polymerase III) are reversed by the annealing of newly synthesized leading- and lagging-strand ends. Reversed forks are reset by the action of RecBC on the DNA double-strand end, and in the absence of RecBC chromosomes are linearized by the Holliday junction resolvase RuvABC. We report here that the UvrD helicase is essential for RuvABC-dependent chromosome linearization in E. coli polymerase III mutants, ...

  1. Insights into the Determination of the Templating Nucleotide at the Initiation of φ29 DNA Replication.

    del Prado, Alicia; Lázaro, José M; Longás, Elisa; Villar, Laurentino; de Vega, Miguel; Salas, Margarita

    2015-11-01

    Bacteriophage φ29 from Bacillus subtilis starts replication of its terminal protein (TP)-DNA by a protein-priming mechanism. To start replication, the DNA polymerase forms a heterodimer with a free TP that recognizes the replication origins, placed at both 5' ends of the linear chromosome, and initiates replication using as primer the OH-group of Ser-232 of the TP. The initiation of φ29 TP-DNA replication mainly occurs opposite the second nucleotide at the 3' end of the template. Earlier analyses of the template position that directs the initiation reaction were performed using single-stranded and double-stranded oligonucleotides containing the replication origin sequence without the parental TP. Here, we show that the parental TP has no influence in the determination of the nucleotide used as template in the initiation reaction. Previous studies showed that the priming domain of the primer TP determines the template position used for initiation. The results obtained here using mutant TPs at the priming loop where Ser-232 is located indicate that the aromatic residue Phe-230 is one of the determinants that allows the positioning of the penultimate nucleotide at the polymerization active site to direct insertion of the initiator dAMP during the initiation reaction. The role of Phe-230 in limiting the internalization of the template strand in the polymerization active site is discussed. PMID:26400085

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

    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.

  3. DNA2 Encodes a DNA Helicase Essential for Replication of Eukaryotic Chromosomes

    Budd, Martin E.; Choe, Won-chae; Campbell, Judith L.

    1995-01-01

    Although a number of eukaryotic DNA helicases have been identified biochemically and still more have been inferred from the amino acid sequences of the products of cloned genes, none of the cellular helicases or putative helicases has to date been implicated in eukaryotic chromosomal DNA replication. By the same token, numerous eukaryotic replication proteins have been identified, but none of these is a helicase. We have recently identified and characterized a temperature-sensitive yeast muta...

  4. Accuracy of replication in the polymerase chain reaction. Comparison between Thermotoga maritima DNA polymerase and Thermus aquaticus DNA polymerase

    Diaz, R S; Sabino, E. C.

    1998-01-01

    For certain applications of the polymerase chain reaction (PCR), it may be necessary to consider the accuracy of replication. The breakthrough that made PCR user friendly was the commercialization of Thermus aquaticus (Taq) DNA polymerase, an enzyme that would survive the high temperatures needed for DNA denaturation. The development of enzymes with an inherent 3' to 5' exonuclease proofreading activity, lacking in Taq polymerase, would be an improvement when higher fidelity is needed. We use...

  5. Chromosome banding and DNA replication patterns in bird karyotypes.

    Schmid, M; Enderle, E; Schindler, D; Schempp, W

    1989-01-01

    The karyotypes of the domestic chicken (Gallus domesticus), Japanese quail (Coturnix coturnix), and griffon vulture (Gyps fulvus) were studied with a variety of banding techniques. The DNA replication patterns of bird chromosomes, analyzed by incorporation of 5-bromodeoxyuridine (BrdU) and deoxythymidine (dT), are presented here for the first time. In particular, the time sequence of replication of the ZZ/ZW sex chromosomes throughout the S-phase was meticulously analyzed. BrdU and dT incorporation are very useful methods to identify homoeologies between karyotypes, as well as rearrangements that occurred in the macroautosomes during speciation. The Z chromosomes of the three birds displayed the same replication patterns, indicating a high degree of evolutionary conservation. In the homogametic male, BrdU and dT incorporation revealed no evidence of asynchronous replication between euchromatic bands in the ZZ pair. The same was true of the three Z chromosomes in a triploid-diploid chimeric chicken embryo. Minor replication asynchronies between the homologous ZZ or ZZZ chromosomes were restricted to heterochromatic C-bands. These results confirm that, in the ZZ male/ZW female sex-determining system of birds, dosage compensation for Z-linked genes does not occur by inactivation of one of the two Z chromosomes in the homogametic male. The heterochromatic W chromosomes of the three species showed bright labeling with distamycin A/mithramycin counterstain-enhanced fluorescence and exhibited significantly delayed DNA replication. The nucleolus organizers of birds, frequently located in microchromosomes, were also distinguished by bright distamycin A/mithramycin fluorescence. PMID:2630186

  6. Plasticity of DNA Replication Initiation in Epstein-Barr Virus Episomes

    Norio Paolo

    2004-01-01

    Full Text Available In mammalian cells, the activity of the sites of initiation of DNA replication appears to be influenced epigenetically, but this regulation is not fully understood. Most studies of DNA replication have focused on the activity of individual initiation sites, making it difficult to evaluate the impact of changes in initiation activity on the replication of entire genomic loci. Here, we used single molecule analysis of replicated DNA (SMARD to study the latent duplication of Epstein-Barr virus (EBV episomes in human cell lines. We found that initiation sites are present throughout the EBV genome and that their utilization is not conserved in different EBV strains. In addition, SMARD shows that modifications in the utilization of multiple initiation sites occur across large genomic regions (tens of kilobases in size. These observations indicate that individual initiation sites play a limited role in determining the replication dynamics of the EBV genome. Long-range mechanisms and the genomic context appear to play much more important roles, affecting the frequency of utilization and the order of activation of multiple initiation sites. Finally, these results confirm that initiation sites are extremely redundant elements of the EBV genome. We propose that these conclusions also apply to mammalian chromosomes.

  7. DnaD Protein of Bacillus subtilis Interacts with DnaA, the Initiator Protein of Replication

    Ishigo-oka, Daisuke; Ogasawara, Naotake; Moriya, Shigeki

    2001-01-01

    The yeast two-hybrid assay revealed that Bacillus subtilis DnaD, a possible component of the primosome and required for replication initiation, interacted with DnaA and DnaD itself. The mutant DnaD23 was incapable of interacting with DnaA but retained interaction with the wild-type DnaD. These results suggest that interaction between DnaD and DnaA is important for replication initiation.

  8. Effects of DNA replication on mRNA noise.

    Peterson, Joseph R; Cole, John A; Fei, Jingyi; Ha, Taekjip; Luthey-Schulten, Zaida A

    2015-12-29

    There are several sources of fluctuations in gene expression. Here we study the effects of time-dependent DNA replication, itself a tightly controlled process, on noise in mRNA levels. Stochastic simulations of constitutive and regulated gene expression are used to analyze the time-averaged mean and variation in each case. The simulations demonstrate that to capture mRNA distributions correctly, chromosome replication must be realistically modeled. Slow relaxation of mRNA from the low copy number steady state before gene replication to the high steady state after replication is set by the transcript's half-life and contributes significantly to the shape of the mRNA distribution. Consequently both the intrinsic kinetics and the gene location play an important role in accounting for the mRNA average and variance. Exact analytic expressions for moments of the mRNA distributions that depend on the DNA copy number, gene location, cell doubling time, and the rates of transcription and degradation are derived for the case of constitutive expression and subsequently extended to provide approximate corrections for regulated expression and RNA polymerase variability. Comparisons of the simulated models and analytical expressions to experimentally measured mRNA distributions show that they better capture the physics of the system than previous theories. PMID:26669443

  9. PARP-1 ensures regulation of replication fork progression by homologous recombination on damaged DNA

    Sugimura, Kazuto; Takebayashi, Shin-ichiro; Taguchi, Hiroshi; Takeda, Shunichi; Okumura, Katsuzumi

    2008-01-01

    Poly-ADP ribose polymerase 1 (PARP-1) is activated by DNA damage and has been implicated in the repair of single-strand breaks (SSBs). Involvement of PARP-1 in other DNA damage responses remains controversial. In this study, we show that PARP-1 is required for replication fork slowing on damaged DNA. Fork progression in PARP-1 −/− DT40 cells is not slowed down even in the presence of DNA damage induced by the topoisomerase I inhibitor camptothecin (CPT). Mammalian cells treated with a PARP in...

  10. Synthetic peptide inhibitors of DNA replication in Staphylococcus aureus

    Løbner-Olesen, Anders; Kjelstrup, Susanne

    of clinically important pathogens and is essential for bacterial proliferation. The bacterial replication apparatus fulfill the requirements for a good drug target. The replisome of S. aureus consists of 5 different subunits (2, PolC2, 4, δ and δ`) who’s organization depends on multiple protein......-protein interactions. Centrally in the replisome is the -clamp where to multiple proteins binds through a conserved motif. We have identified the protein-protein interactions in the replisome of S. aureus by use of a bacterial two-hybrid system. A reverse bacterial two-hybrid system (R-BTH) based on Pyr......N (), DnaB and DnaX (). Three peptides identified as inhibitors of DnaN have been purified. Two of these peptides inhibited growth as well as DNA replication in S. aureus. The minimal inhibitory concentration (MIC) of the peptides was approximately 50 g/ml. Overexpression of DnaN reduced the inhibitory...

  11. Complete in vitro DNA replication of SV40 chromatin in digitonin-treated permeable cells.

    Oda,Takuzo; Watanabe,Sekiko; Hanakawa,Shiro; Nakamura, Takashi

    1980-01-01

    A permeable cell system has been developed by treatment with digitonin for studying in vitro DNA replication of chromatin. DNA replication of simian virus 40 nucleoprotein complexes (SV40 chromatin) in digitonin-treated permeable cells was analyzed by electrophoresis in agarose-gel. Autoradiography of the agarose-gel revealed that [32P]dCTP was incorporated in SV40 DNA I, II and replicating intermediates. The time course of the incorporation indicated the complete replication of SV40 DNA and ...

  12. The Escherichia coli Tus-Ter replication fork barrier causes site-specific DNA replication perturbation in yeast

    Larsen, Nicolai B; Sass, Ehud; Suski, Catherine;

    2014-01-01

    Replication fork (RF) pausing occurs at both 'programmed' sites and non-physiological barriers (for example, DNA adducts). Programmed RF pausing is required for site-specific DNA replication termination in Escherichia coli, and this process requires the binding of the polar terminator protein, Tus...... utilized as a versatile, site-specific, heterologous DNA replication-perturbing system, with a variety of potential applications......., to specific DNA sequences called Ter. Here, we demonstrate that Tus-Ter modules also induce polar RF pausing when engineered into the Saccharomyces cerevisiae genome. This heterologous RF barrier is distinct from a number of previously characterized, protein-mediated, RF pause sites in yeast, as it...

  13. On the Specificity of Interaction between the Saccharomyces cerevisiae Clamp Loader Replication Factor C and Primed DNA Templates during DNA Replication*

    Hingorani, Manju M.; Coman, Maria Magdalena

    2002-01-01

    Replication factor C (RFC) catalyzes assembly of circular proliferating cell nuclear antigen clamps around primed DNA, enabling processive synthesis by DNA polymerase during DNA replication and repair. In order to perform this function efficiently, RFC must rapidly recognize primed DNA as the substrate for clamp assembly, particularly during lagging strand synthesis. Earlier reports as well as quantitative DNA binding experiments from this study indicate, however, that RFC interacts with prim...

  14. A new structural framework for integrating replication protein A into DNA processing machinery

    Brosey, Chris; Yan, Chunli; Tsutakawa, Susan; Heller, William; Rambo, Robert; Tainer, John; Ivanov, Ivaylo; Chazin, Walter

    2013-01-17

    By coupling the protection and organization of single-stranded DNA (ssDNA) with recruitment and alignment of DNA processing factors, replication protein A (RPA) lies at the heart of dynamic multi-protein DNA processing machinery. Nevertheless, how RPA coordinates biochemical functions of its eight domains remains unknown. We examined the structural biochemistry of RPA's DNA-binding activity, combining small-angle X-ray and neutron scattering with all-atom molecular dynamics simulations to investigate the architecture of RPA's DNA-binding core. The scattering data reveal compaction promoted by DNA binding; DNA-free RPA exists in an ensemble of states with inter-domain mobility and becomes progressively more condensed and less dynamic on binding ssDNA. Our results contrast with previous models proposing RPA initially binds ssDNA in a condensed state and becomes more extended as it fully engages the substrate. Moreover, the consensus view that RPA engages ssDNA in initial, intermediate and final stages conflicts with our data revealing that RPA undergoes two (not three) transitions as it binds ssDNA with no evidence for a discrete intermediate state. These results form a framework for understanding how RPA integrates the ssDNA substrate into DNA processing machinery, provides substrate access to its binding partners and promotes the progression and selection of DNA processing pathways.

  15. Cellular DNA repair cofactors affecting hepatitis B virus infection and replication

    Fan Zhao; Ning-Bo Hou; Ting Song; Xiang He; Zi-Rui Zheng; Qing-Jun Ma; Li Li; Yan-Hong Zhang; Hui Zhong

    2008-01-01

    AIM: To investigate whether hepatitis B virus (HBV)infection activates DNA damage response and DNA repair cofactors inhibit HBV infection and replication.METHODS: Human hepatocyte cell line HL7702 was studied. Immunoblotting was performed to test the expression of ataxia telangiectasia-mutated (ATM)-Rad3-related protein (ATR), p21 and the level of phosphorylation of Chk1, p53, H2AX, ATM in HBV-infected or non-infected-cells. Special short RNAi oligos was transfected to induce transient ATR knockdown in HL7702. ATR-ATM chemical inhibitors caffeine (CF) and theophylline (TP), or Chk1 inhibitor 7-hydroxystaurosporine (UCN01) was studied to determine whether they suppress cellular DNA damage response and MG132 inhibits proteasome.RESULTS: The ATR checkpoint pathway, responding to single-strand breaks in DNA, was activated in response to HBV infection. ATR knockdown cells decreased the HBV DNA yields, implying that HBV infection and replication could activate and exploit the activated DNA damage response. CF/TP or UCN01 reduced the HBV DNA yield by 70% and 80%, respectively. HBV abrogated the ATR-dependent DNA damage signaling pathway by degrading p21, and introduction of the p21 protein before HBV infection reduced the HBV DNA yield. Consistent with this result, p21 accumulation after MG132 treatment also sharply decreased the HBV DNA yield.CONCLUSION: HBV infection can be treated with therapeutic approaches targeting host cell proteins by inhibiting a cellular gene required for HBV replication or by restoring a response abrogated by HBV, thus providing a potential approach to the prevention and treatrnent of HBV infection.

  16. Functional redundancy between DNA ligases I and III in DNA replication in vertebrate cells

    Arakawa, Hiroshi; Bednar, Theresa; Wang, Minli; Paul, Katja; Mladenov, Emil; Bencsik-Theilen, Alena A.; Iliakis, George

    2011-01-01

    In eukaryotes, the three families of ATP-dependent DNA ligases are associated with specific functions in DNA metabolism. DNA ligase I (LigI) catalyzes Okazaki-fragment ligation at the replication fork and nucleotide excision repair (NER). DNA ligase IV (LigIV) mediates repair of DNA double strand breaks (DSB) via the canonical non-homologous end-joining (NHEJ) pathway. The evolutionary younger DNA ligase III (LigIII) is restricted to higher eukaryotes and has been associated with base excisio...

  17. Sequence-nonspecific replication of transfected plasmid DNA in poxvirus-infected cells.

    DeLange, A. M.; McFadden, G

    1986-01-01

    A system in which transfected plasmid DNA replicates in the cytoplasm of poxvirus-infected cells is described. A variety of recombinant plasmids was introduced into poxvirus-infected cells by transfection, and replication of input plasmid DNA was monitored by (i) digestion with restriction enzymes that discriminate between input methylated plasmid DNA and unmethylated DNA produced by replication in mammalian cells; (ii) amplification of intracellular plasmid DNA; and (iii) density shift analy...

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

    Federico Comoglio

    2015-05-01

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

  19. Ancient diversification of eukaryotic MCM DNA replication proteins

    Aves Stephen J

    2009-03-01

    Full Text Available Abstract Background Yeast and animal cells require six mini-chromosome maintenance proteins (Mcm2-7 for pre-replication complex formation, DNA replication initiation and DNA synthesis. These six individual MCM proteins form distinct heterogeneous subunits within a hexamer which is believed to form the replicative helicase and which associates with the essential but non-homologous Mcm10 protein during DNA replication. In contrast Archaea generally only possess one MCM homologue which forms a homohexameric MCM helicase. In some eukaryotes Mcm8 and Mcm9 paralogues also appear to be involved in DNA replication although their exact roles are unclear. Results We used comparative genomics and phylogenetics to reconstruct the diversification of the eukaryotic Mcm2-9 gene family, demonstrating that Mcm2-9 were formed by seven gene duplication events before the last common ancestor of the eukaryotes. Mcm2-7 protein paralogues were present in all eukaryote genomes studied suggesting that no gene loss or functional replacements have been tolerated during the evolutionary diversification of eukaryotes. Mcm8 and 9 are widely distributed in eukaryotes and group together on the MCM phylogenetic tree to the exclusion of all other MCM paralogues suggesting co-ancestry. Mcm8 and Mcm9 are absent in some taxa, including Trichomonas and Giardia, and appear to have been secondarily lost in some fungi and some animals. The presence and absence of Mcm8 and 9 is concordant in all taxa sampled with the exception of Drosophila species. Mcm10 is present in most eukaryotes sampled but shows no concordant pattern of presence or absence with Mcm8 or 9. Conclusion A multifaceted and heterogeneous Mcm2-7 hexamer evolved during the early evolution of the eukaryote cell in parallel with numerous other acquisitions in cell complexity and prior to the diversification of extant eukaryotes. The conservation of all six paralogues throughout the eukaryotes suggests that each Mcm2

  20. AcMNPV As A Model for Baculovirus DNA Replication

    Eric B. Carstens

    2009-01-01

    Baculoviruses were first identified as insect-specific pathogens, and it was this specificity that lead to their use as safe, target specific biological pesticides. For the past 30 years, AcMNPV has served as the subject of intense basic molecular research into the baculovirus infectious cycle including the interaction of the virus with a continuous insect cell line derived from Spodoptera frugiperda. The studies on baculoviruese have led to an in-depth understanding of the physical organization of the viral genomes including many complete genomic sequences, the time course of gene expression, and the application of this basic research to the use of baculoviruses not only as insecticides, but also as a universal eukaryotic protein expression system, and a potential vector in gene therapy. A great deal has also been discovered about the viral genes required for the replication of the baculovirus genome, while much remains to be learned about the mechanism of viral DNA replication. This report outlines the current knowledge of the factors involved in baculovirus DNA replication, using data on AcMNPV as a model for most members of the Baculoviridae.

  1. Inferring the Spatiotemporal DNA Replication Program from Noisy Biological Data

    Bechhoefer, John; Baker, Antoine

    2014-03-01

    We generalize a stochastic model of DNA replication to the case where replication-origin-initiation rates vary locally along the genome and with time. Using this generalized model, we address the inverse problem of inferring initiation rates from experimental data concerning replication in cell populations. Previous work based on curve fitting depended on arbitrarily chosen functional forms for the initiation rate, with free parameters that were constrained by the data. We introduce a model-free, non-parametric method of inference that is based on Gaussian process regression. The method replaces specific assumptions about the functional form of initiation rate with more general prior expectations about the smoothness of variation of this rate, along the genome and in time. Using this inference method, we show that we can recover with high precision simulated replication schemes with data that are typical of current experiments. The method of Gaussian process regression can be profitably applied to a wide range of physical and biological problems. Supported by NSERC (Canada).

  2. The logic of DNA replication in double-stranded DNA viruses: insights from global analysis of viral genomes.

    Kazlauskas, Darius; Krupovic, Mart; Venclovas, Česlovas

    2016-06-01

    Genomic DNA replication is a complex process that involves multiple proteins. Cellular DNA replication systems are broadly classified into only two types, bacterial and archaeo-eukaryotic. In contrast, double-stranded (ds) DNA viruses feature a much broader diversity of DNA replication machineries. Viruses differ greatly in both completeness and composition of their sets of DNA replication proteins. In this study, we explored whether there are common patterns underlying this extreme diversity. We identified and analyzed all major functional groups of DNA replication proteins in all available proteomes of dsDNA viruses. Our results show that some proteins are common to viruses infecting all domains of life and likely represent components of the ancestral core set. These include B-family polymerases, SF3 helicases, archaeo-eukaryotic primases, clamps and clamp loaders of the archaeo-eukaryotic type, RNase H and ATP-dependent DNA ligases. We also discovered a clear correlation between genome size and self-sufficiency of viral DNA replication, the unanticipated dominance of replicative helicases and pervasive functional associations among certain groups of DNA replication proteins. Altogether, our results provide a comprehensive view on the diversity and evolution of replication systems in the DNA virome and uncover fundamental principles underlying the orchestration of viral DNA replication. PMID:27112572

  3. Ancient mtDNA Genetic Variants Modulate mtDNA Transcription and Replication

    Suissa, Sarit; Wang, Zhibo; Poole, Jason; Wittkopp, Sharine; Feder, Jeanette; Shutt, Timothy E.; Wallace, Douglas C.; Shadel, Gerald S.; Mishmar, Dan

    2009-01-01

    Although the functional consequences of mitochondrial DNA ( mtDNA) genetic backgrounds (haplotypes, haplogroups) have been demonstrated by both disease association studies and cell culture experiments, it is not clear which of the mutations within the haplogroup carry functional implications and which are "evolutionary silent hitchhikers''. We set forth to study the functionality of haplogroup-defining mutations within the mtDNA transcription/replication regulatory region by in vitro transcri...

  4. Ancient mtDNA genetic variants modulate mtDNA transcription and replication.

    Sarit Suissa; Zhibo Wang; Jason Poole; Sharine Wittkopp; Jeanette Feder; Shutt, Timothy E.; Wallace, Douglas C.; Shadel, Gerald S.; Dan Mishmar

    2009-01-01

    Although the functional consequences of mitochondrial DNA ( mtDNA) genetic backgrounds (haplotypes, haplogroups) have been demonstrated by both disease association studies and cell culture experiments, it is not clear which of the mutations within the haplogroup carry functional implications and which are "evolutionary silent hitchhikers''. We set forth to study the functionality of haplogroup-defining mutations within the mtDNA transcription/replication regulatory region by in vitro transcri...

  5. Plasmid DNA replication and topology as visualized by two-dimensional agarose gel electrophoresis

    Schvartzman, Jorge Bernardo; Martínez-Robles, María Luisa; Krimer, Dora B.

    2010-01-01

    During the last 20 years, two-dimensional agarose gel electrophoresis combined with other techniques such as Polymerase Chain Reaction, helicase assay and electron microscopy, helped to characterize plasmid DNA replication and topology. Here we describe some of the most important findings that were made using this method including the characterization of uni-directional replication, replication origin interference, DNA breakage at the forks, replication fork blockage, replication knotting, re...

  6. The Replication Focus Targeting Sequence (RFTS) Domain Is a DNA-competitive Inhibitor of Dnmt1

    Syeda, Farisa; Fagan, Rebecca L.; Wean, Matthew; Avvakumov, George V.; Walker, John R.; Xue, Sheng; Dhe-Paganon, Sirano; Brenner, Charles (Iowa); (Toronto)

    2015-11-30

    Dnmt1 (DNA methyltransferase 1) is the principal enzyme responsible for maintenance of cytosine methylation at CpG dinucleotides in the mammalian genome. The N-terminal replication focus targeting sequence (RFTS) domain of Dnmt1 has been implicated in subcellular localization, protein association, and catalytic function. However, progress in understanding its function has been limited by the lack of assays for and a structure of this domain. Here, we show that the naked DNA- and polynucleosome-binding activities of Dnmt1 are inhibited by the RFTS domain, which functions by virtue of binding the catalytic domain to the exclusion of DNA. Kinetic analysis with a fluorogenic DNA substrate established the RFTS domain as a 600-fold inhibitor of Dnmt1 enzymatic activity. The crystal structure of the RFTS domain reveals a novel fold and supports a mechanism in which an RFTS-targeted Dnmt1-binding protein, such as Uhrf1, may activate Dnmt1 for DNA binding.

  7. Replication fork progression is paused in two large chromosomal zones flanking the DNA replication origin in Escherichia coli.

    Akiyama, Masahiro Tatsumi; Oshima, Taku; Chumsakul, Onuma; Ishikawa, Shu; Maki, Hisaji

    2016-08-01

    Although the speed of nascent DNA synthesis at individual replication forks is relatively uniform in bacterial cells, the dynamics of replication fork progression on the chromosome are hampered by a variety of natural impediments. Genome replication dynamics can be directly measured from an exponentially growing cell population by sequencing newly synthesized DNA strands that were specifically pulse-labeled with the thymidine analogue 5-bromo-2'-deoxyuridine (BrdU). However, a short pulse labeling with BrdU is impracticable for bacteria because of poor incorporation of BrdU into the cells, and thus, the genomewide dynamics of bacterial DNA replication remain undetermined. Using a new thymidine-requiring Escherichia coli strain, eCOMB, and high-throughput sequencing, we succeeded in determining the genomewide replication profile in bacterial cells. We also found that fork progression is paused in two ~200-kb chromosomal zones that flank the replication origin in the growing cells. This origin-proximal obstruction to fork progression was overcome by an increased thymidine concentration in the culture medium and enhanced by inhibition of transcription. These indicate that DNA replication near the origin is sensitive to the impediments to fork progression, namely a scarcity of the DNA precursor deoxythymidine triphosphate and probable conflicts between replication and transcription machineries. PMID:27353572

  8. Mismatch repair causes the dynamic release of an essential DNA polymerase from the replication fork

    Klocko, Andrew D.; Schroeder, Jeremy W.; Walsh, Brian W.; Lenhart, Justin S.; Evans, Margery L.; Simmons, Lyle A.

    2011-01-01

    Mismatch repair (MMR) corrects DNA polymerase errors occurring during genome replication. MMR is critical for genome maintenance, and its loss increases mutation rates several hundredfold. Recent work has shown that the interaction between the mismatch recognition protein MutS and the replication processivity clamp is important for MMR in Bacillus subtilis. To further understand how MMR is coupled to DNA replication, we examined the subcellular localization of MMR and DNA replication proteins...

  9. Adenovirus DNA replication in vitro is stimulated by RNA from uninfected HeLa cells

    Vliet, P.C. van der; Dam, D. van; Kwant, M.M.

    1984-01-01

    Adenovirus DNA replication was studied in a partially reconstituted system consisting of purified viral proteins (DNA-binding protein, precursor terminal protein and Ad DNA polymerase) and a nuclear extract from uninfected HeLa cells. Optimal DNA replication required the presence of a heat-stable, r

  10. THE TELOMERES OF REPLICATING MACRONUCLEAR DNA-MOLECULES OF THE HYPOTRICHOUS CILIATE STYLONYCHIA LEMNAE

    Juranek, Stefan; Jonsson, Franziska; Maercker, Christian; Lipps, Hans

    2000-01-01

    While the telomeres of higher eukaryotic cells show heterogenity in their length no such length heterogenity is observed in the macronuclear DNA of hypotrichous ciliates. Since the analysis of the telomere structure of replicating DNA molecules might give an insight in the mechanisms of telomere length regulation Stylonychia cell cultures were synchronized, the replicating DNA was labeled with BrdU and isolated by immuno-precipitation. The 3as well as the 5-end of these replicating DNA was la...

  11. Structures of minute virus of mice replication initiator protein N-terminal domain: Insights into DNA nicking and origin binding

    Tewary, Sunil K.; Liang, Lingfei; Lin, Zihan; Lynn, Annie [Department of Molecular Biosciences, University of Kansas, Lawrence, KS 66045 (United States); Cotmore, Susan F. [Departments of Laboratory Medicine, Yale University Medical School, New Haven, CT 06510 (United States); Tattersall, Peter [Departments of Laboratory Medicine, Yale University Medical School, New Haven, CT 06510 (United States); Departments of Genetics, Yale University Medical School, New Haven, CT 06510 (United States); Zhao, Haiyan, E-mail: zhaohy@ku.edu [Department of Molecular Biosciences, University of Kansas, Lawrence, KS 66045 (United States); Tang, Liang, E-mail: tangl@ku.edu [Department of Molecular Biosciences, University of Kansas, Lawrence, KS 66045 (United States)

    2015-02-15

    Members of the Parvoviridae family all encode a non-structural protein 1 (NS1) that directs replication of single-stranded viral DNA, packages viral DNA into capsid, and serves as a potent transcriptional activator. Here we report the X-ray structure of the minute virus of mice (MVM) NS1 N-terminal domain at 1.45 Å resolution, showing that sites for dsDNA binding, ssDNA binding and cleavage, nuclear localization, and other functions are integrated on a canonical fold of the histidine-hydrophobic-histidine superfamily of nucleases, including elements specific for this Protoparvovirus but distinct from its Bocaparvovirus or Dependoparvovirus orthologs. High resolution structural analysis reveals a nickase active site with an architecture that allows highly versatile metal ligand binding. The structures support a unified mechanism of replication origin recognition for homotelomeric and heterotelomeric parvoviruses, mediated by a basic-residue-rich hairpin and an adjacent helix in the initiator proteins and by tandem tetranucleotide motifs in the replication origins. - Highlights: • The structure of a parvovirus replication initiator protein has been determined; • The structure sheds light on mechanisms of ssDNA binding and cleavage; • The nickase active site is preconfigured for versatile metal ligand binding; • The binding site for the double-stranded replication origin DNA is identified; • A single domain integrates multiple functions in virus replication.

  12. Structures of minute virus of mice replication initiator protein N-terminal domain: Insights into DNA nicking and origin binding

    Members of the Parvoviridae family all encode a non-structural protein 1 (NS1) that directs replication of single-stranded viral DNA, packages viral DNA into capsid, and serves as a potent transcriptional activator. Here we report the X-ray structure of the minute virus of mice (MVM) NS1 N-terminal domain at 1.45 Å resolution, showing that sites for dsDNA binding, ssDNA binding and cleavage, nuclear localization, and other functions are integrated on a canonical fold of the histidine-hydrophobic-histidine superfamily of nucleases, including elements specific for this Protoparvovirus but distinct from its Bocaparvovirus or Dependoparvovirus orthologs. High resolution structural analysis reveals a nickase active site with an architecture that allows highly versatile metal ligand binding. The structures support a unified mechanism of replication origin recognition for homotelomeric and heterotelomeric parvoviruses, mediated by a basic-residue-rich hairpin and an adjacent helix in the initiator proteins and by tandem tetranucleotide motifs in the replication origins. - Highlights: • The structure of a parvovirus replication initiator protein has been determined; • The structure sheds light on mechanisms of ssDNA binding and cleavage; • The nickase active site is preconfigured for versatile metal ligand binding; • The binding site for the double-stranded replication origin DNA is identified; • A single domain integrates multiple functions in virus replication

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

    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 damag

  14. Human Replication Protein A Melts a DNA Triple Helix Structure in a Potent and Specific Manner†

    Wu, Yuliang; Rawtani, Nina; Thazhathveetil, Arun Kalliat; Kenny, Mark K.; Seidman, Michael M.; Brosh, Robert M.

    2008-01-01

    Alternate DNA structures other than double-stranded B-form DNA can potentially impede cellular processes such as transcription and replication. The DNA triplex helix and G4 tetraplex structures that form by Hoogsteen hydrogen bonding are two examples of alternate DNA structures that can be a source of genomic instability. In this study, we have examined the ability of human replication protein A (RPA), a single-stranded DNA binding protein that is implicated in all facets of DNA metabolism, t...

  15. Efficient replication bypass of size-expanded DNA base pairs in bacterial cells

    Delaney, James C.; GAO, JIANMIN; Liu, Haibo; Shrivastav, Nidhi; Essigmann, John M.; Kool, Eric T.

    2009-01-01

    Supersize me! Size-expanded DNA bases (xDNA) are able to encode natural DNA sequences in replication. In vitro experiments with a DNA polymerase show nucleotide incorporation opposite the xDNA bases with correct pairing. In vivo experiments using E. coli show that two xDNA bases (xA and xC, see picture) encode the correct replication partners.

  16. Analysis of the DNA replication competence of the xrs-5 mutant cells defective in Ku86.

    Matheos, Diamanto; Novac, Olivia; Price, Gerald B; Zannis-Hadjopoulos, Maria

    2003-01-01

    The radiosensitive mutant xrs-5, a derivative of the Chinese hamster ovary (CHO) K1 cell line, is defective in DNA double-strand break repair and V(D)J recombination. The defective phenotypes of xrs-5 cells are complemented by the 86 kDa subunit of Ku antigen. OBA is a protein, previously purified from HeLa cells, that binds in a sequence-specific manner to mammalian origins of DNA replication. The DNA-binding subunit of OBA has been identified as Ku86. We tested the xrs-5 cell line for its ability to replicate a mammalian origin-containing plasmid, p186, in vivo and in vitro. In vivo, the p186 episomal DNA replication in transfected xrs-5 cells was reduced by 45% when compared with the CHO K1 cells transfected with p186. In vitro, although total and cytoplasmic cell extracts from xrs-5 cells replicated the p186 with the same efficiency as the parental CHO K1 cell extracts, xrs-5 nuclear extracts did not possess any detectable replication activity. Addition of affinity-purified OBA/Ku restored replication in the xrs-5 nuclear extract reaction. Western blot analyses showed that the levels of other replication proteins (Orc2, PCNA, DNA polymerase epsilon and delta, Primase and Topoisomerase IIalpha) were comparable in both the xrs-5 mutant and CHO K1 wild-type cell lines. In addition, the in vivo association of Ku with the DHFR origin-containing sequence (oribeta) was examined in both the CHO K1 and xrs-5 cell lines by a chromatin immunoprecipitation (ChIP) assay. Anti-Ku antibodies did not immunoprecipitate a detectable amount of Ku from the xrs-5 cells in the origin-containing sequence, in contrast to the CHO K1 cells, wherein Ku was found to be associated with the oribeta origin. The data implicate Ku antigen in in vivo and in vitro DNA replication and suggest the existence of another protein with Ku-like functions in the xrs-5 cells. PMID:12456721

  17. Separate Roles of Escherichia coli Replication Proteins in Synthesis and Partitioning of pSC101 Plasmid DNA

    Miller, Christine; Cohen, Stanley N.

    1999-01-01

    We report here that the Escherichia coli replication proteins DnaA, which is required to initiate replication of both the chromosome and plasmid pSC101, and DnaB, the helicase that unwinds strands during DNA replication, have effects on plasmid partitioning that are distinct from their functions in promoting plasmid DNA replication. Temperature-sensitive dnaB mutants cultured under conditions permissive for DNA replication failed to partition plasmids normally, and when cultured under conditi...

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

    Feng, Yunpeng; Vlassis, Arsenios; Roques, Céline;

    2016-01-01

    During DNA replication, thousands of replication origins are activated across the genome. Chromatin architecture contributes to origin specification and usage, yet it remains unclear which chromatin features impact on DNA replication. Here, we perform a RNAi screen for chromatin regulators......45 recruitment, but not MCM2-7 loading, is impaired in BRPF3-depleted cells, identifying a BRPF3-dependent function of HBO1 in origin activation that is complementary to its role in licencing. We thus propose that BRPF3-HBO1 acetylation of histone H3K14 around TSS facilitates efficient activation of...

  19. A role for the weak DnaA binding sites in bacterial replication origins

    Charbon, Godefroid; Løbner-Olesen, Anders

    2011-01-01

    DnaA initiates the chromosomal DNA replication in nearly all bacteria, and replication origins are characterized by binding sites for the DnaA protein (DnaA-boxes) along with an ‘AT-rich’ region. However, great variation in number, spatial organization and specificity of DnaA-boxes is observed...... between species. In the study by Taylor et al. (2011), new and unexpectedly weak DnaA-boxes were identified within the Caulobacter crescentus origin of replication (Cori). The position of weak and stronger DnaA-boxes follows a pattern seen in Escherichia coli oriC. This raises the possibility that...

  20. DNA lesions induced by replication stress trigger mitotic aberration and tetraploidy development.

    Yosuke Ichijima

    Full Text Available During tumorigenesis, cells acquire immortality in association with the development of genomic instability. However, it is still elusive how genomic instability spontaneously generates during the process of tumorigenesis. Here, we show that precancerous DNA lesions induced by oncogene acceleration, which induce situations identical to the initial stages of cancer development, trigger tetraploidy/aneuploidy generation in association with mitotic aberration. Although oncogene acceleration primarily induces DNA replication stress and the resulting lesions in the S phase, these lesions are carried over into the M phase and cause cytokinesis failure and genomic instability. Unlike directly induced DNA double-strand breaks, DNA replication stress-associated lesions are cryptogenic and pass through cell-cycle checkpoints due to limited and ineffective activation of checkpoint factors. Furthermore, since damaged M-phase cells still progress in mitotic steps, these cells result in chromosomal mis-segregation, cytokinesis failure and the resulting tetraploidy generation. Thus, our results reveal a process of genomic instability generation triggered by precancerous DNA replication stress.

  1. DNA replication in ultraviolet light irradiated Chinese hamster cells: the nature of replicon inhibition and post-replication repair

    DNA replication in ultraviolet light irradiated Chinese hamster cells was studied using techniques of DNA fiber autoradiography and alkaline sucrose sedimentation. Bidirectionally growing replicons were observed in the autoradiograms independent of the irradiation conditions. After a dose of 5 J/m2 at 254 nm the rate of fork progression was the same as in unirradiated cells, while the rate of replication was reduced by 50%. After a dose of 10J/m2 the rate of fork progression was reduced 40%, while the replication rate was only 25% of normal. Therefore, at low doses of ultraviolet light irradiation, the inhibition of DNA replication is due to reduction in the number of functioning replicons, while at higher doses the rate of fork progression is also slowed. Those replicons which no longer function after irradiation are blocked in fork movement rather than replicon initiation. After irradiation, pulse label was first incorporated into short nascent strands, the average size of which was approximately equal to the distance between pyrimidine dimers. Under conditions where post-replication repair occurs these short strands were eventually joined into larger pieces. Finally, the data show that slowing post-replication repair with caffeine does not slow fork movement. The results presented here support the post-replication repair model of 'gapped synthesis' and rule out a major role for 'replicative bypass'. (author)

  2. Geminin deploys multiple mechanisms to regulate Cdt1 before cell division thus ensuring the proper execution of DNA replication

    Ballabeni, Andrea; Zamponi, Raffaella; Moore, Jodene K;

    2013-01-01

    accumulation of Cdt1 in mitosis, because decreasing the Geminin levels prevents Cdt1 accumulation and impairs DNA replication. Geminin is known to inhibit Cdt1 function; its depletion during G2 leads to DNA rereplication and checkpoint activation. Here we show that, despite rapid Cdt1 protein turnover in G2...

  3. USP7/HAUSP: A SUMO deubiquitinase at the heart of DNA replication.

    Smits, Veronique A J; Freire, Raimundo

    2016-09-01

    DNA replication is both highly conserved and controlled. Problematic DNA replication can lead to genomic instability and therefore carcinogenesis. Numerous mechanisms work together to achieve this tight control and increasing evidence suggests that post-translational modifications (phosphorylation, ubiquitination, SUMOylation) of DNA replication proteins play a pivotal role in this process. Here we discuss such modifications in the light of a recent article that describes a novel role for the deubiquitinase (DUB) USP7/HAUSP in the control of DNA replication. USP7 achieves this function by an unusual and novel mechanism, namely deubiquitination of SUMOylated proteins at the replication fork, making USP7 also a SUMO DUB (SDUB). This work extends previous observations of increased levels of SUMO and low levels of ubiquitin at the on-going replication fork. Here, we discuss this novel study, its contribution to the DNA replication and genomic stability field and what questions arise from this work. PMID:27374980

  4. Singlet Oxygen-Mediated Oxidation during UVA Radiation Alters the Dynamic of Genomic DNA Replication.

    Dany Graindorge

    Full Text Available UVA radiation (320-400 nm is a major environmental agent that can exert its deleterious action on living organisms through absorption of the UVA photons by endogenous or exogenous photosensitizers. This leads to the production of reactive oxygen species (ROS, such as singlet oxygen (1O2 and hydrogen peroxide (H2O2, which in turn can modify reversibly or irreversibly biomolecules, such as lipids, proteins and nucleic acids. We have previously reported that UVA-induced ROS strongly inhibit DNA replication in a dose-dependent manner, but independently of the cell cycle checkpoints activation. Here, we report that the production of 1O2 by UVA radiation leads to a transient inhibition of replication fork velocity, a transient decrease in the dNTP pool, a quickly reversible GSH-dependent oxidation of the RRM1 subunit of ribonucleotide reductase and sustained inhibition of origin firing. The time of recovery post irradiation for each of these events can last from few minutes (reduction of oxidized RRM1 to several hours (replication fork velocity and origin firing. The quenching of 1O2 by sodium azide prevents the delay of DNA replication, the decrease in the dNTP pool and the oxidation of RRM1, while inhibition of Chk1 does not prevent the inhibition of origin firing. Although the molecular mechanism remains elusive, our data demonstrate that the dynamic of replication is altered by UVA photosensitization of vitamins via the production of singlet oxygen.

  5. Replication Stress-Induced Chromosome Breakage Is Correlated with Replication Fork Progression and Is Preceded by Single-Stranded DNA Formation

    Feng, Wenyi; Di Rienzi, Sara C.; Raghuraman, M. K.; Brewer, Bonita J.

    2011-01-01

    Chromosome breakage as a result of replication stress has been hypothesized to be the direct consequence of defective replication fork progression, or “collapsed” replication forks. However, direct and genome-wide evidence that collapsed replication forks give rise to chromosome breakage is still lacking. Previously we showed that a yeast replication checkpoint mutant mec1-1, after transient exposure to replication impediment imposed by hydroxyurea (HU), failed to complete DNA replication, ac...

  6. Removal of uv-induced pyrimidine dimers from the replicated and unreplicated DNA of human fibroblasts

    Excision repair in uv irradiated human fibroblasts has been examined in portions of DNA replicating after irradiation versus those remaining unreplicated. Two approaches, one using a uv-endonuclease to estimate pyrimidine dimers remaining in DNA, the other using density labeling to measure excision resynthesis, indicate that the extent of repair is the same for both replicated and unreplicated DNA

  7. Kinetics of [32P]orthophosphate and [3H]thymidine incorporation into newly replicating DNA in vivo

    Nuclear DNA can be empirically subdivided into three populations: (1) bulk DNA or low salt-soluble DNA (75%), (2) high salt-soluble DNA (23%), and (3) matrix DNA which remains tightly bound to the nuclear matrix (2%). Newly replicating DNA is associated with the nuclear matrix in regenerating rat liver. To study the incorporation of DNA precursors into replicating DNA via the salvage vs the de novo pathway, 100μCi [3H]thymidine (3H-Thd) and 5mCi [32P]orthophosphate (32P/sub i/) were injected into the hepatic portal vein of partially hepatectomized rats. Increasing time of 3H-Thd incorporation showed the label is chased from matrix DNA to bulk DNA. After a 10 min pulse, 13% of the total specific activity is associated with bulk DNA and 57% with matrix DNA. After 30 min, 32% and 36% of the total specific activity remain associated with bulk and matrix DNA, respectively, indicating that most of the 3H has been chased from the matrix DNA. In contrast, after injection of 32P/sub i/, the amount of label in matrix DNA increases to a maximum at 30 min and only then begins to decrease. At 10 min the specific activity/total specific activity of bulk DNA is 7% and of matrix DNA is 66% vs 8% and 82% after 30 min. The kinetic pattern of 32P/sub i/ incorporation differs dramatically from that of 3H-Thd suggesting (a) the incorporation of de novo precursors lags significantly behind that of precursors entering through the salvage pathway, or (b) there may be two distinct classes of replication forks

  8. Cytotoxicity of psammaplin A from a two-sponge association may correlate with the inhibition of DNA replication

    SV40 DNA replication system is a very useful tool to understand the mechanism of replication, which is a tightly regulated process. Many environmental and cellular factors can induce cell cycle arrest or apoptosis by inhibiting DNA replication. In the course of our search for bioactive metabolites from the marine sponges, psammaplin A was found to have some anticancer properties, the possible mechanism of which was studied. Cell viability was determined by Cell Counting Kit-8 (CCK-8) to count living RAW264.7 cells by combining 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl) -2H-tetrazolium (WST-8) and 1-methoxy-phenazine methosulfate (1-methoxy-PMS). The effect of psammaplin A on DNA replication was carried out in SV40 DNA replication system in vitro. The activities of topoisomerase I and polymerase α-primase were measured by the relaxation of superhelical plasmid DNA and the incorporation of [3H]dTTP to the template respectively. The ssDNA binding activity of RPA was assessed by Gel Mobility Shift Assay (GMSA). We have found that psammaplin A delivers significant cytotoxic activity against the RAW264.7 cell line. It was also found that psammaplin A could substantially inhibit SV40 DNA replication in vitro, in which polymerase α-primase is one of its main targets. Taken together, we suggest that psammaplin A-induced cytotoxicity may correlate with its inhibition on DNA replication. Psammaplin A has the potential to be developed as an anticancer drug

  9. Nutritional Control of DNA Replication Initiation through the Proteolysis and Regulated Translation of DnaA.

    Leslie, David J; Heinen, Christian; Schramm, Frederic D; Thüring, Marietta; Aakre, Christopher D; Murray, Sean M; Laub, Michael T; Jonas, Kristina

    2015-07-01

    Bacteria can arrest their own growth and proliferation upon nutrient depletion and under various stressful conditions to ensure their survival. However, the molecular mechanisms responsible for suppressing growth and arresting the cell cycle under such conditions remain incompletely understood. Here, we identify post-transcriptional mechanisms that help enforce a cell-cycle arrest in Caulobacter crescentus following nutrient limitation and during entry into stationary phase by limiting the accumulation of DnaA, the conserved replication initiator protein. DnaA is rapidly degraded by the Lon protease following nutrient limitation. However, the rate of DnaA degradation is not significantly altered by changes in nutrient availability. Instead, we demonstrate that decreased nutrient availability downregulates dnaA translation by a mechanism involving the 5' untranslated leader region of the dnaA transcript; Lon-dependent proteolysis of DnaA then outpaces synthesis, leading to the elimination of DnaA and the arrest of DNA replication. Our results demonstrate how regulated translation and constitutive degradation provide cells a means of precisely and rapidly modulating the concentration of key regulatory proteins in response to environmental inputs. PMID:26134530

  10. Nutritional Control of DNA Replication Initiation through the Proteolysis and Regulated Translation of DnaA.

    David J Leslie

    2015-07-01

    Full Text Available Bacteria can arrest their own growth and proliferation upon nutrient depletion and under various stressful conditions to ensure their survival. However, the molecular mechanisms responsible for suppressing growth and arresting the cell cycle under such conditions remain incompletely understood. Here, we identify post-transcriptional mechanisms that help enforce a cell-cycle arrest in Caulobacter crescentus following nutrient limitation and during entry into stationary phase by limiting the accumulation of DnaA, the conserved replication initiator protein. DnaA is rapidly degraded by the Lon protease following nutrient limitation. However, the rate of DnaA degradation is not significantly altered by changes in nutrient availability. Instead, we demonstrate that decreased nutrient availability downregulates dnaA translation by a mechanism involving the 5' untranslated leader region of the dnaA transcript; Lon-dependent proteolysis of DnaA then outpaces synthesis, leading to the elimination of DnaA and the arrest of DNA replication. Our results demonstrate how regulated translation and constitutive degradation provide cells a means of precisely and rapidly modulating the concentration of key regulatory proteins in response to environmental inputs.

  11. DNA Structure Specificity Conferred on a Replicative Helicase by Its Loader*

    Gupta, Milind K.; Atkinson, John; McGlynn, Peter

    2009-01-01

    Prokaryotic and eukaryotic replicative helicases can translocate along single-stranded and double-stranded DNA, with the central cavity of these multimeric ring helicases being able to accommodate both forms of DNA. Translocation by such helicases along single-stranded DNA results in the unwinding of forked DNA by steric exclusion and appears critical in unwinding of parental strands at the replication fork, whereas translocation over double-stranded DNA has no well-defined role. We have foun...

  12. Separation of the transcriptional activation and replication functions of the bovine papillomavirus-1 E2 protein.

    Winokur, P L; McBride, A A

    1992-11-01

    Replication of bovine papillomavirus-1 (BPV-1) DNA requires two viral gene products, the E1 protein and the full-length E2 protein. The 48 kDa E2 protein is a site-specific DNA-binding protein that binds to several sites which lie adjacent to the BPV-1 origin of replication. The 85 amino acid C-terminal domain contains the specific DNA binding and dimerization properties of the protein. The approximately 200 amino acid N-terminal domain is crucial for transcriptional activation. Both of these domains are highly conserved among different papillomaviruses. An internal hinge region separates the two functional domains. The region varies in amino acid sequence and length among the E2 proteins of different papillomaviruses. A series of mutations were constructed within the E2 open reading frame which delete various regions of the conserved DNA binding and transactivation domains as well as the internal hinge region. Two mutated E2 proteins that lack portions of the conserved DNA-binding domain but which support DNA replication were identified using transient replication assays. These mutated E2 proteins were unable to function as transcriptional activators. Conversely, two E2 proteins containing large deletions of the hinge region were able to activate transcription, but were defective for replication. Thus, the replication and transactivation functions of the E2 protein are separable. PMID:1327758

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

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

    2014-11-15

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

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

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

  15. The participation of the Fanconi anemia pathway in the replication of UV-damage DNA

    When cells are challenged with genotoxic agents, replicating cells must use damaged DNA as templates. In this way, active replication forks do not collapse and cell viability is protected. After UV irradiation a specialized DNA polymerase pol eta uses UV damaged DNA as template. Intriguingly, Pol eta lost in human cells does not steeply increase UV sensitivity. This suggests that compensatory mechanisms promote cell survival when pol eta is absent. We have found an increase and sustained FANCD2 ubiquitination and focal formation after UV irradiation when pol eta is lost. FANCD2 is a key marker of the activation of the FANCONI ANEMIA (FA) pathway. While there is limited information regarding a role of the FA pathway after UV irradiation, it is well established that FANCD2 ubiquitination is linked to the recruitment of homologous recombination (HR) specific markers to other lesions. We therefore thought that cell viability in the absence of pol eta might result from the activation of FANDC2-dependent HR at collapsed replication forks. We are currently analyzing markers of damage such as γH2AX phosphorylation, markers of HR such as Rad51, markers of double strand breaks accumulation such as 53BP1 and setting up viability assays. This information might allow us to predict if FANCD2 can trigger HR after UV and if this contributes to cell viability when pol eta is absent. (authors)

  16. Structure and Function of the PriC DNA Replication Restart Protein.

    Wessel, Sarah R; Cornilescu, Claudia C; Cornilescu, Gabriel; Metz, Alice; Leroux, Maxime; Hu, Kaifeng; Sandler, Steven J; Markley, John L; Keck, James L

    2016-08-26

    Collisions between DNA replication complexes (replisomes) and barriers such as damaged DNA or tightly bound protein complexes can dissociate replisomes from chromosomes prematurely. Replisomes must be reloaded under these circumstances to avoid incomplete replication and cell death. Bacteria have evolved multiple pathways that initiate DNA replication restart by recognizing and remodeling abandoned replication forks and reloading the replicative helicase. In vitro, the simplest of these pathways is mediated by the single-domain PriC protein, which, along with the DnaC helicase loader, can load the DnaB replicative helicase onto DNA bound by the single-stranded DNA (ssDNA)-binding protein (SSB). Previous biochemical studies have identified PriC residues that mediate interactions with ssDNA and SSB. However, the mechanisms by which PriC drives DNA replication restart have remained poorly defined due to the limited structural information available for PriC. Here, we report the NMR structure of full-length PriC from Cronobacter sakazakii PriC forms a compact bundle of α-helices that brings together residues involved in ssDNA and SSB binding at adjacent sites on the protein surface. Disruption of these interaction sites and of other conserved residues leads to decreased DnaB helicase loading onto SSB-bound DNA. We also demonstrate that PriC can directly interact with DnaB and the DnaB·DnaC complex. These data lead to a model in which PriC acts as a scaffold for recruiting DnaB·DnaC to SSB/ssDNA sites present at stalled replication forks. PMID:27382050

  17. On the origin of DNA genomes: evolution of the division of labor between template and catalyst in model replicator systems.

    Takeuchi, Nobuto; Hogeweg, Paulien; Koonin, Eugene V

    2011-03-01

    The division of labor between template and catalyst is a fundamental property of all living systems: DNA stores genetic information whereas proteins function as catalysts. The RNA world hypothesis, however, posits that, at the earlier stages of evolution, RNA acted as both template and catalyst. Why would such division of labor evolve in the RNA world? We investigated the evolution of DNA-like molecules, i.e. molecules that can function only as template, in minimal computational models of RNA replicator systems. In the models, RNA can function as both template-directed polymerase and template, whereas DNA can function only as template. Two classes of models were explored. In the surface models, replicators are attached to surfaces with finite diffusion. In the compartment models, replicators are compartmentalized by vesicle-like boundaries. Both models displayed the evolution of DNA and the ensuing division of labor between templates and catalysts. In the surface model, DNA provides the advantage of greater resistance against parasitic templates. However, this advantage is at least partially offset by the disadvantage of slower multiplication due to the increased complexity of the replication cycle. In the compartment model, DNA can significantly delay the intra-compartment evolution of RNA towards catalytic deterioration. These results are explained in terms of the trade-off between template and catalyst that is inherent in RNA-only replication cycles: DNA releases RNA from this trade-off by making it unnecessary for RNA to serve as template and so rendering the system more resistant against evolving parasitism. Our analysis of these simple models suggests that the lack of catalytic activity in DNA by itself can generate a sufficient selective advantage for RNA replicator systems to produce DNA. Given the widespread notion that DNA evolved owing to its superior chemical properties as a template, this study offers a novel insight into the evolutionary origin of DNA

  18. Persistent Amplification of DNA Damage Signal Involved in Replicative Senescence of Normal Human Diploid Fibroblasts

    Masatoshi Suzuki

    2012-01-01

    Full Text Available Foci of phosphorylated histone H2AX and ATM are the surrogate markers of DNA double strand breaks. We previously reported that the residual foci increased their size after irradiation, which amplifies DNA damage signals. Here, we addressed whether amplification of DNA damage signal is involved in replicative senescence of normal human diploid fibroblasts. Large phosphorylated H2AX foci (>1.5 μm diameter were specifically detected in presenescent cells. The frequency of cells with large foci was well correlated with that of cells positive for senescence-associated β-galactosidase staining. Hypoxic cell culture condition extended replicative life span of normal human fibroblast, and we found that the formation of large foci delayed in those cells. Our immuno-FISH analysis revealed that large foci partially localized at telomeres in senescent cells. Importantly, large foci of phosphorylated H2AX were always colocalized with phosphorylated ATM foci. Furthermore, Ser15-phosphorylated p53 showed colocalization with the large foci. Since the treatment of senescent cells with phosphoinositide 3-kinase inhibitor, wortmannin, suppressed p53 phosphorylation, it is suggested that amplification of DNA damage signaling sustains persistent activation of ATM-p53 pathway, which is essential for replicative senescence.

  19. Deciphering DNA replication dynamics in eukaryotic cell populations in relation with their averaged chromatin conformations

    Goldar, A.; Arneodo, A.; Audit, B.; Argoul, F.; Rappailles, A.; Guilbaud, G.; Petryk, N.; Kahli, M.; Hyrien, O.

    2016-03-01

    We propose a non-local model of DNA replication that takes into account the observed uncertainty on the position and time of replication initiation in eukaryote cell populations. By picturing replication initiation as a two-state system and considering all possible transition configurations, and by taking into account the chromatin’s fractal dimension, we derive an analytical expression for the rate of replication initiation. This model predicts with no free parameter the temporal profiles of initiation rate, replication fork density and fraction of replicated DNA, in quantitative agreement with corresponding experimental data from both S. cerevisiae and human cells and provides a quantitative estimate of initiation site redundancy. This study shows that, to a large extent, the program that regulates the dynamics of eukaryotic DNA replication is a collective phenomenon that emerges from the stochastic nature of replication origins initiation.

  20. The replication mechanism of kinetoplast DNA networks in several trypanosomatid species.

    Guilbride, D L; Englund, P T

    1998-03-01

    Kinetoplast DNA, a giant network of interlocked DNA circles, replicates by an unusual mechanism. Minicircles are released individually from the network by a topoisomerase II, and then, after replication, their progeny are reattached at antipodal positions on the network periphery. Studies to date have revealed two distinct variations on this model. In Crithidia fasciculata the newly replicated minicircles quickly become uniformly distributed around the network periphery, whereas in Trypanosoma brucei the minicircles accumulate near their two points of attachment. The kinetoplast DNA replication mechanism used by other related trypanosomatid species was until now unknown. Here we used a novel method, involving fluorescence microscopy of isolated networks, to investigate kinetoplast DNA replication in Leishmania tarentolae, Leishmania donovani, Trypanosoma cruzi and Phytomonas serpens. We found that all of these species have a replication mechanism resembling that of C. fasciculata and that the polar replication mechanism observed in T. brucei is so far unique to this species. PMID:9471996

  1. RPA mediates recombination repair during replication stress and is displaced from DNA by checkpoint signalling in human cells

    Sleeth, Kate M; Sørensen, Claus Storgaard; Issaeva, Natalia;

    2007-01-01

    The replication protein A (RPA) is involved in most, if not all, nuclear metabolism involving single-stranded DNA. Here, we show that RPA is involved in genome maintenance at stalled replication forks by the homologous recombination repair system in humans. Depletion of the RPA protein inhibited...... the formation of RAD51 nuclear foci after hydroxyurea-induced replication stalling leading to persistent unrepaired DNA double-strand breaks (DSBs). We demonstrate a direct role of RPA in homology directed recombination repair. We find that RPA is dispensable for checkpoint kinase 1 (Chk1) activation...... and that RPA directly binds RAD52 upon replication stress, suggesting a direct role in recombination repair. In addition we show that inhibition of Chk1 with UCN-01 decreases dissociation of RPA from the chromatin and inhibits association of RAD51 and RAD52 with DNA. Altogether, our data suggest a...

  2. In Vitro Whole Genome DNA Binding Analysis of the Bacterial Replication Initiator and Transcription Factor DnaA

    Smith, Janet L.; Grossman, Alan D.

    2015-01-01

    DnaA, the replication initiation protein in bacteria, is an AAA+ ATPase that binds and hydrolyzes ATP and exists in a heterogeneous population of ATP-DnaA and ADP-DnaA. DnaA binds cooperatively to the origin of replication and several other chromosomal regions, and functions as a transcription factor at some of these regions. We determined the binding properties of Bacillus subtilis DnaA to genomic DNA in vitro at single nucleotide resolution using in vitro DNA affinity purification and deep ...

  3. Interaction of RECQ4 and MCM10 is important for efficient DNA replication origin firing in human cells

    Kliszczak, Maciej; Sedlackova, Hana; Pitchai, Ganesha P;

    2015-01-01

    DNA replication is a highly coordinated process that is initiated at multiple replication origins in eukaryotes. These origins are bound by the origin recognition complex (ORC), which subsequently recruits the Mcm2-7 replicative helicase in a Cdt1/Cdc6-dependent manner. In budding yeast, two...... essential replication factors, Sld2 and Mcm10, are then important for the activation of replication origins. In humans, the putative Sld2 homolog, RECQ4, interacts with MCM10. Here, we have identified two mutants of human RECQ4 that are deficient in binding to MCM10. We show that these RECQ4 variants are...... able to complement the lethality of an avian cell RECQ4 deletion mutant, indicating that the essential function of RECQ4 in vertebrates is unlikely to require binding to MCM10. Nevertheless, we show that the RECQ4-MCM10 interaction is important for efficient replication origin firing....

  4. Quantitative measurement of translesion replication in human cells: Evidence for bypass of abasic sites by a replicative DNA polymerase

    Avkin, Sharon; Adar, Sheera; Blander, Gil; Livneh, Zvi

    2002-01-01

    Mutations in oncogenes and tumor suppressor genes are critical in the development of cancer. A major pathway for the formation of mutations is the replication of unrepaired DNA lesions. To better understand the mechanism of translesion replication (TLR) in mammals, a quantitative assay for TLR in cultured cells was developed. The assay is based on the transient transfection of cultured cells with a gapped plasmid, carrying a site-specific lesion in the gap region. Filling in of the gap by TLR...

  5. A novel class of mutations that affect DNA replication in E. coli

    Nordman, Jared; Skovgaard, Ole; Wright, Andrew

    2007-01-01

    Over-initiation of DNA replication in cells containing the cold-sensitive dnaA(cos) allele has been shown to lead to extensive DNA damage, potentially due to head-to-tail replication fork collisions that ultimately lead to replication fork collapse, growth stasis and/or cell death. Based on the...... assumption that suppressors of the cold-sensitive phenotype of the cos mutant should include mutations that affect the efficiency and/or regulation of DNA replication, we subjected a dnaA(cos) mutant strain to transposon mutagenesis and selected mutant derivatives that could form colonies at 30°C. Four...... suppressors of the dnaA(cos)-mediated cold sensitivity were identified and further characterized. Based on origin to terminus ratios, chromosome content per cell, measured by flow cytometry, and sensitivity to the replication fork inhibitor hydroxyurea, the suppressors fell into two distinct categories: those...

  6. Ancient mtDNA genetic variants modulate mtDNA transcription and replication.

    Sarit Suissa

    2009-05-01

    Full Text Available Although the functional consequences of mitochondrial DNA (mtDNA genetic backgrounds (haplotypes, haplogroups have been demonstrated by both disease association studies and cell culture experiments, it is not clear which of the mutations within the haplogroup carry functional implications and which are "evolutionary silent hitchhikers". We set forth to study the functionality of haplogroup-defining mutations within the mtDNA transcription/replication regulatory region by in vitro transcription, hypothesizing that haplogroup-defining mutations occurring within regulatory motifs of mtDNA could affect these processes. We thus screened >2500 complete human mtDNAs representing all major populations worldwide for natural variation in experimentally established protein binding sites and regulatory regions comprising a total of 241 bp in each mtDNA. Our screen revealed 77/241 sites showing point mutations that could be divided into non-fixed (57/77, 74% and haplogroup/sub-haplogroup-defining changes (i.e., population fixed changes, 20/77, 26%. The variant defining Caucasian haplogroup J (C295T increased the binding of TFAM (Electro Mobility Shift Assay and the capacity of in vitro L-strand transcription, especially of a shorter transcript that maps immediately upstream of conserved sequence block 1 (CSB1, a region associated with RNA priming of mtDNA replication. Consistent with this finding, cybrids (i.e., cells sharing the same nuclear genetic background but differing in their mtDNA backgrounds harboring haplogroup J mtDNA had a >2 fold increase in mtDNA copy number, as compared to cybrids containing haplogroup H, with no apparent differences in steady state levels of mtDNA-encoded transcripts. Hence, a haplogroup J regulatory region mutation affects mtDNA replication or stability, which may partially account for the phenotypic impact of this haplogroup. Our analysis thus demonstrates, for the first time, the functional impact of particular mtDNA

  7. Replication of vertebrate mitochondrial DNA entails transient ribonucleotide incorporation throughout the lagging strand

    Yasukawa, Takehiro; Reyes, Aurelio; Cluett, Tricia J.; Yang, Ming-Yao; Bowmaker, Mark; Jacobs, Howard T.; Holt, Ian J.

    2006-01-01

    Using two-dimensional agarose gel electrophoresis, we show that mitochondrial DNA (mtDNA) replication of birds and mammals frequently entails ribonucleotide incorporation throughout the lagging strand (RITOLS). Based on a combination of two-dimensional agarose gel electrophoretic analysis and mapping of 5′ ends of DNA, initiation of RITOLS replication occurs in the major non-coding region of vertebrate mtDNA and is effectively unidirectional. In some cases, conversion of nascent RNA strands t...

  8. The role of template superhelicity in the initiation of bacteriophage lambda DNA replication.

    Alfano, C; McMacken, R

    1988-01-01

    The prepriming steps in the initiation of bacteriophage lambda DNA replication depend on the action of the lambda O and P proteins and on the DnaB helicase, single-stranded DNA binding protein (SSB), and DnaJ and DnaK heat shock proteins of the E. coli host. The binding of multiple copies of the lambda O protein to the phage replication origin (ori lambda) initiates the ordered assembly of a series of nucleoprotein structures that form at ori lambda prior to DNA unwinding, priming and DNA syn...

  9. Soj/ParA stalls DNA replication by inhibiting helix formation of the initiator protein DnaA.

    Scholefield, Graham; Errington, Jeff; Murray, Heath

    2012-03-21

    Control of DNA replication initiation is essential for normal cell growth. A unifying characteristic of DNA replication initiator proteins across the kingdoms of life is their distinctive AAA+ nucleotide-binding domains. The bacterial initiator DnaA assembles into a right-handed helical oligomer built upon interactions between neighbouring AAA+ domains, that in vitro stretches DNA to promote replication origin opening. The Bacillus subtilis protein Soj/ParA has previously been shown to regulate DnaA-dependent DNA replication initiation; however, the mechanism underlying this control was unknown. Here, we report that Soj directly interacts with the AAA+ domain of DnaA and specifically regulates DnaA helix assembly. We also provide critical biochemical evidence indicating that DnaA assembles into a helical oligomer in vivo and that the frequency of replication initiation correlates with the extent of DnaA oligomer formation. This work defines a significant new regulatory mechanism for the control of DNA replication initiation in bacteria. PMID:22286949

  10. Temporal order of evolution of DNA replication systems inferred by comparison of cellular and viral DNA polymerases

    Koonin Eugene V

    2006-12-01

    Full Text Available Abstract Background The core enzymes of the DNA replication systems show striking diversity among cellular life forms and more so among viruses. In particular, and counter-intuitively, given the central role of DNA in all cells and the mechanistic uniformity of replication, the core enzymes of the replication systems of bacteria and archaea (as well as eukaryotes are unrelated or extremely distantly related. Viruses and plasmids, in addition, possess at least two unique DNA replication systems, namely, the protein-primed and rolling circle modalities of replication. This unexpected diversity makes the origin and evolution of DNA replication systems a particularly challenging and intriguing problem in evolutionary biology. Results I propose a specific succession for the emergence of different DNA replication systems, drawing argument from the differences in their representation among viruses and other selfish replicating elements. In a striking pattern, the DNA replication systems of viruses infecting bacteria and eukaryotes are dominated by the archaeal-type B-family DNA polymerase (PolB whereas the bacterial replicative DNA polymerase (PolC is present only in a handful of bacteriophage genomes. There is no apparent mechanistic impediment to the involvement of the bacterial-type replication machinery in viral DNA replication. Therefore, I hypothesize that the observed, markedly unequal distribution of the replicative DNA polymerases among the known cellular and viral replication systems has a historical explanation. I propose that, among the two types of DNA replication machineries that are found in extant life forms, the archaeal-type, PolB-based system evolved first and had already given rise to a variety of diverse viruses and other selfish elements before the advent of the bacterial, PolC-based machinery. Conceivably, at that stage of evolution, the niches for DNA-viral reproduction have been already filled with viruses replicating with the

  11. The RBBP6/ZBTB38/MCM10 Axis Regulates DNA Replication and Common Fragile Site Stability

    Benoit Miotto

    2014-04-01

    Full Text Available Faithful DNA replication is essential for the maintenance of genome integrity. Incomplete genome replication leads to DNA breaks and chromosomal rearrangements, which are causal factors in cancer and other human diseases. Despite their importance, the molecular mechanisms that control human genome stability are incompletely understood. Here, we report a pathway that is required for human genome replication and stability. This pathway has three components: an E3 ubiquitin ligase, a transcriptional repressor, and a replication protein. The E3 ubiquitin ligase RBBP6 ubiquitinates and destabilizes the transcriptional repressor ZBTB38. This repressor negatively regulates transcription and levels of the MCM10 replication factor on chromatin. Cells lacking RBBP6 experience reduced replication fork progression and increased damage at common fragile sites due to ZBTB38 accumulation and MCM10 downregulation. Our results uncover a pathway that ensures genome-wide DNA replication and chromosomal stability.

  12. The 32 kDa subunit of replication protein A (RPA) participates in the DNA replication of Mung bean yellow mosaic India virus (MYMIV) by interacting with the viral Rep protein.

    Singh, Dharmendra Kumar; Islam, Mohammad Nurul; Choudhury, Nirupam Roy; Karjee, Sumona; Mukherjee, Sunil Kumar

    2007-01-01

    Mung bean yellow mosaic India virus (MYMIV) is a member of genus begomoviridae and its genome comprises of bipartite (two components, namely DNA-A and DNA-B), single-stranded, circular DNA of about 2.7 kb. During rolling circle replication (RCR) of the DNA, the stability of the genome and maintenance of the stem-loop structure of the replication origin is crucial. Hence the role of host single-stranded DNA-binding protein, Replication protein A (RPA), in the RCR of MYMIV was examined. Two RPA subunits, namely the RPA70 kDa and RPA32 kDa, were isolated from pea and their roles were validated in a yeast system in which MYMIV DNA replication has been modelled. Here, we present evidences that only the RPA32 kDa subunit directly interacted with the carboxy terminus of MYMIV-Rep both in vitro as well as in yeast two-hybrid system. RPA32 modulated the functions of Rep by enhancing its ATPase and down regulating its nicking and closing activities. The possible role of these modulations in the context of viral DNA replication has been discussed. Finally, we showed the positive involvement of RPA32 in transient replication of the plasmid DNA bearing MYMIV replication origin using an in planta based assay. PMID:17182628

  13. DNA copy-number control through inhibition of replication fork progression

    J.T. Nordman (Jared T.); E. Kozhevnikova (Elena); C.P. Verrijzer (Peter); A.V. Pindyurin (Alexey); E.N. Andreyeva (Evgeniya); V.V. Shloma (Victor); I.F. Zhimulev (Igor); T. Orr-Weaver (T.)

    2014-01-01

    textabstractProper control of DNA replication is essential to ensure faithful transmission of genetic material and prevent chromosomal aberrations that can drive cancer progression and developmental disorders. DNA replication is regulated primarily at the level of initiation and is under strict cell

  14. Break-induced replication requires DNA damage-induced phosphorylation of Pif1 and leads to telomere lengthening.

    Yulia Vasianovich

    2014-10-01

    Full Text Available Broken replication forks result in DNA breaks that are normally repaired via homologous recombination or break induced replication (BIR. Mild insufficiency in the replicative ligase Cdc9 in budding yeast Saccharomyces cerevisiae resulted in a population of cells with persistent DNA damage, most likely due to broken replication forks, constitutive activation of the DNA damage checkpoint and longer telomeres. This telomere lengthening required functional telomerase, the core DNA damage signaling cascade Mec1-Rad9-Rad53, and the components of the BIR repair pathway - Rad51, Rad52, Pol32, and Pif1. The Mec1-Rad53 induced phosphorylation of Pif1, previously found necessary for inhibition of telomerase at double strand breaks, was also important for the role of Pif1 in BIR and telomere elongation in cdc9-1 cells. Two other mutants with impaired DNA replication, cdc44-5 and rrm3Δ, were similar to cdc9-1: their long telomere phenotype was dependent on the Pif1 phosphorylation locus. We propose a model whereby the passage of BIR forks through telomeres promotes telomerase activity and leads to telomere lengthening.

  15. p53 represses human papillomavirus type 16 DNA replication via the viral E2 protein

    Morgan Iain M; Taylor Ewan R; Kowalczyk Anna M; Brown Craig; Gaston Kevin

    2008-01-01

    Abstract Background Human papillomavirus (HPV) DNA replication can be inhibited by the cellular tumour suppressor protein p53. However, the mechanism through which p53 inhibits viral replication and the role that this might play in the HPV life cycle are not known. The papillomavirus E2 protein is required for efficient HPV DNA replication and also regulates viral gene expression. E2 represses transcription of the HPV E6 and E7 oncogenes and can thereby modulate indirectly host cell prolifera...

  16. Selective Bypass of a Lagging Strand Roadblock by the Eukaryotic Replicative DNA Helicase

    Fu, Yu V.; Yardimci, Hasan; Long, David T.; Ho, The Vinh; Guainazzi, Angelo; Bermudez, Vladimir P.; Hurwitz, Jerard; Oijen, Antoine van; Schärer, Orlando D.; Walter, Johannes C.

    2011-01-01

    The eukaryotic replicative DNA helicase, CMG, unwinds DNA by an unknown mechanism. In some models, CMG encircles and translocates along one strand of DNA while excluding the other strand. In others, CMG encircles and translocates along duplex DNA. To distinguish between these models, replisomes were

  17. DNA Replication Dynamics of the GGGGCC Repeat of the C9orf72 Gene*

    Thys, Ryan Griffin; Wang, Yuh-Hwa

    2015-01-01

    Background: The (GGGGCC)n hexanucleotide repeat expansion of C9orf72 is the most common genetic cause of ALS-FTD. Results: C9orf72 repeat expansion increases instability and decreases replication efficiency by disrupting replication fork progression. Conclusion: C9orf72 repeat length and replication direction contribute to repeat instability in human cells. Significance: DNA replication-induced instability at the C9orf72 GGGGCC repeat can lead to further expansion and more severe disease.

  18. Decreased origin usage and initiation of DNA replication in haploinsufficient HCT116 Ku80+/- cells.

    Sibani, Sahar; Price, Gerald B; Zannis-Hadjopoulos, Maria

    2005-08-01

    One of the functions of the abundant heterodimeric nuclear protein, Ku (Ku70/Ku80), is its involvement in the initiation of DNA replication through its ability to bind to chromosomal replication origins in a sequence-specific and cell cycle dependent manner. Here, using HCT116 Ku80+/- cells, the effect of Ku80 deficiency on cell cycle progression and origin activation was examined. Western blot analyses revealed a 75% and 36% decrease in the nuclear expression of Ku80 and Ku70, respectively. This was concomitant with a 33% and 40% decrease in chromatin binding of both proteins, respectively. Cell cycle analysis of asynchronous and late G1 synchronized Ku80+/- cells revealed a prolonged G1 phase. Furthermore, these Ku-deficient cells had a 4.5-, 3.4- and 4.3-fold decrease in nascent strand DNA abundance at the lamin B2, beta-globin and c-myc replication origins, respectively. Chromatin immunoprecipitation (ChIP) assays showed that the association of Ku80 with the lamin B2, beta-globin and c-myc origins was decreased by 1.5-, 2.3- and 2.5-fold, respectively, whereas that of Ku70 was similarly decreased (by 2.1-, 1.5- and 1.7-fold, respectively) in Ku80+/- cells. The results indicate that a deficiency of Ku80 resulted in a prolonged G1 phase, as well as decreased Ku binding to and activation of origins of DNA replication. PMID:16014376

  19. Separation of the transcriptional activation and replication functions of the bovine papillomavirus-1 E2 protein.

    Winokur, P. L.; McBride, A A

    1992-01-01

    Replication of bovine papillomavirus-1 (BPV-1) DNA requires two viral gene products, the E1 protein and the full-length E2 protein. The 48 kDa E2 protein is a site-specific DNA-binding protein that binds to several sites which lie adjacent to the BPV-1 origin of replication. The 85 amino acid C-terminal domain contains the specific DNA binding and dimerization properties of the protein. The approximately 200 amino acid N-terminal domain is crucial for transcriptional activation. Both of these...

  20. Histone H3 lysine 56 acetylation and the response to DNA replication fork damage

    Wurtele, Hugo; Kaiser, Gitte Schalck; Bacal, Julien;

    2012-01-01

    In Saccharomyces cerevisiae, histone H3 lysine 56 acetylation (H3K56ac) occurs in newly synthesized histones that are deposited throughout the genome during DNA replication. Defects in H3K56ac sensitize cells to genotoxic agents, suggesting that this modification plays an important role in the DNA...... but are only mildly affected by hydroxyurea. We demonstrate that, after exposure to MMS, H3K56ac-deficient cells cannot complete DNA replication and eventually segregate chromosomes with intranuclear foci containing the recombination protein Rad52. In addition, we provide evidence that these...... lesions by recombination and/or from defects in the completion of DNA replication....

  1. Dynamic binding of replication protein a is required for DNA repair

    Chen, Ran; Subramanyam, Shyamal; Elcock, Adrian H.; Spies, Maria; Wold, Marc S.

    2016-01-01

    Replication protein A (RPA), the major eukaryotic single-stranded DNA (ssDNA) binding protein, is essential for replication, repair and recombination. High-affinity ssDNA-binding by RPA depends on two DNA binding domains in the large subunit of RPA. Mutation of the evolutionarily conserved aromatic residues in these two domains results in a separation-of-function phenotype: aromatic residue mutants support DNA replication but are defective in DNA repair. We used biochemical and single-molecule analyses, and Brownian Dynamics simulations to determine the molecular basis of this phenotype. Our studies demonstrated that RPA binds to ssDNA in at least two modes characterized by different dissociation kinetics. We also showed that the aromatic residues contribute to the formation of the longer-lived state, are required for stable binding to short ssDNA regions and are needed for RPA melting of partially duplex DNA structures. We conclude that stable binding and/or the melting of secondary DNA structures by RPA is required for DNA repair, including RAD51 mediated DNA strand exchange, but is dispensable for DNA replication. It is likely that the binding modes are in equilibrium and reflect dynamics in the RPA–DNA complex. This suggests that dynamic binding of RPA to DNA is necessary for different cellular functions. PMID:27131385

  2. Accuracy of replication in the polymerase chain reaction. Comparison between Thermotoga maritima DNA polymerase and Thermus aquaticus DNA polymerase

    R.S. Diaz

    1998-10-01

    Full Text Available For certain applications of the polymerase chain reaction (PCR, it may be necessary to consider the accuracy of replication. The breakthrough that made PCR user friendly was the commercialization of Thermus aquaticus (Taq DNA polymerase, an enzyme that would survive the high temperatures needed for DNA denaturation. The development of enzymes with an inherent 3' to 5' exonuclease proofreading activity, lacking in Taq polymerase, would be an improvement when higher fidelity is needed. We used the forward mutation assay to compare the fidelity of Taq polymerase and Thermotoga maritima (ULTMA™ DNA polymerase, an enzyme that does have proofreading activity. We did not find significant differences in the fidelity of either enzyme, even when using optimal buffer conditions, thermal cycling parameters, and number of cycles (0.2% and 0.13% error rates for ULTMA™ and Taq, respectively, after reading about 3,000 bases each. We conclude that for sequencing purposes there is no difference in using a DNA polymerase that contains an inherent 3' to 5' exonuclease activity for DNA amplification. Perhaps the specificity and fidelity of PCR are complex issues influenced by the nature of the target sequence, as well as by each PCR component.

  3. Interaction of the retinoblastoma protein with Orc1 and its recruitment to human origins of DNA replication.

    Ramiro Mendoza-Maldonado

    Full Text Available BACKGROUND: The retinoblastoma protein (Rb is a crucial regulator of cell cycle progression by binding with E2F transcription factor and repressing the expression of a variety of genes required for the G1-S phase transition. METHODOLOGY/PRINCIPAL FINDINGS: Here we show that Rb and E2F1 directly participate in the control of initiation of DNA replication in human HeLa, U2OS and T98G cells by specifically binding to origins of DNA replication in a cell cycle regulated manner. We show that, both in vitro and inside the cells, the largest subunit of the origin recognition complex (Orc1 specifically binds hypo-phosphorylated Rb and that this interaction is competitive with the binding of Rb to E2F1. The displacement of Rb-bound Orc1 by E2F1 at origins of DNA replication marks the progression of the G1 phase of the cell cycle toward the G1-S border. CONCLUSIONS/SIGNIFICANCE: The participation of Rb and E2F1 in the formation of the multiprotein complex that binds origins of DNA replication in mammalian cells appears to represent an effective mechanism to couple the expression of genes required for cell cycle progression to the activation of DNA replication.

  4. Initiation sites for human DNA replication at a putative ribulose-5-phosphate 3-epimerase gene

    Replication of the human genome requires the activation of thousands of replicons distributed along each one of the chromosomes. Each replicon contains an initiation, or origin, site, at which DNA synthesis begins. However, very little information is known about the nature and positioning of these initiation sites along human chromosomes. We have recently focused our attention to a 1.1 kb region of human chromosome 2 which functioned as an episomal origin in the yeast Saccharomyces cerevisiae. This region corresponded to the largest exon of a putative ribulose-5-phosphate-3-epimerase gene (RPE). In the present study we have used a real-time PCR-based nascent strand DNA abundance assay to map initiation sites for DNA replication in in vivo human chromosomes around a 13.4 kb region encompassing the putative RPE gene. By applying this analysis to a 1-1.4 kb nascent strand DNA fraction isolated from both normal skin fibroblasts, and the breast cell line MCF10; we have identified five initiation sites within the 13.4 kb region of chromosome 2. The initiation sites appear to map to similar positions in both cell lines and occur outside the coding regions of the putative RPE gene

  5. Topoisomerase inhibitors can selectively interfere with different stages of simian virus 40 DNA replication.

    Snapka, R M

    1986-01-01

    I have found that antineoplastic drugs which are known to be inhibitors of mammalian DNA topoisomerases have pronounced and selective effects on simian virus 40 DNA replication. Ellipticine, 4'-(9-acridinylamino)methanesulfon-m-aniside, and Adriamycin blocked decatenation of newly replicated simian virus 40 daughter chromosomes in vivo. The arrested decatenation intermediates produced by these drugs contained single-strand DNA breaks. Ellipticine in particular produced these catenated dimers ...

  6. POLD1: Central mediator of DNA replication and repair, and implication in cancer and other pathologies.

    Nicolas, Emmanuelle; Golemis, Erica A; Arora, Sanjeevani

    2016-09-15

    The evolutionarily conserved human polymerase delta (POLD1) gene encodes the large p125 subunit which provides the essential catalytic activities of polymerase δ (Polδ), mediated by 5'-3' DNA polymerase and 3'-5' exonuclease moieties. POLD1 associates with three smaller subunits (POLD2, POLD3, POLD4), which together with Replication Factor C and Proliferating Nuclear Cell Antigen constitute the polymerase holoenzyme. Polδ function is essential for replication, with a primary role as the replicase for the lagging strand. Polδ also has an important proofreading ability conferred by the exonuclease activity, which is critical for ensuring replicative fidelity, but also serves to repair DNA lesions arising as a result of exposure to mutagens. Polδ has been shown to be important for multiple forms of DNA repair, including nucleotide excision repair, double strand break repair, base excision repair, and mismatch repair. A growing number of studies in the past decade have linked germline and sporadic mutations in POLD1 and the other subunits of Polδ with human pathologies. Mutations in Polδ in mice and humans lead to genomic instability, mutator phenotype and tumorigenesis. The advent of genome sequencing techniques has identified damaging mutations in the proofreading domain of POLD1 as the underlying cause of some inherited cancers, and suggested that mutations in POLD1 may influence therapeutic management. In addition, mutations in POLD1 have been identified in the developmental disorders of mandibular hypoplasia, deafness, progeroid features and lipodystrophy and atypical Werner syndrome, while changes in expression or activity of POLD1 have been linked to senescence and aging. Intriguingly, some recent evidence suggests that POLD1 function may also be altered in diabetes. We provide an overview of critical Polδ activities in the context of these pathologic conditions. PMID:27320729

  7. Inhibition of human Chk1 causes increased initiation of DNA replication, phosphorylation of ATR targets, and DNA breakage

    Syljuåsen, Randi G; Sørensen, Claus Storgaard; Hansen, Lasse Tengbjerg;

    2005-01-01

    -nuclear phosphorylation of histone H2AX, p53, Smc1, replication protein A, and Chk1 itself in human S-phase cells. These phosphorylations were inhibited by ATR siRNA and caffeine, but they occurred independently of ATM. Chk1 inhibition also caused an increased initiation of DNA replication, which was accompanied by...... increased amounts of nonextractable RPA protein, formation of single-stranded DNA, and induction of DNA strand breaks. Moreover, these responses were prevented by siRNA-mediated downregulation of Cdk2 or the replication initiation protein Cdc45, or by addition of the CDK inhibitor roscovitine. We propose...... that Chk1 is required during normal S phase to avoid aberrantly increased initiation of DNA replication, thereby protecting against DNA breakage. These results may help explain why Chk1 is an essential kinase and should be taken into account when drugs to inhibit this kinase are considered for use in...

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

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

    2014-01-01

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

  9. Food storage material silver nanoparticles interfere with DNA replication fidelity and bind with DNA

    Yang, Wenjuan; Shen, Cenchao; Ji, Qiaoli; An, Hongjie; Wang, Jinju; Liu, Qingdai; Zhang, Zhizhou

    2009-02-01

    Nanosilver is increasingly used in the food industry and biomedical applications. A lot of studies have been done to investigate the potential toxicity of nanosilver. But information on whether or how nanosilver particles bring changes in genetic materials remains scant. In this study, the replication fidelity of the rpsL gene was quantified when nanosilver particles were present in polymerase chain reactions (PCRs) or cell cultures of E. coli transformed with the wild-type rpsL gene. Three types of nanosilver (silver nanopowder, SN; silver-copper nanopowder, SCN; and colloidal silver, CS) were tested. The results showed that the replication fidelity of the rpsL gene was differentially compromised by all three kinds of nanosilver particle compared with that without nanosilver. This assay could be expanded and applied to any other materials to preliminarily assess their potential long-term toxicity as a food additive or biomedical reagent. Moreover, we found that nanosilver materials bind with genomic DNA under atomic force microscopy, and this might be an explanation for the compromised DNA replication fidelity.

  10. Food storage material silver nanoparticles interfere with DNA replication fidelity and bind with DNA

    Nanosilver is increasingly used in the food industry and biomedical applications. A lot of studies have been done to investigate the potential toxicity of nanosilver. But information on whether or how nanosilver particles bring changes in genetic materials remains scant. In this study, the replication fidelity of the rpsL gene was quantified when nanosilver particles were present in polymerase chain reactions (PCRs) or cell cultures of E. coli transformed with the wild-type rpsL gene. Three types of nanosilver (silver nanopowder, SN; silver-copper nanopowder, SCN; and colloidal silver, CS) were tested. The results showed that the replication fidelity of the rpsL gene was differentially compromised by all three kinds of nanosilver particle compared with that without nanosilver. This assay could be expanded and applied to any other materials to preliminarily assess their potential long-term toxicity as a food additive or biomedical reagent. Moreover, we found that nanosilver materials bind with genomic DNA under atomic force microscopy, and this might be an explanation for the compromised DNA replication fidelity.

  11. Identification of the Xenopus laevis Homolog of Saccharomyces cerevisiae DNA2 and Its Role in DNA Replication

    Liu, Qingquan; Choe, Won-chae; Campbell, Judith L.

    2000-01-01

    The DNA2 gene of Saccharomyces cerevisiae is essential for growth and appears to be required for a late stage of chromosomal DNA replication. S. cerevisiae Dna2p (ScDna2p) is a DNA helicase and also a nuclease. We have cloned and sequenced the homologous gene from Xenopus (Xenopus Dna2). Xenopus Dna2p (XDna2p) is 32% identical to ScDna2p, and the similarity extends over the entire length, including but not limited to the five conserved helicase motifs. XDna2p is even more closely related (60%...

  12. Structural dynamics and ssDNA binding activity of the three N-terminal domains of the large subunit of Replication Protein A from small angle X-ray scattering

    Pretto, Dalyir I.; Tsutakawa, Susan; Brosey, Chris A.; Castillo, Amalchi; Chagot, Marie-Eve; Smith, Jarrod A.; Tainer, John A.; Chazin, Walter J.

    2010-03-11

    Replication Protein A (RPA) is the primary eukaryotic ssDNA binding protein utilized in diverse DNA transactions in the cell. RPA is a heterotrimeric protein with seven globular domains connected by flexible linkers, which enable substantial inter-domain motion that is essential to its function. Small angle X-ray scattering (SAXS) experiments on two multi-domain constructs from the N-terminus of the large subunit (RPA70) were used to examine the structural dynamics of these domains and their response to the binding of ssDNA. The SAXS data combined with molecular dynamics simulations reveal substantial interdomain flexibility for both RPA70AB (the tandem high affinity ssDNA binding domains A and B connected by a 10-residue linker) and RPA70NAB (RPA70AB extended by a 70-residue linker to the RPA70N protein interaction domain). Binding of ssDNA to RPA70NAB reduces the interdomain flexibility between the A and B domains, but has no effect on RPA70N. These studies provide the first direct measurements of changes in orientation of these three RPA domains upon binding ssDNA. The results support a model in which RPA70N remains structurally independent of RPA70AB in the DNA bound state and therefore freely available to serve as a protein recruitment module.

  13. Dynamic Association of the Replication Initiator and Transcription Factor DnaA with the Bacillus subtilis Chromosome during Replication Stress ▿

    Breier, Adam M.; Grossman, Alan D.

    2008-01-01

    DnaA functions as both a transcription factor and the replication initiator in bacteria. We characterized the DNA binding dynamics of DnaA on a genomic level. Based on cross-linking and chromatin immunoprecipitation data, DnaA binds at least 17 loci, 15 of which are regulated transcriptionally in response to inhibition of replication (replication stress). Six loci, each of which has a cluster of at least nine potential DnaA binding sites, had significant increases in binding by DnaA when repl...

  14. Escherichia coli dnaT gene function is required for pBR322 plasmid replication but not for R1 plasmid replication.

    Masai, H.; Arai, K.

    1989-01-01

    Plasmid pBR322 was unable to replicate in a temperature-sensitive dnaT1 strain at a nonpermissive temperature, whereas a pBR322-derived plasmid carrying the wild-type dnaT+ gene was able to replicate under the same conditions. In contrast to pBR322, plasmid R1 could replicate in the dnaT1 strain at a nonpermissive temperature. In keeping with this finding, in vitro replication of plasmid R1 did not require DnaT protein.

  15. Active DNA Demethylation Mediated by DNA Glycosylases

    Zhu, Jian-Kang

    2009-01-01

    Active DNA demethylation is involved in many vital developmental and physiological processes of plants and animals. Recent genetic and biochemical studies in Arabidopsis have demonstrated that a subfamily of DNA glycosylases function to promote DNA demethylation through a base excision-repair pathway. These specialized bifunctional DNA glycosylases remove the 5-methylcytosine base and then cleave the DNA backbone at the abasic site, resulting in a gap that is then filled with an unmethylated ...

  16. Genesis and wanderings: origins and migrations in asymmetrically replicating mitochondrial DNA.

    Brown, Timothy A; Clayton, David A

    2006-05-01

    Mammalian mitochondria maintain a small circular genome that encodes RNA and polypeptides that are essential for the generation of ATP through oxidative phosphorylation. The mechanism of replication of mammalian mitochondrial DNA (mtDNA) has recently been a topic of controversy. New evidence has led to a modified strand-displacement model that reconciles much of the current data. This revision stems from a new appreciation for alternative light-strand origins. We consider here some of the potential mechanisms for light-strand origin initiation. We also consider further the susceptibility of branch migration within replicating mtDNA molecules. The existence of alternative light-strand origins and a propensity for branch migration in replicating mtDNA molecules exposes a new array of possible configurations of mtDNA. The assortment and assignment of these forms is relevant to the interpretation of experimental data and may also yield insight into the molecular basis of replication errors. PMID:16628009

  17. Effect of 5-fluorodeoxyuridine on DNA replication in ultraviolet-irradiated HeLa cells

    In HeLa cells precultivated for 6 hours with 5-fluorodeoxyuridine (FUdR) and for 18 hours in FUdR-free medium, DNA synthesis was much more resistant to UV irradiation than that of untreated cells. DNA synthesized in FUdR-pretreated and UV irradiated cells represents a semiconservative DNA replication and shows more rapid shift of the pulse-labelled chased DNA to high molecular weight. This DNA synthesis is not induced by synchronization of the cell cycle. It is assumed that either the changes of chromatine structure, or an enhanced level of some enzymes might be involved in the replication of the damaged template. (author)

  18. Human geminin promotes pre-RC formation and DNA replication by stabilizing CDT1 in mitosis

    Ballabeni, Andrea; Melixetian, Marina; Zamponi, Raffaella;

    2004-01-01

    -mediated degradation by inhibiting its ubiquitination. In particular, Geminin ensures basal levels of CDT1 during S phase and its accumulation during mitosis. Consistently, inhibition of Geminin synthesis during M phase leads to impairment of pre-RC formation and DNA replication during the following cell cycle....... Moreover, we show that inhibition of CDK1 during mitosis, and not Geminin depletion, is sufficient for premature formation of pre-RCs, indicating that CDK activity is the major mitotic inhibitor of licensing in human cells. Taken together with recent data from our laboratory, our results demonstrate that...

  19. Rif1 Controls DNA Replication Timing in Yeast through the PP1 Phosphatase Glc7

    Stefano Mattarocci; Maksym Shyian; Laure Lemmens; Pascal Damay; Dogus Murat Altintas; Tianlai Shi; Clinton R. Bartholomew; Thomä, Nicolas H.; Christopher F.J. Hardy; David Shore

    2014-01-01

    The Rif1 protein, originally identified as a telomere-binding factor in yeast, has recently been implicated in DNA replication control from yeast to metazoans. Here, we show that budding yeast Rif1 protein inhibits activation of prereplication complexes (pre-RCs). This inhibitory function requires two N-terminal motifs, RVxF and SILK, associated with recruitment of PP1 phosphatase (Glc7). In G1 phase, we show both that Glc7 interacts with Rif1 in an RVxF/SILK-dependent manner and that two pro...

  20. Replication protein A prevents accumulation of single-stranded telomeric DNA in cells that use alternative lengthening of telomeres

    Grudic, Amra; Jul-Larsen, Åsne; Haring, SJ; Wold, MS; Lønning, Per Eystein; Bjerkvig, Rolf; Bøe, Stig Ove

    2007-01-01

    The activation of a telomere maintenance mechanism is required for cancer development in humans. While most tumors achieve this by expressing the enzyme telomerase, a fraction (5–15%) employs a recombination-based mechanism termed alternative lengthening of telomeres (ALT). Here we show that loss of the single-stranded DNA-binding protein replication protein A (RPA) in human ALT cells, but not in telomerase-positive cells, causes increased exposure of single-stranded G-rich telomeric DNA, cel...

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

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

    2016-05-17

    Early embryonic development features rapid nuclear DNA replication cycles, but lacks mtDNA replication. To meet the high-energy demands of embryogenesis, mature oocytes are furnished with vast amounts of mitochondria and mtDNA However, the cellular machinery driving massive mtDNA replication in ovaries remains unknown. Here, we describe a Drosophila AKAP protein, MDI that recruits a translation stimulator, La-related protein (Larp), to the mitochondrial outer membrane in ovaries. The MDI-Larp complex promotes the synthesis of a subset of nuclear-encoded mitochondrial proteins by cytosolic ribosomes on the mitochondrial surface. MDI-Larp's targets include mtDNA replication factors, mitochondrial ribosomal proteins, and electron-transport chain subunits. Lack of MDI abolishes mtDNA replication in ovaries, which leads to mtDNA deficiency in mature eggs. Targeting Larp to the mitochondrial outer membrane independently of MDI restores local protein synthesis and rescues the phenotypes of mdi mutant flies. Our work suggests that a selective translational boost by the MDI-Larp complex on the outer mitochondrial membrane might be essential for mtDNA replication and mitochondrial biogenesis during oogenesis. PMID:27053724

  2. 遗传物质DNA的复制合成%Synthesis and Replication of DNA

    贺清兰; 索红军

    2011-01-01

    The articles described the nature of DNA replication, the four logical stages of DNA replication process and the repair after the abnormal situation and damage of DNA replication.It maybe help with the genetic information during cell division.%文章结合相关的酶,介绍了DNA复制的性质、DNA复制过程的4个逻辑阶段以及DNA复制过程出现异常或损伤后的修复,为熟悉细胞分裂过程中信息遗传提供帮助.

  3. Complete in vitro replication of SV40 DNA and chromatin in saponin-treated permeable cells.

    Hosogi,Nobuo; Hanakawa,Shiro; Watanabe,Sekiko; Oda,Takuzo

    1981-01-01

    A permeable cell system has been developed by treatment with saponin for studying in vitro replication of DNA and chromatin. DNA replication of simian virus 40 nucleoprotein complexes (SV40 chromatin) in saponin-treated permeable cells was found to be more efficient than that in digitonin-treated permeable cells. Autoradiography of the agarose-gel revealed that [alpha-32P]dCTP was incorporated into SV40 DNA I, II and replicating intermediates. The time course of the incorporation indicated co...

  4. Chromatin-associated degradation is defined by UBXN-3/FAF1 to safeguard DNA replication fork progression.

    Franz, André; Pirson, Paul A; Pilger, Domenic; Halder, Swagata; Achuthankutty, Divya; Kashkar, Hamid; Ramadan, Kristijan; Hoppe, Thorsten

    2016-01-01

    The coordinated activity of DNA replication factors is a highly dynamic process that involves ubiquitin-dependent regulation. In this context, the ubiquitin-directed ATPase CDC-48/p97 recently emerged as a key regulator of chromatin-associated degradation in several of the DNA metabolic pathways that assure genome integrity. However, the spatiotemporal control of distinct CDC-48/p97 substrates in the chromatin environment remained unclear. Here, we report that progression of the DNA replication fork is coordinated by UBXN-3/FAF1. UBXN-3/FAF1 binds to the licensing factor CDT-1 and additional ubiquitylated proteins, thus promoting CDC-48/p97-dependent turnover and disassembly of DNA replication factor complexes. Consequently, inactivation of UBXN-3/FAF1 stabilizes CDT-1 and CDC-45/GINS on chromatin, causing severe defects in replication fork dynamics accompanied by pronounced replication stress and eventually resulting in genome instability. Our work identifies a critical substrate selection module of CDC-48/p97 required for chromatin-associated protein degradation in both Caenorhabditis elegans and humans, which is relevant to oncogenesis and aging. PMID:26842564

  5. The escherichia coli chromosome replication initiator protein, DnaA

    Nyborg, Malene

    The experimental work presented in this thesis involve mutational analysis of the DNA binding domain of the DnaA protein and analysis of the A184V substitution in the ATP area of domain III and other amino acid substitutions found in the DnaA5 and DnaA4G proteins....

  6. A ruthenium polypyridyl intercalator stalls DNA replication forks, radiosensitizes human cancer cells and is enhanced by Chk1 inhibition.

    Gill, Martin R; Harun, Siti Norain; Halder, Swagata; Boghozian, Ramon A; Ramadan, Kristijan; Ahmad, Haslina; Vallis, Katherine A

    2016-01-01

    Ruthenium(II) polypyridyl complexes can intercalate DNA with high affinity and prevent cell proliferation; however, the direct impact of ruthenium-based intercalation on cellular DNA replication remains unknown. Here we show the multi-intercalator [Ru(dppz)2(PIP)](2+) (dppz = dipyridophenazine, PIP = 2-(phenyl)imidazo[4,5-f][1,10]phenanthroline) immediately stalls replication fork progression in HeLa human cervical cancer cells. In response to this replication blockade, the DNA damage response (DDR) cell signalling network is activated, with checkpoint kinase 1 (Chk1) activation indicating prolonged replication-associated DNA damage, and cell proliferation is inhibited by G1-S cell-cycle arrest. Co-incubation with a Chk1 inhibitor achieves synergistic apoptosis in cancer cells, with a significant increase in phospho(Ser139) histone H2AX (γ-H2AX) levels and foci indicating increased conversion of stalled replication forks to double-strand breaks (DSBs). Normal human epithelial cells remain unaffected by this concurrent treatment. Furthermore, pre-treatment of HeLa cells with [Ru(dppz)2(PIP)](2+) before external beam ionising radiation results in a supra-additive decrease in cell survival accompanied by increased γ-H2AX expression, indicating the compound functions as a radiosensitizer. Together, these results indicate ruthenium-based intercalation can block replication fork progression and demonstrate how these DNA-binding agents may be combined with DDR inhibitors or ionising radiation to achieve more efficient cancer cell killing. PMID:27558808

  7. Characterization of human chromosomal DNA sequences which replicate autonomously in Saccharomyces cerevisiae.

    Montiel, J F; Norbury, C. J.; Tuite, M F; Dobson, M J; Mills, J S; Kingsman, A J; Kingsman, S M

    1984-01-01

    We have characterised two restriction fragments, isolated from a "shotgun" collection of human DNA, which function as autonomously replicating sequences (ARSs) in Saccharomyces cerevisiae. Functional domains of these fragments have been defined by subcloning and exonuclease (BAL 31) deletion analysis. Both fragments contain two spatially distinct domains. One is essential for high frequency transformation and is termed the Replication Sequence (RS) domain, the other, termed the Replication En...

  8. A bacterial toxin inhibits DNA replication elongation through a direct interaction with the β sliding clamp.

    Aakre, Christopher D; Phung, Tuyen N; Huang, David; Laub, Michael T

    2013-12-12

    Toxin-antitoxin (TA) systems are ubiquitous on bacterial chromosomes, yet the mechanisms regulating their activity and the molecular targets of toxins remain incompletely defined. Here, we identify SocAB, an atypical TA system in Caulobacter crescentus. Unlike canonical TA systems, the toxin SocB is unstable and constitutively degraded by the protease ClpXP; this degradation requires the antitoxin, SocA, as a proteolytic adaptor. We find that the toxin, SocB, blocks replication elongation through an interaction with the sliding clamp, driving replication fork collapse. Mutations that suppress SocB toxicity map to either the hydrophobic cleft on the clamp that binds DNA polymerase III or a clamp-binding motif in SocB. Our findings suggest that SocB disrupts replication by outcompeting other clamp-binding proteins. Collectively, our results expand the diversity of mechanisms employed by TA systems to regulate toxin activity and inhibit bacterial growth, and they suggest that inhibiting clamp function may be a generalizable antibacterial strategy. PMID:24239291

  9. Allele-specific analysis of DNA replication origins in mammalian cells.

    Bartholdy, Boris; Mukhopadhyay, Rituparna; Lajugie, Julien; Aladjem, Mirit I; Bouhassira, Eric E

    2015-01-01

    The mechanisms that control the location and timing of firing of replication origins are poorly understood. Using a novel functional genomic approach based on the analysis of SNPs and indels in phased human genomes, we observe that replication asynchrony is associated with small cumulative variations in the initiation efficiency of multiple origins between the chromosome homologues, rather than with the activation of dormant origins. Allele-specific measurements demonstrate that the presence of G-quadruplex-forming sequences does not correlate with the efficiency of initiation. Sequence analysis reveals that the origins are highly enriched in sequences with profoundly asymmetric G/C and A/T nucleotide distributions and are almost completely depleted of antiparallel triplex-forming sequences. We therefore propose that although G4-forming sequences are abundant in replication origins, an asymmetry in nucleotide distribution, which increases the propensity of origins to unwind and adopt non-B DNA structure, rather than the ability to form G4, is directly associated with origin activity. PMID:25987481

  10. Polycomb proteins control proliferation and transformation independently of cell cycle checkpoints by regulating DNA replication

    Piunti, Andrea; Rossi, Alessandra; Cerutti, Aurora;

    2014-01-01

    PRCs regulate cellular proliferation and transformation independently of the Ink4a/Arf-pRb-p53 pathway. We provide evidence that PRCs localize at replication forks, and that loss of their function directly affects the progression and symmetry of DNA replication forks. Thus, we have identified a novel...

  11. DNA adenine methylation is required to replicate both Vibrio cholerae chromosomes once per cell cycle.

    Gaëlle Demarre

    2010-05-01

    Full Text Available DNA adenine methylation is widely used to control many DNA transactions, including replication. In Escherichia coli, methylation serves to silence newly synthesized (hemimethylated sister origins. SeqA, a protein that binds to hemimethylated DNA, mediates the silencing, and this is necessary to restrict replication to once per cell cycle. The methylation, however, is not essential for replication initiation per se but appeared so when the origins (oriI and oriII of the two Vibrio cholerae chromosomes were used to drive plasmid replication in E. coli. Here we show that, as in the case of E. coli, methylation is not essential for oriI when it drives chromosomal replication and is needed for once-per-cell-cycle replication in a SeqA-dependent fashion. We found that oriII also needs SeqA for once-per-cell-cycle replication and, additionally, full methylation for efficient initiator binding. The requirement for initiator binding might suffice to make methylation an essential function in V. cholerae. The structure of oriII suggests that it originated from a plasmid, but unlike plasmids, oriII makes use of methylation for once-per-cell-cycle replication, the norm for chromosomal but not plasmid replication.

  12. Inhibition of hepatitis B virus replication with linear DNA sequences expressing antiviral micro-RNA shuttles

    Chattopadhyay, Saket; Ely, Abdullah; Bloom, Kristie; Weinberg, Marc S. [Antiviral Gene Therapy Research Unit, University of the Witwatersrand (South Africa); Arbuthnot, Patrick, E-mail: Patrick.Arbuthnot@wits.ac.za [Antiviral Gene Therapy Research Unit, University of the Witwatersrand (South Africa)

    2009-11-20

    RNA interference (RNAi) may be harnessed to inhibit viral gene expression and this approach is being developed to counter chronic infection with hepatitis B virus (HBV). Compared to synthetic RNAi activators, DNA expression cassettes that generate silencing sequences have advantages of sustained efficacy and ease of propagation in plasmid DNA (pDNA). However, the large size of pDNAs and inclusion of sequences conferring antibiotic resistance and immunostimulation limit delivery efficiency and safety. To develop use of alternative DNA templates that may be applied for therapeutic gene silencing, we assessed the usefulness of PCR-generated linear expression cassettes that produce anti-HBV micro-RNA (miR) shuttles. We found that silencing of HBV markers of replication was efficient (>75%) in cell culture and in vivo. miR shuttles were processed to form anti-HBV guide strands and there was no evidence of induction of the interferon response. Modification of terminal sequences to include flanking human adenoviral type-5 inverted terminal repeats was easily achieved and did not compromise silencing efficacy. These linear DNA sequences should have utility in the development of gene silencing applications where modifications of terminal elements with elimination of potentially harmful and non-essential sequences are required.

  13. Inhibition of hepatitis B virus replication with linear DNA sequences expressing antiviral micro-RNA shuttles

    RNA interference (RNAi) may be harnessed to inhibit viral gene expression and this approach is being developed to counter chronic infection with hepatitis B virus (HBV). Compared to synthetic RNAi activators, DNA expression cassettes that generate silencing sequences have advantages of sustained efficacy and ease of propagation in plasmid DNA (pDNA). However, the large size of pDNAs and inclusion of sequences conferring antibiotic resistance and immunostimulation limit delivery efficiency and safety. To develop use of alternative DNA templates that may be applied for therapeutic gene silencing, we assessed the usefulness of PCR-generated linear expression cassettes that produce anti-HBV micro-RNA (miR) shuttles. We found that silencing of HBV markers of replication was efficient (>75%) in cell culture and in vivo. miR shuttles were processed to form anti-HBV guide strands and there was no evidence of induction of the interferon response. Modification of terminal sequences to include flanking human adenoviral type-5 inverted terminal repeats was easily achieved and did not compromise silencing efficacy. These linear DNA sequences should have utility in the development of gene silencing applications where modifications of terminal elements with elimination of potentially harmful and non-essential sequences are required.

  14. Human Papilloma Viral DNA Replicates as a Stable Episome in Cultured Epidermal Keratinocytes

    Laporta, Robert F.; Taichman, Lorne B.

    1982-06-01

    Human papilloma virus (HPV) is poorly understood because systems for its growth in tissue culture have not been developed. We report here that cultured human epidermal keratinocytes could be infected with HPV from plantar warts and that the viral DNA persisted and replicated as a stable episome. There were 50-200 copies of viral DNA per cell and there was no evidence to indicate integration of viral DNA into the cellular genome. There was also no evidence to suggest that viral DNA underwent productive replication. We conclude that cultured human epidermal keratinocytes may be a model for the study of certain aspects of HPV biology.

  15. Induction of the Sulfolobus shibatae virus SSV1 DNA replication by mitomycin C

    2002-01-01

    The temperate virus SSV1 from the hyperthermophilic archaeon Sulfolobus shibatae provides a useful model system for the study of archaeal DNA replication. Southern hybridization showed that SSV1 existed primarily as a provirus in its host that was grown without shaking. Upon UV or mitomycin C induction, the cellular level of free SSV1 DNA increased drastically whereas that of integrated viral DNA remained unchanged. The results of mitomycin C induction were more reproducible than those of UV induction. We found that, when the cells that had been grown without shaking were shaken, the replication of SSV1 DNA was also induced. Based on our results, we developed a method for the induction of SSV1 DNA replication by mitomycin C. When the S. shibatae virus production was induced using this method, the cellular level of free SSV1 DNA started to increase 10 h after induction, and peaked after 12-15 h. A fully induced S. shibatae cell contained ~50 molecules of free SSV1 DNA. The development of this induction method and the description of the process of SSV1 DNA replication following induction are valuable to the analysis of the origin and mode of replication of the virus.

  16. Identification of Fic-1 as an enzyme that inhibits bacterial DNA replication by AMPylating GyrB, promoting filament formation.

    Lu, Canhua; Nakayasu, Ernesto S; Zhang, Li-Qun; Luo, Zhao-Qing

    2016-01-01

    The morphology of bacterial cells is important for virulence, evasion of the host immune system, and coping with environmental stresses. The widely distributed Fic proteins (filamentation induced by cAMP) are annotated as proteins involved in cell division because of the presence of the HPFx[D/E]GN[G/K]R motif. We showed that the presence of Fic-1 from Pseudomonas fluorescens significantly reduced the yield of plasmid DNA when expressed in Escherichia coli or P. fluorescens. Fic-1 interacted with GyrB, a subunit of DNA gyrase, which is essential for bacterial DNA replication. Fic-1 catalyzed the AMPylation of GyrB at Tyr(109), a residue critical for binding ATP, and exhibited auto-AMPylation activity. Mutation of the Fic-1 auto-AMPylated site greatly reduced AMPylation activity toward itself and toward GyrB. Fic-1-dependent AMPylation of GyrB triggered the SOS response, indicative of DNA replication stress or DNA damage. Fic-1 also promoted the formation of elongated cells when the SOS response was blocked. We identified an α-inhibitor protein that we named anti-Fic-1 (AntF), encoded by a gene immediately upstream of Fic-1. AntF interacted with Fic-1, inhibited the AMPylation activity of Fic-1 for GyrB in vitro, and blocked Fic-1-mediated inhibition of DNA replication in bacteria, suggesting that Fic-1 and AntF comprise a toxin-antitoxin module. Our work establishes Fic-1 as an AMPylating enzyme that targets GyrB to inhibit DNA replication and may target other proteins to regulate bacterial morphology. PMID:26814232

  17. Structural insights into recognition of MDC1 by TopBP1 in DNA replication checkpoint control

    Leung, Charles Chung Yun; Sun, Luxin; Gong, Zihua; Burkat, Michael; Edwards, Ross; Assmus, Mark; Chen, Junjie; Glover, J N Mark

    2013-01-01

    Activation of the DNA replication checkpoint by the ATR kinase requires protein interactions mediated by the ATR activating protein, TopBP1. Accumulation of TopBP1 at stalled replication forks requires the interaction of TopBP1 BRCT5 with the phosphorylated SDT repeats of the adaptor protein MDC1. Here we present the X-ray crystal structures of the tandem BRCT4/5 domains of TopBP1 free and in complex with a MDC1 consensus pSDpT phospho-peptide. TopBP1 BRCT4/5 adopts a variant BRCT-BRCT packin...

  18. The Caenorhabditis elegans Werner syndrome protein functions upstream of ATR and ATM in response to DNA replication inhibition and double-strand DNA breaks.

    Se-Jin Lee

    2010-01-01

    Full Text Available WRN-1 is the Caenorhabditis elegans homolog of the human Werner syndrome protein, a RecQ helicase, mutations of which are associated with premature aging and increased genome instability. Relatively little is known as to how WRN-1 functions in DNA repair and DNA damage signaling. Here, we take advantage of the genetic and cytological approaches in C. elegans to dissect the epistatic relationship of WRN-1 in various DNA damage checkpoint pathways. We found that WRN-1 is required for CHK1 phosphorylation induced by DNA replication inhibition, but not by UV radiation. Furthermore, WRN-1 influences the RPA-1 focus formation, suggesting that WRN-1 functions in the same step or upstream of RPA-1 in the DNA replication checkpoint pathway. In response to ionizing radiation, RPA-1 focus formation and nuclear localization of ATM depend on WRN-1 and MRE-11. We conclude that C. elegans WRN-1 participates in the initial stages of checkpoint activation induced by DNA replication inhibition and ionizing radiation. These functions of WRN-1 in upstream DNA damage signaling are likely to be conserved, but might be cryptic in human systems due to functional redundancy.

  19. Different domains of P21Cip1/waf1 regulate DNA replication and DNA repair-associated processes after UV

    Full text: Many genotoxic insults result in p21 up-regulation and p21-dependent cell cycle arrest but UV irradiation triggers p21 proteolysis. The significance of the increased p21 turnover is unclear and might be associated to DNA repair. While the role of p21 in Nucleotide Excision Repair (NER) remains controversial, two recent reports explore its effect on Translesion DNA Synthesis (TLS), a process that avoids replication blockage during S phase. The first report shows that p21 degradation is required for efficient PCNA ubiquitination, a post transcriptional modification that is relevant for TLS. The second report demonstrates that p21 (-/-) cells have increased TLS-associated mutagenic rates. Herein we analyze the effect of p21 on different PCNA-driven processes including DNA replication, NER and TLS. Whereas only the CDK binding domain of p21 is required for cell cycle arrest in unstressed cells; neither the CDK- nor the PCNA-binding domains of p21 are able to block early and late steps of NER. Intriguingly, through its PCNA binding domain, p21 inhibited recruitment of the TLS-polymerase, polη to PCNA foci after UV. Moreover, this obstruction correlates with accumulation of γH2AX and increased apoptosis. Taking together, our data emphasizes the link between p21 turnover and efficient TLS. This might also suggest a potential effect of p21 on other activities of polζ, a DNA polymerase with central roles in other biological scenarios such as genetic conversion, homologous recombination and modulation of the cellular response to genotoxic agents

  20. Human Cytomegalovirus UL44 Concentrates at the Periphery of Replication Compartments, the Site of Viral DNA Synthesis

    Strang, Blair L.; Boulant, Steeve; Chang, Lynne; Knipe, David M.; Kirchhausen, Tomas; Coen, Donald M

    2012-01-01

    The formation of replication compartments, the subnuclear structures in which the viral DNA genome is replicated, is a hallmark of herpesvirus infections. The localization of proteins and viral DNA within human cytomegalovirus replication compartments is not well characterized. Immunofluorescence analysis demonstrated the accumulation of the viral DNA polymerase subunit UL44 at the periphery of replication compartments and the presence of different populations of UL44 in infected cells. In co...

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

    Majid Eshaghi

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

  2. Yeast 2-microns plasmid DNA replication in vitro: purification of the CDC8 gene product by complementation assay.

    Arendes, J; Kim, K. C.; Sugino, A

    1983-01-01

    Extracts of the yeast Saccharomyces cerevisiae support DNA replication on exogenous yeast 2-microns plasmid DNA templates. A crude extract from a S. cerevisiae cell division cycle mutant, cdc8-1, expressed the temperature-sensitive phenotype since it could be inactivated at 42 degrees C in vitro. This heat-inactivated extract was fully complemented by the addition of either wild-type or cdc8-1 single-stranded DNA binding protein (SSB). restoration by SSB of the activity of the mutant cell ext...

  3. Replication of hepatitis B virus in primary duck hepatocytes transfected with linear viral DNA

    Yun-Qing Yao; Wei-Ping Zhou; Hong Ren; Qi Liu; Shu-Hua Guo; Ding-Feng Zhang; Ni Tang; Ai-Long Huang; Xiao-Yi Zou; Jiang-Feng Xiao; Yun Luo; Da-Zhi Zhang; Bo Wang

    2005-01-01

    AIM: To explore the expression and replication of hepatitis B virus (HBV) DNA in primary duck hepatocytes (PDHs).METHODS: Complete HBV genome was transfected into PDHs by electroporation (transfected group, 1.19×1012copies of linear HBV DNA/1×107 PDHs). After 1-5 d of transfection, HBsAg and HBeAg in the supernatant and lysate of PDHs were measured with the IMX System.Meanwhile, replicative intermediates of HBV DNA were analyzed by Southern blotting and Dot blotting. PDHs electroporated were used as control group.RESULTS: HBsAg in the hepatocyte lysates of transfected group was 15.24 (1 d), 14.55 (3 d) and 5.13 (5 d; P/N values, positive≥2.1) respectively. HBeAg was negative (<2.1). Both HBsAg and HBeAg were negative in the supernatant of transfected group. Dot blotting revealed that HBV DNA was strongly positive in the transfected group and negative in the control group. Southern blot analysis of intracellular total DNA indicated that there were relaxed circular (rc DNA), covalently closed circular (ccc DNA), and single-stranded (ss DNA) HBV DNA replicative intermediates in the transfected group, there was no integrated HBV DNA in the cellular genome. These parameters were negative in control group.CONCLUSION: Expression and replication of HBV genes can occur in hepatocytes from non-mammalian species.HBV replication has no critical species-specificity, and yet hepatic-specific regulating factors in hepatocytes may be essential for viral replication.

  4. Inhibition of hepatitis B virus DNA replicative intermediate forms by recombinant interferon-γ

    Mohammad Khalid Parvez; Deepak Sehgal; Shiv Kumar Sarin; Seemi Farhat Basir; Shahid Jameel

    2006-01-01

    AIM: To evaluate the in vitro anti-HBV activity of recombinant human IFN-γ, alone and in combination with lamivudine.METHODS: A recombinant baculovirus-HBV/HepG2 culture system was developed which could support productive HBV infection in vitro. Expression of HBsAg and HBeAg in infected HepG2 culture medium was detected by commercial enzyme immunoassays. HBV DNA replication intermediates were detected in infected cells by Southern hybridization and viral DNA load was determined by dot hybridization.RESULTS: IFN-γ at 0.1 to 5 μg/L efficiently down regulated HBsAg expression in transduced HepG2 cells.At 5 μg/L, IFN-γ also suppressed HBV DNA replication in these cells. While treatment with a combination of lamivudine and IFN-γ showed no additive effect,sequential treatment first with lamivudine and then IFN-γ was found to be promising. In this culture system the best HBV suppression was observed with a pulse of 2 μmol/L lamivudine for two days, followed by 1 μg/L IFN-γ for another four days. Compared to treatment with lamivudine alone, the sequential use of 0.2 μmol/L lamivudine for two days, followed by 5 μg/L IFN-γ for six days showed a 72% reduction in HBV cccDNA pool.CONCLUSION: This in vitro study warrants further evaluation of a combination of IFN-γ and lamivudine,especially in IFN-α non-responder chronic hepatitis B patients. A reduced duration of lamivudine treatment would also restrict the emergence of drug-resistant HBV mutants.

  5. In Vitro Whole Genome DNA Binding Analysis of the Bacterial Replication Initiator and Transcription Factor DnaA.

    Janet L Smith

    2015-05-01

    Full Text Available DnaA, the replication initiation protein in bacteria, is an AAA+ ATPase that binds and hydrolyzes ATP and exists in a heterogeneous population of ATP-DnaA and ADP-DnaA. DnaA binds cooperatively to the origin of replication and several other chromosomal regions, and functions as a transcription factor at some of these regions. We determined the binding properties of Bacillus subtilis DnaA to genomic DNA in vitro at single nucleotide resolution using in vitro DNA affinity purification and deep sequencing (IDAP-Seq. We used these data to identify 269 binding regions, refine the consensus sequence of the DnaA binding site, and compare the relative affinity of binding regions for ATP-DnaA and ADP-DnaA. Most sites had a slightly higher affinity for ATP-DnaA than ADP-DnaA, but a few had a strong preference for binding ATP-DnaA. Of the 269 sites, only the eight strongest binding ones have been observed to bind DnaA in vivo, suggesting that other cellular factors or the amount of available DnaA in vivo restricts DnaA binding to these additional sites. Conversely, we found several chromosomal regions that were bound by DnaA in vivo but not in vitro, and that the nucleoid-associated protein Rok was required for binding in vivo. Our in vitro characterization of the inherent ability of DnaA to bind the genome at single nucleotide resolution provides a backdrop for interpreting data on in vivo binding and regulation of DnaA, and is an approach that should be adaptable to many other DNA binding proteins.

  6. The β subunit sliding DNA clamp is responsible for unassisted mutagenic translesion replication by DNA polymerase III holoenzyme

    Tomer, Guy; Reuven, Nina Bacher; Livneh, Zvi

    1998-01-01

    The replication of damaged nucleotides that have escaped DNA repair leads to the formation of mutations caused by misincorporation opposite the lesion. In Escherichia coli, this process is under tight regulation of the SOS stress response and is carried out by DNA polymerase III in a process that involves also the RecA, UmuD′ and UmuC proteins. We have shown that DNA polymerase III holoenzyme is able to replicate, unassisted, through a synthetic abasic site in a gapped duplex plasmid. Here, w...

  7. In vitro DNA replication of recombinant plasmid DNAs containing the origin of progeny replicative form DNA synthesis of phage phi X174.

    Zipursky, S. L.; Reinberg, D.; Hurwitz, J

    1980-01-01

    The origin of phage phi X174 progeny replicative form (RF) DNA synthesis has been inserted into the plasmid vector pBR322 and cloned. In direct contrast to pBR322, the recombinant superhelical plasmids can substitute for phi X174 RFI DNA as template in phi X174-specific reactions in vitro. We have shown that the recombinant plasmids: (i) are cleaved by the phi X174 A protein; (ii) support net synthesis of unit-length single-stranded circular DNA in the presence of the phi X174 A protein and E...

  8. Replication and transcription on a collision course: eukaryotic regulation mechanisms and implications for DNA stability.

    Alessandra eBrambati

    2015-04-01

    Full Text Available DNA replication and transcription are vital cellular processes during which the genetic information is copied into complementary DNA and RNA molecules. Highly complex machineries required for DNA and RNA synthesis compete for the same DNA template, therefore being on a collision course. Unscheduled replication-transcription clashes alter the gene transcription program and generate replication stress, reducing fork speed. Molecular pathways and mechanisms that minimize the conflict between replication and transcription have been extensively characterized in prokaryotic cells and recently identified also in eukaryotes. A pathological outcome of replication-transcription collisions is the formation of stable RNA:DNA hybrids in molecular structures called R-loops. Growing evidence suggests that R-loop accumulation promotes both genetic and epigenetic instability, thus severely affecting genome functionality. In the present review, we summarize the current knowledge related to replication and transcription conflicts in eukaryotes, their consequences on genome instability and the pathways involved in their resolution. These findings are relevant to clarify the molecular basis of cancer and neurodegenerative diseases.

  9. Recombination-dependent DNA replication stimulated by double-strand breaks in bacteriophage T4.

    Kreuzer, K N; Saunders, M; Weislo, L J; Kreuzer, H W

    1995-12-01

    We analyzed the mechanism of recombination-dependent DNA replication in bacteriophage T4-infected Escherichia coli using plasmids that have sequence homology to the infecting phage chromosome. Consistent with prior studies, a pBR322 plasmid, initially resident in the infected host cell, does not replicate following infection by T4. However, the resident plasmid can be induced to replicate when an integrated copy of pBR322 vector is present in the phage chromosome. As expected for recombination-dependent DNA replication, the induced replication of pBR322 required the phage-encoded UvsY protein. Therefore, recombination-dependent plasmid replication requires homology between the plasmid and phage genomes but does not depend on the presence of any particular T4 DNA sequence on the test plasmid. We next asked whether T4 recombination-dependent DNA replication can be triggered by a double-strand break (dsb). For these experiments, we generated a novel phage strain that cleaves its own genome within the nonessential frd gene by means of the I-TevI endonuclease (encoded within the intron of the wild-type td gene). The dsb within the phage chromosome substantially increased the replication of plasmids that carry T4 inserts homologous to the region of the dsb (the plasmids are not themselves cleaved by the endonuclease). The dsb stimulated replication when the plasmid was homologous to either or both sides of the break but did not stimulate the replication of plasmids with homology to distant regions of the phage chromosome. As expected for recombination-dependent replication, plasmid replication triggered by dsbs was dependent on T4-encoded recombination proteins. These results confirm two important predictions of the model for T4-encoded recombination-dependent DNA replication proposed by Gisela Mosig (p. 120-130, in C. K. Mathews, E. M. Kutter, G. Mosig, and P. B. Berget (ed.), Bacteriophage T4, 1983). In addition, replication stimulated by dsbs provides a site

  10. The barley EST DNA Replication and Repair Database (bEST-DRRD as a tool for the identification of the genes involved in DNA replication and repair

    Gruszka Damian

    2012-06-01

    Full Text Available Abstract Background The high level of conservation of genes that regulate DNA replication and repair indicates that they may serve as a source of information on the origin and evolution of the species and makes them a reliable system for the identification of cross-species homologs. Studies that had been conducted to date shed light on the processes of DNA replication and repair in bacteria, yeast and mammals. However, there is still much to be learned about the process of DNA damage repair in plants. Description These studies, which were conducted mainly using bioinformatics tools, enabled the list of genes that participate in various pathways of DNA repair in Arabidopsis thaliana (L. Heynh to be outlined; however, information regarding these mechanisms in crop plants is still very limited. A similar, functional approach is particularly difficult for a species whose complete genomic sequences are still unavailable. One of the solutions is to apply ESTs (Expressed Sequence Tags as the basis for gene identification. For the construction of the barley EST DNA Replication and Repair Database (bEST-DRRD, presented here, the Arabidopsis nucleotide and protein sequences involved in DNA replication and repair were used to browse for and retrieve the deposited sequences, derived from four barley (Hordeum vulgare L. sequence databases, including the “Barley Genome version 0.05” database (encompassing ca. 90% of barley coding sequences and from two databases covering the complete genomes of two monocot models: Oryza sativa L. and Brachypodium distachyon L. in order to identify homologous genes. Sequences of the categorised Arabidopsis queries are used for browsing the repositories, which are located on the ViroBLAST platform. The bEST-DRRD is currently used in our project during the identification and validation of the barley genes involved in DNA repair. Conclusions The presented database provides information about the Arabidopsis genes involved in

  11. BENZO(A)PYRENE DIOL EPOXIDE I BINDS TO DNA AT REPLICATION FORKS (JOURNAL VERSION)

    The distribution in replication forks of DNA lesions caused by the treatment of S phase calls with benzo(a)pyrene-diol-epoxide-1 (BPDE-1) was studied in synchronized C3H10T1/2 cells. Sites of carcinogen modification of DNA were identified by polyclonal rabbit antibodies that were...

  12. Tousled-like kinases phosphorylate Asf1 to promote histone supply during DNA replication

    Klimovskaia, Ilnaz M; Young, Clifford; Strømme, Caroline B;

    2014-01-01

    During DNA replication, nucleosomes are rapidly assembled on newly synthesized DNA to restore chromatin organization. Asf1, a key histone H3-H4 chaperone required for this process, is phosphorylated by Tousled-like kinases (TLKs). Here, we identify TLK phosphorylation sites by mass spectrometry and...

  13. Structure of the DNA-bound BRCA1 C-terminal Region from Human Replication Factor C p140 and Model of the Protein-DNA Complex

    2010-01-01

    BRCA1 C-terminal domain (BRCT)-containing proteins are found widely throughout the animal and bacteria kingdoms where they are exclusively involved in cell cycle regulation and DNA metabolism. Whereas most BRCT domains are involved in protein-protein interactions, a small subset has bona fide DNA binding activity. Here, we present the solution structure of the BRCT region of the large subunit of replication factor C bound to DNA and a model of the structure-specific complex with 5′-phosphoryl...

  14. Molecular genetic analysis of a vaccinia virus gene with an essential role in DNA replication

    The poxvirus, vaccinia, is large DNA virus which replicates in the cytoplasma of the host cell. The virus is believed to encode most or all of the functions required for the temporally regulated transcription and replication of its 186 kilobase genome. Physical and genetic autonomy from the host make vaccinia a useful eukaryotic organism in which to study replication genes and proteins, using a combination of biochemical and genetic techniques. Essential viral functions for replication are identified by conditional lethal mutants that fail to synthesize DNA at the non-permissive temperatures. One such group contains the non-complementing alleles ts17, ts24, ts69 (WR strain). Studies were undertaken to define the phenotype of ts mutants, and to identify and characterize the affected gene and protein. Mutant infection was essentially normal at 32 degree C, but at 39 degree C the mutants did not incorporate 3H-thymidine into nascent viral DNA or synthesize late viral proteins. If mutant cultures were shifted to non-permissive conditions at the height of replication, DNA synthesis was halted rapidly, implying that the mutants are defective in DNA elongation. The gene affected in the WR mutants and in ts6389, a DNA-minus mutant of the IHD strain, was mapped by marker rescue and corresponds to open reading frame 5 (orfD5) of the viral HindIII D fragment

  15. A reduction in ribonucleotide reductase activity slows down the chromosome replication fork but does not change its localization.

    Ingvild Odsbu

    Full Text Available BACKGROUND: It has been proposed that the enzymes of nucleotide biosynthesis may be compartmentalized or concentrated in a structure affecting the organization of newly replicated DNA. Here we have investigated the effect of changes in ribonucleotide reductase (RNR activity on chromosome replication and organization of replication forks in Escherichia coli. METHODOLOGY/PRINCIPAL FINDINGS: Reduced concentrations of deoxyribonucleotides (dNTPs obtained by reducing the activity of wild type RNR by treatment with hydroxyurea or by mutation, resulted in a lengthening of the replication period. The replication fork speed was found to be gradually reduced proportionately to moderate reductions in nucleotide availability. Cells with highly extended C periods showed a "delay" in cell division i.e. had a higher cell mass. Visualization of SeqA structures by immunofluorescence indicated no change in organization of the new DNA upon moderate limitation of RNR activity. Severe nucleotide limitation led to replication fork stalling and reversal. Well defined SeqA structures were not found in situations of extensive replication fork repair. In cells with stalled forks obtained by UV irradiation, considerable DNA compaction was observed, possibly indicating a reorganization of the DNA into a "repair structure" during the initial phase of the SOS response. CONCLUSION/SIGNIFICANCE: The results indicate that the replication fork is slowed down in a controlled manner during moderate nucleotide depletion and that a change in the activity of RNR does not lead to a change in the organization of newly replicated DNA. Control of cell division but not control of initiation was affected by the changes in replication elongation.

  16. DNA replication kinetics in x-irradiated Chinese hamster ovary cells

    The kinetics of semiconservative DNA replication have been studied in both asynchronous and synchronized Chinese hamster ovary cells (CHO) irradiated with x-ray doses up to 3000 rad. Amounts of DNA replicated were determined by isopycnic gradient centrifugation of DNA from cells which were incubated after irradiation in medium containing 5-bromodeoxyuridine (50 μg/ml) and 5-fluorodeoxyuridine (0.1 μg/ml). The results confirm that cells irradiated in early G1 phase experience a delay in their entry into S phase. This G1 block is dose independent in the range from 300 to 3000 rad and is 0.5 to 0.7 hr in length. Cells at the G1/S boundary are insensitive to x-ray induced perturbations of bulk DNA synthetic rates when exposed to doses less than 1000 rad. At doses in excess of 1000 rad, these cells are inhibited from replicating their DNA for a time, but ultimately replicate near-control levels of their DNA. Cells irradiated in S phase again show no effects of x-ray doses below 1000 rad on their ability to replicate bulk DNA. After a 3000-rad exposure, however, the rate of DNA replication in these S-phase cells is markedly reduced compared to that of controls. Irradiation of asynchronous cells with doses from 150 to 3000 rad does reduce the rate of semiconservative DNA replication in these cultures in a dose-dependent manner. These results confirm that x-ray doses greater than 1000 rad reduce the rate of DNA synthesis in irradiated S-phase cells, thus prolonging the length of S phase. The combined data from asynchronous or synchronized cultures, irradiated with x-ray doses less than 1000 rad, indicate that at least a portion of the reduction in DNA replication rates in irradiated asynchronous CHO cultures is due to the x-ray induced G1 block, which reduces the overall number of cells in S phase after irradiation

  17. A Network of Multi-Tasking Proteins at the DNA Replication Fork Preserves Genome Stability.

    2005-12-01

    Full Text Available To elucidate the network that maintains high fidelity genome replication, we have introduced two conditional mutant alleles of DNA2, an essential DNA replication gene, into each of the approximately 4,700 viable yeast deletion mutants and determined the fitness of the double mutants. Fifty-six DNA2-interacting genes were identified. Clustering analysis of genomic synthetic lethality profiles of each of 43 of the DNA2-interacting genes defines a network (consisting of 322 genes and 876 interactions whose topology provides clues as to how replication proteins coordinate regulation and repair to protect genome integrity. The results also shed new light on the functions of the query gene DNA2, which, despite many years of study, remain controversial, especially its proposed role in Okazaki fragment processing and the nature of its in vivo substrates. Because of the multifunctional nature of virtually all proteins at the replication fork, the meaning of any single genetic interaction is inherently ambiguous. The multiplexing nature of the current studies, however, combined with follow-up supporting experiments, reveals most if not all of the unique pathways requiring Dna2p. These include not only Okazaki fragment processing and DNA repair but also chromatin dynamics.

  18. Trashing of single-stranded DNA generated during processing of arrested replication fork in E. coli.

    Kohiyama, Masamichi; Contremoulins, Vincent; Baudin, Xavier

    2013-11-29

    We analyzed formation of single-stranded DNA (ssDNA) related to SOS induction in nalidixilate (Nal)-treated Escherichia coli, using immunofluorescence microscopy accompanied by computer analysis. We found enhancement of both ssDNA concentrations and cells having ssDNA foci that often localized around cellpoles. Analyzing several mutants deficient in DNA repair or replication, we found, after Nal treatment, that recN, recA, uvrD and dnaB failed to increase ssDNA concentration and that recG and particularly ruvA only partially enhanced it. In Nal-treated recB mutant, despite its failure in SOS induction, ssDNA foci positive cells increased with a slight enhancement of its concentration. These observations suggest the existence of a cellular process that sequesters genotoxic ssDNA as inert form, offering a new concept for SOS suppressor genes action. PMID:23810902

  19. Phosphopeptide binding by Sld3 links Dbf4-dependent kinase to MCM replicative helicase activation.

    Deegan, Tom D; Yeeles, Joseph Tp; Diffley, John Fx

    2016-05-01

    The initiation of eukaryotic DNA replication requires the assembly of active CMG (Cdc45-MCM-GINS) helicases at replication origins by a set of conserved and essential firing factors. This process is controlled during the cell cycle by cyclin-dependent kinase (CDK) and Dbf4-dependent kinase (DDK), and in response to DNA damage by the checkpoint kinase Rad53/Chk1. Here we show that Sld3, previously shown to be an essential CDK and Rad53 substrate, is recruited to the inactive MCM double hexamer in a DDK-dependent manner. Sld3 binds specifically to DDK-phosphorylated peptides from two MCM subunits (Mcm4, 6) and then recruits Cdc45. MCM mutants that cannot bind Sld3 or Sld3 mutants that cannot bind phospho-MCM or Cdc45 do not support replication. Moreover, phosphomimicking mutants in Mcm4 and Mcm6 bind Sld3 without DDK and facilitate DDK-independent replication. Thus, Sld3 is an essential "reader" of DDK phosphorylation, integrating signals from three distinct protein kinase pathways to coordinate DNA replication during S phase. PMID:26912723

  20. The chromosomal protein HMGBI inhibits DNA replication in vitro. The role of post-synthetic acetylation

    The effect of HMGB1 protein on the replication of closed circular plasmid DNA in cell free extract have been studied using parental form of the protein, post-synthetically acetylated HMGB1 and HMGB1 lacking its acidic C-terminal tail. We have shown that HMGB1 protein inhibits DNA replication and that this effect is eliminated upon either acetylation of the protein or removal of the acidic C-terminal domain. An explanation of these findings suggests interactions of HMGB1 with a protein(s) of the replication complex resulting in reduction of its functional efficiency. Acetylation of HMGB1 affects these interactions in a way that restores the initial replication capacity of the system. The eventual protein-protein interactions are supposed to proceed via the C-terminal domain of HMGB1. (authors)

  1. 14-3-3sigma is a cruciform DNA binding protein and associates in vivo with origins of DNA replication.

    Alvarez, David; Novac, Olivia; Callejo, Mario; Ruiz, Marcia T; Price, Gerald B; Zannis-Hadjopoulos, Maria

    2002-01-01

    A human cruciform binding protein (CBP) was previously shown to bind to cruciform DNA in a structure-specific manner and be a member of the 14-3-3 protein family. CBP had been found to contain the 14-3-3 isoforms beta, gamma, epsilon, and zeta. Here, we show by Western blot analysis that the CBP-cruciform DNA complex eluted from band-shift polyacrylamide gels also contains the 14-3-3sigma isoform, which is present in HeLa cell nuclear extracts. An antibody specific for the 14-3-3sigma isoform was able to interfere with the formation of the CBP-cruciform DNA complex. The effect of the same anti-14-3-3sigma antibody in the in vitro replication of p186, a plasmid containing the minimal replication origin of the monkey origin ors8, was also analyzed. Pre-incubation of total HeLa cell extracts with this antibody decreased p186 in vitro replication to approximately 30% of control levels, while non-specific antibodies had no effect. 14-3-3sigma was found to associate in vivo with the monkey origins of DNA replication ors8 and ors12 in a cell cycle-dependent manner, as assayed by a chromatin immunoprecipitation (ChIP) assay that involved formaldehyde cross-linking, followed by immunoprecipitation with anti-14-3-3sigma antibody and quantitative PCR. The association of 14-3-3sigma with the replication origins was maximal at the G(1)/S phase. The results indicate that 14-3-3sigma is an origin binding protein involved in the regulation of DNA replication via cruciform DNA binding. PMID:12244572

  2. Verifying likelihoods for low template DNA profiles using multiple replicates.

    Steele, C. D.; Greenhalgh, M; Balding, D. J.

    2014-01-01

    To date there is no generally accepted method to test the validity of algorithms used to compute likelihood ratios (LR) evaluating forensic DNA profiles from low-template and/or degraded samples. An upper bound on the LR is provided by the inverse of the match probability, which is the usual measure of weight of evidence for standard DNA profiles not subject to the stochastic effects that are the hallmark of low-template profiles. However, even for low-template profiles the LR in favour of a ...

  3. A subset of herpes simplex virus replication genes induces DNA amplification within the host cell genome.

    Heilbronn, R; zur Hausen, H

    1989-01-01

    Herpes simplex virus (HSV) induces DNA amplification of target genes within the host cell chromosome. To characterize the HSV genes that mediate the amplification effect, combinations of cloned DNA fragments covering the entire HSV genome were transiently transfected into simian virus 40 (SV40)-transformed hamster cells. This led to amplification of the integrated SV40 DNA sequences to a degree comparable to that observed after transfection of intact virion DNA. Transfection of combinations of subclones and of human cytomegalovirus immediate-early promoter-driven expression constructs for individual open reading frames led to the identification of six HSV genes which together were necessary and sufficient for the induction of DNA amplification: UL30 (DNA polymerase), UL29 (major DNA-binding protein), UL5, UL8, UL42, and UL52. All of these genes encode proteins necessary for HSV DNA replication. However, an additional gene coding for an HSV origin-binding protein (UL9) was required for origin-dependent HSV DNA replication but was dispensible for SV40 DNA amplification. Our results show that a subset of HSV replication genes is sufficient for the induction of DNA amplification. This opens the possibility that HSV expresses functions sufficient for DNA amplification but separate from those responsible for lytic viral growth. HSV infection may thereby induce DNA amplification within the host cell genome without killing the host by lytic viral growth. This may lead to persistence of a cell with a new genetic phenotype, which would have implications for the pathogenicity of the virus in vivo. Images PMID:2547992

  4. A subset of herpes simplex virus replication genes induces DNA amplification within the host cell genome

    Heilbronn, R.; zur Hausen, H. (Deutsches Krebsforschungszentrum, Heidelberg (West Germany))

    1989-09-01

    Herpes simplex virus (HSV) induces DNA amplification of target genes within the host cell chromosome. To characterize the HSV genes that mediate the amplification effect, combinations of cloned DNA fragments covering the entire HSV genome were transiently transfected into simian virus 40 (SV40)-transformed hamster cells. This led to amplification of the integrated SV40 DNA sequences to a degree comparable to that observed after transfection of intact virion DNA. Transfection of combinations of subclones and of human cytomegalovirus immediate-early promoter-driven expression constructs for individual open reading frames led to the identification of sic HSV genes which together were necessary and sufficient for the induction of DNA amplification: UL30 (DNA polymerase), UL29 (major DNA-binding protein), UL5, UL8, UL42, and UL52. All of these genes encode proteins necessary for HSV DNA replication. However, an additional gene coding for an HSV origin-binding protein (UL9) was required for origin-dependent HSV DNA replication but was dispensable for SV40 DNA amplification. The results show that a subset of HSV replication genes is sufficient for the induction of DNA amplification. This opens the possibility that HSV expresses functions sufficient for DNA amplification but separate from those responsible for lytic viral growth. HSV infection may thereby induce DNA amplification within the host cell genome without killing the host by lytic viral growth. This may lead to persistence of a cell with a new genetic phenotype, which would have implications for the pathogenicity of the virus in vivo.

  5. Involvement of Escherichia coli DNA Replication Proteins in Phage Lambda Red-Mediated Homologous Recombination.

    Anthony R Poteete

    Full Text Available The Red recombination system of bacteriophage lambda is widely used for genetic engineering because of its ability to promote recombination between bacterial chromosomes or plasmids and linear DNA species introduced by electroporation. The process is known to be intimately tied to replication, but the cellular functions which participate with Red in this process are largely unknown. Here two such functions are identified: the GrpE-DnaK-DnaJ chaperone system, and DNA polymerase I. Mutations in either function are found to decrease the efficiency of Red recombination. grpE and dnaJ mutations which greatly decrease Red recombination with electroporated DNA species have only small effects on Red-mediated transduction. This recombination event specificity suggests that the involvement of GrpE-DnaJ-DnaK is not simply an effect on Red structure or stability.

  6. Mediator proteins orchestrate enzyme-ssDNA assembly during T4 recombination-dependent DNA replication and repair

    Bleuit, Jill S.; Xu, Hang; Ma, Yujie; Wang, Tongsheng; Liu, Jie; Morrical, Scott W.

    2001-01-01

    Studies of recombination-dependent replication (RDR) in the T4 system have revealed the critical roles played by mediator proteins in the timely and productive loading of specific enzymes onto single-stranded DNA (ssDNA) during phage RDR processes. The T4 recombination mediator protein, uvsY, is necessary for the proper assembly of the T4 presynaptic filament (uvsX recombinase cooperatively bound to ssDNA), leading to the recombination-primed initiation of leading strand DNA synthesis. In the...

  7. Bi-directional routing of DNA mismatch repair protein human exonuclease 1 to replication foci and DNA double strand breaks

    Liberti, Sascha Emilie; Andersen, Sofie Dabros; Wang, Jing;

    2011-01-01

    Human exonuclease 1 (hEXO1) is implicated in DNA metabolism, including replication, recombination and repair, substantiated by its interactions with PCNA, DNA helicases BLM and WRN, and several DNA mismatch repair (MMR) proteins. We investigated the sub-nuclear localization of hEXO1 during S......-phase progression and in response to laser-induced DNA double strand breaks (DSBs). We show that hEXO1 and PCNA co-localize in replication foci. This apparent interaction is sustained throughout S-phase. We also demonstrate that hEXO1 is rapidly recruited to DNA DSBs. We have identified a PCNA interacting protein...... 390-490 and 787-846) are required to direct the protein to the DNA damage site. Our results reveal that protein domains in hEXO1 in conjunction with specific protein interactions control bi-directional routing of hEXO1 between on-going DNA replication and repair processes in living cells...

  8. Honey, I Shrunk the DNA : DNA Length as a Probe for Nucleic-Acid Enzyme Activity

    Oijen, Antoine M. van

    2007-01-01

    The replication, recombination, and repair of DNA are processes essential for the maintenance of genomic information and require the activity of numerous enzymes that catalyze the polymerization or digestion of DNA. This review will discuss how differences in elastic properties between single- and d

  9. DNA repair and replication fork helicases are differentially affected by alkyl phosphotriester lesion.

    Suhasini, Avvaru N; Sommers, Joshua A; Yu, Stephen; Wu, Yuliang; Xu, Ting; Kelman, Zvi; Kaplan, Daniel L; Brosh, Robert M

    2012-06-01

    DNA helicases are directly responsible for catalytically unwinding duplex DNA in an ATP-dependent and directionally specific manner and play essential roles in cellular nucleic acid metabolism. It has been conventionally thought that DNA helicases are inhibited by bulky covalent DNA adducts in a strand-specific manner. However, the effects of highly stable alkyl phosphotriester (PTE) lesions that are induced by chemical mutagens and refractory to DNA repair have not been previously studied for their effects on helicases. In this study, DNA repair and replication helicases were examined for unwinding a forked duplex DNA substrate harboring a single isopropyl PTE specifically positioned in the helicase-translocating or -nontranslocating strand within the double-stranded region. A comparison of SF2 helicases (RecQ, RECQ1, WRN, BLM, FANCJ, and ChlR1) with a SF1 DNA repair helicase (UvrD) and two replicative helicases (MCM and DnaB) demonstrates unique differences in the effect of the PTE on the DNA unwinding reactions catalyzed by these enzymes. All of the SF2 helicases tested were inhibited by the PTE lesion, whereas UvrD and the replication fork helicases were fully tolerant of the isopropyl backbone modification, irrespective of strand. Sequestration studies demonstrated that RECQ1 helicase was trapped by the PTE lesion only when it resided in the helicase-translocating strand. Our results are discussed in light of the current models for DNA unwinding by helicases that are likely to encounter sugar phosphate backbone damage during biological DNA transactions. PMID:22500020

  10. Functions of replication factor C and proliferating-cell nuclear antigen: Functional similarity of DNA polymerase accessory proteins from human cells and bacteriophage T4

    The proliferating-cell nuclear antigen (PCNA) and the replication factors A and C (RF-A and RF-C) are cellular proteins essential for complete elongation of DNA during synthesis from the simian virus 40 origin of DNA replication in vitro. All three cooperate to stimulate processive DNA synthesis by DNA polymerase δ on a primed single-stranded M13 template DNA and as such can be categorized as DNA polymerase accessory proteins. Biochemical analyses with highly purified RF-C and PCNA have demonstrated functions that are completely analogous to the functions of bacteriophage T4 DNA polymerase accessory proteins. A primer-template-specific DNA binding activity and a DNA-dependent ATPase activity copurified with the multisubunit protein RF-C and are similar to the functions of the phage T4 gene 44/62 protein complex. Furthermore, PCNA stimulated the RF-C ATPase activity and is, therefore, analogous to the phage T4 gene 45 protein, which stimulates the ATPase function of the gene 44/62 protein complex. Indeed, some primary sequence similarities between human PCNA and the phage T4 gene 45 protein could be detected. These results demonstrate a striking conservation of the DNA replication apparatus in human cells and bacteriophage T4

  11. Artesunate Reduces Proliferation, Interferes DNA Replication and Cell Cycle and Enhances Apoptosis in Vascular Smooth Muscle Cells

    2005-01-01

    This study examined the effect of artesunate (Art) on the proliferation, DNA replication, cell cycles and apoptosis of vascular smooth muscle cells (VSMCs). Primary cultures of VSMCs were established from aortas of mice and artesunate of different concentrations was added into the medium. The number of VSMCs was counted and the curve of cell growth was recorded.The activity of VSMCs was assessed by using MTT method and inhibitory rate was calculated.DNA replication was evaluated by [3 H]-TdR method and apoptosis by DNA laddering and HE staining. Flowmetry was used for simultaneous analysis of cell apoptosis and cell cycles. Compared with the control group, VSMCs proliferation in Art interfering groups were inhibited and [3H]-TdR incorprating rate were decreased as well as cell apoptosis was induced. The progress of cell cycle was blocked in G0/G1 by Art in a dose-dependent manner. It is concluded that Art inhibits VSMCs proliferation by disturbing DNA replication, inducing cell apoptosis and blocking cell cycle in G0/G1 phase.

  12. Direct interaction between DNMT1and G9a coordinates DNA and histone methylation during replication

    Estève, Pierre-Olivier; Chin, Hang Gyeong; Smallwood, Andrea; Feehery, George R; Gangisetty, Omkaram; Karpf, Adam R.; Carey, Michael F.; Pradhan, Sriharsa

    2006-01-01

    Chromatin methylation is necessary for stable repression of gene expression during mammalian development. During cell division, DNMT1 maintains the DNA methylation pattern of the newly synthesized daughter strand, while G9a methylates H3K9. Here, DNMT1 is shown to directly bind G9a both in vivo and in vitro and to colocalize in the nucleus during DNA replication. The complex of DNMT1 and G9a colocalizes with dimethylated H3K9 (H3K9me2) at replication foci. Similarly, another H3K9 histone meth...

  13. DNA Replication Control Is Linked to Genomic Positioning of Control Regions in Escherichia coli.

    Frimodt-Møller, Jakob; Charbon, Godefroid; Krogfelt, Karen A; Løbner-Olesen, Anders

    2016-09-01

    Chromosome replication in Escherichia coli is in part controlled by three non-coding genomic sequences, DARS1, DARS2, and datA that modulate the activity of the initiator protein DnaA. The relative distance from oriC to the non-coding regions are conserved among E. coli species, despite large variations in genome size. Here we use a combination of i) site directed translocation of each region to new positions on the bacterial chromosome and ii) random transposon mediated translocation followed by culture evolution, to show genetic evidence for the importance of position. Here we provide evidence that the genomic locations of these regulatory sequences are important for cell cycle control and bacterial fitness. In addition, our work shows that the functionally redundant DARS1 and DARS2 regions play different roles in replication control. DARS1 is mainly involved in maintaining the origin concentration, whether DARS2 is also involved in maintaining single cell synchrony. PMID:27589233

  14. Peculiarities of the incorporation of [3H]thymidine into AT-rich regions of DNA during replicative synthesis

    The authors studied the role of the AT-rich regions in DNA replication in vivo. The authors selected cells of humans and Drosophila - organisms belonging to different types of alternation of unique and repetitive sequences - as the objects of investigation. The authors then studied the behavior of the AT-rich sequences in replication by the method of thermoelution of [3H]thymidine-labeled DNA, fragmented by ultrasound to 350 nucleotide pairs. By measuring the amount of DNA and the amount of the label in the fractions, the authors were able to construct curves of the change in the specific activity of DNA as a function of the temperature of elution from HAP and, consequently, as a function of the AT composition. The authors call them differential temperature chromatograms (DTC). Human peripheral blood lymphocytes were cultured according to the standard procedure with PHA (Difco P). A culture of D. melanogaster cells was labeled with [3H]thymidine in the logarithmic phase of growth for 1.2 and 42 h. At the end of the labeling, cell DNA was isolated from the lymphocytes and cell and nuclear DNA from a Drosophila tissue culture by the standard methods

  15. FBH1 influences DNA replication fork stability and homologous recombination through ubiquitylation of RAD51

    Chu, Wai Kit; Payne, Miranda J; Beli, Petra;

    2015-01-01

    leads to hyperrecombination, as well as several phenotypes indicative of an altered response to DNA replication stress. These effects are likely to be mediated by the enhanced nuclear matrix association of the ubiquitylation-resistant RAD51. These data are consistent with FBH1 acting as a negative......Unscheduled homologous recombination (HR) can lead to genomic instability, which greatly increases the threat of neoplastic transformation in humans. The F-box DNA helicase 1 (FBH1) is a 3'-5' DNA helicase with a putative function as a negative regulator of HR. It is the only known DNA helicase to...

  16. Pin1 Interacts with the Epstein-Barr Virus DNA Polymerase Catalytic Subunit and Regulates Viral DNA Replication

    Narita, Yohei; Murata, Takayuki; Ryo, Akihide; Kawashima, Daisuke; Sugimoto, Atsuko; Kanda, Teru; Kimura, Hiroshi; Tsurumi, Tatsuya

    2013-01-01

    Peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (Pin1) protein is known as a regulator which recognizes phosphorylated Ser/Thr-Pro motifs and increases the rate of cis and trans amide isomer interconversion, thereby altering the conformation of its substrates. We found that Pin1 knockdown using short hairpin RNA (shRNA) technology resulted in strong suppression of productive Epstein-Barr virus (EBV) DNA replication. We further identified the EBV DNA polymerase catalytic subunit, BALF5,...

  17. Induced excision repair is required for repair of lesions in the vicinity of DNA replication forks

    A technique for resolving DNA fragments containing replication forks from linear DNA fragments by two-dimensional agarose gel electrophoresis is described. The technique is based on the altered mobility in agarose of branched structures relative to linear double-stranded molecules as a function of gel concentration and voltage. When pulse-labeled DNA is isolated, purified, fragmented by digestion with restriction nucleases, and run in the two-dimensional gel system, the bulk of the DNA migrates in a single arc visible by staining with ethidium bromide. However, when autoradiograms are prepared from the gels, it can be seen that the nascent DNA, represented by the radioactive pulse label, is contained in a second distinct arc. We have shown by a variety of criteria that the nascent DNA migrating in this minor arc has properties consistent with replication fork structures. We have now applied this technique to testing the hypothesis that long patch repair occurs at lesions in the vicinity of DNA replication forks. 2 figs

  18. NMR structure of the N-terminal domain of the replication initiator protein DnaA

    Wemmer, David E.; Lowery, Thomas J.; Pelton, Jeffrey G.; Chandonia, John-Marc; Kim, Rosalind; Yokota, Hisao; Wemmer, David E.

    2007-08-07

    DnaA is an essential component in the initiation of bacterial chromosomal replication. DnaA binds to a series of 9 base pair repeats leading to oligomerization, recruitment of the DnaBC helicase, and the assembly of the replication fork machinery. The structure of the N-terminal domain (residues 1-100) of DnaA from Mycoplasma genitalium was determined by NMR spectroscopy. The backbone r.m.s.d. for the first 86 residues was 0.6 +/- 0.2 Angstrom based on 742 NOE, 50 hydrogen bond, 46 backbone angle, and 88 residual dipolar coupling restraints. Ultracentrifugation studies revealed that the domain is monomeric in solution. Features on the protein surface include a hydrophobic cleft flanked by several negative residues on one side, and positive residues on the other. A negatively charged ridge is present on the opposite face of the protein. These surfaces may be important sites of interaction with other proteins involved in the replication process. Together, the structure and NMR assignments should facilitate the design of new experiments to probe the protein-protein interactions essential for the initiation of DNA replication.

  19. The relationship between pyrimidine dimers and replicating DNA in UV-irradiated human fibroblasts

    The relationship between pyrimidine dimers (measured as endonuclease-sensitive sites) and newly-synthesized DNA was examined in UV-irradiated normal and XP variant human fibroblasts. Following irradiation of normal cells, the frequency of pyrimidine dimer sites in sections of DNA which had been synthesized immediately before the UV-irradiation was similar to that in the bulk DNA. The frequency of pyrimidine dimer sites in the parental strands of replicating DNA in irradiated normal cells was similar to that in the bulk DNA. In UV-irradiated XP variant cells, the size of DNA synthesized in the presence of caffeine immediately after irradiation accurately corresponded with the average interdimer distance in the parental DNA. This suggested that in this experimental situation each pyrimidine dimer gives rise to a discontinuity or a termination site in the daughter strand. (author)

  20. Spatial Cross-Talk between Oxidative Stress and DNA Replication in Human Fibroblasts.

    Radulovic, Marko; Baqader, Noor O; Stoeber, Kai; Godovac-Zimmermann, Jasminka

    2016-06-01

    MS-based proteomics has been applied to a differential network analysis of the nuclear-cytoplasmic subcellular distribution of proteins between cell-cycle arrest: (a) at the origin activation checkpoint for DNA replication, or (b) in response to oxidative stress. Significant changes were identified for 401 proteins. Cellular response combines changes in trafficking and in total abundance to vary the local compartmental abundances that are the basis of cellular response. Appreciable changes for both perturbations were observed for 245 proteins, but cross-talk between oxidative stress and DNA replication is dominated by 49 proteins that show strong changes for both. Many nuclear processes are influenced by a spatial switch involving the proteins {KPNA2, KPNB1, PCNA, PTMA, SET} and heme/iron proteins HMOX1 and FTH1. Dynamic spatial distribution data are presented for proteins involved in caveolae, extracellular matrix remodelling, TGFβ signaling, IGF pathways, emerin complexes, mitochondrial protein import complexes, spliceosomes, proteasomes, and so on. The data indicate that for spatially heterogeneous cells cross-compartmental communication is integral to their system biology, that coordinated spatial redistribution for crucial protein networks underlies many functional changes, and that information on dynamic spatial redistribution of proteins is essential to obtain comprehensive pictures of cellular function. We describe how spatial data of the type presented here can provide priorities for further investigation of crucial features of high-level spatial coordination across cells. We suggest that the present data are related to increasing indications that much of subcellular protein transport is constitutive and that perturbation of these constitutive transport processes may be related to cancer and other diseases. A quantitative, spatially resolved nucleus-cytoplasm interaction network is provided for further investigations. PMID:27142241

  1. The X gene of adeno-associated virus 2 (AAV2 is involved in viral DNA replication.

    Maohua Cao

    Full Text Available Adeno-associated virus (AAV (type 2 is a popular human gene therapy vector with a long active transgene expression period and no reported vector-induced adverse reactions. Yet the basic molecular biology of this virus has not been fully addressed. One potential gene at the far 3' end of the AAV2 genome, previously referred to as X (nt 3929 to 4393, overlapping the 3' end of the cap gene, has never been characterized, although we did previously identify a promoter just up-stream (p81. Computer analysis suggested that X was involved in replication and transcription. The X protein was identified during active AAV2 replication using a polyclonal antibody against a peptide starting at amino acid 98. Reagents for the study of X included an AAV2 deletion mutant (dl78-91, a triple nucleotide substitution mutant that destroys all three 5' AUG-initiation products of X, with no effect on the cap coding sequence, and X-positive-293 cell lines. Here, we found that X up-regulated AAV2 DNA replication in differentiating keratinocytes (without helper virus, autonomous replication and in various forms of 293 cell-based assays with help from wild type adenovirus type 5 (wt Ad5 or Ad5 helper plasmid (pHelper. The strongest contribution by X was seen in increasing wt AAV2 DNA replication in keratinocytes and dl78-91 in Ad5-infected X-positive-293 cell lines (both having multi-fold effects. Mutating the X gene in pAAV-RC (pAAV-RC-3Xneg yielded approximately a ∼33% reduction in recombinant AAV vector DNA replication and virion production, but a larger effect was seen when using this same X-knockout AAV helper plasmid in X-positive-293 cell lines versus normal 293 cells (again, multi-fold. Taken together these data strongly suggest that AAV2 X encodes a protein involved in the AAV life cycle, particularly in increasing AAV2 DNA replication, and suggests that further studies are warranted.

  2. Replication protein A: directing traffic at the intersection of replication and repair

    Oakley, Greg G.; Patrick, Steve M.

    2010-01-01

    Since the initial discovery of replication protein A (RPA) as a DNA replication factor, much progress has been made on elucidating critical roles for RPA in other DNA metabolic pathways. RPA has been shown to be required for DNA replication, DNA repair, DNA recombination, and the DNA damage response pathway with roles in checkpoint activation. This review summarizes the current understanding of RPA structure, phosphorylation and protein-protein interactions in mediating these DNA metabolic pr...

  3. Isolation of DNA fragments containing replication forks by two dimensional agarose gel electrophoresis

    Escherichia coli is used as a model system to study DNA repair processes that promote survival after damage from alkylating agents and other processes that affect mutation resulting from such damage. In order to test the hypothesis that the induced incision repair process is able to repair this damage, a two-dimensional gel system has been developed that allows resolution of DNA fragments containing replication forks from the rest of the genome

  4. The linear plastid chromosomes of maize: terminal sequences, structures, and implications for DNA replication.

    Oldenburg, Delene J; Bendich, Arnold J

    2016-05-01

    The structure of a chromosomal DNA molecule may influence the way in which it is replicated and inherited. For decades plastid DNA (ptDNA) was believed to be circular, with breakage invoked to explain linear forms found upon extraction from the cell. Recent evidence indicates that ptDNA in vivo consists of linear molecules with discrete termini, although these ends were not characterized. We report the sequences of two terminal regions, End1 and End2, for maize (Zea mays L.) ptDNA. We describe structural features of these terminal regions and similarities found in other plant ptDNAs. The terminal sequences are within inverted repeat regions (leading to four genomic isomers) and adjacent to origins of replication. Conceptually, stem-loop structures may be formed following melting of the double-stranded DNA ends. Exonuclease digestion indicates that the ends in maize are unobstructed, but tobacco (Nicotiana tabacum L.) ends may have a 5'-protein. If the terminal structure of ptDNA molecules influences the retention of ptDNA, the unprotected molecular ends in mature leaves of maize may be more susceptible to degradation in vivo than the protected ends in tobacco. The terminal sequences and cumulative GC skew profiles are nearly identical for maize, wheat (Triticum aestivum L.) and rice (Oryza sativa L.), with less similarity among other plants. The linear structure is now confirmed for maize ptDNA and inferred for other plants and suggests a virus-like recombination-dependent replication mechanism for ptDNA. Plastid transformation vectors containing the terminal sequences may increase the chances of success in generating transplastomic cereals. PMID:26650613

  5. PARP1 inhibition radiosensitizes HNSCC cells deficient in homologous recombination by disabling the DNA replication fork elongation response.

    Wurster, Stephanie; Hennes, Fabian; Parplys, Ann C; Seelbach, Jasna I; Mansour, Wael Y; Zielinski, Alexandra; Petersen, Cordula; Clauditz, Till S; Münscher, Adrian; Friedl, Anna A; Borgmann, Kerstin

    2016-03-01

    There is a need to develop new, more efficient therapies for head and neck cancer (HNSCC) patients. It is currently unclear whether defects in DNA repair genes play a role in HNSCCs' resistance to therapy. PARP1 inhibitors (PARPi) were found to be "synthetic lethal" in cancers deficient in BRCA1/2 with impaired homologous recombination. Since tumors rarely have these particular mutations, there is considerable interest in finding alternative determinants of PARPi sensitivity. Effectiveness of combined irradiation and PARPi olaparib was evaluated in ten HNSCC cell lines, subdivided into HR-proficient and HR-deficient cell lines using a GFP-based reporter assay. Both groups were equally sensitive to PARPi alone. Combined treatment revealed stronger synergistic interactions in the HR-deficient group. Because HR is mainly active in S-Phase, replication processes were analyzed. A stronger impact of treatment on replication processes (p = 0.04) and an increased number of radial chromosomes (p = 0.003) were observed in the HR-deficient group. We could show that radiosensitization by inhibition of PARP1 strongly correlates with HR competence in a replication-dependent manner. Our observations indicate that PARP1 inhibitors are promising candidates for enhancing the therapeutic ratio achieved by radiotherapy via disabling DNA replication processes in HR-deficient HNSCCs. PMID:26799421

  6. A tale of two HSV-1 helicases: roles of phage and animal virus helicases in DNA replication and recombination.

    Marintcheva, B; Weller, S K

    2001-01-01

    Helicases play essential roles in many important biological processes such as DNA replication, repair, recombination, transcription, splicing, and translation. Many bacteriophages and plant and animal viruses encode one or more helicases, and these enzymes have been shown to play many roles in their respective viral life cycles. In this review we concentrate primarily on the roles of helicases in DNA replication and recombination with special emphasis on the bacteriophages T4, T7, and A as model systems. We explore comparisons between these model systems and the herpesviruses--primarily herpes simplex virus. Bacteriophage utilize various pathways of recombination-dependent DNA replication during the replication of their genomes. In fact the study of recombination in the phage systems has greatly enhanced our understanding of the importance of recombination in the replication strategies of bacteria, yeast, and higher eukaryotes. The ability to "restart" the replication process after a replication fork has stalled or has become disrupted for other reasons is a critical feature in the replication of all organisms studied. Phage helicases and other recombination proteins play critical roles in the "restart" process. Parallels between DNA replication and recombination in phage and in the herpesviruses is explored. We and others have proposed that recombination plays an important role in the life cycle of the herpesviruses, and in this review, we discuss models for herpes simplex virus type 1 (HSV-1) DNA replication. HSV-1 encodes two helicases. UL9 binds specifically to the origins of replication and is believed to initiate HSV DNA replication by unwinding at the origin; the heterotrimeric helicase-primase complex, encoded by UL5, UL8, and UL52 genes, is believed to unwind duplex viral DNA at replication forks. Structure-function analyses of UL9 and the helicase-primase are discussed with attention to the roles these proteins might play during HSV replication. PMID

  7. Susceptibility to superhelically driven DNA duplex destabilization: a highly conserved property of yeast replication origins.

    2005-06-01

    Full Text Available Strand separation is obligatory for several DNA functions, including replication. However, local DNA properties such as A+T content or thermodynamic stability alone do not determine the susceptibility to this transition in vivo. Rather, superhelical stresses provide long-range coupling among the transition behaviors of all base pairs within a topologically constrained domain. We have developed methods to analyze superhelically induced duplex destabilization (SIDD in genomic DNA that take into account both this long-range stress-induced coupling and sequence-dependent local thermodynamic stability. Here we apply this approach to examine the SIDD properties of 39 experimentally well-characterized autonomously replicating DNA sequences (ARS elements, which function as replication origins in the yeast Saccharomyces cerevisiae. We find that these ARS elements have a strikingly increased susceptibility to SIDD relative to their surrounding sequences. On average, these ARS elements require 4.78 kcal/mol less free energy to separate than do their immediately surrounding sequences, making them more than 2,000 times easier to open. Statistical analysis shows that the probability of this strong an association between SIDD sites and ARS elements arising by chance is approximately 4 x 10. This local enhancement of the propensity to separate to single strands under superhelical stress has obvious implications for origin function. SIDD properties also could be used, in conjunction with other known origin attributes, to identify putative replication origins in yeast, and possibly in other metazoan genomes.

  8. Visual Representations of DNA Replication: Middle Grades Students' Perceptions and Interpretations

    Patrick, Michelle D.; Carter, Glenda; Wiebe, Eric N.

    2005-01-01

    Visual representations play a critical role in the communication of science concepts for scientists and students alike. However, recent research suggests that novice students experience difficulty extracting relevant information from representations. This study examined students' interpretations of visual representations of DNA replication. Each…

  9. Bacteriophage resistance of a Delta thyA mutant of Lactococcus lactis blocked in DNA replication

    Pedersen, M.B.; Jensen, Peter Ruhdal; Janzen, T.;

    2002-01-01

    , such as milk, there was no detectable d'ITP pool in the cells. Hence, DNA replication was abolished, and acidification by MBP71 was completely unaffected by the presence of nine different phages tested at a multiplicity of infection (MOI) of 0.1. Nonreplicating MBP71 must be inoculated at a higher...

  10. Guided Practice Software for Teaching DNA Replication to Senior High School Students

    Woods, Eric C.; McKinnon, Alan E.; Hickford, Jonathan G. H.; Abell, Walt A.

    2008-01-01

    The prototype of a guided practice application was developed to instruct year 13 biology students in the process of DNA replication. The application uses a high degree of interaction to engage the student in a guided exploration and problem solving exercise. An evaluation revealed that the students showed considerable enthusiasm and significant…

  11. CHK1 Inhibition Synergizes with Gemcitabine Initially by Destabilizing the DNA Replication Apparatus.

    Koh, Siang-Boon; Courtin, Aurélie; Boyce, Richard J; Boyle, Robert G; Richards, Frances M; Jodrell, Duncan I

    2015-09-01

    Combining cell-cycle checkpoint kinase inhibitors with the DNA-damaging chemotherapeutic agent gemcitabine offers clinical appeal, with a mechanistic rationale based chiefly on abrogation of gemcitabine-induced G2-M checkpoint activation. However, evidence supporting this mechanistic rationale from chemosensitization studies has not been consistent. Here we report a systematic definition of how pancreatic cancer cells harboring mutant p53 respond to this combination therapy, by combining mathematical models with large-scale quantitative biologic analyses of single cells and cell populations. Notably, we uncovered a dynamic range of mechanistic effects at different ratios of gemcitabine and CHK1 inhibitors. Remarkably, effective synergy was attained even where cells exhibited an apparently functional G2-M surveillance mechanism, as exemplified by a lack of both overt premature CDK1 activation and S-phase mitotic entry. Consistent with these findings, S-G2 duration was extended in treated cells, leading to a definable set of lineage-dependent catastrophic fates. At synergistic drug concentrations, global replication stress was a distinct indicator of chemosensitization as characterized molecularly by an accumulation of S-phase cells with high levels of hyperphosphorylated RPA-loaded single-stranded DNA. In a fraction of these cells, persistent genomic damage was observed, including chromosomal fragmentation with a loss of centromeric regions that prevented proper kinetochore-microtubule attachment. Together, our results suggested a "foot-in-the-door" mechanism for drug synergy where cells were destroyed not by frank G2-M phase abrogation but rather by initiating a cumulative genotoxicity that deregulated DNA synthesis. PMID:26141863

  12. An autoradiographic demonstration of nuclear DNA replication by DNA polymerase alpha and of mitochondrial DNA synthesis by DNA polymerase gamma.

    Geuskens, M.; Hardt, N; Pedrali-Noy, G; Spadari, S

    1981-01-01

    The incorporation of thymidine into the DNA of eukaryotic cells is markedly depressed, but not completely inhibited, by aphidicolin, a highly specific inhibitor of DNA polymerase alpha. An electron microscope autoradiographic analysis of the synthesis of nuclear and mitochondrial DNA in vivo in Concanavalin A stimulated rabbit spleen lymphocytes and in Hamster cell cultures, in the absence and in the presence of aphidicolin, revealed that aphidicolin inhibits the nuclear but not the mitochond...

  13. Disruption of mitochondrial DNA replication in Drosophila increases mitochondrial fast axonal transport in vivo.

    Rehan M Baqri

    Full Text Available Mutations in mitochondrial DNA polymerase (pol gamma cause several progressive human diseases including Parkinson's disease, Alper's syndrome, and progressive external ophthalmoplegia. At the cellular level, disruption of pol gamma leads to depletion of mtDNA, disrupts the mitochondrial respiratory chain, and increases susceptibility to oxidative stress. Although recent studies have intensified focus on the role of mtDNA in neuronal diseases, the changes that take place in mitochondrial biogenesis and mitochondrial axonal transport when mtDNA replication is disrupted are unknown. Using high-speed confocal microscopy, electron microscopy and biochemical approaches, we report that mutations in pol gamma deplete mtDNA levels and lead to an increase in mitochondrial density in Drosophila proximal nerves and muscles, without a noticeable increase in mitochondrial fragmentation. Furthermore, there is a rise in flux of bidirectional mitochondrial axonal transport, albeit with slower kinesin-based anterograde transport. In contrast, flux of synaptic vesicle precursors was modestly decreased in pol gamma-alpha mutants. Our data indicate that disruption of mtDNA replication does not hinder mitochondrial biogenesis, increases mitochondrial axonal transport, and raises the question of whether high levels of circulating mtDNA-deficient mitochondria are beneficial or deleterious in mtDNA diseases.

  14. Exonuclease mutations in DNA polymerase epsilon reveal replication strand specific mutation patterns and human origins of replication.

    Shinbrot, Eve; Henninger, Erin E; Weinhold, Nils; Covington, Kyle R; Göksenin, A Yasemin; Schultz, Nikolaus; Chao, Hsu; Doddapaneni, HarshaVardhan; Muzny, Donna M; Gibbs, Richard A; Sander, Chris; Pursell, Zachary F; Wheeler, David A

    2014-11-01

    Tumors with somatic mutations in the proofreading exonuclease domain of DNA polymerase epsilon (POLE-exo*) exhibit a novel mutator phenotype, with markedly elevated TCT→TAT and TCG→TTG mutations and overall mutation frequencies often exceeding 100 mutations/Mb. Here, we identify POLE-exo* tumors in numerous cancers and classify them into two groups, A and B, according to their mutational properties. Group A mutants are found only in POLE, whereas Group B mutants are found in POLE and POLD1 and appear to be nonfunctional. In Group A, cell-free polymerase assays confirm that mutations in the exonuclease domain result in high mutation frequencies with a preference for C→A mutation. We describe the patterns of amino acid substitutions caused by POLE-exo* and compare them to other tumor types. The nucleotide preference of POLE-exo* leads to increased frequencies of recurrent nonsense mutations in key tumor suppressors such as TP53, ATM, and PIK3R1. We further demonstrate that strand-specific mutation patterns arise from some of these POLE-exo* mutants during genome duplication. This is the first direct proof of leading strand-specific replication by human POLE, which has only been demonstrated in yeast so far. Taken together, the extremely high mutation frequency and strand specificity of mutations provide a unique identifier of eukaryotic origins of replication. PMID:25228659

  15. Genome-wide Analysis Reveals Extensive Functional Interaction between DNA Replication Initiation and Transcription in the Genome of Trypanosoma brucei

    Calvin Tiengwe

    2012-07-01

    Full Text Available Identification of replication initiation sites, termed origins, is a crucial step in understanding genome transmission in any organism. Transcription of the Trypanosoma brucei genome is highly unusual, with each chromosome comprising a few discrete transcription units. To understand how DNA replication occurs in the context of such organization, we have performed genome-wide mapping of the binding sites of the replication initiator ORC1/CDC6 and have identified replication origins, revealing that both localize to the boundaries of the transcription units. A remarkably small number of active origins is seen, whose spacing is greater than in any other eukaryote. We show that replication and transcription in T. brucei have a profound functional overlap, as reducing ORC1/CDC6 levels leads to genome-wide increases in mRNA levels arising from the boundaries of the transcription units. In addition, ORC1/CDC6 loss causes derepression of silent Variant Surface Glycoprotein genes, which are critical for host immune evasion.

  16. Soluble histone H2AX is induced by DNA replication stress and sensitizes cells to undergo apoptosis

    Duensing Stefan

    2008-07-01

    Full Text Available Abstract Background Chromatin-associated histone H2AX is a key regulator of the cellular responses to DNA damage. However, non-nucleosomal functions of histone H2AX are poorly characterized. We have recently shown that soluble H2AX can trigger apoptosis but the mechanisms leading to non-chromatin-associated H2AX are unclear. Here, we tested whether stalling of DNA replication, a common event in cancer cells and the underlying mechanism of various chemotherapeutic agents, can trigger increased soluble H2AX. Results Transient overexpression of H2AX was found to lead to a detectable fraction of soluble H2AX and was associated with increased apoptosis. This effect was enhanced by the induction of DNA replication stress using the DNA polymerase α inhibitor aphidicolin. Cells manipulated to stably express H2AX did not contain soluble H2AX, however, short-term treatment with aphidicolin (1 h resulted in detectable amounts of H2AX in the soluble nuclear fraction and enhanced apoptosis. Similarly, soluble endogenous H2AX was detected under these conditions. We found that excessive soluble H2AX causes chromatin aggregation and inhibition of ongoing gene transcription as evidenced by the redistribution and/or loss of active RNA polymerase II as well as the transcriptional co-activators CBP and p300. Conclusion Taken together, these results show that DNA replication stress rapidly leads to increased soluble H2AX and that non-chromatin-associated H2AX can sensitize cells to undergo apoptosis. Our findings encourage further studies to explore H2AX and the cellular pathways that control its expression as anti-cancer drug targets.

  17. Interaction of the replication terminator protein of Bacillus subtilis with DNA probed by NMR spectroscopy

    Termination of DNA replication in Bacillus subtilis involves the polar arrest of replication forks by a specific complex formed between the dimeric 29 kDa replication terminator protein (RTP) and DNA terminator sites. We have used NMR spectroscopy to probe the changes in 1H-15N correlation spectra of a 15N-labelled RTP.C110S mutant upon the addition of a 21 base pair symmetrical DNA binding site. Assignment of the 1H-15N correlations was achieved using a suite of triple resonance NMR experiments with 15N,13C,70% 2H enriched protein recorded at 800 MHz and using TROSY pulse sequences. Perturbations to 1H-15N spectra revealed that the N-termini, α3-helices and several loops are affected by the binding interaction. An analysis of this data in light of the crystallographically determined apo- and DNA-bound forms of RTP.C110S revealed that the NMR spectral perturbations correlate more closely to protein structural changes upon complex formation rather than to interactions at the protein-DNA interface

  18. Reversal of DNA methylation with 5-azacytidine alters chromosome replication patterns in human lymphocyte and fibroblast cultures.

    Shafer, D A; Priest, J H

    1984-01-01

    Prior studies demonstrated that developmental or induced methylation of DNA can inactivate associated gene loci. Such DNA methylation can be reversed and specific genes reactivated by treatment with 5-azacytidine (5- azaC ). The present cytogenetic studies using replication banding methods show that 5- azaC treatment also results in an increase or decrease in replication staining at one or more band locations in human lymphocyte and fibroblast chromosomes. New replication band locations are n...

  19. Ultraviolet-induced cell death is independent of DNA replication in rat kangeroo cells

    Miyaji, E.N.; Menck, C.F.M. [Sao Paulo Univ., SP (Brazil). Inst. de Biociencias

    1995-05-01

    Rat kangaroo (Potorous tridactylus) cells have an efficient repair system for photoreactivation of lethal lesions induced by 254 nm UV. However, this ability is lost with increasing time after UV, being completely ineffective after 24 h. Critical events leading to UV-induced cell death must occur within this period of time. DNA synthesis was inhibited by the DNA polymerase inhibitor aphidicolin and the loss of the capability to photorepair lethal lesions was maintained for replicating cells. Similar data were obtained in synchronized cells UV irradiated immediately before S phase. Under the same conditions, the ability to remove cyclobutane pyrimidine dimers by photoreactivation in these cells remained unchanged 24 h after irradiation. These data indicate that the critical events responsible for UV-induced cell death occur in the absence of DNA replication. (author).

  20. Ultraviolet-induced cell death is independent of DNA replication in rat kangeroo cells

    Rat kangaroo (Potorous tridactylus) cells have an efficient repair system for photoreactivation of lethal lesions induced by 254 nm UV. However, this ability is lost with increasing time after UV, being completely ineffective after 24 h. Critical events leading to UV-induced cell death must occur within this period of time. DNA synthesis was inhibited by the DNA polymerase inhibitor aphidicolin and the loss of the capability to photorepair lethal lesions was maintained for replicating cells. Similar data were obtained in synchronized cells UV irradiated immediately before S phase. Under the same conditions, the ability to remove cyclobutane pyrimidine dimers by photoreactivation in these cells remained unchanged 24 h after irradiation. These data indicate that the critical events responsible for UV-induced cell death occur in the absence of DNA replication. (author)

  1. Suppression of thermosensitive initiation of DNA replication in a dnaR mutant of Escherichia coli by a rifampin resistance mutation in the rpoB gene.

    Sakakibara, Y

    1995-01-01

    The thermosensitivity of the Escherichia coli dnaR130 mutant in initiation of DNA replication was suppressed by a spontaneous rifampin resistance mutation in rpoB, the gene for the beta subunit of RNA polymerase. Among the dnaR-suppressing rpoB alleles obtained was rpoB22, which was able to suppress the thermosensitivity of the dnaA46 or dnaA167 mutant, but not that of the dnaA5 mutant, in initiation of replication. Some dnaA-suppressing rpoB alleles obtained from rifampin-resistant derivativ...

  2. Mechanisms of inhibition of DNA replication by ultraviolet light in normal human and xeroderma pigmentosum fibroblasts

    The inhibition of DNA replication in ultraviolet-irradiated human fibroblasts was characterized by quantitative analysis of radiation-induced alterations in the steady-state distribution of sizes of pulse-labeled, nascent DNA. Low, noncytotoxic fluences rapidly produced an inhibition of DNA synthesis in half-replicon-size replication intermediates. With time, the inhibition produced by low fluences spread progressively to include multi-replicon-size intermediates. The results indicate that ultraviolet radiation inhibits the initiation of DNA synthesis in replicons. Higher cytotoxic fluences inhibited DNA synthesis in operating replicons. Xeroderma pigmentosum fibroblasts with deficiencies in DNA excision repair exhibited an inhibition of replicon initiation after low radiation fluences, indicating the effect was not solely dependent upon operation of the nucleotidyl excision repair pathway. Owing to their inability to remove pyrimidine dimers ahead of DNA growing points, the repair-deficient cells also were more sensitive than normal cells to the ultraviolet-induced inhibition of chain elongation. Xeroderma pigmentosum cells belonging to the variant class were even more sensitive to inhibition of chain elongation despite their ability to remove pyrimidine dimers. The analysis suggested that normal and repair-deficient human fibroblasts either are able to rapidly bypass certain dimers or these dimers are not recognized by the chain elongation machinery. (author)

  3. Intrinsic bent DNA sites in the chromosomal replication origin of Xylella fastidiosa 9a5c

    F. Gimenes

    2008-04-01

    Full Text Available The features of the nucleotide sequences in both replication and promoter regions have been investigated in many organisms. Intrinsically bent DNA sites associated with transcription have been described in several prokaryotic organisms. The aim of the present study was to investigate intrinsic bent DNA sites in the segment that holds the chromosomal replication origin, oriC, of Xylella fastidiosa 9a5c. Electrophoretic behavior analyses, as well as in silico analyses of both the 2-D projection and helical parameters, were performed. The chromosomal segment analyzed contains the initial sequence of the rpmH gene, an intergenic region, the dnaA gene, the oriC sequence, and the 5' partial sequence of the dnaN gene. The analysis revealed fragments with reduced electrophoretic mobility, which indicates the presence of curved DNA segments. The analysis of the helical parameter ENDS ratio revealed three bent DNA sites (b1, b2, and b3 located in the rpmH-dnaA intergenic region, the dnaA gene, and the oriC 5' end, respectively. The chromosomal segment of X. fastidiosa analyzed here is rich in phased AT tracts and in CAnT motifs. The 2-D projection indicated a segment whose structure was determined by the cumulative effect of all bent DNA sites. Further, the in silico analysis of the three different bacterial oriC sequences indicated similar negative roll and twist >34.00° values. The DnaA box sequences, and other motifs in them, may be associated with the intrinsic DNA curvature.

  4. Asynchronous DNA replication within the human β-globin gene locus

    The timing of DNA replication of the human β-globin gene locus has been studied by blot hybridization of newly synthesized BrdUrd-substituted DNA from cells in different stages of the S phase. Using probes that span >120 kilobases across the human β-globin gene locus, the authors show that the majority of this domain replicates in early S phase in the human erythroleukemia cell line K562 and in middle-to-late S phase in the lymphoid cell line Manca. However, in K562 cells three small regions display a strikingly different replication pattern than adjacent sequences. These islands, located in the inter-γ-globin gene region and approximately 20 kilobases 5' to the ε-globin gene and 20 kilobases 3' to the β-globin gene, replicate later and throughout S phase. A similar area is also present in the α-globin gene region in K562 cells. They suggest that these regions may represent sites of termination of replication forks

  5. Mechanism of recruitment of DnaB helicase to the replication origin of the plasmid pSC101

    Datta, Hirock J.; Khatri, Ghan Shyam; Bastia, Deepak

    1999-01-01

    Although many bacterial chromosomes require only one replication initiator protein, e.g., DnaA, most plasmid replicons depend on dual initiators: host-encoded DnaA and plasmid-encoded Rep initiator protein for replication initiation. Using the plasmid pSC101 as a model system, this work investigates the biological rationale for the requirement for dual initiators and shows that the plasmid-encoded RepA specifically interacts with the replicative helicase DnaB. Mutations in DnaB or RepA that d...

  6. Theoretical models for the regulation of DNA replication in fast-growing bacteria

    Creutziger, Martin; Schmidt, Mischa; Lenz, Peter

    2012-09-01

    Growing in always changing environments, Escherichia coli cells are challenged by the task to coordinate growth and division. In particular, adaption of their growth program to the surrounding medium has to guarantee that the daughter cells obtain fully replicated chromosomes. Replication is therefore to be initiated at the right time, which is particularly challenging in media that support fast growth. Here, the mother cell initiates replication not only for the daughter but also for the granddaughter cells. This is possible only if replication occurs from several replication forks that all need to be correctly initiated. Despite considerable efforts during the last 40 years, regulation of this process is still unknown. Part of the difficulty arises from the fact that many details of the relevant molecular processes are not known. Here, we develop a novel theoretical strategy for dealing with this general problem: instead of analyzing a single model, we introduce a wide variety of 128 different models that make different assumptions about the unknown processes. By comparing the predictions of these models we are able to identify the key quantities that allow the experimental discrimination of the different models. Analysis of these quantities yields that out of the 128 models 94 are not consistent with available experimental data. From the remaining 34 models we are able to conclude that mass growth and DNA replication need either to be truly coupled, by coupling DNA replication initiation to the event of cell division, or to the amount of accumulated mass. Finally, we make suggestions for experiments to further reduce the number of possible regulation scenarios.

  7. Catalytic DNA with phosphatase activity

    Chandrasekar, Jagadeeswaran; Silverman, Scott K.

    2013-01-01

    Catalytic DNA sequences (deoxyribozymes, DNA enzymes, or DNAzymes) have been identified by in vitro selection for various catalytic activities. Expanding the limits of DNA catalysis is an important fundamental objective and may facilitate practical utility of catalysts that can be obtained from entirely unbiased (random) sequence populations. In this study, we show that DNA can catalyze Zn2+-dependent phosphomonoester hydrolysis of tyrosine and serine side chains (i.e., exhibit phosphatase ac...

  8. Effect of Bovine Papillomavirus E2 Protein-Specific Monoclonal Antibodies on Papillomavirus DNA Replication

    Kurg, Reet; Parik, Jüri; Juronen, Erkki; Sedman, Tiina; Abroi, Aare; Liiv,Ingrid; Langel, Ülo; Ustav, Mart

    1999-01-01

    The bovine papillomavirus type 1 (BPV-1) E2 protein is the master regulator of papillomavirus replication and transcription. We have raised a panel of monoclonal antibodies (MAbs) against the BPV-1 E2 protein and used them to probe the structure and function of the protein. Five MAbs reacted with linear epitopes, and four MAbs recognized conformation-dependent epitopes which mapped within the C-terminal DNA-binding and dimerization domain. MAb 1E2 was able to recognize the replication- and tr...

  9. Actinophage R4 integrase-based site-specific chromosomal integration of non-replicative closed circular DNA.

    Miura, Takamasa; Nishizawa, Akito; Nishizawa, Tomoyasu; Asayama, Munehiko; Shirai, Makoto

    2016-06-01

    The actinophage R4 integrase (Sre)-based molecular genetic engineering system was developed for the chromosomal integration of multiple genes in Escherichia coli. A cloned DNA fragment containing two attP sites, green fluorescent protein (gfp) as a first transgene, and an antibiotic resistance gene as a selection marker was self-ligated to generate non-replicative closed circular DNA (nrccDNA) for integration. nrccDNA was introduced into attB-inserted E. coli cells harboring the plasmid expressing Sre by electroporation. The expressed Sre catalyzed site-specific integration between one of the two attP sites on nrccDNA and the attB site on the E. coli chromosome. The integration frequency was affected by the chromosomal location of the target site. A second nrccDNA containing two attB sites, lacZα encoding the alpha fragment of β-galactosidase as a transgene, and another antibiotic resistance gene was integrated into the residual attP site on the gfp-integrated E. coli chromosome via one of the two attB sites according to reiterating site-specific recombination. The integrants clearly exhibited β-galactosidase activity and green fluorescence, suggesting the simultaneous expression of multiple recombinant proteins in E. coli. The results of the present study showed that a step-by-step integration procedure using nrccDNA achieved the chromosomal integration of multiple genes. PMID:26870903

  10. Single-molecule FRET and linear dichroism studies of DNA breathing and helicase binding at replication fork junctions

    Phelps, Carey; Lee, Wonbae; Jose, Davis; von Hippel, Peter H.; Marcus, Andrew H.

    2013-01-01

    Unique single-molecule fluorescence techniques were used to monitor DNA “breathing” at and near the junctions of model DNA replication forks on biologically relevant microsecond-to-millisecond time scales. Experiments performed in the absence and presence of helicase complexes addressed the role of these fluctuations in helicase function during DNA replication. These studies simultaneously monitored single-molecule Förster resonance energy transfer and single-molecule fluorescence linear dich...

  11. Cell cycle synchronization of E. coli using the stringent response, with fluorescence labeling assays for DNA content and replication

    Daniel J Ferullo; Cooper, Deani L.; Moore, Hayley R.; Lovett, Susan T.

    2009-01-01

    We describe a method for synchronization of the cell cycle in the bacterium E. coli. Treatment of asynchronous cultures with the amino acid analog, DL-serine hydroxamate, induces the stringent response, with concomitant arrest of DNA replication at initiation. Following release of the stringent response, cells initiate DNA replication in synchrony, as determined by flow cytometry for DNA content, Southern blotting and microscopy. This method has the advantage that it can be used in fully wild...

  12. DNA replication through G-quadruplex motifs is promoted by the S. cerevisiae Pif1 DNA helicase

    Paeschke, Katrin; Capra, John A.; Zakian, Virginia A.

    2011-01-01

    G-quadruplex (G4) DNA structures are extremely stable four-stranded secondary structures held together by non-canonical G-G base pairs. Genome-wide chromatin immuno-precipitation was used to determine the in vivo binding sites of the multi-functional S. cerevisiae Pif1 DNA helicase, a potent unwinder of G4 structures in vitro. G4 motifs were a significant subset of the high confidence Pif1 binding sites. Replication slowed in the vicinity of these motifs, and they were prone to breakage in Pi...

  13. The crystal structure of Neisseria gonorrhoeae PriB reveals mechanistic differences among bacterial DNA replication restart pathways

    Dong, Jinlan; George, Nicholas P.; Duckett, Katrina L.; DeBeer, Madeleine A.P.; Lopper, Matthew E. (UDRI); (UW-MED)

    2010-05-25

    Reactivation of repaired DNA replication forks is essential for complete duplication of bacterial genomes. However, not all bacteria encode homologs of the well-studied Escherichia coli DNA replication restart primosome proteins, suggesting that there might be distinct mechanistic differences among DNA replication restart pathways in diverse bacteria. Since reactivation of repaired DNA replication forks requires coordinated DNA and protein binding by DNA replication restart primosome proteins, we determined the crystal structure of Neisseria gonorrhoeae PriB at 2.7 {angstrom} resolution and investigated its ability to physically interact with DNA and PriA helicase. Comparison of the crystal structures of PriB from N. gonorrhoeae and E. coli reveals a well-conserved homodimeric structure consisting of two oligosaccharide/oligonucleotide-binding (OB) folds. In spite of their overall structural similarity, there is significant species variation in the type and distribution of surface amino acid residues. This correlates with striking differences in the affinity with which each PriB homolog binds single-stranded DNA and PriA helicase. These results provide evidence that mechanisms of DNA replication restart are not identical across diverse species and that these pathways have likely become specialized to meet the needs of individual organisms.

  14. Replication of M13 single—stranded DNA bearing a sitespecific ethenocytosine lesion by Escherichia coil cell extracts

    WANGGE; PAULMDUNMAN; 等

    1997-01-01

    Previous investigation on the mutagenic effects of 3,N4-Ethenocytosine (εC),a nonpairing DNA lesion,revealed the existence of a novel SOS-independent inducible mutagenic mechanism in E.coli termed UVM for UV modulation of mutagenesis.To investigate whether UVM is mediated by an alteration of DNA replication,we have set up an in vitro replication system in which phage M13 viral single-stranded DNA bearing a single site-specific (εC) residue is replicated by soluble protein extracts from E.coli cells.Replication products were analyzed by agarose gel electrophoresis and the frequency of translesion synthesis was determined by restriction endonuclease analyses.Our data indicate that DNA replication is strongly inhibited by εC,but that translesion DNA synthesis does occur in about 14% of the replicated DNA molecules.These results are very similar to those observed previously in vivo,and suggest that this experimental system may be suitable for evaluating alterations in DNA replication in UVM-induced cells.

  15. Collision of Trapped Topoisomerase 2 with Transcription and Replication: Generation and Repair of DNA Double-Strand Breaks with 5′ Adducts

    Hong Yan; Margaret Tammaro; Shuren Liao

    2016-01-01

    Topoisomerase 2 (Top2) is an essential enzyme responsible for manipulating DNA topology during replication, transcription, chromosome organization and chromosome segregation. It acts by nicking both strands of DNA and then passes another DNA molecule through the break. The 5′ end of each nick is covalently linked to the tyrosine in the active center of each of the two subunits of Top2 (Top2cc). In this configuration, the two sides of the nicked DNA are held together by the strong protein-prot...

  16. Restriction of Human Polyomavirus BK Virus DNA Replication in Murine Cells and Extracts

    Mahon, C.; Liang, B.; Tikhanovich, I.; et al

    2009-01-01

    BK virus (BKV) causes persistent and asymptomatic infections in most humans and is the etiologic agent of polyomavirus-associated nephropathy (PVAN) and other pathologies. Unfortunately, there are no animal models with which to study activation of BKV replication in the human kidney and the accompanying PVAN. Here we report studies of the restriction of BKV replication in murine cells and extracts and the cause(s) of this restriction. Upon infection of murine cells, BKV expressed large T anti...

  17. Homologous recombination repairs secondary replication induced DNA double-strand breaks after ionizing radiation

    Groth, Petra; Orta, Manuel Luís; Elvers, Ingegerd; Majumder, Muntasir Mamun; Lagerqvist, Anne; Helleday, Thomas

    2012-01-01

    Ionizing radiation (IR) produces direct two-ended DNA double-strand breaks (DSBs) primarily repaired by non-homologous end joining (NHEJ). It is, however, well established that homologous recombination (HR) is induced and required for repair of a subset of DSBs formed following IR. Here, we find that HR induced by IR is drastically reduced when post-DNA damage replication is inhibited in mammalian cells. Both IR-induced RAD51 foci and HR events in the hprt gene are reduced in the presence of ...

  18. RNA-primed initiation sites of DNA replication in the origin region of bacteriophage lambda genome.

    Yoda, K.; Yasuda, H; Jiang, X W; Okazaki, T

    1988-01-01

    Using DNA molecules synthesized in the early stage of lambda phage infection, deoxynucleotides at the transition sites from primer RNA to DNA synthesis have been mapped in the 1.5 kbase area of the lambda phage genome containing the genetically defined replication origin (ori lambda). Sites in the 1-strand (the polarity of the 1-strand is 5' to 3' from the left to the right direction of the lambda phage genetic map) were distributed both inside and outside of the ori lambda, whereas the sites...

  19. Role of the Escherichia coli Nucleotide Excision Repair Proteins in DNA Replication

    Moolenaar, Geri F.; Moorman, Celine; Goosen, Nora

    2000-01-01

    DNA polymerase I (PolI) functions both in nucleotide excision repair (NER) and in the processing of Okazaki fragments that are generated on the lagging strand during DNA replication. Escherichia coli cells completely lacking the PolI enzyme are viable as long as they are grown on minimal medium. Here we show that viability is fully dependent on the presence of functional UvrA, UvrB, and UvrD (helicase II) proteins but does not require UvrC. In contrast, ΔpolA cells grow even better when the u...

  20. DNA synthesis is initiated at two positions within the origin of replication of plasmid R1162.

    Lin, L S; Meyer, R J

    1987-01-01

    DNA synthesis of broad host-range plasmid R1162 is initiated from two positions, flanking a large (40 bp stem, 40 bp loop) inverted repeat. Each start-point is located within a highly conserved, but oppositely oriented, 10 base-pair sequence. Synthesis from the two positions converges within the intervening inverted repeat. An analysis of deletions suggests that both start positions must be present for synthesis. A model describing possible early events in replication of plasmid R1162 is pres...

  1. Stability of DNA triplexes on shuttle vector plasmids in the replication pool in mammalian cells.

    Lin, F L; Majumdar, A; Klotz, L C; Reszka, A. P.; Neidle, S; Seidman, M M

    2000-01-01

    Triple helix-forming oligonucleotides may be useful as gene-targeting reagents in vivo, for applications such as gene knockout. One important property of these complexes is their often remarkable stability, as demonstrated in solution and in cells following transfection. Although encouraging, these measurements do not necessarily report triplex stability in cellular compartments that support DNA functions such as replication and mutagenesis. We have devised a shuttle vector plasmid assay that...

  2. SETD2 loss-of-function promotes renal cancer branched evolution through replication stress and impaired DNA repair.

    Kanu, N; Grönroos, E; Martinez, P; Burrell, R A; Yi Goh, X; Bartkova, J; Maya-Mendoza, A; Mistrík, M; Rowan, A J; Patel, H; Rabinowitz, A; East, P; Wilson, G; Santos, C R; McGranahan, N; Gulati, S; Gerlinger, M; Birkbak, N J; Joshi, T; Alexandrov, L B; Stratton, M R; Powles, T; Matthews, N; Bates, P A; Stewart, A; Szallasi, Z; Larkin, J; Bartek, J; Swanton, C

    2015-11-12

    Defining mechanisms that generate intratumour heterogeneity and branched evolution may inspire novel therapeutic approaches to limit tumour diversity and adaptation. SETD2 (Su(var), Enhancer of zeste, Trithorax-domain containing 2) trimethylates histone-3 lysine-36 (H3K36me3) at sites of active transcription and is mutated in diverse tumour types, including clear cell renal carcinomas (ccRCCs). Distinct SETD2 mutations have been identified in spatially separated regions in ccRCC, indicative of intratumour heterogeneity. In this study, we have addressed the consequences of SETD2 loss-of-function through an integrated bioinformatics and functional genomics approach. We find that bi-allelic SETD2 aberrations are not associated with microsatellite instability in ccRCC. SETD2 depletion in ccRCC cells revealed aberrant and reduced nucleosome compaction and chromatin association of the key replication proteins minichromosome maintenance complex component (MCM7) and DNA polymerase δ hindering replication fork progression, and failure to load lens epithelium-derived growth factor and the Rad51 homologous recombination repair factor at DNA breaks. Consistent with these data, we observe chromosomal breakpoint locations are biased away from H3K36me3 sites in SETD2 wild-type ccRCCs relative to tumours with bi-allelic SETD2 aberrations and that H3K36me3-negative ccRCCs display elevated DNA damage in vivo. These data suggest a role for SETD2 in maintaining genome integrity through nucleosome stabilization, suppression of replication stress and the coordination of DNA repair. PMID:25728682

  3. Analysis of the Mitochondrial DNA and Its Replicative Capacity in Induced Pluripotent Stem Cells.

    Cagnone, Gael; Vaghjiani, Vijesh; Lee, William; Sun, Claire; Johnson, Jacqueline; Yeung, Ka-Yu; St John, Justin C

    2016-01-01

    The mitochondrial genome resides in the mitochondrion of nearly all mammalian cells. It is important for energy production as it encodes 13 of the key subunits of the electron transfer chain, which generates the vast majority of cellular ATP through the process of oxidative phosphorylation. As cells establish pluripotency, they regulate their mtDNA copy number so that they possess few copies but sufficient that they can be replicated to match the differentiated cell-specific requirements for ATP derived through oxidative phosphorylation. However, the failure to strictly regulate this process prevents pluripotent cells from differentiating. We describe a series of protocols that analyze mtDNA copy number, DNA methylation within the nuclear-encoded mtDNA-specific polymerase, and gene expression of the other factors that drive replication of the mitochondrial genome. We demonstrate how to measure ATP-generating capacity through oxygen respiratory capacity and total cellular ATP and lactate levels. Finally, we also describe how to detect mtDNA variants in pluripotent and differentiating cells using next-generation sequencing protocols and how the variants can be confirmed by high-resolution melt analysis. PMID:25646603

  4. The fidelity of DNA synthesis by eukaryotic replicative and translesion synthesis polymerases

    Scott D McCulloch; Thomas A Kunkel

    2008-01-01

    In their seminal publication describing the structure of the DNA double helix [1], Watson and Crick wrote what may be one of the greatest understatements in the scientific literature, namely that "It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material." Half a century later, we more fully appreciate what a huge challenge it is to replicate six billion nucleotides with the accuracy needed to stably maintain the human genome over many generations. This challenge is perhaps greater than was realized 50 years ago, because subsequent studies have revealed that the genome can be destabilized not only by environmental stresses that generate a large number and variety of potentially cytotoxic and mutagenic lesions in DNA but also by various sequence motifs of normal DNA that present challenges to replication. Towards a better understanding of the many determinants of genome stability, this chapter reviews the fidelity with which undamaged and damaged DNA is copied, with a focus on the eukaryotic B- and Y-family DNA polymerases, and considers how this fidelity is achieved.

  5. Nucleotide insertion initiated by van der Waals interaction during polymerase beta DNA replication

    Arulsamy, Andrew Das

    2011-01-01

    Immortality will remain a fantasy for as long as aging is determined by the erroneous biochemical reactions during a particular DNA replication. The replication and base excision repair mechanism, associated to eukaryotic DNA polymerase-beta enzyme are central to maintaining a healthy cell. Here, we give a series of unambiguous theoretical analyses and prove that the exclusive biochemical reaction involved in a single nucleotide insertion into the DNA primer can be efficiently tracked using the renormalized van der Waals interaction of the stronger type, and the Hermansson blue-shifting hydrogen bond effect. We found that there are two biochemical steps involved to complete the insertion of a single dCTP into the 3' end of a DNA primer. First, the O3' (from a DNA primer) initiates the nucleophilic attack on P_alpha?(from an incoming dCTP), in response, O3_alpha (bonded to P_alpha) retaliates by interacting with H' (bonded to O3'). These interactions are shown to be strongly interdependent and require the form...

  6. Assessment of the Role of the Central DNA Flap in Human Immunodeficiency Virus Type 1 Replication by Using a Single-Cycle Replication System

    Ao, Zhujun; Yao, Xiaojian; Éric A Cohen

    2004-01-01

    In this study, reverse transcriptase (RT)- and integrase (IN)-defective human immunodeficiency virus type 1 (HIV-1) was transcomplemented with Vpr-RT-IN fusion proteins to delineate pol sequences important for HIV-1 replication. Our results reveal that a 194-bp sequence encompassing the 3′end of the IN gene and containing the central DNA flap is necessary and sufficient for efficient HIV-1 single-cycle replication in dividing and nondividing cells. Furthermore, we show that the central DNA fl...

  7. DNA polymerase I is required for premeiotic DNA replication and sporulation but not for X-ray repair in Saccharomyces cerevisiae

    We have used a set of seven temperature-sensitive mutants in the DNA polymerase I gene of Saccharomyces cerevisiae to investigate the role of DNA polymerase I in various aspects of DNA synthesis in vivo. Previously, we showed that DNA polymerase I is required for mitotic DNA replication. Here we extend our studies to several stages of meiosis and repair of X-ray-induced damage. We find that sporulation is blocked in all of the DNA polymerase temperature-sensitive mutants and that premeiotic DNA replication does not occur. Commitment to meiotic recombination is only 2% of wild-type levels. Thus, DNA polymerase I is essential for these steps. However, repair of X-ray-induced single-strand breaks is not defective in the DNA polymerase temperature-sensitive mutants, and DNA polymerase I is therefore not essential for repair of such lesions. These results suggest that DNA polymerase II or III or both, the two other nuclear yeast DNA polymerases for which roles have not yet been established, carry out repair in the absence of DNA polymerase I, but that DNA polymerase II and III cannot compensate for loss of DNA polymerase I in meiotic replication and recombination. These results do not, however, rule out essential roles for DNA polymerase II or III or both in addition to that for DNA polymerase I

  8. Selecting 3D Chaotic Flow States for Accelerated DNA Replication in Micro Scale Convective PCR

    Priye, Aashish; Hassan, Yassin; Ugaz, Victor

    2016-01-01

    Micro-scale flow in cylindrical geometries can harness chaotic advection to perform complex thermally activated biochemical reactions such as the polymerase chain reaction (PCR). We have applied a 3D computational fluid dynamics model to resolve the complex flow patterns in such geometries. The resulting 3D flow trajectories are then used as input to a kinetic model to resolve the time evolution of DNA replication process. A simple mass action kinetic model was developed to couple these biochemical reactions with the intricate flow. Residence time analysis of virtual particles in the flow revealed that the flow has a strong chaotic component in wider geometries in comparison with taller geometries (quasi periodic motion). This work shows, for the first time that the chaotic aspect of the flow field plays a key role in determining the strength of the coupling between the reactions and the flow. Our model can quantify the doubling times of these reactions capturing the lag, exponential and plateau phases of PCR...

  9. Proteome-wide analysis of SUMO2 targets in response to pathological DNA replication stress in human cells

    Bursomanno, Sara; Beli, Petra; Khan, Asif M;

    2015-01-01

    subfamily. SUMO2/3, in contrast to SUMO1, are predominantly involved in the cellular response to certain stresses, including heat shock. Substantial evidence from studies in yeast has shown that SUMOylation plays an important role in the regulation of DNA replication and repair. Here, we report a proteomic...... analysis of proteins modified by SUMO2 in response to DNA replication stress in S phase in human cells. We have identified a panel of 22 SUMO2 targets with increased SUMOylation during DNA replication stress, many of which play key functions within the DNA replication machinery and/or in the cellular...... response to DNA damage. Interestingly, POLD3 was found modified most significantly in response to a low dose aphidicolin treatment protocol that promotes common fragile site (CFS) breakage. POLD3 is the human ortholog of POL32 in budding yeast, and has been shown to act during break-induced recombinational...

  10. NanoPCR observation: different levels of DNA replication fidelity in nanoparticle-enhanced polymerase chain reactions

    Nanoparticle-assisted PCR (polymerase chain reaction) technology is getting more and more attention recently. It is believed that some of the DNA recombinant technologies will be upgraded by nanotechnology in the near future, among which DNA replication is one of the core manipulation techniques. So whether or not the DNA replication fidelity is compromised in nanoparticle-assisted PCR is a question. In this study, a total of 16 different metallic and non-metallic nanoparticles (NPs) were tested for their effects on DNA replication fidelity in vitro and in vivo. Sixteen types of nanomaterials were distinctly different in enhancing the PCR efficiency, and their relative capacity to retain DNA replication fidelity was largely different from each other based on rpsL gene mutation assay. Generally speaking, metallic nanoparticles induced larger error rates in DNA replication fidelity than non-metallic nanoparticles, and non-metallic nanomaterials such as carbon nanopowder or nanotubes were still safe as PCR enhancers because they did not compromise the DNA replication fidelity in the Taq DNA polymerase-based PCR system.

  11. NanoPCR observation: different levels of DNA replication fidelity in nanoparticle-enhanced polymerase chain reactions

    Shen, Cenchao; Yang, Wenjuan; Ji, Qiaoli; Maki, Hisaji; Dong, Anjie; Zhang, Zhizhou

    2009-11-01

    Nanoparticle-assisted PCR (polymerase chain reaction) technology is getting more and more attention recently. It is believed that some of the DNA recombinant technologies will be upgraded by nanotechnology in the near future, among which DNA replication is one of the core manipulation techniques. So whether or not the DNA replication fidelity is compromised in nanoparticle-assisted PCR is a question. In this study, a total of 16 different metallic and non-metallic nanoparticles (NPs) were tested for their effects on DNA replication fidelity in vitro and in vivo. Sixteen types of nanomaterials were distinctly different in enhancing the PCR efficiency, and their relative capacity to retain DNA replication fidelity was largely different from each other based on rpsL gene mutation assay. Generally speaking, metallic nanoparticles induced larger error rates in DNA replication fidelity than non-metallic nanoparticles, and non-metallic nanomaterials such as carbon nanopowder or nanotubes were still safe as PCR enhancers because they did not compromise the DNA replication fidelity in the Taq DNA polymerase-based PCR system.

  12. AAV-mediated delivery of zinc finger nucleases targeting hepatitis B virus inhibits active replication.

    Nicholas D Weber

    Full Text Available Despite an existing effective vaccine, hepatitis B virus (HBV remains a major public health concern. There are effective suppressive therapies for HBV, but they remain expensive and inaccessible to many, and not all patients respond well. Furthermore, HBV can persist as genomic covalently closed circular DNA (cccDNA that remains in hepatocytes even during otherwise effective therapy and facilitates rebound in patients after treatment has stopped. Therefore, the need for an effective treatment that targets active and persistent HBV infections remains. As a novel approach to treat HBV, we have targeted the HBV genome for disruption to prevent viral reactivation and replication. We generated 3 zinc finger nucleases (ZFNs that target sequences within the HBV polymerase, core and X genes. Upon the formation of ZFN-induced DNA double strand breaks (DSB, imprecise repair by non-homologous end joining leads to mutations that inactivate HBV genes. We delivered HBV-specific ZFNs using self-complementary adeno-associated virus (scAAV vectors and tested their anti-HBV activity in HepAD38 cells. HBV-ZFNs efficiently disrupted HBV target sites by inducing site-specific mutations. Cytotoxicity was seen with one of the ZFNs. scAAV-mediated delivery of a ZFN targeting HBV polymerase resulted in complete inhibition of HBV DNA replication and production of infectious HBV virions in HepAD38 cells. This effect was sustained for at least 2 weeks following only a single treatment. Furthermore, high specificity was observed for all ZFNs, as negligible off-target cleavage was seen via high-throughput sequencing of 7 closely matched potential off-target sites. These results show that HBV-targeted ZFNs can efficiently inhibit active HBV replication and suppress the cellular template for HBV persistence, making them promising candidates for eradication therapy.

  13. AAV-mediated delivery of zinc finger nucleases targeting hepatitis B virus inhibits active replication.

    Weber, Nicholas D; Stone, Daniel; Sedlak, Ruth Hall; De Silva Feelixge, Harshana S; Roychoudhury, Pavitra; Schiffer, Joshua T; Aubert, Martine; Jerome, Keith R

    2014-01-01

    Despite an existing effective vaccine, hepatitis B virus (HBV) remains a major public health concern. There are effective suppressive therapies for HBV, but they remain expensive and inaccessible to many, and not all patients respond well. Furthermore, HBV can persist as genomic covalently closed circular DNA (cccDNA) that remains in hepatocytes even during otherwise effective therapy and facilitates rebound in patients after treatment has stopped. Therefore, the need for an effective treatment that targets active and persistent HBV infections remains. As a novel approach to treat HBV, we have targeted the HBV genome for disruption to prevent viral reactivation and replication. We generated 3 zinc finger nucleases (ZFNs) that target sequences within the HBV polymerase, core and X genes. Upon the formation of ZFN-induced DNA double strand breaks (DSB), imprecise repair by non-homologous end joining leads to mutations that inactivate HBV genes. We delivered HBV-specific ZFNs using self-complementary adeno-associated virus (scAAV) vectors and tested their anti-HBV activity in HepAD38 cells. HBV-ZFNs efficiently disrupted HBV target sites by inducing site-specific mutations. Cytotoxicity was seen with one of the ZFNs. scAAV-mediated delivery of a ZFN targeting HBV polymerase resulted in complete inhibition of HBV DNA replication and production of infectious HBV virions in HepAD38 cells. This effect was sustained for at least 2 weeks following only a single treatment. Furthermore, high specificity was observed for all ZFNs, as negligible off-target cleavage was seen via high-throughput sequencing of 7 closely matched potential off-target sites. These results show that HBV-targeted ZFNs can efficiently inhibit active HBV replication and suppress the cellular template for HBV persistence, making them promising candidates for eradication therapy. PMID:24827459

  14. SETD2 loss-of-function promotes renal cancer branched evolution through replication stress and impaired DNA repair

    Kanu, N.; Grönroos, E.; Martinez, P.;

    2015-01-01

    proteins minichromosome maintenance complex component (MCM7) and DNA polymerase δ hindering replication fork progression, and failure to load lens epithelium-derived growth factor and the Rad51 homologous recombination repair factor at DNA breaks. Consistent with these data, we observe chromosomal......, suppression of replication stress and the coordination of DNA repair.......-of-function through an integrated bioinformatics and functional genomics approach. We find that bi-allelic SETD2 aberrations are not associated with microsatellite instability in ccRCC. SETD2 depletion in ccRCC cells revealed aberrant and reduced nucleosome compaction and chromatin association of the key replication...

  15. Autophagic machinery activated by dengue virus enhances virus replication

    Autophagy is a cellular response against stresses which include the infection of viruses and bacteria. We unravel that Dengue virus-2 (DV2) can trigger autophagic process in various infected cell lines demonstrated by GFP-LC3 dot formation and increased LC3-II formation. Autophagosome formation was also observed under the transmission electron microscope. DV2-induced autophagy further enhances the titers of extracellular and intracellular viruses indicating that autophagy can promote viral replication in the infected cells. Moreover, our data show that ATG5 protein is required to execute DV2-induced autophagy. All together, we are the first to demonstrate that DV can activate autophagic machinery that is favorable for viral replication

  16. Hepatitis B virus basal core promoter mutations show lower replication fitness associated with cccDNA acetylation status.

    Koumbi, Lemonica; Pollicino, Teresa; Raimondo, Giovanni; Stampoulis, Dimitrios; Khakoo, Salim; Karayiannis, Peter

    2016-07-15

    In chronic hepatitis B virus (HBV) infection, variants with mutations in the basal core promoter (BCP) and precore region predominate and associate with more severe disease forms. Studies on their effect on viral replication remain controversial. Increasing evidence shows that epigenetic modifications of cccDNA regulate HBV replication and disease outcome. Here we determined the transcription and viral replication efficiency of well-defined BCP and precore mutations and their effect on cccDNA epigenetic control. HBV monomers bearing BCP mutations A1762T/G1764A and A1762T/G1764A/C1766T, and precore mutations G1896A, G1899A and G1896A/G1899A, were transfected into HepG2 cells using a plasmid-free approach. Viral RNA transcripts were detected by Northern blot hybridization and RT PCR, DNA replicative intermediates by Southern blotting and RT PCR, and viral release was measured by ELISA. Acetylation of cccDNA-bound histones was assessed by Chromatin ImmunoPrecipitation (ChIP) assay and methylation of cccDNA by bisulfite sequencing. BCP mutations resulted in low viral release, mRNA transcription and pgRNA/cccDNA ratios that paralleled the acetylation of cccDNA-bound H4 histone and inversely correlated with the HDAC1 recruitment onto cccDNA. Independently of the mutations, cccDNA was a target for methylation, accompanied by the upregulation of DNMT1 expression and DNMT1 recruitment onto cccDNA. Our results suggest that BCP mutations decrease viral replication capacity possibly by modulating the acetylation and deacetylation of cccDNA-bound histones while precore mutations do not have a significant effect on viral replication. These data provide evidence that epigenetic factors contribute to the regulation of HBV viral replication. PMID:27132039

  17. Biochemical analysis of DNA polymerase η fidelity in the presence of replication protein A.

    Samuel C Suarez

    Full Text Available DNA polymerase η (pol η synthesizes across from damaged DNA templates in order to prevent deleterious consequences like replication fork collapse and double-strand breaks. This process, termed translesion synthesis (TLS, is an overall positive for the cell, as cells deficient in pol η display higher mutation rates. This outcome occurs despite the fact that the in vitro fidelity of bypass by pol η alone is moderate to low, depending on the lesion being copied. One possible means of increasing the fidelity of pol η is interaction with replication accessory proteins present at the replication fork. We have previously utilized a bacteriophage based screening system to measure the fidelity of bypass using purified proteins. Here we report on the fidelity effects of a single stranded binding protein, replication protein A (RPA, when copying the oxidative lesion 7,8-dihydro-8-oxo-guanine(8-oxoG and the UV-induced cis-syn thymine-thymine cyclobutane pyrimidine dimer (T-T CPD. We observed no change in fidelity dependent on RPA when copying these damaged templates. This result is consistent in multiple position contexts. We previously identified single amino acid substitution mutants of pol η that have specific effects on fidelity when copying both damaged and undamaged templates. In order to confirm our results, we examined the Q38A and Y52E mutants in the same full-length construct. We again observed no difference when RPA was added to the bypass reaction, with the mutant forms of pol η displaying similar fidelity regardless of RPA status. We do, however, observe some slight effects when copying undamaged DNA, similar to those we have described previously. Our results indicate that RPA by itself does not affect pol η dependent lesion bypass fidelity when copying either 8-oxoG or T-T CPD lesions.

  18. Mitochondrial DNA Replication Defects Disturb Cellular dNTP Pools and Remodel One-Carbon Metabolism.

    Nikkanen, Joni; Forsström, Saara; Euro, Liliya; Paetau, Ilse; Kohnz, Rebecca A; Wang, Liya; Chilov, Dmitri; Viinamäki, Jenni; Roivainen, Anne; Marjamäki, Päivi; Liljenbäck, Heidi; Ahola, Sofia; Buzkova, Jana; Terzioglu, Mügen; Khan, Nahid A; Pirnes-Karhu, Sini; Paetau, Anders; Lönnqvist, Tuula; Sajantila, Antti; Isohanni, Pirjo; Tyynismaa, Henna; Nomura, Daniel K; Battersby, Brendan J; Velagapudi, Vidya; Carroll, Christopher J; Suomalainen, Anu

    2016-04-12

    Mitochondrial dysfunction affects cellular energy metabolism, but less is known about the consequences for cytoplasmic biosynthetic reactions. We report that mtDNA replication disorders caused by TWINKLE mutations-mitochondrial myopathy (MM) and infantile onset spinocerebellar ataxia (IOSCA)-remodel cellular dNTP pools in mice. MM muscle shows tissue-specific induction of the mitochondrial folate cycle, purine metabolism, and imbalanced and increased dNTP pools, consistent with progressive mtDNA mutagenesis. IOSCA-TWINKLE is predicted to hydrolyze dNTPs, consistent with low dNTP pools and mtDNA depletion in the disease. MM muscle also modifies the cytoplasmic one-carbon cycle, transsulfuration, and methylation, as well as increases glucose uptake and its utilization for de novo serine and glutathione biosynthesis. Our evidence indicates that the mitochondrial replication machinery communicates with cytoplasmic dNTP pools and that upregulation of glutathione synthesis through glucose-driven de novo serine biosynthesis contributes to the metabolic stress response. These results are important for disorders with primary or secondary mtDNA instability and offer targets for metabolic therapy. PMID:26924217

  19. RPB5-Mediating Protein Suppresses Hepatitis B Virus (HBV Transcription and Replication by Counteracting the Transcriptional Activation of Hepatitis B virus X Protein in HBV Replication Mouse Model

    Zhou

    2015-09-01

    Full Text Available Background RPB5-Mediating protein (RMP is associated with the RNA polymerase II subunit RPB5. This protein functionally counteracts the transcriptional activation of Hepatitis B Virus X protein (HBx by competitively binding to the RPB5; however, the effects of RMP on Hepatitis B virus (HBV transcription and replication remain unknown. Objectives The purpose of this study was to investigate the effect of RMP on viral transcription and replication in vivo by using the hydrodynamic-based HBV replication mouse model. Materials and Methods Male balb/c mice were transfected with wild type (1.2 wt or the HBx minus HBV plasmids (1.2x (- with or without HBx and RMP, to establish an HBV replication mouse model by hydrodynamic injection through the tail vein. The HBV RNA and HBV DNA replication intermediates (RI were analyzed in the liver. Results RPB5-Mediating protein could inhibit HBV transcription and replication in groups transfected with the 1.2 wt and HBx. The inhibitory effect disappeared in the 1.2x (- groups, yet it reappeared in the groups co-transfected with 1.2x (- and HBx. An inhibitory effect was indicated at a low dose of RMP (0.3 ug, 0.5 ug and 0.7 ug compared to the control group and groups that had received high doses of RMP. Conclusions Our study demonstrated that a low dose of RMP could inhibit HBV transcription and replication, which is dependent on the appearance of HBx in vivo.

  20. DNA polymerase-beta is expressed early in neurons of Alzheimer's disease brain and is loaded into DNA replication forks in neurons challenged with beta-amyloid

    A. Copani; J.J.M. Hoozemans; F. Caraci; M. Calafiore; E.S. van Haastert; R. Veerhuis; A.J.M. Rozemuller; E. Aronica; M.A. Sortino; F. Nicoletti

    2006-01-01

    Cultured neurons exposed to synthetic beta-amyloid (A beta) fragments reenter the cell cycle and initiate a pathway of DNA replication that involves the repair enzyme DNA polymerase-beta (DNA pol-beta) before undergoing apoptotic death. In this study, by performing coimmunoprecipitation experiments

  1. Sequence depth, not PCR replication, improves ecological inference from next generation DNA sequencing.

    Dylan P Smith

    Full Text Available Recent advances in molecular approaches and DNA sequencing have greatly progressed the field of ecology and allowed for the study of complex communities in unprecedented detail. Next generation sequencing (NGS can reveal powerful insights into the diversity, composition, and dynamics of cryptic organisms, but results may be sensitive to a number of technical factors, including molecular practices used to generate amplicons, sequencing technology, and data processing. Despite the popularity of some techniques over others, explicit tests of the relative benefits they convey in molecular ecology studies remain scarce. Here we tested the effects of PCR replication, sequencing depth, and sequencing platform on ecological inference drawn from environmental samples of soil fungi. We sequenced replicates of three soil samples taken from pine biomes in North America represented by pools of either one, two, four, eight, or sixteen PCR replicates with both 454 pyrosequencing and Illumina MiSeq. Increasing the number of pooled PCR replicates had no detectable effect on measures of α- and β-diversity. Pseudo-β-diversity - which we define as dissimilarity between re-sequenced replicates of the same sample - decreased markedly with increasing sampling depth. The total richness recovered with Illumina was significantly higher than with 454, but measures of α- and β-diversity between a larger set of fungal samples sequenced on both platforms were highly correlated. Our results suggest that molecular ecology studies will benefit more from investing in robust sequencing technologies than from replicating PCRs. This study also demonstrates the potential for continuous integration of older datasets with newer technology.

  2. 53BP1 nuclear bodies form around DNA lesions generated by mitotic transmission of chromosomes under replication stress

    Lukas, Claudia; Savic, Velibor; Bekker-Jensen, Simon;

    2011-01-01

    Completion of genome duplication is challenged by structural and topological barriers that impede progression of replication forks. Although this can seriously undermine genome integrity, the fate of DNA with unresolved replication intermediates is not known. Here, we show that mild replication...... bodies shield chromosomal fragile sites sequestered in these compartments against erosion. Together, these data indicate that restoration of DNA or chromatin integrity at loci prone to replication problems requires mitotic transmission to the next cell generations....... increases after genetic ablation of BLM, a DNA helicase associated with dissolution of entangled DNA. Conversely, 53BP1 nuclear bodies are partially suppressed by knocking down SMC2, a condensin subunit required for mechanical stability of mitotic chromosomes. Finally, we provide evidence that 53BP1 nuclear...

  3. ‘The octet’: eight protein kinases that control mammalian DNA replication

    Melvin L. Depamphilis

    2012-09-01

    Full Text Available Development of a fertilized human egg into an average sized adult requires about 29 trillion cell divisions, thereby producing enough DNA to stretch to the Sun and back 200 times (DePamphilis and Bell, 2011! Even more amazing is the fact that throughout these mitotic cell cycles, the human genome is duplicated once and only once each time a cell divides. If a cell accidentally begins to re-replicate its nuclear DNA prior to cell division, checkpoint pathways trigger apoptosis. And yet, some cells are developmentally programmed to respond to environmental cues by switching from mitotic cell cycles to endocycles, a process in which multiple S phases occur in the absence of either mitosis or cytokinesis. Endocycles allow production of viable, differentiated, polyploid cells that no longer proliferate. What is surprising is that among the 516 (Manning et al., 2002 to 557 (BioMart web site protein kinases encoded by the human genome, only eight regulate nuclear DNA replication directly. These are Cdk1, Cdk2, Cdk4, Cdk6, Cdk7, Cdc7, Chk1 and Chk2. Even more remarkable is the fact that only four of these enzymes (Cdk1, Cdk7, Cdc7 and Chk1 are essential for mammalian development. Here we describe how these protein kinases determine when DNA replication occurs during mitotic cell cycles, how mammalian cells switch from mitotic cell cycles to endocycles, and how cancer cells can be selectively targeted for destruction by inducing them to begin a second S phase before mitosis is complete.

  4. RepA and DnaA proteins are required for initiation of R1 plasmid replication in vitro and interact with the oriR sequence.

    Masai, H.; Arai, K.

    1987-01-01

    RepA, an initiation protein of R1 plasmid replication, was purified from an Escherichia coli strain overproducing the protein. The purified RepA protein specifically initiated replication in vitro of plasmid DNA bearing the replication origin of R1 plasmid (oriR). The replication, strictly dependent on added RepA protein, was independent of host RNA polymerase but required other host replication functions (DnaB and DnaC proteins, the single-stranded-DNA-binding protein SSB, and DNA gyrase). T...

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

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

    2014-01-01

    Recent evidence points to a role for Rif1 (Rap1-interacting factor) in DNA replication and genomic stability; however, the mechanism by which Rif1 functions has remained elusive. Hiraga el al. now report that budding yeast Rif1 controls DNA replication genome-wide and describe how Rif1 opposes Dbf4-dependent kinase (DDK) function by directing Protein Phosphatase 1 (PP1)-mediated dephosphorylation of the MCM complex. PP1 interaction sites are evolutionarily conserved within the Rif1 sequence; ...

  6. New origin firing is inhibited by APC/CCdh1 activation in S-phase after severe replication stress.

    Ercilla, Amaia; Llopis, Alba; Feu, Sonia; Aranda, Sergi; Ernfors, Patrik; Freire, Raimundo; Agell, Neus

    2016-06-01

    Defects in DNA replication and repair are known to promote genomic instability, a hallmark of cancer cells. Thus, eukaryotic cells have developed complex mechanisms to ensure accurate duplication of their genomes. While DNA damage response has been extensively studied in tumour cells, the pathways implicated in the response to replication stress are less well understood especially in non-transformed cells. Here we show that in non-transformed cells, APC/C(Cdh1) is activated upon severe replication stress. Activation of APC/C(Cdh1) prevents new origin firing and induces permanent arrest in S-phase. Moreover, Rad51-mediated homologous recombination is also impaired under these conditions. APC/C(Cdh1) activation in S-phase occurs after replication forks have been processed into double strand breaks. Remarkably, this activation, which correlates with decreased Emi1 levels, is not prevented by ATR/ATM inhibition, but it is abrogated in cells depleted of p53 or p21. Importantly, we found that the lack of APC/C(Cdh1) activity correlated with an increase in genomic instability. Taken together, our results define a new APC/C(Cdh1) function that prevents cell cycle resumption after prolonged replication stress by inhibiting origin firing, which may act as an additional mechanism in safeguarding genome integrity. PMID:26939887

  7. A study of the topoisomerase II activity in HIV-1 replication using the ferrocene derivatives as probes.

    Kondapi, Anand K; Satyanarayana, Nathamu; Saikrishna, A D

    2006-06-15

    Human Topoisomerase II is present in two isoforms, 170KDa alpha and 180KDa beta. Both the isoforms play a crucial role in maintenance of topological changes during DNA replication and recombination. It has been shown that Topoisomerase II activity is required for HIV-1 replication and the enzyme is phosphorylated during early time points of HIV-1 replication. In the present study, we have studied the molecular action of Topoisomerase II inhibitors, azalactone ferrocene (AzaFecp), Thiomorpholide amido methyl ferrocene (ThioFecp), and Ruthenium benzene amino pyridine (Ru(ben)Apy) on cell proliferation and also on various events of HIV-1 replication cycle. The Topoisomerase II beta over-expressing neuroblastoma cell line shows a higher sensitivity to these compounds compared to the Sup-T1 cell line. All the three Topoisomerase II inhibitors show significant anti-HIV activity at nanomolar concentrations against an Indian isolate of HIV-1(93IN101) in Sup-T1 cell line. An analysis of action of these compounds on proviral DNA synthesis at 5h of post-infection shows that they inhibit proviral DNA synthesis as well as the formation of pre-integration complexes completely. Further analysis, using polymerase chain reaction and western blot, showed that both the Topoisomerase II alpha and beta isoforms are present in the pre-integration complexes, suggesting their significant role in HIV-1 replication. PMID:16712776

  8. DNA replication factor C1 mediates genomic stability and transcriptional gene silencing in Arabidopsis

    Liu, Qian

    2010-07-01

    Genetic screening identified a suppressor of ros1-1, a mutant of REPRESSOR OF SILENCING1 (ROS1; encoding a DNA demethylation protein). The suppressor is a mutation in the gene encoding the largest subunit of replication factor C (RFC1). This mutation of RFC1 reactivates the unlinked 35S-NPTII transgene, which is silenced in ros1 and also increases expression of the pericentromeric Athila retrotransposons named transcriptional silent information in a DNA methylationindependent manner. rfc1 is more sensitive than the wild type to the DNA-damaging agent methylmethane sulphonate and to the DNA inter- and intra- cross-linking agent cisplatin. The rfc1 mutant constitutively expresses the G2/M-specific cyclin CycB1;1 and other DNA repair-related genes. Treatment with DNA-damaging agents mimics the rfc1 mutation in releasing the silenced 35S-NPTII, suggesting that spontaneously induced genomic instability caused by the rfc1 mutation might partially contribute to the released transcriptional gene silencing (TGS). The frequency of somatic homologous recombination is significantly increased in the rfc1 mutant. Interestingly, ros1 mutants show increased telomere length, but rfc1 mutants show decreased telomere length and reduced expression of telomerase. Our results suggest that RFC1 helps mediate genomic stability and TGS in Arabidopsis thaliana. © 2010 American Society of Plant Biologists.

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

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

    2016-01-01

    Disruption of DNA replication, recombination, and repair (DNA-RRR) systems has been hypothesized to cause highly elevated nucleotide substitution rates and genome rearrangements in the plastids of angiosperms, but this theory remains untested. To investigate nuclear-plastid genome (plastome) coevolution in Geraniaceae, four different measures of plastome complexity (rearrangements, repeats, nucleotide insertions/deletions, and substitution rates) were evaluated along with substitution rates of 12 nuclear-encoded, plastid-targeted DNA-RRR genes from 27 Geraniales species. Significant correlations were detected for nonsynonymous (dN) but not synonymous (dS) substitution rates for three DNA-RRR genes (uvrB/C, why1, and gyrA) supporting a role for these genes in accelerated plastid genome evolution in Geraniaceae. Furthermore, correlation between dN of uvrB/C and plastome complexity suggests the presence of nucleotide excision repair system in plastids. Significant correlations were also detected between plastome complexity and 13 of the 90 nuclear-encoded organelle-targeted genes investigated. Comparisons revealed significant acceleration of dN in plastid-targeted genes of Geraniales relative to Brassicales suggesting this correlation may be an artifact of elevated rates in this gene set in Geraniaceae. Correlation between dN of plastid-targeted DNA-RRR genes and plastome complexity supports the hypothesis that the aberrant patterns in angiosperm plastome evolution could be caused by dysfunction in DNA-RRR systems. PMID:26893456

  10. Signaling from Mus81-Eme2-Dependent DNA Damage Elicited by Chk1 Deficiency Modulates Replication Fork Speed and Origin Usage

    Hervé Técher

    2016-02-01

    Full Text Available Mammalian cells deficient in ATR or Chk1 display moderate replication fork slowing and increased initiation density, but the underlying mechanisms have remained unclear. We show that exogenous deoxyribonucleosides suppress both replication phenotypes in Chk1-deficient, but not ATR-deficient, cells. Thus, in the absence of exogenous stress, depletion of either protein impacts the replication dynamics through different mechanisms. In addition, Chk1 deficiency, but not ATR deficiency, triggers nuclease-dependent DNA damage. Avoiding damage formation through invalidation of Mus81-Eme2 and Mre11, or preventing damage signaling by turning off the ATM pathway, suppresses the replication phenotypes of Chk1-deficient cells. Damage and resulting DDR activation are therefore the cause, not the consequence, of replication dynamics modulation in these cells. Together, we identify moderate reduction of precursors available for replication as an additional outcome of DDR activation. We propose that resulting fork slowing, and subsequent firing of backup origins, helps replication to proceed along damaged templates.

  11. Photodynamic effect of proflavine on 0X174 bacteriophage, its DNA replicative form and its isolated single-stranded DNA

    In contrast to that what is observed with most inactivating agents, proflavine-mediated photoinactivation is about 10 times more efficient on double-stranded 0X174 replicative form DNA (RFI) than on isolated single-stranded 0X174 DNA. Both 0XRFI DNA and encapsidated DNA have similar sensitivities to proflavine and light treatment. With the three substrates studied, reactivation can occur through high multiplicity of infection and depends upon the cellular rec A gene product. No effect of the pol A, uvr A or lex A gene mutations has been found on either phage of DNA inactivation rates. The photodynamically induced lesions can be repaired, at least in part, by the SOS repair system induced in the host-cells by a 100 J x m-2 UV irradiation. SOS repair does not occur with bacteria (or spheroplasts) irradiated in the presence of chloramphenicol. Reversion frequency of the 0X174 amber mutations indicates that 1) photodynamically induced lesions are mutagenic whether the rec A gene product is present or not in the indicator bacteria; 2) induction of the SOS repair system is accompanied by a mutagenic process which results in a almost twofold increase of the reversion frequency; and 3) multiplicity reactivation occurs through a re ombinational process and is not mutagenic per se. (orig./AJ)

  12. Heat shock proteins DnaJ, DnaK, and GrpE stimulate P1 plasmid replication by promoting initiator binding to the origin.

    Sozhamannan, S; Chattoraj, D K

    1993-01-01

    Binding of the P1-encoded protein RepA to the origin of P1 plasmid replication is essential for initiation of DNA replication and for autoregulatory repression of the repA promoter. Previous studies have shown defects in both initiation and repression in hosts lacking heat shock proteins DnaJ, DnaK, and GrpE and have suggested that these proteins play a role in the RepA-DNA binding required for initiation and repression. In this study, using in vivo dimethyl sulfate footprinting, we have conf...

  13. Regulation of mitochondrial genome replication by hypoxia: The role of DNA oxidation in D-loop region.

    Pastukh, Viktor M; Gorodnya, Olena M; Gillespie, Mark N; Ruchko, Mykhaylo V

    2016-07-01

    Mitochondria of mammalian cells contain multiple copies of mitochondrial (mt) DNA. Although mtDNA copy number can fluctuate dramatically depending on physiological and pathophysiologic conditions, the mechanisms regulating mitochondrial genome replication remain obscure. Hypoxia, like many other physiologic stimuli that promote growth, cell proliferation and mitochondrial biogenesis, uses reactive oxygen species as signaling molecules. Emerging evidence suggests that hypoxia-induced transcription of nuclear genes requires controlled DNA damage and repair in specific sequences in the promoter regions. Whether similar mechanisms are operative in mitochondria is unknown. Here we test the hypothesis that controlled oxidative DNA damage and repair in the D-loop region of the mitochondrial genome are required for mitochondrial DNA replication and transcription in hypoxia. We found that hypoxia had little impact on expression of mitochondrial proteins in pulmonary artery endothelial cells, but elevated mtDNA content. The increase in mtDNA copy number was accompanied by oxidative modifications in the D-loop region of the mitochondrial genome. To investigate the role of this sequence-specific oxidation of mitochondrial genome in mtDNA replication, we overexpressed mitochondria-targeted 8-oxoguanine glycosylase Ogg1 in rat pulmonary artery endothelial cells, enhancing the mtDNA repair capacity of transfected cells. Overexpression of Ogg1 resulted in suppression of hypoxia-induced mtDNA oxidation in the D-loop region and attenuation of hypoxia-induced mtDNA replication. Ogg1 overexpression also reduced binding of mitochondrial transcription factor A (TFAM) to both regulatory and coding regions of the mitochondrial genome without altering total abundance of TFAM in either control or hypoxic cells. These observations suggest that oxidative DNA modifications in the D-loop region during hypoxia are important for increased TFAM binding and ensuing replication of the mitochondrial

  14. Studies on DNA replication in vitro by DNA polymerase-α/primase complex from embryonic chicken brain

    DNA polymerase - α activity, characterized by its sensitivity to N-ethylmaleimide, high sedimentation coefficient, and acidic isoelectric point was found to decline with increasing embryonic age. Primase activity, the enzyme responsible for the initiation of DNA synthesis, was found to co-sediment with DNA polymerase - α activity on a continuous glycerol velocity gradient. Of all the single-stranded DNA templates tested, primase activity was found to be maximally active with poly dC. In the presence of [α-32P] GTP, primase activity was found to catalyze the formation of an alkali-labile oligoriboguanylate, 15-20 nucleotides long. The chain length of the products were not altered by the presence or absence of dGTP. Primase activity was not inhibited by a high concentration of α- amanitin. Consistent with the finding of a decline of the activity level of DNA polymerase - α, the level of DNA polymerase - α antigen was also found to decrease with embryonic age, as evidenced by ELISA with a non-neutralizing monoclonal antibody, SJK 237-71

  15. Positive roles of SAS2 in DNA replication and transcriptional silencing in yeast

    Zou, Yanfei; Bi, Xin

    2008-01-01

    Sas2p is a histone acetyltransferase implicated in the regulation of transcriptional silencing, and ORC is the six-subunit origin recognition complex involved in the initiation of DNA replication and the establishment of transcriptionally silent chromatin by silencers in yeast. We show here that SAS2 deletion (sas2Δ) exacerbates the temperature sensitivity of the ORC mutants orc2-1 and orc5-1. Moreover, sas2Δ and orc2-1 have a synthetic effect on cell cycle progression through S phase and ini...

  16. A conserved amphipathic helix in the N-terminal regulatory region of the papillomavirus E1 helicase is required for efficient viral DNA replication.

    Morin, Geneviève; Fradet-Turcotte, Amélie; Di Lello, Paola; Bergeron-Labrecque, Fanny; Omichinski, James G; Archambault, Jacques

    2011-06-01

    The papillomavirus E1 helicase, with the help of E2, assembles at the viral origin into a double hexamer that orchestrates replication of the viral genome. The N-terminal region (NTR) of E1 is essential for DNA replication in vivo but dispensable in vitro, suggesting that it has a regulatory function. By deletion analysis, we identified a conserved region of the E1 NTR needed for efficient replication of viral DNA. This region is predicted to form an amphipathic α-helix (AH) and shows sequence similarity to portions of the p53 and herpes simplex virus (HSV) VP16 transactivation domains known as transactivation domain 2 (TAD2) and VP16C, which fold into α-helices upon binding their target proteins, including the Tfb1/p62 (Saccharomyces cerevisiae/human) subunit of general transcription factor TFIIH. By nuclear magnetic resonance (NMR) spectroscopy and isothermal titration calorimetry (ITC), we found that a peptide spanning the E1 AH binds Tfb1 on the same surface as TAD2/VP16C and with a comparable affinity, suggesting that it does bind as an α-helix. Furthermore, the E1 NTRs from several human papillomavirus (HPV) types could activate transcription in yeast, and to a lesser extent in mammalian cells, when fused to a heterologous DNA-binding domain. Mutation of the three conserved hydrophobic residues in the E1 AH, analogous to those in TAD2/VP16C that directly contact their target proteins, decreased transactivation activity and, importantly, also reduced by 50% the ability of E1 to support transient replication of DNA in C33A cells, at a step following assembly of the E1-E2-ori preinitiation complex. These results demonstrate the existence of a conserved TAD2/VP16C-like AH in E1 that is required for efficient replication of viral DNA. PMID:21450828

  17. Structural Determinant for Switching between the Polymerase and Exonuclease Modes in the PCNA-Replicative DNA Polymerase Complex

    Nishida, Hirokazu; Mayanagi, Kouta; Ishino, Yoshizumi; Morikawa, Kosuke

    Proliferating cell nuclear antigen (PCNA) is responsible for the processivity of DNA polymerase. We determined the crystal structure of Pyrococcus furiosus DNA polymerase (PfuPol) complexed with a cognate monomeric PCNA, which allowed us to construct a convincing model of the polymerase-PCNA ring interaction. Electron microscopy analyses confirmed that this complex structure exists among the multiple functional configurations in solution. Together with data from mutational analyses, this structural study indicated that the novel interaction between a stretched loop of PCNA and the PfuPol Thumb domain is quite important, in addition to the authentic PCNA-polymerase recognition site (PIP box). A comparison of the present structures with the previously reported structures of polymerases complexed with DNA suggested that the second interaction site plays a crucial role in switching between the polymerase and exonuclease modes, by stabilizing only the polymerase mode. This proposed mechanism of fidelity control of replicative DNA polymerases was supported by experiments, in which a mutation within the second interaction site caused an enhancement in the exonuclease activity in the presence of PCNA.

  18. In vitro-reconstituted nucleoids can block mitochondrial DNA replication and transcription

    Farge, Géraldine; Mehmedovic, Majda; Baclayon, Marian; van den Wildenberg, Siet M J L; Roos, Wouter H; Gustafsson, Claes M; Wuite, Gijs J L; Falkenberg, Maria

    2014-01-01

    The mechanisms regulating the number of active copies of mtDNA are still unclear. A mammalian cell typically contains 1,000-10,000 copies of mtDNA, which are packaged into nucleoprotein complexes termed nucleoids. The main protein component of these structures is mitochondrial transcription factor A

  19. RNA1-Independent Replication and GFP Expression from Tomato marchitez virus Isolate M Cloned cDNA.

    Ferriol, I; Turina, M; Zamora-Macorra, E J; Falk, B W

    2016-05-01

    Tomato marchitez virus (ToMarV; synonymous with Tomato apex necrosis virus) is a positive-strand RNA virus in the genus Torradovirus within the family Secoviridae. ToMarV is an emergent whitefly-transmitted virus that causes important diseases in tomato (Solanum lycopersicum) in Mexico. Here, the genome sequence of the ToMarV isolate M (ToMarV-M) was determined. We engineered full-length cDNA clones of the ToMarV-M genomic RNA (RNA1 and RNA2), separately, into a binary vector. Coinfiltration of both triggered systemic infections in Nicotiana benthamiana, tomato, and tomatillo (Physalis philadelphica) plants and recapitulated the biological activity of the wild-type virus. The viral progeny generated from tomato and tomatillo plants were transmissible by the whitefly Bemisia tabaci biotype B. Also, we assessed whether these infectious clones could be used for screening tomato cultivars for resistance to ToMarV and our results allowed us to differentiate resistant and susceptible tomato lines. We demonstrated that RNA1 of ToMarV-M is required for the replication of RNA2, and it can replicate independently of RNA2. From this, ToMarV-M RNA2 was used to express the green fluorescent protein in N. benthamiana plants, which allowed us to track cell-to-cell movement. The construction of full-length infectious cDNA clones of ToMarV-M provides an excellent tool to investigate virus-host-vector interactions and elucidate the functions of torradovirus-encoded proteins or the mechanisms of replication of torradovirus genomic RNA. PMID:26756828

  20. A prophage-encoded actin-like protein required for efficient viral DNA replication in bacteria.

    Donovan, Catriona; Heyer, Antonia; Pfeifer, Eugen; Polen, Tino; Wittmann, Anja; Krämer, Reinhard; Frunzke, Julia; Bramkamp, Marc

    2015-05-26

    In host cells, viral replication is localized at specific subcellular sites. Viruses that infect eukaryotic and prokaryotic cells often use host-derived cytoskeletal structures, such as the actin skeleton, for intracellular positioning. Here, we describe that a prophage, CGP3, integrated into the genome of Corynebacterium glutamicum encodes an actin-like protein, AlpC. Biochemical characterization confirms that AlpC is a bona fide actin-like protein and cell biological analysis shows that AlpC forms filamentous structures upon prophage induction. The co-transcribed adaptor protein, AlpA, binds to a consensus sequence in the upstream promoter region of the alpAC operon and also interacts with AlpC, thus connecting circular phage DNA to the actin-like filaments. Transcriptome analysis revealed that alpA and alpC are among the early induced genes upon excision of the CGP3 prophage. Furthermore, qPCR analysis of mutant strains revealed that both AlpA and AlpC are required for efficient phage replication. Altogether, these data emphasize that AlpAC are crucial for the spatio-temporal organization of efficient viral replication. This is remarkably similar to actin-assisted membrane localization of eukaryotic viruses that use the actin cytoskeleton to concentrate virus particles at the egress sites and provides a link of evolutionary conserved interactions between intracellular virus transport and actin. PMID:25916847

  1. Replication protein A and γ-H2AX foci assembly is triggered by cellular response to DNA double-strand breaks

    Human replication protein A (RPA p34), a crucial component of diverse DNA excision repair pathways, is implicated in DNA double-strand break (DSB) repair. To evaluate its role in DSB repair, the intranuclear dynamics of RPA was investigated after DNA damage and replication blockage in human cells. Using two different agents [ionizing radiation (IR) and hydroxyurea (HU)] to generate DSBs, we found that RPA relocated into distinct nuclear foci and colocalized with a well-known DSB binding factor, γ-H2AX, at the sites of DNA damage in a time-dependent manner. Colocalization of RPA and γ-H2AX foci peaked at 2 h after IR treatment and subsequently declined with increasing postrecovery times. The time course of RPA and γ-H2AX foci association correlated well with the DSB repair activity detected by a neutral comet assay. A phosphatidylinositol-3 (PI-3) kinase inhibitor, wortmannin, completely abolished both RPA and γ-H2AX foci formation triggered by IR. Additionally, radiosensitive ataxia telangiectasia (AT) cells harboring mutations in ATM gene product were found to be deficient in RPA and γ-H2AX colocalization after IR. Transfection of AT cells with ATM cDNA fully restored the association of RPA foci with γ-H2AX illustrating the requirement of ATM gene product for this process. The exact coincidence of RPA and γ-H2AX in response to HU specifically in S-phase cells supports their role in DNA replication checkpoint control. Depletion of RPA by small interfering RNA (SiRNA) substantially elevated the frequencies of IR-induced micronuclei (MN) and apoptosis in human cells suggestive of a role for RPA in DSB repair. We propose that RPA in association with γ-H2AX contributes to both DNA damage checkpoint control and repair in response to strand breaks and stalled replication forks in human cells

  2. Regulation of chromosomal replication by DnaA protein availability in Escherichia coli: effects of the datA region

    Morigen, H.; Boye, E.; Skarstad, K.;

    2001-01-01

    Initiation of chromosomal replication in Escherichia coli is dependent on availability of the initiator protein DnaA. We have introduced into E coli cells plasmids carrying the chromosomal locus datA, which has a high affinity for DnaA. To be able to monitor oriC initiation as a function of dat......A copy number, we introduced a minichromosome which only replicates from oriC., using a host cell which replicates its chromosome independently of oriC. Our data show that a moderate increase in datA copy number is accompanied by increased DnaA protein synthesis that allows oriC initiation to occur...... normally, as measured by minichromosome copy number. As datA gene dosage is increased dnaA expression cannot be further derepressed, and the minichromosome copy number is dramatically reduced. Under these conditions the minichromosome was maintained by integration into the chromosome. These findings...

  3. The 32 kDa subunit of replication protein A (RPA) participates in the DNA replication of Mung bean yellow mosaic India virus (MYMIV) by interacting with the viral Rep protein

    Singh, Dharmendra Kumar; Islam, Mohammad Nurul; Choudhury, Nirupam Roy; Karjee, Sumona; Mukherjee, Sunil Kumar

    2006-01-01

    Mung bean yellow mosaic India virus (MYMIV) is a member of genus begomoviridae and its genome comprises of bipartite (two components, namely DNA-A and DNA-B), single-stranded, circular DNA of about 2.7 kb. During rolling circle replication (RCR) of the DNA, the stability of the genome and maintenance of the stem–loop structure of the replication origin is crucial. Hence the role of host single-stranded DNA-binding protein, Replication protein A (RPA), in the RCR of MYMIV was examined. Two RPA...

  4. Regulation of the activity of the dual-function DnaA protein in Caulobacter crescentus.

    Carmen Fernandez-Fernandez

    Full Text Available DnaA is a conserved essential bacterial protein that acts as the initiator of chromosomal replication as well as a master transcriptional regulator in Caulobacter crescentus. Thus, the intracellular levels of active DnaA need to be tightly regulated during the cell cycle. Our previous work suggested that DnaA may be regulated at the level of its activity by the replisome-associated protein HdaA. Here, we describe the construction of a mutant DnaA protein [DnaA(R357A]. The R357 residue in the AAA+ domain of the C. crescentus DnaA protein is equivalent to the R334 residue of the E. coli DnaA protein, which is required for the Regulatory Inactivation of DnaA (RIDA. We found that the expression of the DnaA(R357A mutant protein in C. crescentus, but not the expression of the wild-type DnaA protein at similar levels, causes a severe phenotype of over-initiation of chromosomal replication and that it blocks cell division. Thus, the mutant DnaA(R357A protein is hyper-active to promote the initiation of DNA replication, compared to the wild-type DnaA protein. DnaA(R357A could not replace DnaA in vivo, indicating that the switch in DnaA activity once chromosomal replication has started may be an essential process in C. crescentus. We propose that the inactivation of DnaA is the main mechanism ensuring that chromosomal replication starts only once per cell cycle. We further observed that the R357A substitution in DnaA does not promote the activity of DnaA as a direct transcriptional activator of four important genes, encoding HdaA, the GcrA master cell cycle regulator, the FtsZ cell division protein and the MipZ spatial regulator of cell division. Thus, the AAA+ domain of DnaA may play a role in temporally regulating the bifunctionality of DnaA by reallocating DnaA molecules from initiating DNA replication to transcribing genes within the unique DnaA regulon of C. crescentus.

  5. Leishmania replication protein A-1 binds in vivo single-stranded telomeric DNA

    Replication protein A (RPA) is a highly conserved heterotrimeric single-stranded DNA-binding protein involved in different events of DNA metabolism. In yeast, subunits 1 (RPA-1) and 2 (RPA-2) work also as telomerase recruiters and, in humans, the complex unfolds G-quartet structures formed by the 3' G-rich telomeric strand. In most eukaryotes, RPA-1 and RPA-2 bind DNA using multiple OB fold domains. In trypanosomatids, including Leishmania, RPA-1 has a canonical OB fold and a truncated RFA-1 structural domain. In Leishmania amazonensis, RPA-1 alone can form a complex in vitro with the telomeric G-rich strand. In this work, we show that LaRPA-1 is a nuclear protein that associates in vivo with Leishmania telomeres. We mapped the boundaries of the OB fold DNA-binding domain using deletion mutants. Since Leishmania and other trypanosomatids lack homologues of known telomere end binding proteins, our results raise questions about the function of RPA-1 in parasite telomeres

  6. Residue profile in predivergence sequences as a guide to the origin of DNA replication

    Davis, B K

    1999-01-01

    DNA dependent RNA polymerase core subunits abb' conserved residues at frequencies most closely matched with codons at stage 10.4-11.1 in code evolution. An excess of acidic residues (stage 2 additions) lowered this estimate, but not by more than 2.3 stages. With 1 tryptophan (stage 14 addition) in 529 conserved residues, abb' significantly under-represented this amino acid, consistent with a cut-off in its residue profile before completion of the genetic code. Residue profiles in FEN-1 homologs and DNA topoisomerase I-5' placed their origin at stage 11-13. Prokaryote septation protein, FtsZ, arose earlier, between stage 8-11. Proteolipid in an ATP-driven proton pump served as a marker for cell membrane formation. It indicated this event took place near stage 7. Cell division, DNA replication and transcription were inferred to have originated in a protocell antecedent of the last common ancestor. Late-forming residue profiles characterized RNA dependent RNA replicase, DNA polymerase, reverse transcriptase and ...

  7. Hyperthermia Synergizes with Chemotherapy by Inhibiting PARP1-Dependent DNA Replication Arrest.

    Schaaf, Lea; Schwab, Matthias; Ulmer, Christoph; Heine, Simon; Mürdter, Thomas E; Schmid, Jens O; Sauer, Georg; Aulitzky, Walter E; van der Kuip, Heiko

    2016-05-15

    Although hyperthermia offers clinical appeal to sensitize cells to chemotherapy, this approach has been limited in terms of long-term outcome as well as economic and technical burden. Thus, a more detailed knowledge about how hyperthermia exerts its effects on chemotherapy may illuminate ways to improve the approach. Here, we asked whether hyperthermia alters the response to chemotherapy-induced DNA damage and whether this mechanism is involved in its sensitizing effect in BRCA-competent models of ovarian and colon cancer. Notably, we found that hyperthermia delayed the repair of DNA damage caused by cisplatin or doxorubicin, acting upstream of different repair pathways to block histone polyADP-ribosylation (PARylation), a known effect of chemotherapy. Furthermore, hyperthermia blocked this histone modification as efficiently as pharmacologic inhibitors of PARP (PARPi), producing comparable delay in DNA repair, induction of double-strand breaks (DSB), and cell cytotoxicity after chemotherapy. Mechanistic investigations indicated that inhibiting PARylation by either hyperthermia or PARPi induced lethal DSB upon chemotherapy treatment not only by reducing DNA repair but also by preventing replication fork slowing. Overall, our work reveals how PARP blockade, either by hyperthermia or small-molecule inhibition, can increase chemotherapy-induced damage in BRCA-competent cells. Cancer Res; 76(10); 2868-75. ©2016 AACR. PMID:27013194

  8. Loss-of-function mutations in MGME1 impair mtDNA replication and cause multi-systemic mitochondrial disease

    Kornblum, Cornelia; Nicholls, Thomas J.; Haack, Tobias B.; Schöler, Susanne; Peeva, Viktoriya; Danhauser, Katharina; Hallmann, Kerstin; Zsurka, Gábor; Rorbach, Joanna; Iuso, Arcangela; Wieland, Thomas; Sciacco, Monica; Ronchi, Dario; Comi, Giacomo P; Moggio, Maurizio

    2013-01-01

    Known disease mechanisms in mitochondrial DNA (mtDNA) maintenance disorders alter either the mitochondrial replication machinery (POLG1, POLG22 and C10orf23) or the biosynthesis pathways of deoxyribonucleoside 5′-triphosphates for mtDNA synthesis4–11. However, in many of these disorders, the underlying genetic defect has not yet been discovered. Here, we identified homozygous nonsense and missense mutations in the orphan gene C20orf72 in three families with a mitochondrial syndrome characteri...

  9. Translesion replication by DNA polymerase beta is modulated by sequence context and stimulated by fork-like flap structures in DNA.

    Daube, S S; Arad, G; Livneh, Z

    2000-01-18

    Mutations in the human genome are clustered in hot-spot regions, suggesting that some sequences are more prone to accumulate mutations than others. These regions are therefore more likely to lead to the development of cancer. Several pathways leading to the creation of mutations may be influenced by the DNA sequence, including sensitivity to DNA damaging agents, and repair mechanisms. We have analyzed sequence context effects on translesion replication, the error-prone repair of single-stranded DNA regions carrying lesions. By using synthetic oligonucleotides containing systematic variations of sequences flanking a synthetic abasic site, we show that translesion replication by the repair polymerase DNA polymerase beta is stimulated to a moderate extent by low stacking levels of the template nucleotides downstream of the lesion, combined with homopolymeric runs flanking the lesion both upstream and downstream. A strong stimulation of translesion replication by DNA polymerase beta was seen when fork-like flap structures were introduced into the DNA substrate downstream of the lesion. Unlike for gapped substrates, this stimulation was independent of the presence of a phosphate group at the 5' terminus of the flap. These results suggest that DNA polymerase beta may participate in cellular DNA transactions involving higher order structures. The significance of these results for in vivo translesion replication is discussed. PMID:10631001

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

    Zhigang Yi

    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

  11. Inhibition of Replication and Transcription Activator and Latency-Associated Nuclear Antigen of Kaposi’s Sarcoma-Associated Herpesvirus by Morpholino Oligomers

    Zhang, Yan-Jin; Wang, Kai-Yu; Stein, David A.; Patel, Deendayal; Watkins, Rheba; Moulton, Hong M.; Iversen, Patrick L.; Matson, David O.

    2006-01-01

    Kaposi’s sarcoma-associated herpesvirus (KSHV) is associated with Kaposi’s sarcoma and primary effusion lymphoma (PEL). The KSHV replication and transcription activator (RTA) and latency-associated nuclear antigen (LANA) play key roles in activating KSHV lytic replication and maintaining KSHV latency, respectively. Phosphorodiamidate morpholino oligomers (PMO) are similar to short single-stranded DNA oligomers, but possess a modified backbone that confers highly specific binding and resistanc...

  12. Cell cycle synchronization of Escherichia coli using the stringent response, with fluorescence labeling assays for DNA content and replication.

    Ferullo, Daniel J; Cooper, Deani L; Moore, Hayley R; Lovett, Susan T

    2009-05-01

    We describe a method for synchronization of the cell cycle in the bacterium Escherichia coli. Treatment of asynchronous cultures with the amino acid analog, dl-serine hydroxamate, induces the stringent response, with concomitant arrest of DNA replication at initiation. Following release of the stringent response, cells initiate DNA replication in synchrony, as determined by flow cytometry for DNA content, Southern blotting and microscopy. This method has the advantage that it can be used in fully wild-type cells, at different growth rates, and may be applicable to other bacterial species with replication control by the stringent response. We also elaborate other methods useful for establishing cell cycle parameters in bacterial populations. We describe flow cytometric methods for analyzing bacterial populations for DNA content using the DNA-specific dye PicoGreen, readily detected by most commercial flow cytometers. We also present an method for incorporation of the nucleotide ethynyl-deoxyuridine, EdU, followed by "click" labeling with fluorescent dyes, which allows us to measure and visualize newly replicated DNA in fixed E. coli K-12 cells under non-denaturing conditions. PMID:19245839

  13. Cell cycle synchronization of E. coli using the stringent response, with fluorescence labeling assays for DNA content and replication

    Ferullo, Daniel J.; Cooper, Deani L.; Moore, Hayley R.; Lovett, Susan T.

    2009-01-01

    We describe a method for synchronization of the cell cycle in the bacterium E. coli. Treatment of asynchronous cultures with the amino acid analog, DL-serine hydroxamate, induces the stringent response, with concomitant arrest of DNA replication at initiation. Following release of the stringent response, cells initiate DNA replication in synchrony, as determined by flow cytometry for DNA content, Southern blotting and microscopy. This method has the advantage that it can be used in fully wild-type cells, at different growth rates, and may be applicable to other bacterial species with replication control by the stringent response. We also elaborate other methods useful for establishing cell cycle parameters in bacterial populations. We describe flow cytometric methods for analyzing bacterial populations for DNA content using the DNA-specific dye PicoGreen, readily detected by most commercial flow cytometers. We also present an method for incorporation of the nucleotide ethynyl-deoxyuridine, EdU, followed by “click” labeling with fluorescent dyes, which allows us to measure and visualize newly replicated DNA in fixed E. coli K-12 cells under non-denaturing conditions. PMID:19245839

  14. Simian virus 40 (SV40) large tumor antigen causes stepwise changes in SV40 origin structure during initiation of DNA replication.

    Roberts, J.M.

    1989-01-01

    We have studied structural changes in the simian virus 40 (SV40) replication origin induced by SV40 large tumor antigen (T antigen). T-antigen-induced changes in origin DNA conformation can be visualized as specific and discrete topologic changes in origin DNA minicircles. We discovered three origin-T-antigen complexes defined by changes in DNA linking number. These complexes probably reflected essential early steps in the initiation of DNA replication since their formation required DNA seque...

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

    David S. Shin

    2014-01-01

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

  16. Efficiency, specificity and DNA polymerase-dependence of translesion replication across the oxidative DNA lesion 8-oxoguanine in human cells

    The oxidation product of guanine, 8-oxoguanine, is a major lesion formed in DNA by intracellular metabolism, ionizing radiation, and tobacco smoke. Using a recently developed method for the quantitative analysis of translesion replication, we have studied the bypass of 8-oxoguanine in vivo by transfecting human cells with a gapped plasmid carrying a site-specific 8-oxoguanine in the ssDNA region. The efficiency of bypass in the human large-cell lung carcinoma cell line H1299 was 80%, and it was similar when assayed in the presence of aphidicolin, an inhibitor of DNA polymerases α, δ and ε. A similar extent of bypass was observed also in XP-V cells, defective in pol η, both in the absence and presence of aphidicolin. DNA sequence analysis indicated that the major nucleotide inserted opposite the 8-oxoguanine was the correct nucleotide C, both in H1299 cells (81%) and in XP-V cells (77%). The major mutagenic event was the insertion of an A, both in H1299 and XP-V cells, and it occurred at a frequency of 16-17%, significantly higher than previously reported. Interestingly, the misinsertion frequency of A opposite 8-oxoguanine was decreased in XP-V cells in the presence of aphidicolin, and misinsertion of G was observed. This modulation of the mutagenic specificity at 8-oxoguanine is consistent with the notion that while not essential for the bypass reaction, pol η and pol δ, when present, are involved in bypass of 8-oxoguanine in vivo

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

    Riber, Leise; Fujimitsu, K.; Katayama, T.;

    2009-01-01

    Initiation of chromosome replication in Escherichia coli is limited by the initiator protein DnaA associated with ATP. Within the replication origin, binding sites for DnaA associated with ATP or ADP (R boxes) and the DnaA(ATP) specific sites (I-boxes, tau-boxes and 6-mer sites) are found. We...

  18. TWINKLE is an essential mitochondrial helicase required for synthesis of nascent D-loop strands and complete mtDNA replication.

    Milenkovic, Dusanka; Matic, Stanka; Kühl, Inge; Ruzzenente, Benedetta; Freyer, Christoph; Jemt, Elisabeth; Park, Chan Bae; Falkenberg, Maria; Larsson, Nils-Göran

    2013-05-15

    Replication of the mammalian mitochondrial DNA (mtDNA) is dependent on the minimal replisome, consisting of the heterotrimeric mtDNA polymerase (POLG), the hexameric DNA helicase TWINKLE and the tetrameric single-stranded DNA-binding protein (mtSSB). TWINKLE has been shown to unwind DNA during the replication process and many disease-causing mutations have been mapped to its gene. Patients carrying Twinkle mutations develop multiple deletions of mtDNA, deficient respiratory chain function and neuromuscular symptoms. Despite its importance in human disease, it has been unclear whether TWINKLE is the only replicative DNA helicase in mammalian mitochondria. Furthermore, a substantial portion of mtDNA replication events is prematurely terminated at the end of mitochondrial control region (D-loop) and it is unknown whether TWINKLE also has a role in this abortive replication. Here, we present a conditional mouse knockout for Twinkle and demonstrate that TWINKLE is essential for mouse embryonic development and thus is the only replicative DNA helicase in mammalian mitochondria. Conditional knockout of Twinkle results in severe and rapid mtDNA depletion in heart and skeletal muscle. No replication intermediates or deleted mtDNA molecules are observed after Twinkle knockout, suggesting that TWINKLE once loaded is very processive. We also demonstrate that TWINKLE is essential for nascent H-strand synthesis in the D-loop, thus showing that there is no separate DNA helicase responsible for replication of this region. Our data thus suggest that the relative levels of abortive D-loop synthesis versus complete mtDNA replication are regulated and may provide a mechanism to control progression to complete mtDNA replication. PMID:23393161

  19. Gene dosage imbalance during DNA replication controls bacterial cell-fate decision

    Igoshin, Oleg

    Genes encoding proteins in a common regulatory network are frequently located close to one another on the chromosome to facilitate co-regulation or couple gene expression to growth rate. Contrasting with these observations, here we demonstrate a functional role for the arrangement of Bacillus subtilis sporulation network genes on opposite sides of the chromosome. We show that the arrangement of two sporulation network genes, one located close to the origin, the other close to the terminus leads to a transient gene dosage imbalance during chromosome replication. This imbalance is detected by the sporulation network to produce cell-cycle coordinated pulses of the sporulation master regulator Spo0A~P. This pulsed response allows cells to decide between sporulation and continued vegetative growth during each cell-cycle spent in starvation. Furthermore, changes in DNA replication and cell-cycle parameters with decreased growth rate in starvation conditions enable cells to indirectly detect starvation without the need for evaluating specific metabolites. The simplicity of the uncovered coordination mechanism and starvation sensing suggests that it may be widely applicable in a variety of gene regulatory and stress-response settings. This work is supported by National Science Foundation Grants MCB-1244135, EAGER-1450867, MCB-1244423, NIH NIGMS Grant R01 GM088428 and HHMI International Student Fellowship.

  20. The DNA replication priming protein, PriA, is required for homologous recombination and double-strand break repair.

    Kogoma, T; Cadwell, G W; Barnard, K G; Asai, T

    1996-01-01

    The PriA protein, a component of the phiX174-type primosome, was previously shown to be essential for damage-inducible DNA replication in Escherichia coli, termed inducible stable DNA replication. Here, we show that priA::kan null mutants are defective in transductional and conjugational homologous recombination and are hypersensitive to mitomycin C and gamma rays, which cause double-strand breaks. The introduction of a plasmid carrying the priA300 allele, which encodes a mutant PriA protein ...

  1. Dynamic regulation of genes involved in mitochondrial DNA replication and transcription during mouse brown fat cell differentiation and recruitment

    Murholm, Maria; Dixen, Karen; Qvortrup, Klaus;

    2009-01-01

    ) and a remarkably higher mitochondrial abundance in brown adipocytes. METHODOLOGY/PRINCIPAL FINDINGS: Here we report a comprehensive characterisation of gene expression linked to mitochondrial DNA replication, transcription and function during white and brown fat cell differentiation in vitro as well...... in brown adipocyte precursor cells promotes mitochondrial DNA replication, and that silencing of PRDM16 expression during brown fat cell differentiation blunts mitochondrial biogenesis and expression of brown fat cell markers. CONCLUSIONS/SIGNIFICANCE: Using both in vitro and in vivo model systems of...

  2. Identification of a lytic-phase origin of DNA replication in human herpesvirus 6B strain Z29.

    Dewhurst, S; Dollard, S C; Pellett, P E; Dambaugh, T R

    1993-01-01

    DNA sequences which have structural features suggestive of their functioning as an origin of lytic-phase DNA replication were previously identified in both human herpesvirus 6B strain Z29 [HHV-6B (Z29)] and in HHV-6A (U1102). Plasmid constructs containing the putative HHV-6B (Z29) oriLyt element were replicated after transfection into permissive T cells, when trans-acting factors were provided by HHV-6B (R-1) infection. By using this assay, the HHV-6B (Z29) oriLyt was mapped to a minimal regi...

  3. Small-molecule inhibitors identify the RAD52-ssDNA interaction as critical for recovery from replication stress and for survival of BRCA2 deficient cells

    Hengel, Sarah R; Malacaria, Eva; Folly da Silva Constantino, Laura; Bain, Fletcher E; Diaz, Andrea; Koch, Brandon G; Yu, Liping; Wu, Meng; Pichierri, Pietro; Spies, M Ashley; Spies, Maria

    2016-01-01

    The DNA repair protein RAD52 is an emerging therapeutic target of high importance for BRCA-deficient tumors. Depletion of RAD52 is synthetically lethal with defects in tumor suppressors BRCA1, BRCA2 and PALB2. RAD52 also participates in the recovery of the stalled replication forks. Anticipating that ssDNA binding activity underlies the RAD52 cellular functions, we carried out a high throughput screening campaign to identify compounds that disrupt the RAD52-ssDNA interaction. Lead compounds were confirmed as RAD52 inhibitors in biochemical assays. Computational analysis predicted that these inhibitors bind within the ssDNA-binding groove of the RAD52 oligomeric ring. The nature of the inhibitor-RAD52 complex was validated through an in silico screening campaign, culminating in the discovery of an additional RAD52 inhibitor. Cellular studies with our inhibitors showed that the RAD52-ssDNA interaction enables its function at stalled replication forks, and that the inhibition of RAD52-ssDNA binding acts additively with BRCA2 or MUS81 depletion in cell killing. DOI: http://dx.doi.org/10.7554/eLife.14740.001 PMID:27434671

  4. Two potential Petunia hybrida mitochondrial DNA replication origins show structural and in vitro functional homology with the animal mitochondrial DNA heavy and light strand replication origins

    Haas, Jan M. de; Hille, Jacques; Kors, Frank; Meer, Bert van der; Kool, Ad J.; Folkerts, Otto; Nijkamp, H. John J.

    1991-01-01

    Four Petunia hybrida mitochondrial (mt) DNA fragments have been isolated, sequenced, localized on the physical map and analyzed for their ability to initiate specific DNA synthesis. When all four mtDNA fragments were tested as templates in an in vitro DNA synthesizing lysate system, developed from p

  5. Biochemical evidence for the requirement of Hoogsteen base pairing for replication by human DNA polymerase iota.

    Johnson, Robert E; Prakash, Louise; Prakash, Satya

    2005-07-26

    Because of the near geometric identity of Watson-Crick (W-C) GxC and AxT base pairs, a given DNA polymerase forms the four possible correct base pairs with nearly identical catalytic efficiencies. However, human DNA polymerase iota (Pol iota), a member of the Y family of DNA polymerases, exhibits a marked template specificity, being more efficient at incorporating the correct nucleotide opposite template purines than opposite pyrimidines. By using 7-deazaadenine and 7-deazaguanine as the templating residues, which disrupt Hoogsteen base pair formation, we show that, unlike the other DNA polymerases belonging to the A, B, or Y family, DNA synthesis by Pol iota is severely inhibited by these N7-modified bases. These observations provide biochemical evidence that, during normal DNA synthesis, template purines adopt a syn conformation in the Pol iota active site, enabling the formation of a Hoogsteen base pair with the incoming pyrimidine nucleotide. Additionally, mutational studies with Leu-62, which lies in close proximity to the templating residue in the Pol iota ternary complex, have indicated that both factors, steric constraints within the active site and the stability provided by the hydrogen bonds in the Hoogsteen base pair, contribute to the efficiency of correct nucleotide incorporation opposite template purines by Pol iota. PMID:16014707

  6. Modulation of UvrD Helicase Activity by Covalent DNA-Protein Cross-links*

    Kumari, Anuradha; Minko, Irina G.; Smith, Rebecca L.; Lloyd, R. Stephen; McCullough, Amanda K.

    2010-01-01

    UvrD (DNA helicase II) has been implicated in DNA replication, DNA recombination, nucleotide excision repair, and methyl-directed mismatch repair. The enzymatic function of UvrD is to translocate along a DNA strand in a 3′ to 5′ direction and unwind duplex DNA utilizing a DNA-dependent ATPase activity. In addition, UvrD interacts with many other proteins involved in the above processes and is hypothesized to facilitate protein turnover, thus promoting further DNA processing. Although UvrD int...

  7. DNA replication and repair in a human melanoma cell-line resistant to ultra-violet-radiation

    The effect of ultra-violet (U.V.)-irradiation on DNA replication was studied in a U.V.-resistant, human melanoma cell-line (MM96). Semi-conservative synthesis of DNA was decreased about five-fold by a U.V.-dose of 100 ergs/mm2. The size of DNA fragments synthesized in irradiated cells at short times after U.V. was smaller than those synthesized in unirradiated cells. Elongation of these fragments occurred with time, and 6 hours after irradiation cells synthesized DNA in fragments of the same size as obtained in unirradiated cells. In this post-replication repair process, elongation appeared to involve de novo synthesis and was not inhibited by theophylline. (author)

  8. miR-30a can inhibit DNA replication by targeting RPA1 thus slowing cancer cell proliferation.

    Zou, Zhenyou; Ni, Mengjie; Zhang, Jing; Chen, Yongfeng; Ma, Hongyu; Qian, Shihan; Tang, Longhua; Tang, Jiamei; Yao, Hailun; Zhao, Chengbin; Lu, Xiongwen; Sun, Hongyang; Qian, Jue; Mao, Xiaoting; Lu, Xulin; Liu, Qun; Zen, Juping; Wu, Hanbing; Bao, Zhaosheng; Lin, Shudan; Sheng, Hongyu; Li, Yunlong; Liang, Yong; Chen, Zhiqiang; Zong, Dan

    2016-07-15

    Cell proliferation was inhibited following forced over-expression of miR-30a in the ovary cancer cell line A2780DX5 and the gastric cancer cell line SGC7901R. Interestingly, miR-30a targets the DNA replication protein RPA1, hinders the replication of DNA and induces DNA fragmentation. Furthermore, ataxia telangiectasia mutated (ATM) and checkpoint kinase 2 (CHK2) were phosphorylated after DNA damage, which induced p53 expression, thus triggering the S-phase checkpoint, arresting cell cycle progression and ultimately initiating cancer cell apoptosis. Therefore, forced miR-30a over-expression in cancer cells can be a potential way to inhibit tumour development. PMID:27208176

  9. Bacillus subtilis strain deficient for the protein-tyrosine kinase PtkA exhibits impaired DNA replication

    Petranovic, Dina; Michelsen, Ole; Zahradka, K;

    2007-01-01

    study. We were unable to identify any striking phenotypes related to control of UDP-glucose dehydrogenases, natural competence and DNA lesion repair; however, a very strong phenotype of ΔptkA emerged with respect to DNA replication and cell cycle control, as revealed by flow cytometry and fluorescent...... microscopy. B. subtilis cells lacking the kinase PtkA accumulated extra chromosome equivalents, exhibited aberrant initiation mass for DNA replication and an unusually long D period....... previously shown to regulate the phosphorylation state of UDP-glucose dehydrogenases and single-stranded DNA-binding proteins. This promiscuity towards substrates is reminiscent of eukaryal kinases and has prompted us to investigate possible physiological effects of ptkA and ptpZ gene inactivations in this...

  10. Inhibition of human cytomegalovirus DNA replication by small interfering RNAs targeted to UL49

    Kezhen Wang; Yueqin Li; Gaoxiang Zhao; Yingzi Wu; Xin Zhang; Hongjian Li; Tianhong Zhou

    2013-01-01

    Human cytomegalovirus (HCMV) is a ubiquitous virus.Although the infection in healthy children and adults is usually asymptomatic,in immunocompromised individuals and newborns it is a significant cause of morbidity and mortality.UL49,an essential gene of HCMV,is highly conserved among various HCMV strains.The expression of UL49 is correlated with the production of virions.When UL49 is inhibited in the HCMV,the production of virions is reduced severely.In this study,RNA interference was applied to further investigate the roles of UL49 in viral replication.Two effective small interfering RNAs against UL49 were selected.Silencing of UL49 in HCMV-infected human foreskin fibroblast cells reduced the transcription levels of early and late genes,but not immediate-early ones.In addition,the viral DNA content was significantly reduced.This is the first time to uncover the role of UL49 in viral DNA synthesis,which indicates that UL49 might play an important role in this period.So the down-regulation of UL49 mRNA using RNAi might be a potential clinical therapy against the virus.

  11. Host DNA replication or excision repair requirement for ultraviolet induction of bacteriophage lambda lysogens

    It is stated that the mechanism for prophage induction by radiation, or chemical agents, is not known, although a variety of hypothesis have been advanced during recent years. Biochemical data have been described that seem to favour the suggestion that DNA intermediates in the repair of DNA damage compete with prophage operators for repressor binding. When sufficient repressor is bound none remains for prophage repression and induction occurs. If this is so the prediction may be made that induction should not occur in the absence of normal repair processes. Some experimental work is described with a view to testing and verifying this prediction. A bacteriophage lambda lysogen was used in the work. Irradiation was at 420C, and samples were removed at intervals and assayed for free plaque-forming units, little induction was observed over a wide range of UV doses in non-replicating non-excising lysogens, in contradiction with some earlier results. Competition between prophage operators and repair intermediates for lambda repressor appears to be the simplest way to account for the observed results, although other possibilities are discussed. (U.K.)

  12. Cloning human herpes virus 6A genome into bacterial artificial chromosomes and study of DNA replication intermediates

    Borenstein, Ronen; Frenkel, Niza

    2009-01-01

    Cloning of large viral genomes into bacterial artificial chromosomes (BACs) facilitates analyses of viral functions and molecular mutagenesis. Previous derivations of viral BACs involved laborious recombinations within infected cells. We describe a single-step production of viral BACs by direct cloning of unit length genomes, derived from circular or head-to-tail concatemeric DNA replication intermediates. The BAC cloning is independent of intracellular recombinations and DNA packaging constr...

  13. Interaction of the replication terminator protein (RTP) with DNA probed by NMR spectroscopy and x-ray crystallography

    Full text: The arrest of replication forks during the termination of DNA replication in Bacillus subtilis is dependent upon the binding of the 30 kDa replication terminator protein (RTP) to its cognate Ter binding site. Two adjacently bound dimers of RTP form a termination complex that can prevent the progression of a replication fork approaching from one direction, but not the other. The crystal structure of free RTP has previously been solved, but the precise orientation with which it binds to Ter sites remains unknown. This information is important for understanding the molecular mechanism of replication fork arrest. We have used NMR spectroscopy to observe 1H-15N correlations arising from 15N-labelled RTP mutant, and to track their perturbations upon the addition of DNA. This showed that 60% of the amino acid residues are affected by the DNA interaction, and also that the complex is symmetrical. Assignment of the 1H-15N correlations was achieved using a suite of triple resonance NMR experiments with 15N,13C,2H enriched protein recorded at 800 MHz and using TROSY pulse sequences. This revealed that α3-helices are involved in the binding interaction, and that the 'wings' of RTP may not be contributing to binding. Crystals of the complex have been grown from the NMR sample, and data collected to 3.1 Angstroms is anticipated to provide further molecular detail

  14. Study of the activity of DNA polymerases β and λ using 5-formyluridine containing DNA substrates

    Lavrik O. I.

    2012-06-01

    Full Text Available Aim. To investigate the TLS-activity of human DNA polymerases β and λ (pols β and λ using 5-formyluridine (5-foU containing DNA duplexes which are imitating the intermediates during replication of the leading DNA strand, and to study the influence of replication factors hRPA and hPCNA on this activity. Methods. The EMSA and the methods of enzyme’s kinetics were used. Results. The capability of pols β and λ to catalyze DNA synthesis across 5-foU was investigated and the kinetic characteristics of this process in the presence and in the absence of protein factors hRPA and hPCNA were evaluated. Conclusions. It was shown that: (i both proteins are able to catalyze TLS on used DNA substrates regardless of the reaction conditions, however, pol λ was more accurate enzyme; (ii hRPA can stimulate the efficacy of the nonmutagenic TLS catalyzed by pol at the nucleotide incorporation directly opposite of 5-foU, at the same time it doesn’t influence the incorporation efficacy if the damage displaced into the duplex; (iii hPCNA doesn’t influence the efficacy of TLS catalyzed by both enzymes.

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

    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...

  16. nfi-1 affects behavior and life-span in C. elegans but is not essential for DNA replication or survival

    Hirono Keiko

    2005-10-01

    Full Text Available Abstract Background The Nuclear Factor I (one (NFI family of transcription/replication factors plays essential roles in mammalian gene expression and development and in adenovirus DNA replication. Because of its role in viral DNA replication NFI has long been suspected to function in host DNA synthesis. Determining the requirement for NFI proteins in mammalian DNA replication is complicated by the presence of 4 NFI genes in mice and humans. Loss of individual NFI genes in mice cause defects in brain, lung and tooth development, but the presence of 4 homologous NFI genes raises the issue of redundant roles for NFI genes in DNA replication. No NFI genes are present in bacteria, fungi or plants. However single NFI genes are present in several simple animals including Drosophila and C. elegans, making it possible to test for a requirement for NFI in multicellular eukaryotic DNA replication and development. Here we assess the functions of the single nfi-1 gene in C. elegans. Results C. elegans NFI protein (CeNFI binds specifically to the same NFI-binding site recognized by vertebrate NFIs. nfi-1 encodes alternatively-spliced, maternally-inherited transcripts that are expressed at the single cell stage, during embryogenesis, and in adult muscles, neurons and gut cells. Worms lacking nfi-1 survive but have defects in movement, pharyngeal pumping and egg-laying and have a reduced life-span. Expression of the muscle gene Ce titin is decreased in nfi-1 mutant worms. Conclusion NFI gene function is not needed for survival in C. elegans and thus NFI is likely not essential for DNA replication in multi-cellular eukaryotes. The multiple defects in motility, egg-laying, pharyngeal pumping, and reduced lifespan indicate that NFI is important for these processes. Reduction in Ce titin expression could affect muscle function in multiple tissues. The phenotype of nfi-1 null worms indicates that NFI functions in multiple developmental and behavioral systems in C

  17. Molecular cloning and DNA sequence analysis of Escherichia coli priA, the gene encoding the primosomal protein replication factor Y.

    Nurse, P; DiGate, R J; Zavitz, K H; Marians, K J

    1990-01-01

    Escherichia coli replication factor Y (protein n') functions in the assembly of a mobile multiprotein replication-priming complex called the primosome. Although the role of factor Y in primosome assembly during replication in vitro of bacteriophage phi X174 and plasmid pBR322 DNA is clear, its role in E. coli chromosomal replication is not. To address this issue, the gene for factor Y has been cloned molecularly and its DNA sequence has been determined. The cloned fragment of DNA contained an...

  18. FBH1 co-operates with MUS81 in inducing DNA double-strand breaks and cell death following replication stress

    Fugger, Kasper; Chu, Wai Kit; Haahr, Peter;

    2013-01-01

    The molecular events occurring following the disruption of DNA replication forks are poorly characterized, despite extensive use of replication inhibitors such as hydroxyurea in the treatment of malignancies. Here, we identify a key role for the FBH1 helicase in mediating DNA double-strand break ...

  19. Local chromatin microenvironment determines DNMT activity : from DNA methyltransferase to DNA demethylase or DNA dehydroxymethylase

    van der Wijst, Monique G. P.; Venkiteswaran, Muralidhar; Chen, Hui; Xu, Guo-Liang; Plosch, Torsten; Rots, Marianne G.

    2015-01-01

    Insights on active DNA demethylation disproved the original assumption that DNA methylation is a stable epigenetic modification. Interestingly, mammalian DNA methyltransferases 3A and 3B (DNMT-3A and -3B) have also been reported to induce active DNA demethylation, in addition to their well-known fun

  20. A DNA binding winged helix domain in CAF-1 functions with PCNA to stabilize CAF-1 at replication forks

    Zhang, Kuo; Gao, Yuan; Li, Jingjing; Burgess, Rebecca; Han, Junhong; Liang, Huanhuan; Zhang, Zhiguo; Liu, Yingfang

    2016-01-01

    Chromatin assembly factor 1 (CAF-1) is a histone H3–H4 chaperone that deposits newly synthesized histone (H3–H4)2 tetramers during replication-coupled nucleosome assembly. However, how CAF-1 functions in this process is not yet well understood. Here, we report the crystal structure of C terminus of Cac1 (Cac1C), a subunit of yeast CAF-1, and the function of this domain in stabilizing CAF-1 at replication forks. We show that Cac1C forms a winged helix domain (WHD) and binds DNA in a sequence-independent manner. Mutations in Cac1C that abolish DNA binding result in defects in transcriptional silencing and increased sensitivity to DNA damaging agents, and these defects are exacerbated when combined with Cac1 mutations deficient in PCNA binding. Similar phenotypes are observed for corresponding mutations in mouse CAF-1. These results reveal a mechanism conserved in eukaryotic cells whereby the ability of CAF-1 to bind DNA is important for its association with the DNA replication forks and subsequent nucleosome assembly. PMID:26908650

  1. The spatiotemporal program of DNA replication is associated with specific combinations of chromatin marks in human cells.

    Franck Picard

    2014-05-01

    Full Text Available The duplication of mammalian genomes is under the control of a spatiotemporal program that orchestrates the positioning and the timing of firing of replication origins. The molecular mechanisms coordinating the activation of about [Formula: see text] predicted origins remain poorly understood, partly due to the intrinsic rarity of replication bubbles, making it difficult to purify short nascent strands (SNS. The precise identification of origins based on the high-throughput sequencing of SNS constitutes a new methodological challenge. We propose a new statistical method with a controlled resolution, adapted to the detection of replication origins from SNS data. We detected an average of 80,000 replication origins in different cell lines. To evaluate the consistency between different protocols, we compared SNS detections with bubble trapping detections. This comparison demonstrated a good agreement between genome-wide methods, with 65% of SNS-detected origins validated by bubble trapping, and 44% of bubble trapping origins validated by SNS origins, when compared at the same resolution. We investigated the interplay between the spatial and the temporal programs of replication at fine scales. We show that most of the origins detected in regions replicated in early S phase are shared by all the cell lines investigated whereas cell-type-specific origins tend to be replicated in late S phase. We shed a new light on the key role of CpG islands, by showing that 80% of the origins associated with CGIs are constitutive. Our results further show that at least 76% of CGIs are origins of replication. The analysis of associations with chromatin marks at different timing of cell division revealed new potential epigenetic regulators driving the spatiotemporal activity of replication origins. We highlight the potential role of H4K20me1 and H3K27me3, the coupling of which is correlated with increased efficiency of replication origins, clearly identifying those

  2. Replication independent DNA double-strand break retention may prevent genomic instability

    Pornthanakasem Wichai

    2010-03-01

    Full Text Available Abstract Background Global hypomethylation and genomic instability are cardinal features of cancers. Recently, we established a method for the detection of DNA methylation levels at sites close to endogenous DNA double strand breaks (EDSBs, and found that those sites have a higher level of methylation than the rest of the genome. Interestingly, the most significant differences between EDSBs and genomes were observed when cells were cultured in the absence of serum. DNA methylation levels on each genomic location are different. Therefore, there are more replication-independent EDSBs (RIND-EDSBs located in methylated genomic regions. Moreover, methylated and unmethylated RIND-EDSBs are differentially processed. Euchromatins respond rapidly to DSBs induced by irradiation with the phosphorylation of H2AX, γ-H2AX, and these initiate the DSB repair process. During G0, most DSBs are repaired by non-homologous end-joining repair (NHEJ, mediated by at least two distinct pathways; the Ku-mediated and the ataxia telangiectasia-mutated (ATM-mediated. The ATM-mediated pathway is more precise. Here we explored how cells process methylated RIND-EDSBs and if RIND-EDSBs play a role in global hypomethylation-induced genomic instability. Results We observed a significant number of methylated RIND-EDSBs that are retained within deacetylated chromatin and free from an immediate cellular response to DSBs, the γ-H2AX. When cells were treated with tricostatin A (TSA and the histones became hyperacetylated, the amount of γ-H2AX-bound DNA increased and the retained RIND-EDSBs were rapidly repaired. When NHEJ was simultaneously inhibited in TSA-treated cells, more EDSBs were detected. Without TSA, a sporadic increase in unmethylated RIND-EDSBs could be observed when Ku-mediated NHEJ was inhibited. Finally, a remarkable increase in RIND-EDSB methylation levels was observed when cells were depleted of ATM, but not of Ku86 and RAD51. Conclusions Methylated RIND-EDSBs are

  3. Role of bacterial and phage recombination systems and of DNA replication in genetic recombination of UV-irradiated phage lambda

    A study was made in E. coli K12 of the influence of the bacterial Rec and phage lambda Red recombination systems on the rescue of the 0+ gene from the prophage by a superinfecting 0- phage, UV irradiated or not. In the absence of UV irradiation the Red system produces more recombinants than does the Rec system, and its action requires DNA replication. The presence of UV lesions in the lambda DNA facilitates the action of the Rec system, which is more efficient in this instance than the Red system and can act in the absence of DNA replication. In all cases, there is a cooperation between the two generalized recombination systems. (orig.)

  4. Effect of the interaction between M-CSF and MCM7 on DNA replication in HeLa cells

    ZHANG Meng-xia; WU Hai-yan; TU Jian; ZHANG Xiao-hong; LE Xiao-yong; TANG Sheng-song

    2008-01-01

    Objective To explore the effect of the interaction between microphage colony-stimulating factor (M-CSF) and minichromosome maintenance protein-7(Mcm7) on DNA replication in HeLa cells. Methods pCMV/nuc/mye, pCMV/nuc/GFP and pCMV/nuc/M-CSF vector were stably transfected into HeLa cells by Lipofectarnine, respectively. After screening with G418, the expression and localization of M-CSF in HeLa cells were verified by RT-PCR, Western blot and immunofluoreseence staining. The statue and interaction between intracellular M-CSF and Mcm7 in HeLa cells was analyzed by co-immunoprecipitation. The effect of the interaction between M-CSF and Mcm7 on DNA replication was analyzed by a mammalian cell or cell-free DNA replication system in vitro. Results The results indicated that the M-CSF-transfected HeLa cells stably express both M-CSF mRNA and protein, and that M-CSF protein is located to the nuclei of HeLa cells mentioned above. To further analyze the status and interaction between intracellular M-CSF and Mcm7, the Mcm7 from HeLa cells was precipitated with anti-Mcm7 antibody and followed by Protein A/G PLUS agarose. The precipitation was blotted with anti-M-CSF monoclonal antibody. The results show that M-CSF was coprecipitated with Mcm7, so intracellular M-CSF existed in Mcm7-bound state. The DNA replication experiments reveal that a higher percentage of the replicating nuclei is present either in unsyn-chronized or in both synchronized G1 and S phase M-CSF-transfected HeLa cells, compared with both pCMV/nuc-transfeeted and un-transfected HeLa cells, which suggests that interaction between M-CSF and Mcm7 promote both the initiation and elongation of DNA replication. Conclusions M-CSF directly interacts with McmT. The interaction between M-CSF and Mcm7 promotes both the initiation and elongation of DNA replication.

  5. Kinetics of reparative DNA replication induced in CBA and C57BL/6 mouse spleen cells by urethane and influenza virus

    The authors investigated the reparative replication of DNA, induced by urethane and influenza virus in C57BL/6 and CBA mice which have marked differences in tumor formation. Reparative DNA replication, induced by urethane and influenza virus was investigated by a liquid scintillation method based on measuring incorporation of 3H-thymidine into the total mass of mouse spleen cells, when replicative DNA synthesis was inhibited by hydroxyurea. The intensity of reparative DNA replication was judged from the value of the stimulation index which is the ratio of the radioactivity in the spleen cells of the experimental animals to the corresponding parameters in cells of intact animals. The phenomenon of the stimulating and inhibitory effects of influenza virus on reparative DNA replication in cells depending on their genotype and sensitivity to virus is established

  6. Rapid, Semiquantitative Assay To Discriminate among Compounds with Activity against Replicating or Nonreplicating Mycobacterium tuberculosis

    Gold, Ben; Roberts, Julia; Ling, Yan; Quezada, Landys Lopez; Glasheen, Jou; Ballinger, Elaine; Somersan-Karakaya, Selin; Warrier, Thulasi; Warren, J. David; Nathan, Carl

    2015-01-01

    The search for drugs that can kill replicating and nonreplicating Mycobacterium tuberculosis faces practical bottlenecks. Measurement of CFU and discrimination of bacteriostatic from bactericidal activity are costly in compounds, supplies, labor, and time. Testing compounds against M. tuberculosis under conditions that prevent the replication of M. tuberculosis often involves a second phase of the test in which conditions are altered to permit the replication of bacteria that survived the fir...

  7. Informatics and Physics Models of Recognitions of DNA Replication and Their Biological Applications

    Kang Cheng

    2006-01-01

    Full Text Available We initially propose concepts of Informative Intensity, Informative Response Intensity and Informative Flux, with different expressions. In a special expression of electrostatics, we describe Informative Intensity, Informative Response Intensity and Informative Flux in terms of electric field intensity, electric field force and electric field flux respectively. In a special expression of quantum mechanics, we originally propose a concept of Probability Flux. Then we present Informative Intensity in terms of a wave function of Schrödinger equation and Informative Response Intensity in terms of an interactive force between or among objects, Informative Flux in terms of a probability flux. Based on these concepts, we develop our Informatics and physics models of recognitions between a DNA polymerase and an initiation site and of pairing deoxyribonucleotides, for a natural DNA replication, beyond lengths of chemical bounds and half quantitatively explain a probability of the wrong paring. We originally hypothesize the information is, stored in structured charges or masses, transmitted with Informative Intensity in a media and recognized with an Informative Response Intensity by other structured charges or masses. We further generalize, Informative Intensity as multiple layers of fields, potentials or waves, Informative Response Intensity as multiple layers of forces and Informative Flux as an integration of Informative Intensity. We also initially define Transverse Information: Informative Intensity is perpendicular to the direction of information propagation, e.g. electromagnetic wave or magnetic field; and Longitudinal Information: Informative Intensity is parallel or antiparalell to the direction of information propagation, e.g. electric field or gravitational field.

  8. Replicative bypass repair of ultraviolet damage to DNA of mammalian cells: caffeine sensitive and caffeine resistant mechanism

    Replicative bypass repair of UV damage to DNA was studied in a wide variaty of human, mouse and hamster cells in culture. Survival curve analysis revealed that in established cell lines (mouse L, Chinese hamster V79, HeLa S3 and SV40-transformed xeroderma pigmentosum (XP), post-UV caffeine treatment potentiated cell killing by reducing the extrapolation number and mean lethal UV fluence (Do). In the Do reduction as the result of random inactivation by caffeine of sensitive repair there were marked clonal differences among such cell lines, V79 being most sensitive to caffeine potentiation. However, other diploid cell lines (normal human, excision-defective XP and Syrian hamster) exhibited no obvious reduction in Do by caffeine. In parallel, alkaline sucrose sedimentation results showed that the conversion of initially smaller segments of DNA synthesized after irradiation with 10 J/m2 to high-molecular-weight DNA was inhibited by caffeine in transformed XP cells, but not in the diploid human cell lines. Exceptionally, diploid XP variants had a retarded ability of bypass repair which was drastically prevented by caffeine, so that caffeine enhanced the lethal effect of UV. Neutral CsCl study on the bypass repair mechanism by use of bromodeoxyuridine for DNA synthesis on damaged template suggests that the pyrimodine dimer acts as a block to replication and subsequently it is circumvented presumably by a new process involving replicative bypassing following strand displacement, rather than by gap-filling de novo. This mechanism worked similarly in normal and XP cells, whether or not caffeine was present, indicating that excision of dimer is not always necessary. However, replicative bypassing became defective in XP variant and transformed XP cells when caffeine was present. It appears, therefore, that the replicative bypass repair process is either caffeine resistant or sensitive, depending on the cell type used, but not necessarily on the excision repair capability

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

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

    2005-01-01

    Human high mobility group box (HMGB) 1 and -2 proteins are highly conserved and abundant chromosomal proteins that regulate chromatin structure and DNA metabolism. HMGB proteins bind preferentially to DNA that is bent or underwound and to DNA damaged by agents such as cisplatin, UVC radiation, and...... benzo[a]pyrenediol epoxide (BPDE). Binding of HMGB1 to DNA adducts is thought to inhibit nucleotide excision repair (NER), leading to cell death, but the biological roles of these proteins remain obscure. We have used psoralen-modified triplex-forming oligonucleotides (TFOs) to direct a psoralen-DNA...... interstrand cross-link (ICL) to a specific site to determine the effect of HMGB proteins on recognition of these lesions. Our results reveal that human HMGB1 (but not HMGB2) binds with high affinity and specificity to psoralen ICLs, and interacts with the essential NER protein, replication protein A (RPA), at...

  10. Replication Fidelity of Escherichia Coli DNA Polymerase III Holoenzyme in Vitro and Repair of Heteroduplex DNA with Multibase Loops in Vivo.

    Carraway, Margaretha Bernardina Maria

    The genetic integrity of an organism is maintained by accurate replication and correction of asymmetry in the DNA. To study replication fidelity, single-stranded plasmid DNA containing the mnt gene, was replicated in vitro with DNA polymerase III holoenzyme by extension of a complimentary annealed primer. On this plasmid the mnt region is fused to a promoterless tet gene. Accurate replication of mnt generates a tetracycline sensitive phenotype, errors in replication are identified by mutation to tetracycline resistance. Mismatch repair deficient mutH cells were transformed to ampicillin-resistance by replicated circles. The mutations in mnt were identified by replica plating and selecting for tetracycline resistant cells. The mutation rate was 1 in 100,000. DNA sequence analysis of 65 isolates identified 33 single base changes, 20 deletions and 12 concurrent deletions and insertions. Except for the deletions and substitutions, identical mutations were isolated in vivo in mismatch repair deficient cells. Therefore, in vitro replication errors resemble those isolated in vivo. Heteroduplexes with loops occur as a result of replication or recombination. To examine if E. coli converts these molecules to a homoduplex via DNA repair, plasmid heteroduplexes with loops of 5, 7, 9, 192, 410 or 514 bases in mnt were constructed. Conversion was examined by tranforming the plasmid heteroduplexes into E. coli lysogens which had a non-functional mnt gene fused to a promoterless lac gene. Repair of the heteroduplex to wild type yields white/tetracycline sensitive colonies; repair to the mutant yields red/tetracycline resistant colonies and no repair results in red-white (mixed)/tetracycline resistant colonies. No significant change in colony color distribution was observed when the heteroduplexes were transformed into wild type and the following mutant strains: pcnB, mutS, recA, recD, recBC sbcBC, recF, recJ, recR, recN, recO, recG ruvC, ruvB, lexA3, lexA51, uvrA, recBC sbcBC rec

  11. Molecular docking of the anticancer bioactive compound proceraside with macromolecules involved in the cell cycle and DNA replication.

    Gurung, A B; Ali, M A; Bhattacharjee, A; AbulFarah, M; Al-Hemaid, F; Abou-Tarboush, F M; Al-Anazi, K M; Al-Anazi, F S M; Lee, J

    2016-01-01

    The bioactive compounds proceraside A, frugoside and calotropin, which were extracted from the root bark of Calotropis procera (Aiton) W.T. Aiton (family Asclepiadaceae), were recently reported to inhibit the growth of inhibition against various human cancer cell lines in vitro. However, their modes of action have not been clearly defined. Therefore, we attempted an in silico approach to gain insights into their binding modes against the following selected molecular targets: CDK-2, CDK-6, topoisomerase I, BCL-2, VEGFR-2, telomere: G-quadruplex, and topoisomerase II. These targets were selected based on their key roles in cancer progression via the regulation of the cell cycle and DNA replication. Molecular-docking analyses revealed that proceraside A was the best docked ligand against all the targets, with the exception of telomere-G: quadruplex. Furthermore, it displayed the lowest binding energies and inhibition constants, and critical hydrogen bonds and hydrophobic interactions with the targets were also revealed. The present study may aid in the identification of possible targets for proceraside A, and might provide a plausible explanation for its proven anti-tumor activities. Moreover, the result of this study may further guide structure-activity relationship studies used to generate more potent target-specific inhibitors. PMID:27173346

  12. Biological activity of SV40 DNA

    This thesis deals with a study on the biological activity of SV40 DNA. The transforming activity of SV40 DNA and DNA fragments is investigated in order to define as precisely as possible the area of the viral genome that is involved in the transformation. The infectivity of SV40 DNA is used to study the defective repair mechanisms of radiation damages of human xeroderma pigmentosum cells. (C.F.)

  13. Activation of nucleotide oligomerization domain 2 (NOD2 by human cytomegalovirus initiates innate immune responses and restricts virus replication.

    Arun Kapoor

    Full Text Available Nucleotide-binding oligomerization domain 2 (NOD2 is an important innate immune sensor of bacterial pathogens. Its induction results in activation of the classic NF-κB pathway and alternative pathways including type I IFN and autophagy. Although the importance of NOD2 in recognizing RNA viruses has recently been identified, its role in sensing DNA viruses has not been studied. We report that infection with human cytomegalovirus (HCMV results in significant induction of NOD2 expression, beginning as early as 2 hours post infection and increasing steadily 24 hours post infection and afterwards. Infection with human herpesvirus 1 and 2 does not induce NOD2 expression. While the HCMV-encoded glycoprotein B is not required for NOD2 induction, a replication competent virion is necessary. Lentivirus-based NOD2 knockdown in human foreskin fibroblasts (HFFs and U373 glioma cells leads to enhanced HCMV replication along with decreased levels of interferon beta (IFN-β and the pro-inflammatory cytokine, IL8. NOD2 induction in HCMV-infected cells activates downstream NF-κB and interferon pathways supported by reduced nuclear localization of NF-κB and pIRF3 in NOD2 knockdown HFFs. Stable overexpression of NOD2 in HFFs restricts HCMV replication in association with increased levels of IFN-β and IL8. Similarly, transient overexpression of NOD2 in U373 cells or its downstream kinase, RIPK2, results in decreased HCMV replication and enhanced cytokine responses. However, overexpression of a mutant NOD2, 3020insC, associated with severe Crohn's disease, results in enhanced HCMV replication and decreased levels of IFN-β in U373 cells. These results show for the first time that NOD2 plays a significant role in HCMV replication and may provide a model for studies of HCMV recognition by the host cell and HCMV colitis in Crohn's disease.

  14. Abnormal ultraviolet mutagenic spectrum in plasmid DNA replicated in cultured fibroblasts from a patient with the skin cancer-prone disease, xeroderma pigmentosum.

    Seetharam, S; Protić-Sabljić, M; Seidman, M M; Kraemer, K H

    1987-01-01

    A shuttle vector plasmid, pZ189, was utilized to assess the types of mutations that cells from a patient with xeroderma pigmentosum, complementation group D, introduce into ultraviolet (UV) damaged, replicating DNA. Patients with xeroderma pigmentosum have clinical and cellular UV hypersensitivity, increased frequency of sun-induced skin cancer, and deficient DNA repair. In comparison to UV-treated pZ189 replicated in DNA repair-proficient cells, there were fewer surviving plasmids, a higher ...

  15. Single-molecule imaging of DNA polymerase I (Klenow fragment) activity by atomic force microscopy

    Chao, J.; Zhang, P.; Wang, Q.; Wu, N.; Zhang, F.; Hu, J.; Fan, C. H.; Li, B.

    2016-03-01

    We report a DNA origami-facilitated single-molecule platform that exploits atomic force microscopy to study DNA replication. We imaged several functional activities of the Klenow fragment of E. coli DNA polymerase I (KF) including binding, moving, and dissociation from the template DNA. Upon completion of these actions, a double-stranded DNA molecule was formed. Furthermore, the direction of KF activities was captured and then confirmed by shifting the KF binding sites on the template DNA.We report a DNA origami-facilitated single-molecule platform that exploits atomic force microscopy to study DNA replication. We imaged several functional activities of the Klenow fragment of E. coli DNA polymerase I (KF) including binding, moving, and dissociation from the template DNA. Upon completion of these actions, a double-stranded DNA molecule was formed. Furthermore, the direction of KF activities was captured and then confirmed by shifting the KF binding sites on the template DNA. Electronic supplementary information (ESI) available. See DOI: 10.1039/c5nr06544e

  16. Hemi-methylated DNA regulates DNA methylation inheritance through allosteric activation of H3 ubiquitylation by UHRF1

    Harrison, Joseph S; Cornett, Evan M; Goldfarb, Dennis; DaRosa, Paul A; Li, Zimeng M; Yan, Feng; Dickson, Bradley M; Guo, Angela H; Cantu, Daniel V; Kaustov, Lilia; Brown, Peter J; Arrowsmith, Cheryl H; Erie, Dorothy A; Major, Michael B; Klevit, Rachel E; Krajewski, Krzysztof; Kuhlman, Brian; Strahl, Brian D; Rothbart, Scott B

    2016-01-01

    The epigenetic inheritance of DNA methylation requires UHRF1, a histone- and DNA-binding RING E3 ubiquitin ligase that recruits DNMT1 to sites of newly replicated DNA through ubiquitylation of histone H3. UHRF1 binds DNA with selectivity towards hemi-methylated CpGs (HeDNA); however, the contribution of HeDNA sensing to UHRF1 function remains elusive. Here, we reveal that the interaction of UHRF1 with HeDNA is required for DNA methylation but is dispensable for chromatin interaction, which is governed by reciprocal positive cooperativity between the UHRF1 histone- and DNA-binding domains. HeDNA recognition activates UHRF1 ubiquitylation towards multiple lysines on the H3 tail adjacent to the UHRF1 histone-binding site. Collectively, our studies are the first demonstrations of a DNA-protein interaction and an epigenetic modification directly regulating E3 ubiquitin ligase activity. They also define an orchestrated epigenetic control mechanism involving modifications both to histones and DNA that facilitate UHRF1 chromatin targeting, H3 ubiquitylation, and DNA methylation inheritance. DOI: http://dx.doi.org/10.7554/eLife.17101.001 PMID:27595565

  17. DNA polymerase-α regulates the activation of type I interferons through cytosolic RNA:DNA synthesis.

    Starokadomskyy, Petro; Gemelli, Terry; Rios, Jonathan J; Xing, Chao; Wang, Richard C; Li, Haiying; Pokatayev, Vladislav; Dozmorov, Igor; Khan, Shaheen; Miyata, Naoteru; Fraile, Guadalupe; Raj, Prithvi; Xu, Zhe; Xu, Zigang; Ma, Lin; Lin, Zhimiao; Wang, Huijun; Yang, Yong; Ben-Amitai, Dan; Orenstein, Naama; Mussaffi, Huda; Baselga, Eulalia; Tadini, Gianluca; Grunebaum, Eyal; Sarajlija, Adrijan; Krzewski, Konrad; Wakeland, Edward K; Yan, Nan; de la Morena, Maria Teresa; Zinn, Andrew R; Burstein, Ezra

    2016-05-01

    Aberrant nucleic acids generated during viral replication are the main trigger for antiviral immunity, and mutations that disrupt nucleic acid metabolism can lead to autoinflammatory disorders. Here we investigated the etiology of X-linked reticulate pigmentary disorder (XLPDR), a primary immunodeficiency with autoinflammatory features. We discovered that XLPDR is caused by an intronic mutation that disrupts the expression of POLA1, which encodes the catalytic subunit of DNA polymerase-α. Unexpectedly, POLA1 deficiency resulted in increased production of type I interferons. This enzyme is necessary for the synthesis of RNA:DNA primers during DNA replication and, strikingly, we found that POLA1 is also required for the synthesis of cytosolic RNA:DNA, which directly modulates interferon activation. Together this work identifies POLA1 as a critical regulator of the type I interferon response. PMID:27019227

  18. Plasmid DNA initiates replication of yellow fever vaccine in vitro and elicits virus-specific immune response in mice

    Tretyakova, Irina; Nickols, Brian; Hidajat, Rachmat [Medigen, Inc., 8420 Gas House Pike, Suite S, Frederick, MD 21701 (United States); Jokinen, Jenny; Lukashevich, Igor S. [Department of Pharmacology and Toxicology, School of Medicine, Center for Predictive Medicine and Emerging Infectious Diseases, University of Louisville, Louisville, KY (United States); Pushko, Peter, E-mail: ppushko@medigen-usa.com [Medigen, Inc., 8420 Gas House Pike, Suite S, Frederick, MD 21701 (United States)

    2014-11-15

    Yellow fever (YF) causes an acute hemorrhagic fever disease in tropical Africa and Latin America. To develop a novel experimental YF vaccine, we applied iDNA infectious clone technology. The iDNA represents plasmid that encodes the full-length RNA genome of 17D vaccine downstream from a cytomegalovirus (CMV) promoter. The vaccine was designed to transcribe the full-length viral RNA and to launch 17D vaccine virus in vitro and in vivo. Transfection with 10 ng of iDNA plasmid was sufficient to start replication of vaccine virus in vitro. Safety of the parental 17D and iDNA-derived 17D viruses was confirmed in AG129 mice deficient in receptors for IFN-α/β/γ. Finally, direct vaccination of BALB/c mice with a single 20 μg dose of iDNA plasmid resulted in seroconversion and elicitation of virus-specific neutralizing antibodies in animals. We conclude that iDNA immunization approach combines characteristics of DNA and attenuated vaccines and represents a promising vaccination strategy for YF. - Highlights: • The iDNA{sup ®} platform combines advantages of DNA and live attenuated vaccines. • Yellow fever (YF) 17D vaccine was launched from iDNA plasmid in vitro and in vivo. • Safety of iDNA-generated 17D virus was confirmed in AG129 mice. • BALB/c mice seroconverted after a single-dose vaccination with iDNA. • YF virus-neutralizing response was elicited in iDNA-vaccinated mice.

  19. Role of Fanconi Anemia FANCG in Preventing Double-Strand Breakage and Chromosomal Rearrangement during DNA Replication

    Tebbs, R S; Hinz, J M; Yamada, N A; Wilson, J B; Jones, N J; Salazar, E P; Thomas, C B; Jones, I M; Thompson, L H

    2003-10-04

    The Fanconi anemia (FA) proteins overlap with those of homologous recombination through FANCD1/BRCA2, but the biochemical functions of other FA proteins are unknown. By constructing and characterizing a null fancg mutant of hamster CHO cells, we present several new insights for FA. The fancg cells show a broad sensitivity to genotoxic agents, not supporting the conventional concept of sensitivity to only DNA crosslinking agents. The aprt mutation rate is normal, but hprt mutations are reduced, which we ascribe to the lethality of large deletions. CAD and dhfr gene amplification rates are increased, implying excess chromosomal breakage during DNA replication, and suggesting amplification as a contributing factor to cancer-proneness in FA patients. In S-phase cells, both spontaneous and mutagen-induced Rad51 nuclear foci are elevated. These results support a model in which FancG protein helps to prevent collapse of replication forks by allowing translesion synthesis or lesion bypass through homologous recombination.

  20. DNA Sequences Proximal to Human Mitochondrial DNA Deletion Breakpoints Prevalent in Human Disease Form G-quadruplexes, a Class of DNA Structures Inefficiently Unwound by the Mitochondrial Replicative Twinkle Helicase

    Bharti, S.K.; Sommers, J.A.; Zhou, J.; Kaplan, D.L.; Spelbrink, J.N.; Mergny, J.L.; Brosh, R.M., Jr.

    2014-01-01

    Mitochondrial DNA deletions are prominent in human genetic disorders, cancer, and aging. It is thought that stalling of the mitochondrial replication machinery during DNA synthesis is a prominent source of mitochondrial genome instability; however, the precise molecular determinants of defective mit