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Sample records for replication fork progression

  1. TRAIP regulates replication fork recovery and progression via PCNA

    PubMed Central

    Feng, Wanjuan; Guo, Yingying; Huang, Jun; Deng, Yiqun; Zang, Jianye; Huen, Michael Shing-Yan

    2016-01-01

    PCNA is a central scaffold that coordinately assembles replication and repair machineries at DNA replication forks for faithful genome duplication. Here, we describe TRAIP (RNF206) as a novel PCNA-interacting factor that has important roles during mammalian replicative stress responses. We show that TRAIP encodes a nucleolar protein that migrates to stalled replication forks, and that this is accomplished by its targeting of PCNA via an evolutionarily conserved PIP box on its C terminus. Accordingly, inactivation of TRAIP or its interaction with the PCNA clamp compromised replication fork recovery and progression, and leads to chromosome instability. Together, our findings establish TRAIP as a component of the mammalian replicative stress response network, and implicate the TRAIP-PCNA axis in recovery of stalled replication forks. PMID:27462463

  2. Mitomycin C reduces abundance of replication forks but not rates of fork progression in primary and transformed human cells

    PubMed Central

    Kehrli, Keffy; Sidorova, Julia M.

    2014-01-01

    DNA crosslinks can block replication in vitro and slow down S phase progression in vivo. We characterized the effect of mitomycin C crosslinker on S phase globally and on individual replication forks in wild type and FANCD2-deficient human cells. FANCD2 is critical to crosslink repair, and is also implicated in facilitating DNA replication. We used DNA fiber analysis to demonstrate persistent reduction in abundance but not progression rate of replication forks during an S phase of MMC-treated cells. FANCD2 deficiency did not eliminate this phenotype. Immunoprecipitation of EdU-labeled DNA indicated that replication was not suppressed in the domains that were undergoing response to MMC as marked by the presence of γH2AX, and in fact γH2AX was overrepresented on DNA that had replicated immediately after MMC in wild type through less so in FANCD2-depleted cells. FANCD2-depleted cells also produced fewer tracks of uninterrupted replication of up to 240Kb long, regardless of MMC treatment. Overall, the data suggest that crosslinks may not pose a block to S phase as a whole, but instead profoundly change its progress by reducing density of replication forks and causing at least a fraction of forks to operate within a DNA damage response-altered chromatin. PMID:25580447

  3. Replication-Fork Dynamics

    PubMed Central

    Duderstadt, Karl E.; Reyes-Lamothe, Rodrigo; van Oijen, Antoine M.; Sherratt, David J.

    2014-01-01

    The proliferation of all organisms depends on the coordination of enzymatic events within large multiprotein replisomes that duplicate chromosomes. Whereas the structure and function of many core replisome components have been clarified, the timing and order of molecular events during replication remains obscure. To better understand the replication mechanism, new methods must be developed that allow for the observation and characterization of short-lived states and dynamic events at single replication forks. Over the last decade, great progress has been made toward this goal with the development of novel DNA nanomanipulation and fluorescence imaging techniques allowing for the direct observation of replication-fork dynamics both reconstituted in vitro and in live cells. This article reviews these new single-molecule approaches and the revised understanding of replisome operation that has emerged. PMID:23881939

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

    PubMed

    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

  5. HERC2 Interacts with Claspin and regulates DNA origin firing and replication fork progression.

    PubMed

    Izawa, Naoki; Wu, Wenwen; Sato, Ko; Nishikawa, Hiroyuki; Kato, Akihiro; Boku, Narikazu; Itoh, Fumio; Ohta, Tomohiko

    2011-09-01

    DNA replication, recombination, and repair are highly interconnected processes the disruption of which must be coordinated in cancer. HERC2, a large HECT protein required for homologous recombination repair, is an E3 ubiquitin ligase that targets breast cancer suppressor BRCA1 for degradation. Here, we show that HERC2 is a component of the DNA replication fork complex that plays a critical role in DNA elongation and origin firing. In the presence of BRCA1, endogenous HERC2 interacts with Claspin, a protein essential for G(2)-M checkpoint activation and replication fork stability. Claspin depletion slowed S-phase progression and additional HERC2 depletion reduced the effect of Claspin depletion. In addition, HERC2 interacts with replication fork complex proteins. Depletion of HERC2 alleviated the slow replication fork progression in Claspin-deficient cells, suppressed enhanced origin firing, and led to a decrease in MCM2 phosphorylation. In a HERC2-dependent manner, treatment of cells with replication inhibitor aphidicolin enhanced MCM2 phosphorylation. Taken together, our results suggest that HERC2 regulates DNA replication progression and origin firing by facilitating MCM2 phosphorylation. These findings establish HERC2 as a critical function in DNA repair, checkpoint activation, and DNA replication. PMID:21775519

  6. Excess Cdt1 inhibits nascent strand elongation by repressing the progression of replication forks in Xenopus egg extracts.

    PubMed

    Nakazaki, Yuta; Tsuyama, Takashi; Seki, Masayuki; Takahashi, Mikiko; Enomoto, Takemi; Tada, Shusuke

    2016-02-01

    Cdt1 is a protein essential for initiation of DNA replication; it recruits MCM helicase, a core component of the replicative DNA helicase, onto replication origins. In our previous study, we showed that addition of excess Cdt1 inhibits nascent strand elongation during DNA replication in Xenopus egg extracts. In the present study, we investigated the mechanism behind the inhibitory effect of Cdt1. We found that addition of recombinant Cdt1 inhibited nascent DNA synthesis in a reinitiation-independent manner. To identify the mechanism by which Cdt1 inhibits nascent strand elongation, the effect of Cdt1 on loading of Mcm4 and Rpa70 onto chromatin was examined. The results showed that Cdt1 suppressed the excessive Rpa70 binding caused by extensive, aphidicolin-induced DNA unwinding; this unwinding occurs between stalled DNA polymerases and advancing replication forks. These findings suggested that excess Cdt1 suppressed the progression of replication forks. PMID:26773501

  7. Distinct functions of human RECQ helicases WRN and BLM in replication fork recovery and progression after hydroxyurea-induced stalling

    PubMed Central

    Sidorova, Julia M; Kehrli, Keffy; Mao, Frances; Monnat, Raymond

    2012-01-01

    Human WRN and BLM genes are members of the conserved RECQ helicase family. Mutations in these genes are associated with Werner and Bloom syndromes. WRN and BLM proteins are implicated in DNA replication, recombination, repair, telomere maintenance, and transcription. Using microfluidics-assisted display of DNA for replication track analysis (ma-RTA), we show that WRN and BLM contribute additively to normal replication fork progression, and non-additively, in a RAD51-dependent pathway, to resumption of replication after arrest by hydroxyurea (HU), a replication-stalling drug. WRN but not BLM is required to support fork progression after HU. Resumption of replication by forks may be necessary but is not sufficient for timely completion of the cell cycle after HU arrest, as depletion of WRN or BLM compromises fork recovery to a similar degree, but only BLM depletion leads to extensive delay of cell division after HU, as well as more pronounced chromatin bridging. Finally, we show that recovery from HU includes apparent removal of some of the DNA that was synthesized immediately after release from HU, a novel phenomenon that we refer to as nascent strand processing, NSP. PMID:23253856

  8. Concerted activities of Mcm4, Sld3, and Dbf4 in control of origin activation and DNA replication fork progression.

    PubMed

    Sheu, Yi-Jun; Kinney, Justin B; Stillman, Bruce

    2016-03-01

    Eukaryotic chromosomes initiate DNA synthesis from multiple replication origins in a temporally specific manner during S phase. The replicative helicase Mcm2-7 functions in both initiation and fork progression and thus is an important target of regulation. Mcm4, a helicase subunit, possesses an unstructured regulatory domain that mediates control from multiple kinase signaling pathways, including the Dbf4-dependent Cdc7 kinase (DDK). Following replication stress in S phase, Dbf4 and Sld3, an initiation factor and essential target of Cyclin-Dependent Kinase (CDK), are targets of the checkpoint kinase Rad53 for inhibition of initiation from origins that have yet to be activated, so-called late origins. Here, whole-genome DNA replication profile analysis is used to access under various conditions the effect of mutations that alter the Mcm4 regulatory domain and the Rad53 targets, Sld3 and Dbf4. Late origin firing occurs under genotoxic stress when the controls on Mcm4, Sld3, and Dbf4 are simultaneously eliminated. The regulatory domain of Mcm4 plays an important role in the timing of late origin firing, both in an unperturbed S phase and in dNTP limitation. Furthermore, checkpoint control of Sld3 impacts fork progression under replication stress. This effect is parallel to the role of the Mcm4 regulatory domain in monitoring fork progression. Hypomorph mutations in sld3 are suppressed by a mcm4 regulatory domain mutation. Thus, in response to cellular conditions, the functions executed by Sld3, Dbf4, and the regulatory domain of Mcm4 intersect to control origin firing and replication fork progression, thereby ensuring genome stability. PMID:26733669

  9. DNA damage tolerance pathway involving DNA polymerase ι and the tumor suppressor p53 regulates DNA replication fork progression

    PubMed Central

    Hampp, Stephanie; Kiessling, Tina; Buechle, Kerstin; Mansilla, Sabrina F.; Thomale, Jürgen; Rall, Melanie; Ahn, Jinwoo; Pospiech, Helmut; Gottifredi, Vanesa; Wiesmüller, Lisa

    2016-01-01

    DNA damage tolerance facilitates the progression of replication forks that have encountered obstacles on the template strands. It involves either translesion DNA synthesis initiated by proliferating cell nuclear antigen monoubiquitination or less well-characterized fork reversal and template switch mechanisms. Herein, we characterize a novel tolerance pathway requiring the tumor suppressor p53, the translesion polymerase ι (POLι), the ubiquitin ligase Rad5-related helicase-like transcription factor (HLTF), and the SWI/SNF catalytic subunit (SNF2) translocase zinc finger ran-binding domain containing 3 (ZRANB3). This novel p53 activity is lost in the exonuclease-deficient but transcriptionally active p53(H115N) mutant. Wild-type p53, but not p53(H115N), associates with POLι in vivo. Strikingly, the concerted action of p53 and POLι decelerates nascent DNA elongation and promotes HLTF/ZRANB3-dependent recombination during unperturbed DNA replication. Particularly after cross-linker–induced replication stress, p53 and POLι also act together to promote meiotic recombination enzyme 11 (MRE11)-dependent accumulation of (phospho-)replication protein A (RPA)-coated ssDNA. These results implicate a direct role of p53 in the processing of replication forks encountering obstacles on the template strand. Our findings define an unprecedented function of p53 and POLι in the DNA damage response to endogenous or exogenous replication stress. PMID:27407148

  10. DNA damage tolerance pathway involving DNA polymerase ι and the tumor suppressor p53 regulates DNA replication fork progression.

    PubMed

    Hampp, Stephanie; Kiessling, Tina; Buechle, Kerstin; Mansilla, Sabrina F; Thomale, Jürgen; Rall, Melanie; Ahn, Jinwoo; Pospiech, Helmut; Gottifredi, Vanesa; Wiesmüller, Lisa

    2016-07-26

    DNA damage tolerance facilitates the progression of replication forks that have encountered obstacles on the template strands. It involves either translesion DNA synthesis initiated by proliferating cell nuclear antigen monoubiquitination or less well-characterized fork reversal and template switch mechanisms. Herein, we characterize a novel tolerance pathway requiring the tumor suppressor p53, the translesion polymerase ι (POLι), the ubiquitin ligase Rad5-related helicase-like transcription factor (HLTF), and the SWI/SNF catalytic subunit (SNF2) translocase zinc finger ran-binding domain containing 3 (ZRANB3). This novel p53 activity is lost in the exonuclease-deficient but transcriptionally active p53(H115N) mutant. Wild-type p53, but not p53(H115N), associates with POLι in vivo. Strikingly, the concerted action of p53 and POLι decelerates nascent DNA elongation and promotes HLTF/ZRANB3-dependent recombination during unperturbed DNA replication. Particularly after cross-linker-induced replication stress, p53 and POLι also act together to promote meiotic recombination enzyme 11 (MRE11)-dependent accumulation of (phospho-)replication protein A (RPA)-coated ssDNA. These results implicate a direct role of p53 in the processing of replication forks encountering obstacles on the template strand. Our findings define an unprecedented function of p53 and POLι in the DNA damage response to endogenous or exogenous replication stress. PMID:27407148

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

    PubMed

    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

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

    PubMed Central

    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

  13. Pfh1 Is an Accessory Replicative Helicase that Interacts with the Replisome to Facilitate Fork Progression and Preserve Genome Integrity.

    PubMed

    McDonald, Karin R; Guise, Amanda J; Pourbozorgi-Langroudi, Parham; Cristea, Ileana M; Zakian, Virginia A; Capra, John A; Sabouri, Nasim

    2016-09-01

    Replicative DNA helicases expose the two strands of the double helix to the replication apparatus, but accessory helicases are often needed to help forks move past naturally occurring hard-to-replicate sites, such as tightly bound proteins, RNA/DNA hybrids, and DNA secondary structures. Although the Schizosaccharomyces pombe 5'-to-3' DNA helicase Pfh1 is known to promote fork progression, its genomic targets, dynamics, and mechanisms of action are largely unknown. Here we address these questions by integrating genome-wide identification of Pfh1 binding sites, comprehensive analysis of the effects of Pfh1 depletion on replication and DNA damage, and proteomic analysis of Pfh1 interaction partners by immunoaffinity purification mass spectrometry. Of the 621 high confidence Pfh1-binding sites in wild type cells, about 40% were sites of fork slowing (as marked by high DNA polymerase occupancy) and/or DNA damage (as marked by high levels of phosphorylated H2A). The replication and integrity of tRNA and 5S rRNA genes, highly transcribed RNA polymerase II genes, and nucleosome depleted regions were particularly Pfh1-dependent. The association of Pfh1 with genomic integrity at highly transcribed genes was S phase dependent, and thus unlikely to be an artifact of high transcription rates. Although Pfh1 affected replication and suppressed DNA damage at discrete sites throughout the genome, Pfh1 and the replicative DNA polymerase bound to similar extents to both Pfh1-dependent and independent sites, suggesting that Pfh1 is proximal to the replication machinery during S phase. Consistent with this interpretation, Pfh1 co-purified with many key replisome components, including the hexameric MCM helicase, replicative DNA polymerases, RPA, and the processivity clamp PCNA in an S phase dependent manner. Thus, we conclude that Pfh1 is an accessory DNA helicase that interacts with the replisome and promotes replication and suppresses DNA damage at hard-to-replicate sites. These data

  14. DNA polymerase η modulates replication fork progression and DNA damage responses in platinum-treated human cells

    NASA Astrophysics Data System (ADS)

    Sokol, Anna M.; Cruet-Hennequart, Séverine; Pasero, Philippe; Carty, Michael P.

    2013-11-01

    Human cells lacking DNA polymerase η (polη) are sensitive to platinum-based cancer chemotherapeutic agents. Using DNA combing to directly investigate the role of polη in bypass of platinum-induced DNA lesions in vivo, we demonstrate that nascent DNA strands are up to 39% shorter in human cells lacking polη than in cells expressing polη. This provides the first direct evidence that polη modulates replication fork progression in vivo following cisplatin and carboplatin treatment. Severe replication inhibition in individual platinum-treated polη-deficient cells correlates with enhanced phosphorylation of the RPA2 subunit of replication protein A on serines 4 and 8, as determined using EdU labelling and immunofluorescence, consistent with formation of DNA strand breaks at arrested forks in the absence of polη. Polη-mediated bypass of platinum-induced DNA lesions may therefore represent one mechanism by which cancer cells can tolerate platinum-based chemotherapy.

  15. ING2 controls the progression of DNA replication forks to maintain genome stability

    PubMed Central

    Larrieu, Delphine; Ythier, Damien; Binet, Romuald; Brambilla, Christian; Brambilla, Elisabeth; Sengupta, Sagar; Pedeux, Rémy

    2009-01-01

    Inhibitor of growth 2 (ING2) is a candidate tumour suppressor gene the expression of which is frequently lost in tumours. Here, we identified a new function for ING2 in the control of DNA replication and in the maintenance of genome stability. Global replication rate was markedly reduced during normal S-phase in small interfering RNA (siRNA) ING2 cells, as seen in a DNA fibre spreading experiment. Accordingly, we found that ING2 interacts with proliferating cell nuclear antigen and regulates its amount to the chromatin fraction, allowing normal replication progression and normal cell proliferation. Deregulation of DNA replication has been previously associated with genome instability. Hence, a high proportion of siRNA ING2 cells presented endoreduplication of their genome as well as an increased frequency of sister chromatid exchange. Thus, we propose for the first time that ING2 might function as a tumour suppressor gene by directly maintaining DNA integrity. PMID:19730436

  16. Stability of blocked replication forks in vivo

    PubMed Central

    Mettrick, Karla A.; Grainge, Ian

    2016-01-01

    Replication of chromosomal DNA must be carried out to completion in order for a cell to proliferate. However, replication forks can stall during this process for a variety of reasons, including nucleoprotein ‘roadblocks’ and DNA lesions. In these circumstances the replisome copying the DNA may disengage from the chromosome to allow various repair processes to restore DNA integrity and enable replication to continue. Here, we report the in vivo stability of the replication fork when it encounters a nucleoprotein blockage in Escherichia coli. Using a site-specific and reversible protein block system in conjunction with the temperature sensitive DnaC helicase loader and DnaB replicative helicase, we monitored the disappearance of the Y-shaped DNA replication fork structures using neutral-neutral 2D agarose gels. We show the replication fork collapses within 5 min of encountering the roadblock. Therefore, the stalled replication fork does not pause at a block in a stable confirmation for an extended period of time as previously postulated. PMID:26490956

  17. Are the SSB-Interacting Proteins RecO, RecG, PriA and the DnaB-Interacting Protein Rep Bound to Progressing Replication Forks in Escherichia coli?

    PubMed

    Bentchikou, Esma; Chagneau, Carine; Long, Emilie; Matelot, Mélody; Allemand, Jean-François; Michel, Bénédicte

    2015-01-01

    In all organisms several enzymes that are needed upon replication impediment are targeted to replication forks by interaction with a replication protein. In most cases these proteins interact with the polymerase clamp or with single-stranded DNA binding proteins (SSB). In Escherichia coli an accessory replicative helicase was also shown to interact with the DnaB replicative helicase. Here we have used cytological observation of Venus fluorescent fusion proteins expressed from their endogenous loci in live E. coli cells to determine whether DNA repair and replication restart proteins that interact with a replication protein travel with replication forks. A custom-made microscope that detects active replisome molecules provided that they are present in at least three copies was used. Neither the recombination proteins RecO and RecG, nor the replication accessory helicase Rep are detected specifically in replicating cells in our assay, indicating that either they are not present at progressing replication forks or they are present in less than three copies. The Venus-PriA fusion protein formed foci even in the absence of replication forks, which prevented us from reaching a conclusion. PMID:26244508

  18. The E. coli DNA Replication Fork.

    PubMed

    Lewis, J S; Jergic, S; Dixon, N E

    2016-01-01

    DNA replication in Escherichia coli initiates at oriC, the origin of replication and proceeds bidirectionally, resulting in two replication forks that travel in opposite directions from the origin. Here, we focus on events at the replication fork. The replication machinery (or replisome), first assembled on both forks at oriC, contains the DnaB helicase for strand separation, and the DNA polymerase III holoenzyme (Pol III HE) for DNA synthesis. DnaB interacts transiently with the DnaG primase for RNA priming on both strands. The Pol III HE is made up of three subassemblies: (i) the αɛθ core polymerase complex that is present in two (or three) copies to simultaneously copy both DNA strands, (ii) the β2 sliding clamp that interacts with the core polymerase to ensure its processivity, and (iii) the seven-subunit clamp loader complex that loads β2 onto primer-template junctions and interacts with the α polymerase subunit of the core and the DnaB helicase to organize the two (or three) core polymerases. Here, we review the structures of the enzymatic components of replisomes, and the protein-protein and protein-DNA interactions that ensure they remain intact while undergoing substantial dynamic changes as they function to copy both the leading and lagging strands simultaneously during coordinated replication. PMID:27241927

  19. Non-replicative helicases at the replication fork.

    PubMed

    Heller, Ryan C; Marians, Kenneth J

    2007-07-01

    Reactivation of stalled or collapsed replication forks is an essential process in bacteria. Restart systems operate to restore the 5'-->3' replicative helicase, DnaB, to the lagging-strand template. However, other non-replicative 3'-->5' helicases play an important role in the restart process as well. Here we examine the DNA-binding specificity of three of the latter group, PriA, Rep, and UvrD. Only PriA and Rep display structure-specific fork binding. Interestingly, their specificity is opposite: PriA binds a leading-strand fork, presumably reflecting its restart activity in directing loading of DnaB to the lagging-strand template. Rep binds a lagging-strand fork, presumably reflecting its role in partially displacing Okazaki fragments that originate near the fork junction. This activity is necessary for generating a single-stranded landing pad for DnaB. While UvrD shows little structure-specificity, there is a slight preference for lagging-strand forks, suggesting that there might be some redundancy between Rep and UvrD and possibly explaining the observed synthetic lethality that occurs when mutations in the genes encoding these two proteins are combined. PMID:17382604

  20. The Replication Checkpoint Protects Fork Stability by Releasing Transcribed Genes from Nuclear Pores

    PubMed Central

    Bermejo, Rodrigo; Capra, Thelma; Jossen, Rachel; Colosio, Arianna; Frattini, Camilla; Carotenuto, Walter; Cocito, Andrea; Doksani, Ylli; Klein, Hannah; Gómez-González, Belén; Aguilera, Andrés; Katou, Yuki; Shirahige, Katsuhiko; Foiani, Marco

    2011-01-01

    Summary Transcription hinders replication fork progression and stability, and the Mec1/ATR checkpoint protects fork integrity. Examining checkpoint-dependent mechanisms controlling fork stability, we find that fork reversal and dormant origin firing due to checkpoint defects are rescued in checkpoint mutants lacking THO, TREX-2, or inner-basket nucleoporins. Gene gating tethers transcribed genes to the nuclear periphery and is counteracted by checkpoint kinases through phosphorylation of nucleoporins such as Mlp1. Checkpoint mutants fail to detach transcribed genes from nuclear pores, thus generating topological impediments for incoming forks. Releasing this topological complexity by introducing a double-strand break between a fork and a transcribed unit prevents fork collapse. Mlp1 mutants mimicking constitutive checkpoint-dependent phosphorylation also alleviate checkpoint defects. We propose that the checkpoint assists fork progression and stability at transcribed genes by phosphorylating key nucleoporins and counteracting gene gating, thus neutralizing the topological tension generated at nuclear pore gated genes. PMID:21784245

  1. Recovery of arrested replication forks by homologous recombination is error-prone.

    PubMed

    Iraqui, Ismail; Chekkal, Yasmina; Jmari, Nada; Pietrobon, Violena; Fréon, Karine; Costes, Audrey; Lambert, Sarah A E

    2012-01-01

    Homologous recombination is a universal mechanism that allows repair of DNA and provides support for DNA replication. Homologous recombination is therefore a major pathway that suppresses non-homology-mediated genome instability. Here, we report that recovery of impeded replication forks by homologous recombination is error-prone. Using a fork-arrest-based assay in fission yeast, we demonstrate that a single collapsed fork can cause mutations and large-scale genomic changes, including deletions and translocations. Fork-arrest-induced gross chromosomal rearrangements are mediated by inappropriate ectopic recombination events at the site of collapsed forks. Inverted repeats near the site of fork collapse stimulate large-scale genomic changes up to 1,500 times over spontaneous events. We also show that the high accuracy of DNA replication during S-phase is impaired by impediments to fork progression, since fork-arrest-induced mutation is due to erroneous DNA synthesis during recovery of replication forks. The mutations caused are small insertions/duplications between short tandem repeats (micro-homology) indicative of replication slippage. Our data establish that collapsed forks, but not stalled forks, recovered by homologous recombination are prone to replication slippage. The inaccuracy of DNA synthesis does not rely on PCNA ubiquitination or trans-lesion-synthesis DNA polymerases, and it is not counteracted by mismatch repair. We propose that deletions/insertions, mediated by micro-homology, leading to copy number variations during replication stress may arise by progression of error-prone replication forks restarted by homologous recombination. PMID:23093942

  2. WRNIP1 protects stalled forks from degradation and promotes fork restart after replication stress.

    PubMed

    Leuzzi, Giuseppe; Marabitti, Veronica; Pichierri, Pietro; Franchitto, Annapaola

    2016-07-01

    Accurate handling of stalled replication forks is crucial for the maintenance of genome stability. RAD51 defends stalled replication forks from nucleolytic attack, which otherwise can threaten genome stability. However, the identity of other factors that can collaborate with RAD51 in this task is poorly elucidated. Here, we establish that human Werner helicase interacting protein 1 (WRNIP1) is localized to stalled replication forks and cooperates with RAD51 to safeguard fork integrity. We show that WRNIP1 is directly involved in preventing uncontrolled MRE11-mediated degradation of stalled replication forks by promoting RAD51 stabilization on ssDNA We further demonstrate that replication fork protection does not require the ATPase activity of WRNIP1 that is however essential to achieve the recovery of perturbed replication forks. Loss of WRNIP1 or its catalytic activity causes extensive DNA damage and chromosomal aberrations. Intriguingly, downregulation of the anti-recombinase FBH1 can compensate for loss of WRNIP1 activity, since it attenuates replication fork degradation and chromosomal aberrations in WRNIP1-deficient cells. Therefore, these findings unveil a unique role for WRNIP1 as a replication fork-protective factor in maintaining genome stability. PMID:27242363

  3. Regression of Replication Forks Stalled by Leading-strand Template Damage

    PubMed Central

    Gupta, Sankalp; Yeeles, Joseph T. P.; Marians, Kenneth J.

    2014-01-01

    The orderly progression of replication forks formed at the origin of replication in Escherichia coli is challenged by encounters with template damage, slow moving RNA polymerases, and frozen DNA-protein complexes that stall the fork. These stalled forks are foci for genomic instability and must be reactivated. Many models of replication fork reactivation invoke nascent strand regression as an intermediate in the processing of the stalled fork. We have investigated the replication fork regression activity of RecG and RuvAB, two proteins commonly thought to be involved in the process, using a reconstituted DNA replication system where the replisome is stalled by collision with leading-strand template damage. We find that both RecG and RuvAB can regress the stalled fork in the presence of the replisome and SSB; however, RuvAB generates a completely unwound product consisting of the paired nascent leading and lagging strands, whereas RuvC cleaves the Holliday junction generated by RecG-catalyzed fork regression. We also find that RecG stimulates RuvAB-catalyzed regression, presumably because it is more efficient at generating the initial Holliday junction from the stalled fork. PMID:25138216

  4. THE FORK AND THE KINASE: A DNA REPLICATION TALE FROM A CHK1 PERSPECTIVE

    PubMed Central

    González Besteiro, Marina A.; Gottifredi, Vanesa

    2014-01-01

    Replication fork progression is being continuously hampered by exogenously introduced and naturally occurring DNA lesions and other physical obstacles. The checkpoint kinase 1 (Chk1) is activated at replication forks that encounter damaged-DNA. Chk1 inhibits the initiation of new replication factories and stimulates the firing of dormant origins (those in the vicinity of stalled forks). Chk1 also avoids fork collapse into DSBs (double strand breaks) and promotes fork elongation. At the molecular level, the current model considers stalled forks as the site of Chk1 activation and the nucleoplasm as the location where Chk1 phosphorylates target proteins. This model certainly serves to explain how Chk1 modulates origin firing, but how Chk1 controls the fate of stalled forks is less clear. Interestingly, recent reports demonstrating that Chk1 phosphorylates chromatin-bound proteins and even holds kinase-independent functions might shed light on how Chk1 contributes to the elongation of damaged DNA. Such findings unveil a puzzling connection between Chk1 and DNA-lesion bypass, which might be central to promoting fork elongation and checkpoint attenuation. In summary, the multifaceted and versatile functions of Chk1 at ongoing forks and replication origins determine the extent and quality of the cellular response to replication stress. PMID:25795119

  5. Replication fork movement and methylation govern SeqA binding to the Escherichia coli chromosome

    PubMed Central

    Waldminghaus, Torsten; Weigel, Christoph; Skarstad, Kirsten

    2012-01-01

    In Escherichia coli, the SeqA protein binds specifically to GATC sequences which are methylated on the A of the old strand but not on the new strand. Such hemimethylated DNA is produced by progression of the replication forks and lasts until Dam methyltransferase methylates the new strand. It is therefore believed that a region of hemimethylated DNA covered by SeqA follows the replication fork. We show that this is, indeed, the case by using global ChIP on Chip analysis of SeqA in cells synchronized regarding DNA replication. To assess hemimethylation, we developed the first genome-wide method for methylation analysis in bacteria. Since loss of the SeqA protein affects growth rate only during rapid growth when cells contain multiple replication forks, a comparison of rapid and slow growth was performed. In cells with six replication forks per chromosome, the two old forks were found to bind surprisingly little SeqA protein. Cell cycle analysis showed that loss of SeqA from the old forks did not occur at initiation of the new forks, but instead occurs at a time point coinciding with the end of SeqA-dependent origin sequestration. The finding suggests simultaneous origin de-sequestration and loss of SeqA from old replication forks. PMID:22373925

  6. Stalled DNA Replication Forks at the Endogenous GAA Repeats Drive Repeat Expansion in Friedreich's Ataxia Cells.

    PubMed

    Gerhardt, Jeannine; Bhalla, Angela D; Butler, Jill Sergesketter; Puckett, James W; Dervan, Peter B; Rosenwaks, Zev; Napierala, Marek

    2016-08-01

    Friedreich's ataxia (FRDA) is caused by the expansion of GAA repeats located in the Frataxin (FXN) gene. The GAA repeats continue to expand in FRDA patients, aggravating symptoms and contributing to disease progression. The mechanism leading to repeat expansion and decreased FXN transcription remains unclear. Using single-molecule analysis of replicated DNA, we detected that expanded GAA repeats present a substantial obstacle for the replication machinery at the FXN locus in FRDA cells. Furthermore, aberrant origin activation and lack of a proper stress response to rescue the stalled forks in FRDA cells cause an increase in 3'-5' progressing forks, which could enhance repeat expansion and hinder FXN transcription by head-on collision with RNA polymerases. Treatment of FRDA cells with GAA-specific polyamides rescues DNA replication fork stalling and alleviates expansion of the GAA repeats, implicating DNA triplexes as a replication impediment and suggesting that fork stalling might be a therapeutic target for FRDA. PMID:27425605

  7. Rad51-mediated replication fork reversal is a global response to genotoxic treatments in human cells

    PubMed Central

    Zellweger, Ralph; Dalcher, Damian; Mutreja, Karun; Berti, Matteo; Schmid, Jonas A.; Herrador, Raquel; Vindigni, Alessandro

    2015-01-01

    Replication fork reversal protects forks from breakage after poisoning of Topoisomerase 1. We here investigated fork progression and chromosomal breakage in human cells in response to a panel of sublethal genotoxic treatments, using other topoisomerase poisons, DNA synthesis inhibitors, interstrand cross-linking inducers, and base-damaging agents. We used electron microscopy to visualize fork architecture under these conditions and analyzed the association of specific molecular features with checkpoint activation. Our data identify replication fork uncoupling and reversal as global responses to genotoxic treatments. Both events are frequent even after mild treatments that do not affect fork integrity, nor activate checkpoints. Fork reversal was found to be dependent on the central homologous recombination factor RAD51, which is consistently present at replication forks independently of their breakage, and to be antagonized by poly (ADP-ribose) polymerase/RECQ1-regulated restart. Our work establishes remodeling of uncoupled forks as a pivotal RAD51-regulated response to genotoxic stress in human cells and as a promising target to potentiate cancer chemotherapy. PMID:25733714

  8. Rad51-mediated replication fork reversal is a global response to genotoxic treatments in human cells.

    PubMed

    Zellweger, Ralph; Dalcher, Damian; Mutreja, Karun; Berti, Matteo; Schmid, Jonas A; Herrador, Raquel; Vindigni, Alessandro; Lopes, Massimo

    2015-03-01

    Replication fork reversal protects forks from breakage after poisoning of Topoisomerase 1. We here investigated fork progression and chromosomal breakage in human cells in response to a panel of sublethal genotoxic treatments, using other topoisomerase poisons, DNA synthesis inhibitors, interstrand cross-linking inducers, and base-damaging agents. We used electron microscopy to visualize fork architecture under these conditions and analyzed the association of specific molecular features with checkpoint activation. Our data identify replication fork uncoupling and reversal as global responses to genotoxic treatments. Both events are frequent even after mild treatments that do not affect fork integrity, nor activate checkpoints. Fork reversal was found to be dependent on the central homologous recombination factor RAD51, which is consistently present at replication forks independently of their breakage, and to be antagonized by poly (ADP-ribose) polymerase/RECQ1-regulated restart. Our work establishes remodeling of uncoupled forks as a pivotal RAD51-regulated response to genotoxic stress in human cells and as a promising target to potentiate cancer chemotherapy. PMID:25733714

  9. Checkpoint-dependent RNR induction promotes fork restart after replicative stress.

    PubMed

    Morafraile, Esther C; Diffley, John F X; Tercero, José Antonio; Segurado, Mónica

    2015-01-01

    The checkpoint kinase Rad53 is crucial to regulate DNA replication in the presence of replicative stress. Under conditions that interfere with the progression of replication forks, Rad53 prevents Exo1-dependent fork degradation. However, although EXO1 deletion avoids fork degradation in rad53 mutants, it does not suppress their sensitivity to the ribonucleotide reductase (RNR) inhibitor hydroxyurea (HU). In this case, the inability to restart stalled forks is likely to account for the lethality of rad53 mutant cells after replication blocks. Here we show that Rad53 regulates replication restart through the checkpoint-dependent transcriptional response, and more specifically, through RNR induction. Thus, in addition to preventing fork degradation, Rad53 prevents cell death in the presence of HU by regulating RNR-expression and localization. When RNR is induced in the absence of Exo1 and RNR negative regulators, cell viability of rad53 mutants treated with HU is increased and the ability of replication forks to restart after replicative stress is restored. PMID:25601385

  10. Checkpoint-dependent RNR induction promotes fork restart after replicative stress

    PubMed Central

    Morafraile, Esther C.; Diffley, John F. X.; Tercero, José Antonio; Segurado, Mónica

    2015-01-01

    The checkpoint kinase Rad53 is crucial to regulate DNA replication in the presence of replicative stress. Under conditions that interfere with the progression of replication forks, Rad53 prevents Exo1-dependent fork degradation. However, although EXO1 deletion avoids fork degradation in rad53 mutants, it does not suppress their sensitivity to the ribonucleotide reductase (RNR) inhibitor hydroxyurea (HU). In this case, the inability to restart stalled forks is likely to account for the lethality of rad53 mutant cells after replication blocks. Here we show that Rad53 regulates replication restart through the checkpoint-dependent transcriptional response, and more specifically, through RNR induction. Thus, in addition to preventing fork degradation, Rad53 prevents cell death in the presence of HU by regulating RNR-expression and localization. When RNR is induced in the absence of Exo1 and RNR negative regulators, cell viability of rad53 mutants treated with HU is increased and the ability of replication forks to restart after replicative stress is restored. PMID:25601385

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

    PubMed Central

    Xing, Meichun; Wang, Xiaohui; Palmai-Pallag, Timea; Shen, Huahao; Helleday, Thomas; Hickson, Ian D.; Ying, Songmin

    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. PMID:26415217

  12. The Fork in the Road: Histone Partitioning During DNA Replication

    PubMed Central

    Annunziato, Anthony T.

    2015-01-01

    In the following discussion the distribution of histones at the replication fork is examined, with specific attention paid to the question of H3/H4 tetramer "splitting." After a presentation of early experiments surrounding this topic, more recent contributions are detailed. The implications of these findings with respect to the transmission of histone modifications and epigenetic models are also addressed. PMID:26110314

  13. Mitochondrial transcription termination factor 1 directs polar replication fork pausing

    PubMed Central

    Shi, Yonghong; Posse, Viktor; Zhu, Xuefeng; Hyvärinen, Anne K.; Jacobs, Howard T.; Falkenberg, Maria; Gustafsson, Claes M.

    2016-01-01

    During replication of nuclear ribosomal DNA (rDNA), clashes with the transcription apparatus can cause replication fork collapse and genomic instability. To avoid this problem, a replication fork barrier protein is situated downstream of rDNA, there preventing replication in the direction opposite rDNA transcription. A potential candidate for a similar function in mitochondria is the mitochondrial transcription termination factor 1 (MTERF1, also denoted mTERF), which binds to a sequence just downstream of the ribosomal transcription unit. Previous studies have shown that MTERF1 prevents antisense transcription over the ribosomal RNA genes, a process which we here show to be independent of the transcription elongation factor TEFM. Importantly, we now demonstrate that MTERF1 arrests mitochondrial DNA (mtDNA) replication with distinct polarity. The effect is explained by the ability of MTERF1 to act as a directional contrahelicase, blocking mtDNA unwinding by the mitochondrial helicase TWINKLE. This conclusion is also supported by in vivo evidence that MTERF1 stimulates TWINKLE pausing. We conclude that MTERF1 can direct polar replication fork arrest in mammalian mitochondria. PMID:27112570

  14. Termination of DNA replication forks: “Breaking up is hard to do”

    PubMed Central

    Bailey, Rachael; Priego Moreno, Sara; Gambus, Agnieszka

    2015-01-01

    To ensure duplication of the entire genome, eukaryotic DNA replication initiates from thousands of replication origins. The replication forks move through the chromatin until they encounter forks from neighboring origins. During replication fork termination forks converge, the replisomes disassemble and topoisomerase II resolves the daughter DNA molecules. If not resolved efficiently, terminating forks result in genomic instability through the formation of pathogenic structures. Our recent findings shed light onto the mechanism of replisome disassembly upon replication fork termination. We have shown that termination-specific polyubiquitylation of the replicative helicase component – Mcm7, leads to dissolution of the active helicase in a process dependent on the p97/VCP/Cdc48 segregase. The inhibition of terminating helicase disassembly resulted in a replication termination defect. In this extended view we present hypothetical models of replication fork termination and discuss remaining and emerging questions in the DNA replication termination field. PMID:25835602

  15. Ubiquitinated Fancd2 recruits Fan1 to stalled replication forks to prevent genome instability.

    PubMed

    Lachaud, Christophe; Moreno, Alberto; Marchesi, Francesco; Toth, Rachel; Blow, J Julian; Rouse, John

    2016-02-19

    Mono-ubiquitination of Fancd2 is essential for repairing DNA interstrand cross-links (ICLs), but the underlying mechanisms are unclear. The Fan1 nuclease, also required for ICL repair, is recruited to ICLs by ubiquitinated (Ub) Fancd2. This could in principle explain how Ub-Fancd2 promotes ICL repair, but we show that recruitment of Fan1 by Ub-Fancd2 is dispensable for ICL repair. Instead, Fan1 recruitment--and activity--restrains DNA replication fork progression and prevents chromosome abnormalities from occurring when DNA replication forks stall, even in the absence of ICLs. Accordingly, Fan1 nuclease-defective knockin mice are cancer-prone. Moreover, we show that a Fan1 variant in high-risk pancreatic cancers abolishes recruitment by Ub-Fancd2 and causes genetic instability without affecting ICL repair. Therefore, Fan1 recruitment enables processing of stalled forks that is essential for genome stability and health. PMID:26797144

  16. Replication Termination: Containing Fork Fusion-Mediated Pathologies in Escherichia coli.

    PubMed

    Dimude, Juachi U; Midgley-Smith, Sarah L; Stein, Monja; Rudolph, Christian J

    2016-01-01

    Duplication of bacterial chromosomes is initiated via the assembly of two replication forks at a single defined origin. Forks proceed bi-directionally until they fuse in a specialised termination area opposite the origin. This area is flanked by polar replication fork pause sites that allow forks to enter but not to leave. The precise function of this replication fork trap has remained enigmatic, as no obvious phenotypes have been associated with its inactivation. However, the fork trap becomes a serious problem to cells if the second fork is stalled at an impediment, as replication cannot be completed, suggesting that a significant evolutionary advantage for maintaining this chromosomal arrangement must exist. Recently, we demonstrated that head-on fusion of replication forks can trigger over-replication of the chromosome. This over-replication is normally prevented by a number of proteins including RecG helicase and 3' exonucleases. However, even in the absence of these proteins it can be safely contained within the replication fork trap, highlighting that multiple systems might be involved in coordinating replication fork fusions. Here, we discuss whether considering the problems associated with head-on replication fork fusion events helps us to better understand the important role of the replication fork trap in cellular metabolism. PMID:27463728

  17. Replication Termination: Containing Fork Fusion-Mediated Pathologies in Escherichia coli

    PubMed Central

    Dimude, Juachi U.; Midgley-Smith, Sarah L.; Stein, Monja; Rudolph, Christian J.

    2016-01-01

    Duplication of bacterial chromosomes is initiated via the assembly of two replication forks at a single defined origin. Forks proceed bi-directionally until they fuse in a specialised termination area opposite the origin. This area is flanked by polar replication fork pause sites that allow forks to enter but not to leave. The precise function of this replication fork trap has remained enigmatic, as no obvious phenotypes have been associated with its inactivation. However, the fork trap becomes a serious problem to cells if the second fork is stalled at an impediment, as replication cannot be completed, suggesting that a significant evolutionary advantage for maintaining this chromosomal arrangement must exist. Recently, we demonstrated that head-on fusion of replication forks can trigger over-replication of the chromosome. This over-replication is normally prevented by a number of proteins including RecG helicase and 3’ exonucleases. However, even in the absence of these proteins it can be safely contained within the replication fork trap, highlighting that multiple systems might be involved in coordinating replication fork fusions. Here, we discuss whether considering the problems associated with head-on replication fork fusion events helps us to better understand the important role of the replication fork trap in cellular metabolism. PMID:27463728

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

    PubMed

    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

  19. A case for sliding SeqA tracts at anchored replication forks during Escherichia coli chromosome replication and segregation

    PubMed Central

    Brendler, Therese; Sawitzke, Jim; Sergueev, Kirill; Austin, Stuart

    2000-01-01

    SeqA is an Escherichia coli DNA-binding protein that acts at replication origins and controls DNA replication. However, binding is not exclusive to origins. Many fragments containing two or more hemi-methylated GATC sequences bind efficiently. Binding was optimal when two such sequences were closely apposed or up to 31 bases apart on the same face of the DNA helix. Binding studies suggest that neighboring bound proteins contact each other to form a complex with the intervening DNA looped out. There are many potential binding sites distributed around the E.coli chromosome. As replication produces a transient wave of hemi-methylation, tracts of SeqA binding are likely to associate with each fork as replication progresses. The number and positions of green fluorescent protein–SeqA foci seen in living cells suggest that they correspond to these tracts, and that the forks are tethered to planes of cell division. SeqA may help to tether the forks or to organize newly replicated DNA into a structure that aids DNA to segregate away from the replication machinery. PMID:11080170

  20. Overexpression of the Replicative Helicase in Escherichia coli Inhibits Replication Initiation and Replication Fork Reloading

    PubMed Central

    Brüning, Jan-Gert; Myka, Kamila Katarzyna; McGlynn, Peter

    2016-01-01

    Replicative helicases play central roles in chromosome duplication and their assembly onto DNA is regulated via initiators and helicase loader proteins. The Escherichia coli replicative helicase DnaB and the helicase loader DnaC form a DnaB6–DnaC6 complex that is required for loading DnaB onto single-stranded DNA. Overexpression of dnaC inhibits replication by promoting continual rebinding of DnaC to DnaB and consequent prevention of helicase translocation. Here we show that overexpression of dnaB also inhibits growth and chromosome duplication. This inhibition is countered by co-overexpression of wild-type DnaC but not of a DnaC mutant that cannot interact with DnaB, indicating that a reduction in DnaB6–DnaC6 concentration is responsible for the phenotypes associated with elevated DnaB concentration. Partial defects in the oriC-specific initiator DnaA and in PriA-specific initiation away from oriC during replication repair sensitise cells to dnaB overexpression. Absence of the accessory replicative helicase Rep, resulting in increased replication blockage and thus increased reinitiation away from oriC, also exacerbates DnaB-induced defects. These findings indicate that elevated levels of helicase perturb replication initiation not only at origins of replication but also during fork repair at other sites on the chromosome. Thus, imbalances in levels of the replicative helicase and helicase loader can inhibit replication both via inhibition of DnaB6–DnaC6 complex formation with excess DnaB, as shown here, and promotion of formation of DnaB6–DnaC6 complexes with excess DnaC [Allen GC, Jr., Kornberg A. Fine balance in the regulation of DnaB helicase by DnaC protein in replication in Escherichia coli. J. Biol. Chem. 1991;266:22096–22101; Skarstad K, Wold S. The speed of the Escherichia coli fork in vivo depends on the DnaB:DnaC ratio. Mol. Microbiol. 1995;17:825–831]. Thus, there are two mechanisms by which an imbalance in the replicative helicase and its

  1. Response of the bacteriophage T4 replisome to non-coding lesions and regression of a stalled replication fork

    PubMed Central

    Nelson, Scott W.; Benkovic, Stephen J.

    2010-01-01

    DNA is constantly damaged by endogenous and exogenous agents. The resulting DNA lesions have the potential to halt the progression of the replisome, possibly leading to replication fork collapse. Here, we examine the effect of a non-coding DNA lesion in either the leading or lagging strand template on the bacteriophage T4 replisome. A damaged base in the lagging strand template does not affect the progression of the replication fork. Instead, the stalled lagging strand polymerase recycles from the lesion and initiates synthesis of the new Okazaki fragment upstream from the damaged base. In contrast, when the replisome encounters a blocking lesion in the leading strand template, the replication fork only travels approximately 1 kb beyond the point of the DNA lesion before complete replication fork collapse. The primosome and lagging strand polymerase remain active during this period and an Okazaki fragment is synthesized beyond the point of the leading strand lesion. There is no evidence for a new priming event on the leading strand template. Instead, the DNA structure that is produced by the stalled replication fork is a substrate for the DNA repair helicase, UvsW. UvsW catalyzes the regression of a stalled replication fork into a “chicken foot” structure that has been postulated to be an intermediate in an error-free lesion bypass pathway. PMID:20600127

  2. Stalled replication forks within heterochromatin require ATRX for protection.

    PubMed

    Huh, M S; Ivanochko, D; Hashem, L E; Curtin, M; Delorme, M; Goodall, E; Yan, K; Picketts, D J

    2016-01-01

    Expansive growth of neural progenitor cells (NPCs) is a prerequisite to the temporal waves of neuronal differentiation that generate the six-layered neocortex, while also placing a heavy burden on proteins that regulate chromatin packaging and genome integrity. This problem is further reflected by the growing number of developmental disorders caused by mutations in chromatin regulators. ATRX gene mutations cause a severe intellectual disability disorder (α-thalassemia mental retardation X-linked (ATRX) syndrome; OMIM no. 301040), characterized by microcephaly, urogenital abnormalities and α-thalassemia. Although the ATRX protein is required for the maintenance of repetitive DNA within heterochromatin, how this translates to disease pathogenesis remain poorly understood and was a focus of this study. We demonstrate that Atrx(FoxG1Cre) forebrain-specific conditional knockout mice display poly(ADP-ribose) polymerase-1 (Parp-1) hyperactivation during neurogenesis and generate fewer late-born Cux1- and Brn2-positive neurons that accounts for the reduced cortical size. Moreover, DNA damage, induced Parp-1 and Atm activation is elevated in progenitor cells and contributes to their increased level of cell death. ATRX-null HeLa cells are similarly sensitive to hydroxyurea-induced replication stress, accumulate DNA damage and proliferate poorly. Impaired BRCA1-RAD51 colocalization and PARP-1 hyperactivation indicated that stalled replication forks are not efficiently protected. DNA fiber assays confirmed that MRE11 degradation of stalled replication forks was rampant in the absence of ATRX or DAXX. Indeed, fork degradation in ATRX-null cells could be attenuated by treatment with the MRE11 inhibitor mirin, or exacerbated by inhibiting PARP-1 activity. Taken together, these results suggest that ATRX is required to limit replication stress during cellular proliferation, whereas upregulation of PARP-1 activity functions as a compensatory mechanism to protect stalled forks

  3. Stalled replication forks within heterochromatin require ATRX for protection

    PubMed Central

    Huh, M S; Ivanochko, D; Hashem, L E; Curtin, M; Delorme, M; Goodall, E; Yan, K; Picketts, D J

    2016-01-01

    Expansive growth of neural progenitor cells (NPCs) is a prerequisite to the temporal waves of neuronal differentiation that generate the six-layered neocortex, while also placing a heavy burden on proteins that regulate chromatin packaging and genome integrity. This problem is further reflected by the growing number of developmental disorders caused by mutations in chromatin regulators. ATRX gene mutations cause a severe intellectual disability disorder (α-thalassemia mental retardation X-linked (ATRX) syndrome; OMIM no. 301040), characterized by microcephaly, urogenital abnormalities and α-thalassemia. Although the ATRX protein is required for the maintenance of repetitive DNA within heterochromatin, how this translates to disease pathogenesis remain poorly understood and was a focus of this study. We demonstrate that AtrxFoxG1Cre forebrain-specific conditional knockout mice display poly(ADP-ribose) polymerase-1 (Parp-1) hyperactivation during neurogenesis and generate fewer late-born Cux1- and Brn2-positive neurons that accounts for the reduced cortical size. Moreover, DNA damage, induced Parp-1 and Atm activation is elevated in progenitor cells and contributes to their increased level of cell death. ATRX-null HeLa cells are similarly sensitive to hydroxyurea-induced replication stress, accumulate DNA damage and proliferate poorly. Impaired BRCA1-RAD51 colocalization and PARP-1 hyperactivation indicated that stalled replication forks are not efficiently protected. DNA fiber assays confirmed that MRE11 degradation of stalled replication forks was rampant in the absence of ATRX or DAXX. Indeed, fork degradation in ATRX-null cells could be attenuated by treatment with the MRE11 inhibitor mirin, or exacerbated by inhibiting PARP-1 activity. Taken together, these results suggest that ATRX is required to limit replication stress during cellular proliferation, whereas upregulation of PARP-1 activity functions as a compensatory mechanism to protect stalled forks

  4. Functional Analysis of DNA Replication Fork Reversal Catalyzed by Mycobacterium tuberculosis RuvAB Proteins*

    PubMed Central

    Khanduja, Jasbeer Singh; Muniyappa, K.

    2012-01-01

    Initially discovered in Escherichia coli, RuvAB proteins are ubiquitous in bacteria and play a dual role as molecular motor proteins responsible for branch migration of the Holliday junction(s) and reversal of stalled replication forks. Despite mounting genetic evidence for a crucial role of RuvA and RuvB proteins in reversal of stalled replication forks, the mechanistic aspects of this process are still not fully understood. Here, we elucidate the ability of Mycobacterium tuberculosis RuvAB (MtRuvAB) complex to catalyze the reversal of replication forks using a range of DNA replication fork substrates. Our studies show that MtRuvAB, unlike E. coli RuvAB, is able to drive replication fork reversal via the formation of Holliday junction intermediates, suggesting that RuvAB-catalyzed fork reversal involves concerted unwinding and annealing of nascent leading and lagging strands. We also demonstrate the reversal of replication forks carrying hemi-replicated DNA, indicating that MtRuvAB complex-catalyzed fork reversal is independent of symmetry at the fork junction. The fork reversal reaction catalyzed by MtRuvAB is coupled to ATP hydrolysis, is processive, and culminates in the formation of an extended reverse DNA arm. Notably, we found that sequence heterology failed to impede the fork reversal activity of MtRuvAB. We discuss the implications of these results in the context of recognition and processing of varied types of replication fork structures by RuvAB proteins. PMID:22094465

  5. Replication fork stability confers chemoresistance in BRCA-deficient cells.

    PubMed

    Ray Chaudhuri, Arnab; Callen, Elsa; Ding, Xia; Gogola, Ewa; Duarte, Alexandra A; Lee, Ji-Eun; Wong, Nancy; Lafarga, Vanessa; Calvo, Jennifer A; Panzarino, Nicholas J; John, Sam; Day, Amanda; Crespo, Anna Vidal; Shen, Binghui; Starnes, Linda M; de Ruiter, Julian R; Daniel, Jeremy A; Konstantinopoulos, Panagiotis A; Cortez, David; Cantor, Sharon B; Fernandez-Capetillo, Oscar; Ge, Kai; Jonkers, Jos; Rottenberg, Sven; Sharan, Shyam K; Nussenzweig, André

    2016-07-21

    Cells deficient in the Brca1 and Brca2 genes have reduced capacity to repair DNA double-strand breaks by homologous recombination and consequently are hypersensitive to DNA-damaging agents, including cisplatin and poly(ADP-ribose) polymerase (PARP) inhibitors. Here we show that loss of the MLL3/4 complex protein, PTIP, protects Brca1/2-deficient cells from DNA damage and rescues the lethality of Brca2-deficient embryonic stem cells. However, PTIP deficiency does not restore homologous recombination activity at double-strand breaks. Instead, its absence inhibits the recruitment of the MRE11 nuclease to stalled replication forks, which in turn protects nascent DNA strands from extensive degradation. More generally, acquisition of PARP inhibitors and cisplatin resistance is associated with replication fork protection in Brca2-deficient tumour cells that do not develop Brca2 reversion mutations. Disruption of multiple proteins, including PARP1 and CHD4, leads to the same end point of replication fork protection, highlighting the complexities by which tumour cells evade chemotherapeutic interventions and acquire drug resistance. PMID:27443740

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

    PubMed

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

    2004-10-01

    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, whereas its partners in DNA repair (UvrA/B and MutL/S) are not. We conclude that UvrD participates in replication fork reversal in E. coli. PMID:15375374

  7. Mammalian RAD51 paralogs protect nascent DNA at stalled forks and mediate replication restart

    PubMed Central

    Somyajit, Kumar; Saxena, Sneha; Babu, Sharath; Mishra, Anup; Nagaraju, Ganesh

    2015-01-01

    Mammalian RAD51 paralogs are implicated in the repair of collapsed replication forks by homologous recombination. However, their physiological roles in replication fork maintenance prior to fork collapse remain obscure. Here, we report on the role of RAD51 paralogs in short-term replicative stress devoid of DSBs. We show that RAD51 paralogs localize to nascent DNA and common fragile sites upon replication fork stalling. Strikingly, RAD51 paralogs deficient cells exhibit elevated levels of 53BP1 nuclear bodies and increased DSB formation, the latter being attributed to extensive degradation of nascent DNA at stalled forks. RAD51C and XRCC3 promote the restart of stalled replication in an ATP hydrolysis dependent manner by disengaging RAD51 and other RAD51 paralogs from the halted forks. Notably, we find that Fanconi anemia (FA)-like disorder and breast and ovarian cancer patient derived mutations of RAD51C fails to protect replication fork, exhibit under-replicated genomic regions and elevated micro-nucleation. Taken together, RAD51 paralogs prevent degradation of stalled forks and promote the restart of halted replication to avoid replication fork collapse, thereby maintaining genomic integrity and suppressing tumorigenesis. PMID:26354865

  8. Fork rotation and DNA precatenation are restricted during DNA replication to prevent chromosomal instability

    PubMed Central

    Schalbetter, Stephanie A.; Mansoubi, Sahar; Chambers, Anna L.; Downs, Jessica A.; Baxter, Jonathan

    2015-01-01

    Faithful genome duplication and inheritance require the complete resolution of all intertwines within the parental DNA duplex. This is achieved by topoisomerase action ahead of the replication fork or by fork rotation and subsequent resolution of the DNA precatenation formed. Although fork rotation predominates at replication termination, in vitro studies have suggested that it also occurs frequently during elongation. However, the factors that influence fork rotation and how rotation and precatenation may influence other replication-associated processes are unknown. Here we analyze the causes and consequences of fork rotation in budding yeast. We find that fork rotation and precatenation preferentially occur in contexts that inhibit topoisomerase action ahead of the fork, including stable protein–DNA fragile sites and termination. However, generally, fork rotation and precatenation are actively inhibited by Timeless/Tof1 and Tipin/Csm3. In the absence of Tof1/Timeless, excessive fork rotation and precatenation cause extensive DNA damage following DNA replication. With Tof1, damage related to precatenation is focused on the fragile protein–DNA sites where fork rotation is induced. We conclude that although fork rotation and precatenation facilitate unwinding in hard-to-replicate contexts, they intrinsically disrupt normal chromosome duplication and are therefore restricted by Timeless/Tipin. PMID:26240319

  9. Mechanism of asymmetric polymerase assembly at the eukaryotic replication fork.

    PubMed

    Georgescu, Roxana E; Langston, Lance; Yao, Nina Y; Yurieva, Olga; Zhang, Dan; Finkelstein, Jeff; Agarwal, Tani; O'Donnell, Mike E

    2014-08-01

    Eukaryotes use distinct polymerases for leading- and lagging-strand replication, but how they target their respective strands is uncertain. We reconstituted Saccharomyces cerevisiae replication forks and found that CMG helicase selects polymerase (Pol) ɛ to the exclusion of Pol δ on the leading strand. Even if Pol δ assembles on the leading strand, Pol ɛ rapidly replaces it. Pol δ-PCNA is distributive with CMG, in contrast to its high stability on primed ssDNA. Hence CMG will not stabilize Pol δ, instead leaving the leading strand accessible for Pol ɛ and stabilizing Pol ɛ. Comparison of Pol ɛ and Pol δ on a lagging-strand model DNA reveals the opposite. Pol δ dominates over excess Pol ɛ on PCNA-primed ssDNA. Thus, PCNA strongly favors Pol δ over Pol ɛ on the lagging strand, but CMG over-rides and flips this balance in favor of Pol ɛ on the leading strand. PMID:24997598

  10. Mechanism of asymmetric polymerase assembly at the eukaryotic replication fork

    PubMed Central

    Georgescu, Roxana E; Langston, Lance; Yao, Nina Y; Yurieva, Olga; Zhang, Dan; Finkelstein, Jeff; Agarwal, Tani; O’Donnell, Mike E

    2015-01-01

    Eukaryotes use distinct polymerases for leading- and lagging-strand replication, but how they target their respective strands is uncertain. We reconstituted Saccharomyces cerevisiae replication forks and found that CMG helicase selects polymerase (Pol) ε to the exclusion of Pol δ on the leading strand. Even if Pol δ assembles on the leading strand, Pol ε rapidly replaces it. Pol δ–PCNA is distributive with CMG, in contrast to its high stability on primed ssDNA. Hence CMG will not stabilize Pol δ, instead leaving the leading strand accessible for Pol ε and stabilizing Pol ε. Comparison of Pol ε and Pol δ on a lagging-strand model DNA reveals the opposite. Pol δ dominates over excess Pol ε on PCNA-primed ssDNA. Thus, PCNA strongly favors Pol δ over Pol ε on the lagging strand, but CMG over-rides and flips this balance in favor of Pol ε on the leading strand. PMID:24997598

  11. Tim/Timeless, a member of the replication fork protection complex, operates with the Warsaw breakage syndrome DNA helicase DDX11 in the same fork recovery pathway.

    PubMed

    Calì, Federica; Bharti, Sanjay Kumar; Perna, Roberta Di; Brosh, Robert M; Pisani, Francesca M

    2016-01-29

    We present evidence that Tim establishes a physical and functional interaction with DDX11, a super-family 2 iron-sulfur cluster DNA helicase genetically linked to the chromosomal instability disorder Warsaw breakage syndrome. Tim stimulates DDX11 unwinding activity on forked DNA substrates up to 10-fold and on bimolecular anti-parallel G-quadruplex DNA structures and three-stranded D-loop approximately 4-5-fold. Electrophoretic mobility shift assays revealed that Tim enhances DDX11 binding to DNA, suggesting that the observed stimulation derives from an improved ability of DDX11 to interact with the nucleic acid substrate. Surface plasmon resonance measurements indicate that DDX11 directly interacts with Tim. DNA fiber track assays with HeLa cells exposed to hydroxyurea demonstrated that Tim or DDX11 depletion significantly reduced replication fork progression compared to control cells; whereas no additive effect was observed by co-depletion of both proteins. Moreover, Tim and DDX11 are epistatic in promoting efficient resumption of stalled DNA replication forks in hydroxyurea-treated cells. This is consistent with the finding that association of the two endogenous proteins in the cell extract chromatin fraction is considerably increased following hydroxyurea exposure. Overall, our studies provide evidence that Tim and DDX11 physically and functionally interact and act in concert to preserve replication fork progression in perturbed conditions. PMID:26503245

  12. BRCA1 controls homologous recombination at Tus/Ter-stalled mammalian replication forks.

    PubMed

    Willis, Nicholas A; Chandramouly, Gurushankar; Huang, Bin; Kwok, Amy; Follonier, Cindy; Deng, Chuxia; Scully, Ralph

    2014-06-26

    Replication fork stalling can promote genomic instability, predisposing to cancer and other diseases. Stalled replication forks may be processed by sister chromatid recombination (SCR), generating error-free or error-prone homologous recombination (HR) outcomes. In mammalian cells, a long-standing hypothesis proposes that the major hereditary breast/ovarian cancer predisposition gene products, BRCA1 and BRCA2, control HR/SCR at stalled replication forks. Although BRCA1 and BRCA2 affect replication fork processing, direct evidence that BRCA gene products regulate homologous recombination at stalled chromosomal replication forks is lacking, due to a dearth of tools for studying this process. Here we report that the Escherichia coli Tus/Ter complex can be engineered to induce site-specific replication fork stalling and chromosomal HR/SCR in mouse cells. Tus/Ter-induced homologous recombination entails processing of bidirectionally arrested forks. We find that the Brca1 carboxy (C)-terminal tandem BRCT repeat and regions of Brca1 encoded by exon 11-two Brca1 elements implicated in tumour suppression-control Tus/Ter-induced homologous recombination. Inactivation of either Brca1 or Brca2 increases the absolute frequency of 'long-tract' gene conversions at Tus/Ter-stalled forks, an outcome not observed in response to a site-specific endonuclease-mediated chromosomal double-strand break. Therefore, homologous recombination at stalled forks is regulated differently from homologous recombination at double-strand breaks arising independently of a replication fork. We propose that aberrant long-tract homologous recombination at stalled replication forks contributes to genomic instability and breast/ovarian cancer predisposition in BRCA mutant cells. PMID:24776801

  13. Active Control of Repetitive Structural Transitions between Replication Forks and Holliday Junctions by Werner Syndrome Helicase.

    PubMed

    Shin, Soochul; Lee, Jinwoo; Yoo, Sangwoon; Kulikowicz, Tomasz; Bohr, Vilhelm A; Ahn, Byungchan; Hohng, Sungchul

    2016-08-01

    The reactivation of stalled DNA replication via fork regression invokes Holliday junction formation, branch migration, and the recovery of the replication fork after DNA repair or error-free DNA synthesis. The coordination mechanism for these DNA structural transitions by molecular motors, however, remains unclear. Here we perform single-molecule fluorescence experiments with Werner syndrome protein (WRN) and model replication forks. The Holliday junction is readily formed once the lagging arm is unwound, and migrated unidirectionally with 3.2 ± 0.03 bases/s velocity. The recovery of the replication fork was controlled by branch migration reversal of WRN, resulting in repetitive fork regression. The Holliday junction formation, branch migration, and migration direction reversal are all ATP dependent, revealing that WRN uses the energy of ATP hydrolysis to actively coordinate the structural transitions of DNA. PMID:27427477

  14. DNA2 drives processing and restart of reversed replication forks in human cells

    PubMed Central

    Thangavel, Saravanabhavan; Berti, Matteo; Levikova, Maryna; Pinto, Cosimo; Gomathinayagam, Shivasankari; Vujanovic, Marko; Zellweger, Ralph; Moore, Hayley; Lee, Eu Han; Hendrickson, Eric A.; Cejka, Petr; Stewart, Sheila; Lopes, Massimo

    2015-01-01

    Accurate processing of stalled or damaged DNA replication forks is paramount to genomic integrity and recent work points to replication fork reversal and restart as a central mechanism to ensuring high-fidelity DNA replication. Here, we identify a novel DNA2- and WRN-dependent mechanism of reversed replication fork processing and restart after prolonged genotoxic stress. The human DNA2 nuclease and WRN ATPase activities functionally interact to degrade reversed replication forks with a 5′-to-3′ polarity and promote replication restart, thus preventing aberrant processing of unresolved replication intermediates. Unexpectedly, EXO1, MRE11, and CtIP are not involved in the same mechanism of reversed fork processing, whereas human RECQ1 limits DNA2 activity by preventing extensive nascent strand degradation. RAD51 depletion antagonizes this mechanism, presumably by preventing reversed fork formation. These studies define a new mechanism for maintaining genome integrity tightly controlled by specific nucleolytic activities and central homologous recombination factors. PMID:25733713

  15. RNF4 and PLK1 are required for replication fork collapse in ATR-deficient cells.

    PubMed

    Ragland, Ryan L; Patel, Sima; Rivard, Rebecca S; Smith, Kevin; Peters, Ashley A; Bielinsky, Anja-Katrin; Brown, Eric J

    2013-10-15

    The ATR-CHK1 axis stabilizes stalled replication forks and prevents their collapse into DNA double-strand breaks (DSBs). Here, we show that fork collapse in Atr-deleted cells is mediated through the combined effects the sumo targeted E3-ubiquitin ligase RNF4 and activation of the AURKA-PLK1 pathway. As indicated previously, Atr-deleted cells exhibited a decreased ability to restart DNA replication following fork stalling in comparison with control cells. However, suppression of RNF4, AURKA, or PLK1 returned the reinitiation of replication in Atr-deleted cells to near wild-type levels. In RNF4-depleted cells, this rescue directly correlated with the persistence of sumoylation of chromatin-bound factors. Notably, RNF4 repression substantially suppressed the accumulation of DSBs in ATR-deficient cells, and this decrease in breaks was enhanced by concomitant inhibition of PLK1. DSBs resulting from ATR inhibition were also observed to be dependent on the endonuclease scaffold protein SLX4, suggesting that RNF4 and PLK1 either help activate the SLX4 complex or make DNA replication fork structures accessible for subsequent SLX4-dependent cleavage. Thus, replication fork collapse following ATR inhibition is a multistep process that disrupts replisome function and permits cleavage of the replication fork. PMID:24142876

  16. Regression of Replication Forks Stalled by Leading-strand Template Damage

    PubMed Central

    Gupta, Sankalp; Yeeles, Joseph T. P.; Marians, Kenneth J.

    2014-01-01

    Stalled replication forks are sites of chromosome breakage and the formation of toxic recombination intermediates that undermine genomic stability. Thus, replication fork repair and reactivation are essential processes. Among the many models of replication fork reactivation is one that invokes fork regression catalyzed by the strand exchange protein RecA as an intermediate in the processing of the stalled fork. We have investigated the replication fork regression activity of RecA using a reconstituted DNA replication system where the replisome is stalled by collision with leading-strand template damage. We find that RecA is unable to regress the stalled fork in the presence of the replisome and SSB. If the replication proteins are removed from the stalled fork, RecA will catalyze net regression as long as the Okazaki fragments are sealed. RecA-generated Holliday junctions can be detected by RuvC cleavage, although this is not a robust reaction. On the other hand, extensive branch migration by RecA, where a completely unwound product consisting of the paired nascent leading and lagging strands is produced, is observed under conditions where RuvC activity is suppressed. This branch migration reaction is inhibited by SSB, possibly accounting for the failure of RecA to generate products in the presence of the replication proteins. Interestingly, we find that the RecA-RuvC reaction is supported to differing extents, depending on the template damage; templates carrying a cyclopyrimidine dimer elicit more RecA-RuvC product than those carrying a synthetic abasic site. This difference could be ascribed to a higher affinity of RecA binding to DNAs carrying a thymidine dimer than to those with an abasic site. PMID:25138217

  17. Regulating telomere length from the inside out: the replication fork model

    PubMed Central

    2016-01-01

    Telomere length is regulated around an equilibrium set point. Telomeres shorten during replication and are lengthened by telomerase. Disruption of the length equilibrium leads to disease; thus, it is important to understand the mechanisms that regulate length at the molecular level. The prevailing protein-counting model for regulating telomerase access to elongate the telomere does not explain accumulating evidence of a role of DNA replication in telomere length regulation. Here I present an alternative model: the replication fork model that can explain how passage of a replication fork and regulation of origin firing affect telomere length. PMID:27401551

  18. A short G1 phase imposes constitutive replication stress and fork remodelling in mouse embryonic stem cells.

    PubMed

    Ahuja, Akshay K; Jodkowska, Karolina; Teloni, Federico; Bizard, Anna H; Zellweger, Ralph; Herrador, Raquel; Ortega, Sagrario; Hickson, Ian D; Altmeyer, Matthias; Mendez, Juan; Lopes, Massimo

    2016-01-01

    Embryonic stem cells (ESCs) represent a transient biological state, where pluripotency is coupled with fast proliferation. ESCs display a constitutively active DNA damage response (DDR), but its molecular determinants have remained elusive. Here we show in cultured ESCs and mouse embryos that H2AX phosphorylation is dependent on Ataxia telangiectasia and Rad3 related (ATR) and is associated with chromatin loading of the ssDNA-binding proteins RPA and RAD51. Single-molecule analysis of replication intermediates reveals massive ssDNA gap accumulation, reduced fork speed and frequent fork reversal. All these marks of replication stress do not impair the mitotic process and are rapidly lost at differentiation onset. Delaying the G1/S transition in ESCs allows formation of 53BP1 nuclear bodies and suppresses ssDNA accumulation, fork slowing and reversal in the following S-phase. Genetic inactivation of fork slowing and reversal leads to chromosomal breakage in unperturbed ESCs. We propose that rapid cell cycle progression makes ESCs dependent on effective replication-coupled mechanisms to protect genome integrity. PMID:26876348

  19. A short G1 phase imposes constitutive replication stress and fork remodelling in mouse embryonic stem cells

    PubMed Central

    Ahuja, Akshay K.; Jodkowska, Karolina; Teloni, Federico; Bizard, Anna H.; Zellweger, Ralph; Herrador, Raquel; Ortega, Sagrario; Hickson, Ian D.; Altmeyer, Matthias; Mendez, Juan; Lopes, Massimo

    2016-01-01

    Embryonic stem cells (ESCs) represent a transient biological state, where pluripotency is coupled with fast proliferation. ESCs display a constitutively active DNA damage response (DDR), but its molecular determinants have remained elusive. Here we show in cultured ESCs and mouse embryos that H2AX phosphorylation is dependent on Ataxia telangiectasia and Rad3 related (ATR) and is associated with chromatin loading of the ssDNA-binding proteins RPA and RAD51. Single-molecule analysis of replication intermediates reveals massive ssDNA gap accumulation, reduced fork speed and frequent fork reversal. All these marks of replication stress do not impair the mitotic process and are rapidly lost at differentiation onset. Delaying the G1/S transition in ESCs allows formation of 53BP1 nuclear bodies and suppresses ssDNA accumulation, fork slowing and reversal in the following S-phase. Genetic inactivation of fork slowing and reversal leads to chromosomal breakage in unperturbed ESCs. We propose that rapid cell cycle progression makes ESCs dependent on effective replication-coupled mechanisms to protect genome integrity. PMID:26876348

  20. Restriction of Replication Fork Regression Activities by a Conserved SMC Complex

    PubMed Central

    Xue, Xiaoyu; Choi, Koyi; Bonner, Jaclyn; Chiba, Tamara; Kwon, Youngho; Xu, Yuanyuan; Sanchez, Humberto; Wyman, Claire; Niu, Hengyao; Zhao, Xiaolan; Sung, Patrick

    2014-01-01

    SUMMARY Conserved, multi-tasking DNA helicases mediate diverse DNA transactions and are relevant for human disease pathogenesis. These helicases and their regulation help maintain genome stability during DNA replication and repair. We show that the structural maintenance of chromosome complex Smc5-Smc6 restrains the replication fork regression activity of Mph1 helicase, but not its D-loop disruptive activity. This regulatory mechanism enables flexibility in replication fork repair without interfering with DNA break repair. In vitro studies find that Smc5-Smc6 binds to a Mph1 region required for efficient fork regression, preventing assembly of Mph1 oligomers at the junction of DNA forks. In vivo impairment of this regulatory mechanism compensates for the inactivation of another fork regression helicase and increases reliance on joint DNA structure removal or avoidance. Our findings provide molecular insights into replication fork repair regulation and uncover a role of Smc5-Smc6 in directing Mph1 activity towards a specific biochemical outcome. PMID:25439736

  1. Cyclin E Is Stabilized in Response to Replication Fork Barriers Leading to Prolonged S Phase Arrest*

    PubMed Central

    Lu, Xiaoyan; Liu, Jia; Legerski, Randy J.

    2009-01-01

    Cyclin E is a regulator of cyclin-dependent protein kinases (Cdks) and is involved in mediating the cell cycle transition from G1 to S phase. Here, we describe a novel function for cyclin E in the long term maintenance of checkpoint arrest in response to replication barriers. Exposure of cells to mitomycin C or UV irradiation, but not ionizing radiation, induces stabilization of cyclin E. Stabilization of cyclin E reduces the activity of Cdk2-cyclin A, resulting in a slowing of S phase progression and arrest. In addition, cyclin E is shown to be required for stabilization of Cdc6, which is required for activation of Chk1 and the replication checkpoint pathway. Furthermore, the stabilization of cyclin E in response to replication fork barriers depends on ATR, but not Nbs1 or Chk1. These results indicate that in addition to its well studied role in promoting cell cycle progression, cyclin E also has a role in regulating cell cycle arrest in response to DNA damage. PMID:19812034

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

    PubMed

    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

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

    PubMed Central

    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

  4. Recombinational repair and restart of damaged replication forks.

    PubMed

    McGlynn, Peter; Lloyd, Robert G

    2002-11-01

    Genome duplication necessarily involves the replication of imperfect DNA templates and, if left to their own devices, replication complexes regularly run into problems. The details of how cells overcome these replicative 'hiccups' are beginning to emerge, revealing a complex interplay between DNA replication, recombination and repair that ensures faithful passage of the genetic material from one generation to the next. PMID:12415303

  5. Slow Replication Fork Velocity of Homologous Recombination-Defective Cells Results from Endogenous Oxidative Stress.

    PubMed

    Wilhelm, Therese; Ragu, Sandrine; Magdalou, Indiana; Machon, Christelle; Dardillac, Elodie; Técher, Hervé; Guitton, Jérôme; Debatisse, Michelle; Lopez, Bernard S

    2016-05-01

    Replications forks are routinely hindered by different endogenous stresses. Because homologous recombination plays a pivotal role in the reactivation of arrested replication forks, defects in homologous recombination reveal the initial endogenous stress(es). Homologous recombination-defective cells consistently exhibit a spontaneously reduced replication speed, leading to mitotic extra centrosomes. Here, we identify oxidative stress as a major endogenous source of replication speed deceleration in homologous recombination-defective cells. The treatment of homologous recombination-defective cells with the antioxidant N-acetyl-cysteine or the maintenance of the cells at low O2 levels (3%) rescues both the replication fork speed, as monitored by single-molecule analysis (molecular combing), and the associated mitotic extra centrosome frequency. Reciprocally, the exposure of wild-type cells to H2O2 reduces the replication fork speed and generates mitotic extra centrosomes. Supplying deoxynucleotide precursors to H2O2-exposed cells rescued the replication speed. Remarkably, treatment with N-acetyl-cysteine strongly expanded the nucleotide pool, accounting for the replication speed rescue. Remarkably, homologous recombination-defective cells exhibit a high level of endogenous reactive oxygen species. Consistently, homologous recombination-defective cells accumulate spontaneous γH2AX or XRCC1 foci that are abolished by treatment with N-acetyl-cysteine or maintenance at 3% O2. Finally, oxidative stress stimulated homologous recombination, which is suppressed by supplying deoxynucleotide precursors. Therefore, the cellular redox status strongly impacts genome duplication and transmission. Oxidative stress should generate replication stress through different mechanisms, including DNA damage and nucleotide pool imbalance. These data highlight the intricacy of endogenous replication and oxidative stresses, which are both evoked during tumorigenesis and senescence initiation

  6. Slow Replication Fork Velocity of Homologous Recombination-Defective Cells Results from Endogenous Oxidative Stress

    PubMed Central

    Magdalou, Indiana; Machon, Christelle; Dardillac, Elodie; Técher, Hervé; Guitton, Jérôme; Debatisse, Michelle; Lopez, Bernard S.

    2016-01-01

    Replications forks are routinely hindered by different endogenous stresses. Because homologous recombination plays a pivotal role in the reactivation of arrested replication forks, defects in homologous recombination reveal the initial endogenous stress(es). Homologous recombination-defective cells consistently exhibit a spontaneously reduced replication speed, leading to mitotic extra centrosomes. Here, we identify oxidative stress as a major endogenous source of replication speed deceleration in homologous recombination-defective cells. The treatment of homologous recombination-defective cells with the antioxidant N-acetyl-cysteine or the maintenance of the cells at low O2 levels (3%) rescues both the replication fork speed, as monitored by single-molecule analysis (molecular combing), and the associated mitotic extra centrosome frequency. Reciprocally, the exposure of wild-type cells to H2O2 reduces the replication fork speed and generates mitotic extra centrosomes. Supplying deoxynucleotide precursors to H2O2-exposed cells rescued the replication speed. Remarkably, treatment with N-acetyl-cysteine strongly expanded the nucleotide pool, accounting for the replication speed rescue. Remarkably, homologous recombination-defective cells exhibit a high level of endogenous reactive oxygen species. Consistently, homologous recombination-defective cells accumulate spontaneous γH2AX or XRCC1 foci that are abolished by treatment with N-acetyl-cysteine or maintenance at 3% O2. Finally, oxidative stress stimulated homologous recombination, which is suppressed by supplying deoxynucleotide precursors. Therefore, the cellular redox status strongly impacts genome duplication and transmission. Oxidative stress should generate replication stress through different mechanisms, including DNA damage and nucleotide pool imbalance. These data highlight the intricacy of endogenous replication and oxidative stresses, which are both evoked during tumorigenesis and senescence initiation

  7. Host DNA replication forks are not preferred targets for bacteriophage Mu transposition.

    PubMed Central

    Nakai, H; Taylor, A L

    1985-01-01

    Bacteriophage Mu DNA integration in Escherichia coli strains infected after alignment of chromosomal replication was analyzed by a sandwich hybridization assay. The results indicated that Mu integrated into chromosomal segments at various distances from oriC with similar kinetics. In an extension of these studies, various Hfr strains were infected after alignment of chromosomal replication, and Mu transposition was shut down early after infection. The positions of integrated Mu copies were inferred from the transfer kinetics of Mu to an F- strain. Our analysis indicated that the location of Mu DNA in the host chromosome was not dependent on the positions of host replication forks at the time of infection. However, the procedure for aligning chromosomal replication affected DNA transfer by various Hfr strains differently, and this effect could account for prior results suggesting preferential integration of Mu at host replication forks. Images PMID:3159718

  8. Human HLTF mediates postreplication repair by its HIRAN domain-dependent replication fork remodelling

    SciTech Connect

    Achar, Yathish Jagadheesh; Balogh, David; Neculai, Dante; Juhasz, Szilvia; Morocz, Monika; Gali, Himabindu; Dhe-Paganon, Sirano; Venclovas, Česlovas; Haracska, Lajos

    2015-09-08

    Defects in the ability to respond properly to an unrepaired DNA lesion blocking replication promote genomic instability and cancer. Human HLTF, implicated in error-free replication of damaged DNA and tumour suppression, exhibits a HIRAN domain, a RING domain, and a SWI/SNF domain facilitating DNA-binding, PCNA-polyubiquitin-ligase, and dsDNA-translocase activities, respectively. Here, we investigate the mechanism of HLTF action with emphasis on its HIRAN domain. We found that in cells HLTF promotes the filling-in of gaps left opposite damaged DNA during replication, and this postreplication repair function depends on its HIRAN domain. Our biochemical assays show that HIRAN domain mutant HLTF proteins retain their ubiquitin ligase, ATPase and dsDNA translocase activities but are impaired in binding to a model replication fork. These data and our structural study indicate that the HIRAN domain recruits HLTF to a stalled replication fork, and it also provides the direction for the movement of the dsDNA translocase motor domain for fork reversal. We suggest functional similarities between the HIRAN, the OB, the HARP2, and other domains found in certain motor proteins, which may explain why only a subset of DNA translocases can carry out fork reversal.

  9. Human HLTF mediates postreplication repair by its HIRAN domain-dependent replication fork remodelling.

    PubMed

    Achar, Yathish Jagadheesh; Balogh, David; Neculai, Dante; Juhasz, Szilvia; Morocz, Monika; Gali, Himabindu; Dhe-Paganon, Sirano; Venclovas, Česlovas; Haracska, Lajos

    2015-12-01

    Defects in the ability to respond properly to an unrepaired DNA lesion blocking replication promote genomic instability and cancer. Human HLTF, implicated in error-free replication of damaged DNA and tumour suppression, exhibits a HIRAN domain, a RING domain, and a SWI/SNF domain facilitating DNA-binding, PCNA-polyubiquitin-ligase, and dsDNA-translocase activities, respectively. Here, we investigate the mechanism of HLTF action with emphasis on its HIRAN domain. We found that in cells HLTF promotes the filling-in of gaps left opposite damaged DNA during replication, and this postreplication repair function depends on its HIRAN domain. Our biochemical assays show that HIRAN domain mutant HLTF proteins retain their ubiquitin ligase, ATPase and dsDNA translocase activities but are impaired in binding to a model replication fork. These data and our structural study indicate that the HIRAN domain recruits HLTF to a stalled replication fork, and it also provides the direction for the movement of the dsDNA translocase motor domain for fork reversal. In more general terms, we suggest functional similarities between the HIRAN, the OB, the HARP2, and other domains found in certain motor proteins, which may explain why only a subset of DNA translocases can carry out fork reversal. PMID:26350214

  10. Human HLTF mediates postreplication repair by its HIRAN domain-dependent replication fork remodelling

    DOE PAGESBeta

    Achar, Yathish Jagadheesh; Balogh, David; Neculai, Dante; Juhasz, Szilvia; Morocz, Monika; Gali, Himabindu; Dhe-Paganon, Sirano; Venclovas, Česlovas; Haracska, Lajos

    2015-09-08

    Defects in the ability to respond properly to an unrepaired DNA lesion blocking replication promote genomic instability and cancer. Human HLTF, implicated in error-free replication of damaged DNA and tumour suppression, exhibits a HIRAN domain, a RING domain, and a SWI/SNF domain facilitating DNA-binding, PCNA-polyubiquitin-ligase, and dsDNA-translocase activities, respectively. Here, we investigate the mechanism of HLTF action with emphasis on its HIRAN domain. We found that in cells HLTF promotes the filling-in of gaps left opposite damaged DNA during replication, and this postreplication repair function depends on its HIRAN domain. Our biochemical assays show that HIRAN domain mutant HLTF proteinsmore » retain their ubiquitin ligase, ATPase and dsDNA translocase activities but are impaired in binding to a model replication fork. These data and our structural study indicate that the HIRAN domain recruits HLTF to a stalled replication fork, and it also provides the direction for the movement of the dsDNA translocase motor domain for fork reversal. We suggest functional similarities between the HIRAN, the OB, the HARP2, and other domains found in certain motor proteins, which may explain why only a subset of DNA translocases can carry out fork reversal.« less

  11. Human HLTF mediates postreplication repair by its HIRAN domain-dependent replication fork remodelling

    PubMed Central

    Achar, Yathish Jagadheesh; Balogh, David; Neculai, Dante; Juhasz, Szilvia; Morocz, Monika; Gali, Himabindu; Dhe-Paganon, Sirano; Venclovas, Česlovas; Haracska, Lajos

    2015-01-01

    Defects in the ability to respond properly to an unrepaired DNA lesion blocking replication promote genomic instability and cancer. Human HLTF, implicated in error-free replication of damaged DNA and tumour suppression, exhibits a HIRAN domain, a RING domain, and a SWI/SNF domain facilitating DNA-binding, PCNA-polyubiquitin-ligase, and dsDNA-translocase activities, respectively. Here, we investigate the mechanism of HLTF action with emphasis on its HIRAN domain. We found that in cells HLTF promotes the filling-in of gaps left opposite damaged DNA during replication, and this postreplication repair function depends on its HIRAN domain. Our biochemical assays show that HIRAN domain mutant HLTF proteins retain their ubiquitin ligase, ATPase and dsDNA translocase activities but are impaired in binding to a model replication fork. These data and our structural study indicate that the HIRAN domain recruits HLTF to a stalled replication fork, and it also provides the direction for the movement of the dsDNA translocase motor domain for fork reversal. In more general terms, we suggest functional similarities between the HIRAN, the OB, the HARP2, and other domains found in certain motor proteins, which may explain why only a subset of DNA translocases can carry out fork reversal. PMID:26350214

  12. UvrD controls the access of recombination proteins to blocked replication forks.

    PubMed

    Lestini, Roxane; Michel, Bénédicte

    2007-08-22

    Blocked replication forks often need to be processed by recombination proteins prior to replication restart. In Escherichia coli, the UvrD repair helicase was recently shown to act at inactivated replication forks, where it counteracts a deleterious action of RecA. Using two mutants affected for different subunits of the polymerase III holoenzyme (Pol IIIh), we show here that the anti-RecA action of UvrD at blocked forks reflects two different activities of this enzyme. A defective UvrD mutant is able to antagonize RecA in cells affected for the Pol IIIh catalytic subunit DnaE. In this mutant, RecA action at blocked forks specifically requires the protein RarA (MgsA). We propose that UvrD prevents RecA binding, possibly by counteracting RarA. In contrast, at forks affected for the Pol IIIh clamp (DnaN), RarA is not required for RecA binding and the ATPase function of UvrD is essential to counteract RecA, supporting the idea that UvrD removes RecA from DNA. UvrD action on RecA is conserved in evolution as it can be performed in E. coli by the UvrD homologue from Bacillus subtilis, PcrA. PMID:17641684

  13. UvrD controls the access of recombination proteins to blocked replication forks

    PubMed Central

    Lestini, Roxane; Michel, Bénédicte

    2007-01-01

    Blocked replication forks often need to be processed by recombination proteins prior to replication restart. In Escherichia coli, the UvrD repair helicase was recently shown to act at inactivated replication forks, where it counteracts a deleterious action of RecA. Using two mutants affected for different subunits of the polymerase III holoenzyme (Pol IIIh), we show here that the anti-RecA action of UvrD at blocked forks reflects two different activities of this enzyme. A defective UvrD mutant is able to antagonize RecA in cells affected for the Pol IIIh catalytic subunit DnaE. In this mutant, RecA action at blocked forks specifically requires the protein RarA (MgsA). We propose that UvrD prevents RecA binding, possibly by counteracting RarA. In contrast, at forks affected for the Pol IIIh clamp (DnaN), RarA is not required for RecA binding and the ATPase function of UvrD is essential to counteract RecA, supporting the idea that UvrD removes RecA from DNA. UvrD action on RecA is conserved in evolution as it can be performed in E. coli by the UvrD homologue from Bacillus subtilis, PcrA. PMID:17641684

  14. Replication Fork Polarity Gradients Revealed by Megabase-Sized U-Shaped Replication Timing Domains in Human Cell Lines

    PubMed Central

    Baker, Antoine; Audit, Benjamin; Chen, Chun-Long; Moindrot, Benoit; Leleu, Antoine; Guilbaud, Guillaume; Rappailles, Aurélien; Vaillant, Cédric; Goldar, Arach; Mongelard, Fabien; d'Aubenton-Carafa, Yves; Hyrien, Olivier; Thermes, Claude; Arneodo, Alain

    2012-01-01

    In higher eukaryotes, replication program specification in different cell types remains to be fully understood. We show for seven human cell lines that about half of the genome is divided in domains that display a characteristic U-shaped replication timing profile with early initiation zones at borders and late replication at centers. Significant overlap is observed between U-domains of different cell lines and also with germline replication domains exhibiting a N-shaped nucleotide compositional skew. From the demonstration that the average fork polarity is directly reflected by both the compositional skew and the derivative of the replication timing profile, we argue that the fact that this derivative displays a N-shape in U-domains sustains the existence of large-scale gradients of replication fork polarity in somatic and germline cells. Analysis of chromatin interaction (Hi-C) and chromatin marker data reveals that U-domains correspond to high-order chromatin structural units. We discuss possible models for replication origin activation within U/N-domains. The compartmentalization of the genome into replication U/N-domains provides new insights on the organization of the replication program in the human genome. PMID:22496629

  15. Replication stalling at unstable inverted repeats: Interplay between DNA hairpins and fork stabilizing proteins

    PubMed Central

    Voineagu, Irina; Narayanan, Vidhya; Lobachev, Kirill S.; Mirkin, Sergei M.

    2008-01-01

    DNA inverted repeats (IRs) are hotspots of genomic instability in both prokaryotes and eukaryotes. This feature is commonly attributed to their ability to fold into hairpin- or cruciform-like DNA structures interfering with DNA replication and other genetic processes. However, direct evidence that IRs are replication stall sites in vivo is currently lacking. Here, we show by 2D electrophoretic analysis of replication intermediates that replication forks stall at IRs in bacteria, yeast, and mammalian cells. We found that DNA hairpins, rather than DNA cruciforms, are responsible for the replication stalling by comparing the effects of specifically designed imperfect IRs with varying lengths of their central spacer. Finally, we report that yeast fork-stabilizing proteins, Tof1 and Mrc1, are required to counteract repeat-mediated replication stalling. We show that the function of the Tof1 protein at DNA structure-mediated stall sites is different from its previously described effect on protein-mediated replication fork barriers. PMID:18632578

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

    EPA Science Inventory

    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...

  17. DNA Replication Forks Pause at Silent Origins near the HML Locus in Budding Yeast

    PubMed Central

    Wang, Yangzhou; Vujcic, Marija; Kowalski, David

    2001-01-01

    Chromosomal replicators in budding yeast contain an autonomously replicating sequence (ARS) that functions in a plasmid, but certain ARSs are silent as replication origins in their natural chromosomal context. In chromosome III, the HML ARS cluster (ARS302-ARS303-ARS320) and ARS301 flank the transcriptionally silent mating-type locus HML, and all of these ARSs are silent as replication origins. ARS301 and ARS302 function in transcriptional silencing mediated by the origin recognition complex (ORC) and a heterochromatin structure, while the functions of ARS303 and ARS320 are not known. In this work, we discovered replication fork pause sites at the HML ARS cluster and ARS301 by analyzing DNA replication intermediates from the chromosome via two-dimensional gel electrophoresis. The replication fork pause at the HML ARS cluster was independent of cis- and trans-acting mutations that abrogate transcriptional silencing at HML. Deletion of the HML ARS cluster led to loss of the pause site. Insertion of a single, heterologous ARS (ARS305) in place of the HML ARS cluster reconstituted the pause site, as did multiple copies of DNA elements (A and B1) that bind ORC. The orc2-1 mutation, known to alter replication timing at origins, did not detectably affect the pause but activated the silent origin at the HML ARS cluster in a minority of cells. Delaying the time of fork arrival at HML led to the elimination of the pause sites at the HML ARS cluster and at the copy of ARS305 inserted in place of the cluster. Loss of the pause sites was accompanied by activation of the silent origins in the majority of cells. Thus, replication fork movement near HML pauses at a silent origin which is competent for replication initiation but kept silent through Orc2p, a component of the replication initiator. Possible functions for replication fork pause sites in checkpoints, S-phase regulation, mating-type switching, and transcriptionally silent heterochromatin are discussed. PMID:11438651

  18. Structure of the replication fork in ultraviolet light-irradiated human cells.

    PubMed Central

    Cordeiro-Stone, M; Schumacher, R I; Meneghini, R

    1979-01-01

    The DNA extracted from xeroderma pigmentosum human fibroblasts previously irradiated with 12.5 J/m2 of UV light and pulse-labeled for 45 min with radioactive and (or) heavy precursors, was used to determine the structural characteristics of the replication fork. Density equilibrium centrifugation experiments showed that a fork moved 6 micrometer in 45 min and bypassed 3 pyrimidine dimers in both strands. The same length was covered in 15-20 min in control cells. The delay in irradiated cells was apparently due to pyrimidine dimers acting as temporary blocks to the fork movement. Evidence for this interpretation comes from kinetics of incorporation of [3H]thymidine into DNA, which show that the time necessary to attain a new stable level of DNA synthesis in irradiated cells is equivalent to that required for the replication fork to cover the interdimer distance in one strand. On the other hand, the action of S1 nuclease on DNA synthesized soon after irradiation gives rise to a bimodal distribution in neutral sucrose gradients, one peak corresponding to 43 X 10(6) daltons and the other to 3 X 10(6) daltons. These two DNA species are generated by the attack of the S1 nuclease on single-stranded regions associated with the replication fork. A possible explanation for these results is given by a model according to which there is a delayed bypass of the dimer in the leading strand and the appearance of gaps opposite pyrimidine dimers in the lagging strand, as a direct consequence of the discontinuous mode of DNA replication. In terms of the model, the DNA of 43 X 10(6) daltons corresponds to the leading strand, linked to the unreplicated branch of the forks, whereas the piece of 3 X 10(6) daltons is the intergap DNA coming from the lagging strand. Pulse and chase experiments reveal that the low molecular weight DNA grows in a pattern that suggests that more than one gap may be formed per replication fork. PMID:233582

  19. EEPD1 Rescues Stressed Replication Forks and Maintains Genome Stability by Promoting End Resection and Homologous Recombination Repair

    PubMed Central

    Wu, Yuehan; Lee, Suk-Hee; Williamson, Elizabeth A.; Reinert, Brian L.; Cho, Ju Hwan; Xia, Fen; Jaiswal, Aruna Shanker; Srinivasan, Gayathri; Patel, Bhavita; Brantley, Alexis; Zhou, Daohong; Shao, Lijian; Pathak, Rupak; Hauer-Jensen, Martin; Singh, Sudha; Kong, Kimi; Wu, Xaiohua; Kim, Hyun-Suk; Beissbarth, Timothy; Gaedcke, Jochen; Burma, Sandeep; Nickoloff, Jac A.; Hromas, Robert A.

    2015-01-01

    Replication fork stalling and collapse is a major source of genome instability leading to neoplastic transformation or cell death. Such stressed replication forks can be conservatively repaired and restarted using homologous recombination (HR) or non-conservatively repaired using micro-homology mediated end joining (MMEJ). HR repair of stressed forks is initiated by 5’ end resection near the fork junction, which permits 3’ single strand invasion of a homologous template for fork restart. This 5’ end resection also prevents classical non-homologous end-joining (cNHEJ), a competing pathway for DNA double-strand break (DSB) repair. Unopposed NHEJ can cause genome instability during replication stress by abnormally fusing free double strand ends that occur as unstable replication fork repair intermediates. We show here that the previously uncharacterized Exonuclease/Endonuclease/Phosphatase Domain-1 (EEPD1) protein is required for initiating repair and restart of stalled forks. EEPD1 is recruited to stalled forks, enhances 5’ DNA end resection, and promotes restart of stalled forks. Interestingly, EEPD1 directs DSB repair away from cNHEJ, and also away from MMEJ, which requires limited end resection for initiation. EEPD1 is also required for proper ATR and CHK1 phosphorylation, and formation of gamma-H2AX, RAD51 and phospho-RPA32 foci. Consistent with a direct role in stalled replication fork cleavage, EEPD1 is a 5’ overhang nuclease in an obligate complex with the end resection nuclease Exo1 and BLM. EEPD1 depletion causes nuclear and cytogenetic defects, which are made worse by replication stress. Depleting 53BP1, which slows cNHEJ, fully rescues the nuclear and cytogenetic abnormalities seen with EEPD1 depletion. These data demonstrate that genome stability during replication stress is maintained by EEPD1, which initiates HR and inhibits cNHEJ and MMEJ. PMID:26684013

  20. Nek1 Regulates Rad54 to Orchestrate Homologous Recombination and Replication Fork Stability.

    PubMed

    Spies, Julian; Waizenegger, Anja; Barton, Olivia; Sürder, Michael; Wright, William D; Heyer, Wolf-Dietrich; Löbrich, Markus

    2016-06-16

    Never-in-mitosis A-related kinase 1 (Nek1) has established roles in apoptosis and cell cycle regulation. We show that human Nek1 regulates homologous recombination (HR) by phosphorylating Rad54 at Ser572 in late G2 phase. Nek1 deficiency as well as expression of unphosphorylatable Rad54 (Rad54-S572A) cause unresolved Rad51 foci and confer a defect in HR. Phospho-mimic Rad54 (Rad54-S572E), in contrast, promotes HR and rescues the HR defect associated with Nek1 loss. Although expression of phospho-mimic Rad54 is beneficial for HR, it causes Rad51 removal from chromatin and degradation of stalled replication forks in S phase. Thus, G2-specific phosphorylation of Rad54 by Nek1 promotes Rad51 chromatin removal during HR in G2 phase, and its absence in S phase is required for replication fork stability. In summary, Nek1 regulates Rad51 removal to orchestrate HR and replication fork stability. PMID:27264870

  1. Saccharomyces cerevisiae Genetics Predicts Candidate Therapeutic Genetic Interactions at the Mammalian Replication Fork

    PubMed Central

    van Pel, Derek M.; Stirling, Peter C.; Minaker, Sean W.; Sipahimalani, Payal; Hieter, Philip

    2013-01-01

    The concept of synthetic lethality has gained popularity as a rational guide for predicting chemotherapeutic targets based on negative genetic interactions between tumor-specific somatic mutations and a second-site target gene. One hallmark of most cancers that can be exploited by chemotherapies is chromosome instability (CIN). Because chromosome replication, maintenance, and segregation represent conserved and cell-essential processes, they can be modeled effectively in simpler eukaryotes such as Saccharomyces cerevisiae. Here we analyze and extend genetic networks of CIN cancer gene orthologs in yeast, focusing on essential genes. This identifies hub genes and processes that are candidate targets for synthetic lethal killing of cancer cells with defined somatic mutations. One hub process in these networks is DNA replication. A nonessential, fork-associated scaffold, CTF4, is among the most highly connected genes. As Ctf4 lacks enzymatic activity, potentially limiting its development as a therapeutic target, we exploited its function as a physical interaction hub to rationally predict synthetic lethal interactions between essential Ctf4-binding proteins and CIN cancer gene orthologs. We then validated a subset of predicted genetic interactions in a human colorectal cancer cell line, showing that siRNA-mediated knockdown of MRE11A sensitizes cells to depletion of various replication fork-associated proteins. Overall, this work describes methods to identify, predict, and validate in cancer cells candidate therapeutic targets for tumors with known somatic mutations in CIN genes using data from yeast. We affirm not only replication stress but also the targeting of DNA replication fork proteins themselves as potential targets for anticancer therapeutic development. PMID:23390603

  2. The annealing helicase SMARCAL1 maintains genome integrity at stalled replication forks

    PubMed Central

    Bansbach, Carol E.; Bétous, Rémy; Lovejoy, Courtney A.; Glick, Gloria G.; Cortez, David

    2009-01-01

    Mutations in SMARCAL1 (HARP) cause Schimke immunoosseous dysplasia (SIOD). The mechanistic basis for this disease is unknown. Using functional genomic screens, we identified SMARCAL1 as a genome maintenance protein. Silencing and overexpression of SMARCAL1 leads to activation of the DNA damage response during S phase in the absence of any genotoxic agent. SMARCAL1 contains a Replication protein A (RPA)-binding motif similar to that found in the replication stress response protein TIPIN (Timeless-Interacting Protein), which is both necessary and sufficient to target SMARCAL1 to stalled replication forks. RPA binding is critical for the cellular function of SMARCAL1; however, it is not necessary for the annealing helicase activity of SMARCAL1 in vitro. An SIOD-associated SMARCAL1 mutant fails to prevent replication-associated DNA damage from accumulating in cells in which endogenous SMARCAL1 is silenced. Ataxia-telangiectasia mutated (ATM), ATM and Rad3-related (ATR), and DNA-dependent protein kinase (DNA-PK) phosphorylate SMARCAL1 in response to replication stress. Loss of SMARCAL1 activity causes increased RPA loading onto chromatin and persistent RPA phosphorylation after a transient exposure to replication stress. Furthermore, SMARCAL1-deficient cells are hypersensitive to replication stress agents. Thus, SMARCAL1 is a replication stress response protein, and the pleiotropic phenotypes of SIOD are at least partly due to defects in genome maintenance during DNA replication. PMID:19793861

  3. The RecQ DNA helicase Rqh1 constrains Exonuclease 1-dependent recombination at stalled replication forks.

    PubMed

    Osman, Fekret; Ahn, Jong Sook; Lorenz, Alexander; Whitby, Matthew C

    2016-01-01

    DNA double-strand break (DSB) repair by homologous recombination (HR) involves resection of the break to expose a 3' single-stranded DNA tail. In budding yeast, resection occurs in two steps: initial short-range resection, performed by Mre11-Rad50-Xrs2 and Sae2; and long-range resection catalysed by either Exo1 or Sgs1-Dna2. Here we use genetic assays to investigate the importance of Exo1 and the Sgs1 homologue Rqh1 for DNA repair and promotion of direct repeat recombination in the fission yeast Schizosaccharomyces pombe. We find that Exo1 and Rqh1 function in alternative redundant pathways for promoting survival following replication fork breakage. Exo1 promotes replication fork barrier-induced direct repeat recombination but intriguingly limits recombination induced by fork breakage. Direct repeat recombination induced by ultraviolet light depends on either Exo1 or Rqh1. Finally, we show that Rqh1 plays a major role in limiting Exo1-dependent direct repeat recombination induced by replication fork stalling but only a minor role in constraining recombination induced by fork breakage. The implications of our findings are discussed in the context of the benefits that long-range resection may bring to processing perturbed replication forks. PMID:26957021

  4. The RecQ DNA helicase Rqh1 constrains Exonuclease 1-dependent recombination at stalled replication forks

    PubMed Central

    Osman, Fekret; Ahn, Jong Sook; Lorenz, Alexander; Whitby, Matthew C.

    2016-01-01

    DNA double-strand break (DSB) repair by homologous recombination (HR) involves resection of the break to expose a 3′ single-stranded DNA tail. In budding yeast, resection occurs in two steps: initial short-range resection, performed by Mre11-Rad50-Xrs2 and Sae2; and long-range resection catalysed by either Exo1 or Sgs1-Dna2. Here we use genetic assays to investigate the importance of Exo1 and the Sgs1 homologue Rqh1 for DNA repair and promotion of direct repeat recombination in the fission yeast Schizosaccharomyces pombe. We find that Exo1 and Rqh1 function in alternative redundant pathways for promoting survival following replication fork breakage. Exo1 promotes replication fork barrier-induced direct repeat recombination but intriguingly limits recombination induced by fork breakage. Direct repeat recombination induced by ultraviolet light depends on either Exo1 or Rqh1. Finally, we show that Rqh1 plays a major role in limiting Exo1-dependent direct repeat recombination induced by replication fork stalling but only a minor role in constraining recombination induced by fork breakage. The implications of our findings are discussed in the context of the benefits that long-range resection may bring to processing perturbed replication forks. PMID:26957021

  5. Xenopus Mcm10 is a CDK-substrate required for replication fork stability

    PubMed Central

    Chadha, Gaganmeet Singh; Gambus, Agnieszka; Gillespie, Peter J.; Blow, J. Julian

    2016-01-01

    ABSTRACT During S phase, following activation of the S phase CDKs and the DBF4-dependent kinases (DDK), double hexamers of Mcm2-7 at licensed replication origins are activated to form the core replicative helicase. Mcm10 is one of several proteins that have been implicated from work in yeasts to play a role in forming a mature replisome during the initiation process. Mcm10 has also been proposed to play a role in promoting replisome stability after initiation has taken place. The role of Mcm10 is particularly unclear in metazoans, where conflicting data has been presented. Here, we investigate the role and regulation of Mcm10 in Xenopus egg extracts. We show that Xenopus Mcm10 is recruited to chromatin late in the process of replication initiation and this requires prior action of DDKs and CDKs. We also provide evidence that Mcm10 is a CDK substrate but does not need to be phosphorylated in order to associate with chromatin. We show that in extracts depleted of more than 99% of Mcm10, the bulk of DNA replication still occurs, suggesting that Mcm10 is not required for the process of replication initiation. However, in extracts depleted of Mcm10, the replication fork elongation rate is reduced. Furthermore, the absence of Mcm10 or its phosphorylation by CDK results in instability of replisome proteins on DNA, which is particularly important under conditions of replication stress. PMID:27327991

  6. Spatial separation of replisome arrest sites influences homologous recombination quality at a Tus/Ter-mediated replication fork barrier.

    PubMed

    Willis, Nicholas A; Scully, Ralph

    2016-07-17

    The Escherichia coli replication fork arrest complex Tus/Ter mediates site-specific replication fork arrest and homologous recombination (HR) on a mammalian chromosome, inducing both conservative "short tract" gene conversion (STGC) and error-prone "long tract" gene conversion (LTGC) products. We showed previously that bidirectional fork arrest is required for the generation of STGC products at Tus/Ter-stalled replication forks and that the HR mediators BRCA1, BRCA2 and Rad51 mediate STGC but suppress LTGC at Tus/Ter-arrested forks. Here, we report the impact of Ter array length on Tus/Ter-induced HR, comparing HR reporters containing arrays of 6, 9, 15 or 21 Ter sites-each targeted to the ROSA26 locus of mouse embryonic stem (ES) cells. Increasing Ter copy number within the array beyond 6 did not affect the magnitude of Tus/Ter-induced HR but biased HR in favor of LTGC. A "lock"-defective Tus mutant, F140A, known to exhibit higher affinity than wild type (wt)Tus for duplex Ter, reproduced these effects. In contrast, increasing Ter copy number within the array reduced HR induced by the I-SceI homing endonuclease, but produced no consistent bias toward LTGC. Thus, the mechanisms governing HR at Tus/Ter-arrested replication forks are distinct from those governing HR at an enzyme-induced chromosomal double strand break (DSB). We propose that increased spatial separation of the 2 arrested forks encountering an extended Tus/Ter barrier impairs the coordination of DNA ends generated by the processing of the stalled forks, thereby favoring aberrant LTGC over conservative STGC. PMID:27136113

  7. Modulation of mutagenesis in eukaryotes by DNA replication fork dynamics and quality of nucleotide pools

    PubMed Central

    Waisertreiger, Irina S.-R.; Liston, Victoria G.; Menezes, Miriam R.; Kim, Hyun-Min; Lobachev, Kirill S.; Stepchenkova, Elena I.; Tahirov, Tahir H.; Rogozin, Igor B.; Pavlov, Youri. I.

    2014-01-01

    The rate of mutations in eukaryotes depends on a plethora of factors and is not immediately derived from the fidelity of DNA polymerases (Pols). Replication of chromosomes containing the anti-parallel strands of duplex DNA occurs through the copying of leading and lagging strand templates by a trio of Pols α, δ and ε, with the assistance of Pol ζ and Y-family Pols at difficult DNA template structures or sites of DNA damage. The parameters of the synthesis at a given location are dictated by the quality and quantity of nucleotides in the pools, replication fork architecture, transcription status, regulation of Pol switches, and structure of chromatin. The result of these transactions is a subject of survey and editing by DNA repair. PMID:23055184

  8. Stalled replication fork repair and misrepair during thymineless death in Escherichia coli.

    PubMed

    Kuong, Kawai J; Kuzminov, Andrei

    2010-06-01

    Starvation for DNA precursor dTTP, known as 'thymineless death' (TLD), kills bacterial and eukaryotic cells alike. Despite numerous investigations, toxic mechanisms behind TLD remain unknown, although wrong nucleotide incorporation with subsequent excision dominates the explanations. We show that kinetics of TLD in Escherichia coli is not affected by mutations in DNA repair, ruling out excision after massive misincorporation as the cause of TLD. We found that the rate of DNA synthesis in thymine-starved cells decreases exponentially, indicating replication fork stalling. Processing of stalled replication forks by recombinational repair is known to fragment the chromosome, and we detect significant chromosomal fragmentation during TLD. Moreover, we report that, out of major recombinational repair functions, only inactivation of recF and recO relieves TLD, identifying the poisoning mechanism. Inactivation of recJ and rep has slight effect, while the recA, recBC, ruvABC, recG and uvrD mutations all accelerate TLD, identifying the protection mechanisms. Our epistatic analysis argues for two distinct pathways protecting against TLD: RecABCD/Ruv repairs the double-strand breaks, whereas UvrD counteracts RecAFO-catalyzed toxic single-strand gap processing. PMID:20465561

  9. Stalled fork rescue via dormant replication origins in unchallenged S phase promotes proper chromosome segregation and tumor suppression

    PubMed Central

    Kawabata, Tsuyoshi; Luebben, Spencer W.; Yamaguchi, Satoru; Ilves, Ivar; Matise, Ilze; Buske, Tavanna; Botchan, Michael R.; Shima, Naoko

    2011-01-01

    Summary Eukaryotic cells license far more origins than are actually used for DNA replication, thereby generating a large number of dormant origins. Accumulating evidence suggests that such origins play a role in chromosome stability and tumor suppression, though the underlying mechanism is largely unknown. Here, we show that a loss of dormant origins results in an increased number of stalled replication forks even in unchallenged S phase in primary mouse fibroblasts derived from embryos homozygous for the Mcm4Chaos3 allele. We found that this allele reduces the stability of the MCM2-7 complex, but confers normal helicase activity in vitro. Despite the activation of multiple fork recovery pathways, replication intermediates in these cells persist into M phase, increasing the number of abnormal anaphase cells with lagging chromosomes and/or acentric fragments. These findings suggest that dormant origins constitute a major pathway for stalled fork recovery, contributing to faithful chromosome segregation and tumor suppression. PMID:21362550

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

    PubMed

    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

  11. Chk1 and p21 Cooperate to Prevent Apoptosis during DNA Replication Fork StressD⃞

    PubMed Central

    Rodriguez, Rene; Meuth, Mark

    2006-01-01

    Cells respond to DNA replication stress by triggering cell cycle checkpoints, repair, or death. To understand the role of the DNA damage response pathways in determining whether cells survive replication stress or become committed to death, we examined the effect of loss of these pathways on cellular response to agents that slow or arrest DNA synthesis. We show that replication inhibitors such as excess thymidine, hydroxyurea, and camptothecin are normally poor inducers of apoptosis. However, these agents become potent inducers of death in S-phase cells upon small interfering RNA-mediated depletion of the checkpoint kinase Chk1. This death response is independent of p53 and Chk2. p21-deficient cells, on the other hand, produce a more robust apoptotic response upon Chk1 depletion. p21 is normally induced only late after thymidine treatment. In Chk1-depleted cells p21 induction occurs earlier and does not require p53. Thus, Chk1 plays a primary role in the protection of cells from death induced by replication fork stress, whereas p21 mediates through its role in regulating entry into S phase. These findings are of potential importance to cancer therapy because we demonstrate that the efficacy of clinically relevant agents can be enhanced by manipulation of these signaling pathways. PMID:16280359

  12. Poly(ADP-ribose) binding to Chk1 at stalled replication forks is required for S-phase checkpoint activation

    NASA Astrophysics Data System (ADS)

    Min, Wookee; Bruhn, Christopher; Grigaravicius, Paulius; Zhou, Zhong-Wei; Li, Fu; Krüger, Anja; Siddeek, Bénazir; Greulich, Karl-Otto; Popp, Oliver; Meisezahl, Chris; Calkhoven, Cornelis F.; Bürkle, Alexander; Xu, Xingzhi; Wang, Zhao-Qi

    2013-12-01

    Damaged replication forks activate poly(ADP-ribose) polymerase 1 (PARP1), which catalyses poly(ADP-ribose) (PAR) formation; however, how PARP1 or poly(ADP-ribosyl)ation is involved in the S-phase checkpoint is unknown. Here we show that PAR, supplied by PARP1, interacts with Chk1 via a novel PAR-binding regulatory (PbR) motif in Chk1, independent of ATR and its activity. iPOND studies reveal that Chk1 associates readily with the unperturbed replication fork and that PAR is required for efficient retention of Chk1 and phosphorylated Chk1 at the fork. A PbR mutation, which disrupts PAR binding, but not the interaction with its partners Claspin or BRCA1, impairs Chk1 and the S-phase checkpoint activation, and mirrors Chk1 knockdown-induced hypersensitivity to fork poisoning. We find that long chains, but not short chains, of PAR stimulate Chk1 kinase activity. Collectively, we disclose a previously unrecognized mechanism of the S-phase checkpoint by PAR metabolism that modulates Chk1 activity at the replication fork.

  13. The Deubiquitinase USP9X Maintains DNA Replication Fork Stability and DNA Damage Checkpoint Responses by Regulating CLASPIN during S-Phase.

    PubMed

    McGarry, Edel; Gaboriau, David; Rainey, Michael D; Restuccia, Umberto; Bachi, Angela; Santocanale, Corrado

    2016-04-15

    Coordination of the multiple processes underlying DNA replication is key for maintaining genome stability and preventing tumorigenesis. CLASPIN, a critical player in replication fork stabilization and checkpoint responses, must be tightly regulated during the cell cycle to prevent the accumulation of DNA damage. In this study, we used a quantitative proteomics approach and identified USP9X as a novel CLASPIN-interacting protein. USP9X is a deubiquitinase involved in multiple signaling and survival pathways whose tumor suppressor or oncogenic activity is highly context dependent. We found that USP9X regulated the expression and stability of CLASPIN in an S-phase-specific manner. USP9X depletion profoundly impairs the progression of DNA replication forks, causing unscheduled termination events with a frequency similar to CLASPIN depletion, resulting in excessive endogenous DNA damage. Importantly, restoration of CLASPIN expression in USP9X-depleted cells partially suppressed the accumulation of DNA damage. Furthermore, USP9X depletion compromised CHK1 activation in response to hydroxyurea and UV, thus promoting hypersensitivity to drug-induced replication stress. Taken together, our results reveal a novel role for USP9X in the maintenance of genomic stability during DNA replication and provide potential mechanistic insights into its tumor suppressor role in certain malignancies. Cancer Res; 76(8); 2384-93. ©2016 AACR. PMID:26921344

  14. A DNA binding winged helix domain in CAF-1 functions with PCNA to stabilize CAF-1 at replication forks.

    PubMed

    Zhang, Kuo; Gao, Yuan; Li, Jingjing; Burgess, Rebecca; Han, Junhong; Liang, Huanhuan; Zhang, Zhiguo; Liu, Yingfang

    2016-06-20

    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

  15. A DNA binding winged helix domain in CAF-1 functions with PCNA to stabilize CAF-1 at replication forks

    PubMed Central

    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

  16. Three different proteins recognize a multifunctional determinant that controls replication initiation, fork arrest and transcription in Tetrahymena.

    PubMed

    Mohammad, M; Saha, S; Kapler, G M

    2000-02-01

    Type I elements regulate the initiation of DNA replication, elongation of replication forks and transcription of the Tetrahymena thermophila rDNA minichromosome. Previous studies identified a 24 kDa protein, ssA-TIBF, which binds the A-rich strand of type I elements. Here we describe two additional type I element binding activities (native mol. wt approximately 65 and approximately 250 kDa) that interact with DNA via previously unidentified 32 and 110 kDa polypeptides. The 65 kDa activity was purified to homogeneity and consists of a homodimer of a 32 kDa polypeptide. In contrast to the other type I element binding factors, the 65 kDa activity partitions preferentially to the nuclear fraction during isolation. Levels of the 65 kDa activity increase dramatically in starved cells, raising the possibility that it might negatively regulate replication or transcription. By comparison, the other two binding activities were elevated slightly during macronuclear development, when the rDNA was undergoing DNA replication. Previous studies indicate that the initiation of rDNA replication is regulated by long range interactions between dispersed type I elements. Competitive DNA binding or cooperative protein-protein interactions between the factors described here may play a regulatory role in replication or expression of the rDNA minichromosome. PMID:10637338

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

    PubMed

    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

  18. Segmental Duplications Arise from Pol32-Dependent Repair of Broken Forks through Two Alternative Replication-Based Mechanisms

    PubMed Central

    Dujon, Bernard; Fischer, Gilles

    2008-01-01

    The propensity of segmental duplications (SDs) to promote genomic instability is of increasing interest since their involvement in numerous human genomic diseases and cancers was revealed. However, the mechanism(s) responsible for their appearance remain mostly speculative. Here, we show that in budding yeast, replication accidents, which are most likely transformed into broken forks, play a causal role in the formation of SDs. The Pol32 subunit of the major replicative polymerase Polδ is required for all SD formation, demonstrating that SDs result from untimely DNA synthesis rather than from unequal crossing-over. Although Pol32 is known to be required for classical (Rad52-dependant) break-induced replication, only half of the SDs can be attributed to this mechanism. The remaining SDs are generated through a Rad52-independent mechanism of template switching between microsatellites or microhomologous sequences. This new mechanism, named microhomology/microsatellite-induced replication (MMIR), differs from all known DNA double-strand break repair pathways, as MMIR-mediated duplications still occur in the combined absence of homologous recombination, microhomology-mediated, and nonhomologous end joining machineries. The interplay between these two replication-based pathways explains important features of higher eukaryotic genomes, such as the strong, but not strict, association between SDs and transposable elements, as well as the frequent formation of oncogenic fusion genes generating protein innovations at SD junctions. PMID:18773114

  19. Polyubiquitination of proliferating cell nuclear antigen by HLTF and SHPRH prevents genomic instability from stalled replication forks

    PubMed Central

    Motegi, Akira; Liaw, Hung-Jiun; Lee, Kyoo-Young; Roest, Henk P.; Maas, Alex; Wu, Xiaoli; Moinova, Helen; Markowitz, Sanford D.; Ding, Hao; Hoeijmakers, Jan H. J.; Myung, Kyungjae

    2008-01-01

    Chronic stalling of DNA replication forks caused by DNA damage can lead to genomic instability. Cells have evolved lesion bypass pathways such as postreplication repair (PRR) to resolve these arrested forks. In yeast, one branch of PRR involves proliferating cell nuclear antigen (PCNA) polyubiquitination mediated by the Rad5-Ubc13-Mms2 complex that allows bypass of DNA lesion by a template-switching mechanism. Previously, we identified human SHPRH as a functional homologue of yeast Rad5 and revealed the existence of RAD5-like pathway in human cells. Here we report the identification of HLTF as a second RAD5 homologue in human cells. HLTF, like SHPRH, shares a unique domain architecture with Rad5 and promotes lysine 63-linked polyubiquitination of PCNA. Similar to yeast Rad5, HLTF is able to interact with UBC13 and PCNA, as well as SHPRH; and the reduction of either SHPRH or HLTF expression enhances spontaneous mutagenesis. Moreover, Hltf-deficient mouse embryonic fibroblasts show elevated chromosome breaks and fusions after methyl methane sulfonate treatment. Our results suggest that HLTF and SHPRH are functional homologues of yeast Rad5 that cooperatively mediate PCNA polyubiquitination and maintain genomic stability. PMID:18719106

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

    PubMed

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

    2016-05-27

    Examples of dynamic polymerase exchange have been previously characterized in model systems provided by coliphages T4 and T7. Using a dominant negative D403E polymerase (Pol) III α that can form initiation complexes and sequester primer termini but not elongate, we investigated the possibility of exchange at the Escherichia coli replication fork on a rolling circle template. Unlike other systems, addition of polymerase alone did not lead to exchange. Only when D403E Pol III was bound to a τ-containing DnaX complex did exchange occur. In contrast, addition of Pol IV led to rapid exchange in the absence of bound DnaX complex. Examination of Pol III* with varying composition of τ or the alternative shorter dnaX translation product γ showed that τ-, τ2-, or τ3-DnaX complexes supported equivalent levels of synthesis, identical Okazaki fragment size, and gaps between fragments, possessed the ability to challenge pre-established replication forks, and displayed equivalent susceptibility to challenge by exogenous D403E Pol III*. These findings reveal that redundant interactions at the replication fork must stabilize complexes containing only one τ. Previously, it was thought that at least two τs in the trimeric DnaX complex were required to couple the leading and lagging strand polymerases at the replication fork. Possible mechanisms of exchange are discussed. PMID:27056333

  1. Modeling Inhomogeneous DNA Replication Kinetics

    PubMed Central

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

    2012-01-01

    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. PMID:22412853

  2. Mcm10 coordinates the timely assembly and activation of the replication fork helicase

    PubMed Central

    Perez-Arnaiz, Patricia; Bruck, Irina; Kaplan, Daniel L.

    2016-01-01

    Mcm10 is an essential replication factor that is required for DNA replication in eukaryotes. Two key steps in the initiation of DNA replication are the assembly and activation of Cdc45–Mcm2–7-GINS (CMG) replicative helicase. However, it is not known what coordinates helicase assembly with helicase activation. We show in this manuscript, using purified proteins from budding yeast, that Mcm10 directly interacts with the Mcm2–7 complex and Cdc45. In fact, Mcm10 recruits Cdc45 to Mcm2–7 complex in vitro. To study the role of Mcm10 in more detail in vivo we used an auxin inducible degron in which Mcm10 is degraded upon addition of auxin. We show in this manuscript that Mcm10 is required for the timely recruitment of Cdc45 and GINS recruitment to the Mcm2–7 complex in vivo during early S phase. We also found that Mcm10 stimulates Mcm2 phosphorylation by DDK in vivo and in vitro. These findings indicate that Mcm10 plays a critical role in coupling replicative helicase assembly with helicase activation. Mcm10 is first involved in the recruitment of Cdc45 to the Mcm2–7 complex. After Cdc45–Mcm2–7 complex assembly, Mcm10 promotes origin melting by stimulating DDK phosphorylation of Mcm2, which thereby leads to GINS attachment to Mcm2–7. PMID:26582917

  3. Conserved mechanism for coordinating replication fork helicase assembly with phosphorylation of the helicase

    PubMed Central

    Bruck, Irina; Kaplan, Daniel L.

    2015-01-01

    Dbf4-dependent kinase (DDK) phosphorylates minichromosome maintenance 2 (Mcm2) during S phase in yeast, and Sld3 recruits cell division cycle 45 (Cdc45) to minichromosome maintenance 2-7 (Mcm2-7). We show here DDK-phosphoryled Mcm2 preferentially interacts with Cdc45 in vivo, and that Sld3 stimulates DDK phosphorylation of Mcm2 by 11-fold. We identified a mutation of the replication initiation factor Sld3, Sld3-m16, that is specifically defective in stimulating DDK phosphorylation of Mcm2. Wild-type expression levels of sld3-m16 result in severe growth and DNA replication defects. Cells expressing sld3-m16 exhibit no detectable Mcm2 phosphorylation in vivo, reduced replication protein A-ChIP signal at an origin, and diminished Go, Ichi, Ni, and San association with Mcm2-7. Treslin, the human homolog of Sld3, stimulates human DDK phosphorylation of human Mcm2 by 15-fold. DDK phosphorylation of human Mcm2 decreases the affinity of Mcm5 for Mcm2, suggesting a potential mechanism for helicase ring opening. These data suggest a conserved mechanism for replication initiation: Sld3/Treslin coordinates Cdc45 recruitment to Mcm2-7 with DDK phosphorylation of Mcm2 during S phase. PMID:26305950

  4. Formation of a stable RuvA protein double tetramer is required for efficient branch migration in vitro and for replication fork reversal in vivo.

    PubMed

    Bradley, Alison S; Baharoglu, Zeynep; Niewiarowski, Andrew; Michel, Bénédicte; Tsaneva, Irina R

    2011-06-24

    In bacteria, RuvABC is required for the resolution of Holliday junctions (HJ) made during homologous recombination. The RuvAB complex catalyzes HJ branch migration and replication fork reversal (RFR). During RFR, a stalled fork is reversed to form a HJ adjacent to a DNA double strand end, a reaction that requires RuvAB in certain Escherichia coli replication mutants. The exact structure of active RuvAB complexes remains elusive as it is still unknown whether one or two tetramers of RuvA support RuvB during branch migration and during RFR. We designed an E. coli RuvA mutant, RuvA2(KaP), specifically impaired for RuvA tetramer-tetramer interactions. As expected, the mutant protein is impaired for complex II (two tetramers) formation on HJs, although the binding efficiency of complex I (a single tetramer) is as wild type. We show that although RuvA complex II formation is required for efficient HJ branch migration in vitro, RuvA2(KaP) is fully active for homologous recombination in vivo. RuvA2(KaP) is also deficient at forming complex II on synthetic replication forks, and the binding affinity of RuvA2(KaP) for forks is decreased compared with wild type. Accordingly, RuvA2(KaP) is inefficient at processing forks in vitro and in vivo. These data indicate that RuvA2(KaP) is a separation-of-function mutant, capable of homologous recombination but impaired for RFR. RuvA2(KaP) is defective for stimulation of RuvB activity and stability of HJ·RuvA·RuvB tripartite complexes. This work demonstrates that the need for RuvA tetramer-tetramer interactions for full RuvAB activity in vitro causes specifically an RFR defect in vivo. PMID:21531731

  5. RNA polymerase mutations that facilitate replication progression in the rep uvrD recF mutant lacking two accessory replicative helicases.

    PubMed

    Baharoglu, Zeynep; Lestini, Roxane; Duigou, Stéphane; Michel, Bénédicte

    2010-07-01

    We observed that cells lacking Rep and UvrD, two replication accessory helicases, and the recombination protein RecF are cryo-sensitive on rich medium. We isolated five mutations that suppress this Luria-Bertani (LB)-cryo-sensitivity and show that they map in the genes encoding the RNA polymerase subunits RpoB and RpoC. These rpoB (D444G, H447R and N518D) and rpoC mutants (H113R and P451L) were characterized. rpoB(H447R) and rpoB(D444G) prevent activation of the Prrn core promoter in rich medium, but only rpoB(H447R) also suppresses the auxotrophy of a relA spoT mutant (stringent-like phenotype). rpoC(H113R) suppresses the thermo-sensitivity of a greA greB mutant, suggesting that it destabilizes stalled elongation complexes. All mutations but rpoC(P451L) prevent R-loop formation. We propose that these rpo mutations allow replication in the absence of Rep and UvrD by destabilizing RNA Pol upon replication-transcription collisions. In a RecF(+) context, they improve growth of rep uvrD cells only if DinG is present, supporting the hypothesis that Rep, UvrD and DinG facilitate progression of the replication fork across transcribed sequences. They rescue rep uvrD dinG recF cells, indicating that in a recF mutant replication forks arrested by unstable transcription complexes can restart without any of the three known replication accessory helicases Rep, UvrD and DinG. PMID:20497334

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

    PubMed

    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

  7. DNA Damage Responses in Prokaryotes: Regulating Gene Expression, Modulating Growth Patterns, and Manipulating Replication Forks

    PubMed Central

    Kreuzer, Kenneth N.

    2013-01-01

    Recent advances in the area of bacterial DNA damage responses are reviewed here. The SOS pathway is still the major paradigm of bacterial DNA damage response, and recent studies have clarified the mechanisms of SOS induction and key physiological roles of SOS including a very major role in genetic exchange and variation. When considering diverse bacteria, it is clear that SOS is not a uniform pathway with one purpose, but rather a platform that has evolved for differing functions in different bacteria. Relating in part to the SOS response, the field has uncovered multiple apparent cell-cycle checkpoints that assist cell survival after DNA damage and remarkable pathways that induce programmed cell death in bacteria. Bacterial DNA damage responses are also much broader than SOS, and several important examples of LexA-independent regulation will be reviewed. Finally, some recent advances that relate to the replication and repair of damaged DNA will be summarized. PMID:24097899

  8. DNA-PK Phosphorylation of RPA32 Ser4/Ser8 Regulates Replication Stress Checkpoint Activation, Fork Restart, Homologous Recombination and Mitotic Catastrophe

    PubMed Central

    Ashley, Amanda K.; Shrivastav, Meena; Nie, Jingyi; Amerin, Courtney; Troksa, Kyle; Glanzer, Jason G.; Liu, Shengqin; Opiyo, Stephen O.; Dimitrova, Diana D.; Le, Phuong; Sishc, Brock; Bailey, Susan M.; Oakley, Greg G.; Nickoloff, Jac A.

    2014-01-01

    Genotoxins and other factors cause replication stress that activate the DNA damage response (DDR), comprising checkpoint and repair systems. The DDR suppresses cancer by promoting genome stability, and it regulates tumor resistance to chemo- and radiotherapy. Three members of the phosphatidylinositol 3-kinase-related kinase (PIKK) family, ATM, ATR, and DNA-PK, are important DDR proteins. A key PIKK target is replication protein A (RPA), which binds single-stranded DNA and functions in DNA replication, DNA repair, and checkpoint signaling. An early response to replication stress is ATR activation, which occurs when RPA accumulates on ssDNA. Activated ATR phosphorylates many targets, including the RPA32 subunit of RPA, leading to Chk1 activation and replication arrest. DNA-PK also phosphorylates RPA32 in response to replication stress, and we demonstrate that cells with DNA-PK defects, or lacking RPA32 Ser4/Ser8 targeted by DNA-PK, confer similar phenotypes, including defective replication checkpoint arrest, hyper-recombination, premature replication fork restart, failure to block late origin firing, and increased mitotic catastrophe. We present evidence that hyper-recombination in these mutants is ATM-dependent, but the other defects are ATM-independent. These results indicate that DNA-PK and ATR signaling through RPA32 plays a critical role in promoting genome stability and cell survival in response to replication stress. PMID:24819595

  9. FANC Pathway Promotes UV-Induced Stalled Replication Forks Recovery by Acting Both Upstream and Downstream Polη and Rev1

    PubMed Central

    Renaud, Emilie; Rosselli, Filippo

    2013-01-01

    To cope with ultraviolet C (UVC)-stalled replication forks and restart DNA synthesis, cells either undergo DNA translesion synthesis (TLS) by specialised DNA polymerases or tolerate the lesions using homologous recombination (HR)-based mechanisms. To gain insight into how cells manage UVC-induced stalled replication forks, we analysed the molecular crosstalk between the TLS DNA polymerases Polη and Rev1, the double-strand break repair (DSB)-associated protein MDC1 and the FANC pathway. We describe three novel functional interactions that occur in response to UVC-induced DNA lesions. First, Polη and Rev1, whose optimal expression and/or relocalisation depend on the FANC core complex, act upstream of FANCD2 and are required for the proper relocalisation of monoubiquitinylated FANCD2 (Ub-FANCD2) to subnuclear foci. Second, during S-phase, Ub-FANCD2 and MDC1 relocalise to UVC-damaged nuclear areas or foci simultaneously but independently of each other. Third, Ub-FANCD2 and MDC1 are independently required for optimal BRCA1 relocalisation. While RPA32 phosphorylation (p-RPA32) and RPA foci formation were reduced in parallel with increasing levels of H2AX phosphorylation and MDC1 foci in UVC-irradiated FANC pathway-depleted cells, MDC1 depletion was associated with increased UVC-induced Ub-FANCD2 and FANCD2 foci as well as p-RPA32 levels and p-RPA32 foci. On the basis of the previous observations, we propose that the FANC pathway participates in the rescue of UVC-stalled replication forks in association with TLS by maintaining the integrity of ssDNA regions and by preserving genome stability and preventing the formation of DSBs, the resolution of which would require the intervention of MDC1. PMID:23365640

  10. 3D replicon distributions arise from stochastic initiation and domino-like DNA replication progression

    PubMed Central

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

    2016-01-01

    DNA replication dynamics in cells from higher eukaryotes follows very complex but highly efficient mechanisms. However, the principles behind initiation of potential replication origins and emergence of typical patterns of nuclear replication sites remain unclear. Here, we propose a comprehensive model of DNA replication in human cells that is based on stochastic, proximity-induced replication initiation. Critical model features are: spontaneous stochastic firing of individual origins in euchromatin and facultative heterochromatin, inhibition of firing at distances below the size of chromatin loops and a domino-like effect by which replication forks induce firing of nearby origins. The model reproduces the empirical temporal and chromatin-related properties of DNA replication in human cells. We advance the one-dimensional DNA replication model to a spatial model by taking into account chromatin folding in the nucleus, and we are able to reproduce the spatial and temporal characteristics of the replication foci distribution throughout S-phase. PMID:27052359

  11. HUWE1 interacts with PCNA to alleviate replication stress.

    PubMed

    Choe, Katherine N; Nicolae, Claudia M; Constantin, Daniel; Imamura Kawasawa, Yuka; Delgado-Diaz, Maria Rocio; De, Subhajyoti; Freire, Raimundo; Smits, Veronique Aj; Moldovan, George-Lucian

    2016-06-01

    Defects in DNA replication, DNA damage response, and DNA repair compromise genomic stability and promote cancer development. In particular, unrepaired DNA lesions can arrest the progression of the DNA replication machinery during S-phase, causing replication stress, mutations, and DNA breaks. HUWE1 is a HECT-type ubiquitin ligase that targets proteins involved in cell fate, survival, and differentiation. Here, we report that HUWE1 is essential for genomic stability, by promoting replication of damaged DNA We show that HUWE1-knockout cells are unable to mitigate replication stress, resulting in replication defects and DNA breakage. Importantly, we find that this novel role of HUWE1 requires its interaction with the replication factor PCNA, a master regulator of replication fork restart, at stalled replication forks. Finally, we provide evidence that HUWE1 mono-ubiquitinates H2AX to promote signaling at stalled forks. Altogether, our work identifies HUWE1 as a novel regulator of the replication stress response. PMID:27146073

  12. Failure of origin activation in response to fork stalling leads to chromosomal instability at fragile sites.

    PubMed

    Ozeri-Galai, Efrat; Lebofsky, Ronald; Rahat, Ayelet; Bester, Assaf C; Bensimon, Aaron; Kerem, Batsheva

    2011-07-01

    Perturbed DNA replication in early stages of cancer development induces chromosomal instability preferentially at fragile sites. However, the molecular basis for this instability is unknown. Here, we show that even under normal growth conditions, replication fork progression along the fragile site, FRA16C, is slow and forks frequently stall at AT-rich sequences, leading to activation of additional origins to enable replication completion. Under mild replication stress, the frequency of stalling at AT-rich sequences is further increased. Strikingly, unlike in the entire genome, in the FRA16C region additional origins are not activated, suggesting that all potential origins are already activated under normal conditions. Thus, the basis for FRA16C fragility is replication fork stalling at AT-rich sequences and inability to activate additional origins under replication stress. Our results provide a mechanism explaining the replication stress sensitivity of fragile sites and thus, the basis for genomic instability during early stages of cancer development. PMID:21726815

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

    NASA Astrophysics Data System (ADS)

    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.

  14. Polyploid cells rewire DNA damage response networks to overcome replication stress-induced barriers for tumour progression

    PubMed Central

    Zheng, Li; Dai, Huifang; Zhou, Mian; Li, Xiaojin; Liu, Changwei; Guo, Zhigang; Wu, Xiwei; Wu, Jun; Wang, Charles; Zhong, John; Huang, Qin; Garcia-Aguilar, Julio; Pfeifer, Gerd P.; Shen, Binghui

    2012-01-01

    Mutations in genes involved in DNA replication such as FEN1, can cause single-stranded DNA breaks (SSBs) and subsequent collapse of DNA replication forks leading to DNA replication stresses. Persistent replication stresses normally induce p53-mediated senescence or apoptosis to prevent tumor progression. It is unclear how some mutant cells can overcome persistent replication stresses and bypass the p53-mediated pathways to develop malignancy. Here we show that formation of polyploidy, which is often observed in human cancers, leads to overexpression of BRCA1, p19arf and other DNA repair genes in FEN1 mutant cells. This overexpression triggers SSB repair and non-homologous end joining pathways to increase DNA repair activity, but at the cost of frequent chromosomal translocations. Meanwhile, DNA methylation silences p53 target genes, to bypass the p53-mediated senescence and apoptosis. These molecular changes rewire DNA damage response and repair gene networks in polyploid tumor cells, enabling them to escape replication stress-induced senescence barriers. PMID:22569363

  15. Replication Forks Stalled at Ultraviolet Lesions Are Rescued via RecA and RuvABC Protein-catalyzed Disintegration in Escherichia coli*

    PubMed Central

    Khan, Sharik R.; Kuzminov, Andrei

    2012-01-01

    Ultraviolet (UV) irradiation is not known to induce chromosomal fragmentation in sublethal doses, and yet UV irradiation causes genetic instability and cancer, suggesting that chromosomes are fragmented. Here we show that UV irradiation induces fragmentation in sublethal doses, but the broken chromosomes are repaired or degraded by RecBCD; therefore, to observe full fragmentation, RecBCD enzyme needs to be inactivated. Using quantitative pulsed field gel electrophoresis and sensitive DNA synthesis measurements, we investigated the mechanisms of UV radiation-induced chromosomal fragmentation in recBC mutants, comparing five existing models of DNA damage-induced fragmentation. We found that fragmentation depends on active DNA synthesis before, but not after, UV irradiation. At low UV irradiation doses, fragmentation does not need excision repair or daughter strand gap repair. Fragmentation absolutely depends on both RecA-catalyzed homologous strand exchange and RuvABC-catalyzed Holliday junction resolution. Thus, chromosomes fragment when replication forks stall at UV lesions and regress, generating Holliday junctions. Remarkably, cells specifically utilize fork breakage to rescue stalled replication and avoid lethality. PMID:22194615

  16. Translesion replication by DNA polymerase beta is modulated by sequence context and stimulated by fork-like flap structures in DNA.

    PubMed

    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

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

    NASA Astrophysics Data System (ADS)

    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).

  18. Cycling of the E. coli lagging strand polymerase is triggered exclusively by the availability of a new primer at the replication fork

    PubMed Central

    Yuan, Quan; McHenry, Charles S.

    2014-01-01

    Two models have been proposed for triggering release of the lagging strand polymerase at the replication fork, enabling cycling to the primer for the next Okazaki fragment—either collision with the 5′-end of the preceding fragment (collision model) or synthesis of a new primer by primase (signaling model). Specific perturbation of lagging strand elongation on minicircles with a highly asymmetric G:C distribution with ddGTP or dGDPNP yielded results that confirmed the signaling model and ruled out the collision model. We demonstrated that the presence of a primer, not primase per se, provides the signal that triggers cycling. Lagging strand synthesis proceeds much faster than leading strand synthesis, explaining why gaps between Okazaki fragments are not found under physiological conditions. PMID:24234450

  19. Low doses of ultraviolet radiation and oxidative damage induce dramatic accumulation of mitochondrial DNA replication intermediates, fork regression, and replication initiation shift

    PubMed Central

    Torregrosa-Muñumer, Rubén; Goffart, Steffi; Haikonen, Juha A.; Pohjoismäki, Jaakko L. O.

    2015-01-01

    Mitochondrial DNA is prone to damage by various intrinsic as well as environmental stressors. DNA damage can in turn cause problems for replication, resulting in replication stalling and double-strand breaks, which are suspected to be the leading cause of pathological mtDNA rearrangements. In this study, we exposed cells to subtle levels of oxidative stress or UV radiation and followed their effects on mtDNA maintenance. Although the damage did not influence mtDNA copy number, we detected a massive accumulation of RNA:DNA hybrid–containing replication intermediates, followed by an increase in cruciform DNA molecules, as well as in bidirectional replication initiation outside of the main replication origin, OH. Our results suggest that mitochondria maintain two different types of replication as an adaptation to different cellular environments; the RNA:DNA hybrid–involving replication mode maintains mtDNA integrity in tissues with low oxidative stress, and the potentially more error tolerant conventional strand-coupled replication operates when stress is high. PMID:26399294

  20. FANCD2 limits replication stress and genome instability in cells lacking BRCA2.

    PubMed

    Michl, Johanna; Zimmer, Jutta; Buffa, Francesca M; McDermott, Ultan; Tarsounas, Madalena

    2016-08-01

    The tumor suppressor BRCA2 plays a key role in genome integrity by promoting replication-fork stability and homologous recombination (HR) DNA repair. Here we report that human cancer cells lacking BRCA2 rely on the Fanconi anemia protein FANCD2 to limit replication-fork progression and genomic instability. Our results identify a new role of FANCD2 in limiting constitutive replication stress in BRCA2-deficient cells, thereby affecting cell survival and treatment responses. PMID:27322732

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

    PubMed

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

    2015-08-01

    During DNA replication, chromatin is reassembled by recycling of modified old histones and deposition of new ones. How histone dynamics integrates with DNA replication to maintain genome and epigenome information remains unclear. Here, we reveal how human MCM2, part of the replicative helicase, chaperones histones H3-H4. Our first structure shows an H3-H4 tetramer bound by two MCM2 histone-binding domains (HBDs), which hijack interaction sites used by nucleosomal DNA. Our second structure reveals MCM2 and ASF1 cochaperoning an H3-H4 dimer. Mutational analyses show that the MCM2 HBD is required for MCM2-7 histone-chaperone function and normal cell proliferation. Further, we show that MCM2 can chaperone both new and old canonical histones H3-H4 as well as H3.3 and CENPA variants. The unique histone-binding mode of MCM2 thus endows the replicative helicase with ideal properties for recycling histones genome wide during DNA replication. PMID:26167883

  2. DNA Replication Catalyzed by Herpes Simplex Virus Type 1 Proteins Reveals Trombone Loops at the Fork*♦

    PubMed Central

    Bermek, Oya; Willcox, Smaranda; Griffith, Jack D.

    2015-01-01

    Using purified replication factors encoded by herpes simplex virus type 1 and a 70-base minicircle template, we obtained robust DNA synthesis with leading strand products of >20,000 nucleotides and lagging strand fragments from 600 to 9,000 nucleotides as seen by alkaline gel electrophoresis. ICP8 was crucial for the synthesis on both strands. Visualization of the deproteinized products using electron microscopy revealed long, linear dsDNAs, and in 87%, one end, presumably the end with the 70-base circle, was single-stranded. The remaining 13% had multiple single-stranded segments separated by dsDNA segments 500 to 1,000 nucleotides in length located at one end. These features are diagnostic of the trombone mechanism of replication. Indeed, when the products were examined with the replication proteins bound, a dsDNA loop was frequently associated with the replication complex located at one end of the replicated DNA. Furthermore, the frequency of loops correlated with the fraction of DNA undergoing Okazaki fragment synthesis. PMID:25471368

  3. Src family kinases maintain the balance between replication stress and the replication checkpoint.

    PubMed

    Miura, Takahito; Fukumoto, Yasunori; Morii, Mariko; Honda, Takuya; Yamaguchi, Noritaka; Nakayama, Yuji; Yamaguchi, Naoto

    2016-01-01

    Progression of DNA replication is tightly controlled by replication checkpoints to ensure the accurate and rapid duplication of genetic information. Upon replication stress, the replication checkpoint slows global DNA replication by inhibiting the late-firing origins and by slowing replication fork progression. Activation of the replication checkpoint has been studied in depth; however, little is known about the termination of the replication checkpoint. Here, we show that Src family kinases promote the recovery from replication checkpoints. shRNA knockdown of a Src family kinase, Lyn, and acute chemical inhibition of Src kinases prevented inactivation of Chk1 after removal of replication stress. Consistently, Src inhibition slowed resumption of DNA replication, after the removal of replication blocks. The effect of Src inhibition was not observed in the presence of an ATM/ATR inhibitor caffeine. These data indicate that Src kinases promote the resumption of DNA replication by suppressing ATR-dependent replication checkpoints. Surprisingly, the resumption of replication was delayed by caffeine. In addition, Src inhibition delayed recovery from replication fork collapse. We propose that Src kinases maintain the balance between replication stress and the activity of the replication checkpoint. PMID:26194897

  4. Top2 and Sgs1-Top3 Act Redundantly to Ensure rDNA Replication Termination

    PubMed Central

    Fredsøe, Jacob; Nielsen, Ida; Pedersen, Jakob Madsen; Bentsen, Iben Bach; Lisby, Michael; Bjergbaek, Lotte; Andersen, Anni H

    2015-01-01

    Faithful DNA replication with correct termination is essential for genome stability and transmission of genetic information. Here we have investigated the potential roles of Topoisomerase II (Top2) and the RecQ helicase Sgs1 during late stages of replication. We find that cells lacking Top2 and Sgs1 (or Top3) display two different characteristics during late S/G2 phase, checkpoint activation and accumulation of asymmetric X-structures, which are both independent of homologous recombination. Our data demonstrate that checkpoint activation is caused by a DNA structure formed at the strongest rDNA replication fork barrier (RFB) during replication termination, and consistently, checkpoint activation is dependent on the RFB binding protein, Fob1. In contrast, asymmetric X-structures are formed independent of Fob1 at less strong rDNA replication fork barriers. However, both checkpoint activation and formation of asymmetric X-structures are sensitive to conditions, which facilitate fork merging and progression of replication forks through replication fork barriers. Our data are consistent with a redundant role of Top2 and Sgs1 together with Top3 (Sgs1-Top3) in replication fork merging at rDNA barriers. At RFB either Top2 or Sgs1-Top3 is essential to prevent formation of a checkpoint activating DNA structure during termination, but at less strong rDNA barriers absence of the enzymes merely delays replication fork merging, causing an accumulation of asymmetric termination structures, which are solved over time. PMID:26630413

  5. DNA replication: keep moving and don't mind the gap.

    PubMed

    Langston, Lance D; O'Donnell, Mike

    2006-07-21

    As the replication fork progresses, synthesis of the discontinuous lagging strand requires frequent priming and cycling of the lagging strand polymerase to the new primers. It appears that this mechanism also permits bypass of template lesions on both strands, leaving the damage behind in a single-strand gap and precluding fork stalling or collapse. PMID:16857582

  6. PARP1 inhibition radiosensitizes HNSCC cells deficient in homologous recombination by disabling the DNA replication fork elongation response.

    PubMed

    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

  7. PARP1 inhibition radiosensitizes HNSCC cells deficient in homologous recombination by disabling the DNA replication fork elongation response

    PubMed Central

    Parplys, Ann C.; Seelbach, Jasna I.; Mansour, Wael Y.; Zielinski, Alexandra; Petersen, Cordula; Clauditz, Till S.; Münscher, Adrian; Friedl, Anna A.; Borgmann, Kerstin

    2016-01-01

    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

  8. Direct Evidence for the Formation of Precatenanes during DNA Replication*

    PubMed Central

    Cebrián, Jorge; Castán, Alicia; Martínez, Víctor; Kadomatsu-Hermosa, Maridian J.; Parra, Cristina; Fernández-Nestosa, María José; Schaerer, Christian; Hernández, Pablo; Krimer, Dora B.; Schvartzman, Jorge B.

    2015-01-01

    The dynamics of DNA topology during replication are still poorly understood. Bacterial plasmids are negatively supercoiled. This underwinding facilitates strand separation of the DNA duplex during replication. Leading the replisome, a DNA helicase separates the parental strands that are to be used as templates. This strand separation causes overwinding of the duplex ahead. If this overwinding persists, it would eventually impede fork progression. In bacteria, DNA gyrase and topoisomerase IV act ahead of the fork to keep DNA underwound. However, the processivity of the DNA helicase might overcome DNA gyrase and topoisomerase IV. It was proposed that the overwinding that builds up ahead of the fork could force it to swivel and diffuse this positive supercoiling behind the fork where topoisomerase IV would also act to maintain replicating the DNA underwound. Putative intertwining of sister duplexes in the replicated region are called precatenanes. Fork swiveling and the formation of precatenanes, however, are still questioned. Here, we used classical genetics and high resolution two-dimensional agarose gel electrophoresis to examine the torsional tension of replication intermediates of three bacterial plasmids with the fork stalled at different sites before termination. The results obtained indicated that precatenanes do form as replication progresses before termination. PMID:25829493

  9. Replication Stress: A Lifetime of Epigenetic Change

    PubMed Central

    Khurana, Simran; Oberdoerffer, Philipp

    2015-01-01

    DNA replication is essential for cell division. Challenges to the progression of DNA polymerase can result in replication stress, promoting the stalling and ultimately collapse of replication forks. The latter involves the formation of DNA double-strand breaks (DSBs) and has been linked to both genome instability and irreversible cell cycle arrest (senescence). Recent technological advances have elucidated many of the factors that contribute to the sensing and repair of stalled or broken replication forks. In addition to bona fide repair factors, these efforts highlight a range of chromatin-associated changes at and near sites of replication stress, suggesting defects in epigenome maintenance as a potential outcome of aberrant DNA replication. Here, we will summarize recent insight into replication stress-induced chromatin-reorganization and will speculate on possible adverse effects for gene expression, nuclear integrity and, ultimately, cell function. PMID:26378584

  10. Human Heart Mitochondrial DNA Is Organized in Complex Catenated Networks Containing Abundant Four-way Junctions and Replication Forks*

    PubMed Central

    Pohjoismäki, Jaakko L. O.; Goffart, Steffi; Tyynismaa, Henna; Willcox, Smaranda; Ide, Tomomi; Kang, Dongchon; Suomalainen, Anu; Karhunen, Pekka J.; Griffith, Jack D.; Holt, Ian J.; Jacobs, Howard T.

    2009-01-01

    Analysis of human heart mitochondrial DNA (mtDNA) by electron microscopy and agarose gel electrophoresis revealed a complete absence of the θ-type replication intermediates seen abundantly in mtDNA from all other tissues. Instead only Y- and X-junctional forms were detected after restriction digestion. Uncut heart mtDNA was organized in tangled complexes of up to 20 or more genome equivalents, which could be resolved to genomic monomers, dimers, and linear fragments by treatment with the decatenating enzyme topoisomerase IV plus the cruciform-cutting T7 endonuclease I. Human and mouse brain also contained a population of such mtDNA forms, which were absent, however, from mouse, rabbit, or pig heart. Overexpression in transgenic mice of two proteins involved in mtDNA replication, namely human mitochondrial transcription factor A or the mouse Twinkle DNA helicase, generated abundant four-way junctions in mtDNA of heart, brain, and skeletal muscle. The organization of mtDNA of human heart as well as of mouse and human brain in complex junctional networks replicating via a presumed non-θ mechanism is unprecedented in mammals. PMID:19525233

  11. Quarterly Progress Report - Biological Monitoring Program for East Fork Poplar Creek

    SciTech Connect

    Adams, S.M.; Christensen, S.W.; Greeley, M.S. jr; Hill, W.R.; McCarthy, J.F.; Peterson, M.J.; Ryon, M.G.; Smith, J.G.; Southworth, G.R.; Stewart, A.J.

    2000-07-18

    In May 1985, a National Pollutant Discharge Elimination System (NPDES) permit was issued for the Oak Ridge Y-12 Plant. As a condition of the permit, a Biological Monitoring and Abatement Program (BMAP) was developed to demonstrate that the effluent limitations established for the Y-12 Plant protect the classified uses of the receiving stream (East Fork Poplar Creek; EFPC), in particular, the growth and propagation of aquatic life (Loar et al. 1989). A second objective of the BMAP is to document the ecological effects resulting from the implementation of a water pollution control program designed to eliminate direct discharges of wastewaters to EFPC and to minimize the inadvertent release of pollutants to the environment. Because of the complex nature of the discharges to EFPC and the temporal and spatial variability in the composition of the discharges, a comprehensive, integrated approach to biological monitoring was developed. A new permit was issued to the Y-12 Plant on April 28, 1995 and became effective on July 1, 1995. Biological monitoring continues to be required under the new permit. The BMAP consists of four major tasks that reflect different but complementary approaches to evaluating the effects of the Y-12 Plant discharges on the aquatic integrity of EFPC. These tasks are (1) toxicity monitoring, (2) biological indicator studies, (3) bioaccumulation studies, and (4) ecological surveys of the periphyton, benthic macroinvertebrate, and fish communities. Monitoring is currently being conducted at five primary EFPC sites, although sites may be excluded or added depending upon the specific objectives of the various tasks. Criteria used in selecting the sites include: (1) location of sampling sites used in other studies, (2) known or suspected sources of downstream impacts, (3) proximity to U.S. Department of Energy (DOE) Oak Ridge Reservation (ORR) boundaries, (4) concentration of mercury in the adjacent floodplain, (5) appropriate habitat distribution, and

  12. Quarterly Progress Report - Biological Monitoring Program for East Fork Poplar Creek

    SciTech Connect

    Adams, S.M.; Christensen, S.W.; Greeley, M.S.jr; Hill, W.R.; McCarthy, J.F.; Peterson, M.J.; Ryon, M.G.; Smith, J.G.; Southworth, G.R.; Stewart, A.J.

    2000-10-18

    In May 1985, a National Pollutant Discharge Elimination System (NPDES) permit was issued for the Oak Ridge Y-12 Plant. As a condition of the permit, a Biological Monitoring and Abatement Program (BMAP) was developed to demonstrate that the effluent limitations established for the Y-12 Plant protect the classified uses of the receiving stream (East Fork Poplar Creek; EFPC), in particular, the growth and propagation of aquatic life (Loar et al. 1989). A second objective of the BMAP is to document the ecological effects resulting from the implementation of a water pollution control program designed to eliminate direct discharges of wastewaters to EFPC and to minimize the inadvertent release of pollutants to the environment. Because of the complex nature of the discharges to EFPC and the temporal and spatial variability in the composition of the discharges, a comprehensive, integrated approach to biological monitoring was developed. A new permit was issued to the Y-12 Plant on April 28, 1995 and became effective on July 1, 1995. Biological monitoring continues to be required under the new permit. The BMAP consists of four major tasks that reflect different but complementary approaches to evaluating the effects of the Y-12 Plant discharges on the aquatic integrity of EFPC. These tasks are (1) toxicity monitoring, (2) biological indicator studies, (3) bioaccumulation studies, and (4) ecological surveys of the periphyton, benthic macroinvertebrate, and fish communities. Monitoring is currently being conducted at five primary EFPC sites, although sites may be excluded or added depending upon the specific objectives of the various tasks. Criteria used in selecting the sites include: (1) location of sampling sites used in other studies, (2) known or suspected sources of downstream impacts, (3) proximity to U.S. Department of Energy (DOE) Oak Ridge Reservation (ORR) boundaries, (4) concentration of mercury in the adjacent floodplain, (5) appropriate habitat distribution, and

  13. Quarterly Progress Report - Biological Monitoring Program for East Fork Poplar Creek

    SciTech Connect

    Adams, S.M.; Christensen, S.W.; Greeley, M.S.jr; Hill, W.R.; McCarthy, J.F.; Peterson, M.J.; Ryon, M.G.; Smith, J.G.; Southworth, G.R.; Stewart, A.J.

    2000-04-18

    In May 1985, a National Pollutant Discharge Elimination System (NPDES) permit was issued for the Oak Ridge Y-12 Plant. As a condition of the permit, a Biological Monitoring and Abatement Program (BMAP) was developed to demonstrate that the effluent limitations established for the Y-12 Plant protect the classified uses of the receiving stream (East Fork Poplar Creek; EFPC), in particular, the growth and propagation of aquatic life (Loar et al. 1989). A second objective of the BMAP is to document the ecological effects resulting from the implementation of a water pollution control program designed to eliminate direct discharges of wastewaters to EFPC and to minimize the inadvertent release of pollutants to the environment. Because of the complex nature of the discharges to EFPC and the temporal and spatial variability in the composition of the discharges, a comprehensive, integrated approach to biological monitoring was developed. A new permit was issued to the Y-12 Plant on April 28, 1995 and became effective on July 1, 1995. Biological monitoring continues to be required under the new permit. The BMAP consists of four major tasks that reflect different but complementary approaches to evaluating the effects of the Y-12 Plant discharges on the aquatic integrity of EFPC. These tasks are (1) toxicity monitoring, (2) biological indicator studies, (3) bioaccumulation studies, and (4) ecological surveys of the periphyton, benthic macroinvertebrate, and fish communities. Monitoring is currently being conducted at five primary EFPC sites, although sites may be excluded or added depending upon the specific objectives of the various tasks. Criteria used in selecting the sites include: (1) location of sampling sites used in other studies, (2) known or suspected sources of downstream impacts, (3) proximity to U.S. Department of Energy (DOE) Oak Ridge Reservation (ORR) boundaries, (4) concentration of mercury in the adjacent floodplain, (5) appropriate habitat distribution, and

  14. LAKE FORK

    EPA Science Inventory

    The Lake Fork of the Arkansas River Watershed has been adversely affected through mining, water diversion and storage projects, grazing, logging, and other human influences over the past 120 years. It is the goals of the LFWWG to improve the health of Lake fork by addressing th...

  15. Regulation of Unperturbed DNA Replication by Ubiquitylation

    PubMed Central

    Priego Moreno, Sara; Gambus, Agnieszka

    2015-01-01

    Posttranslational modification of proteins by means of attachment of a small globular protein ubiquitin (i.e., ubiquitylation) represents one of the most abundant and versatile mechanisms of protein regulation employed by eukaryotic cells. Ubiquitylation influences almost every cellular process and its key role in coordination of the DNA damage response is well established. In this review we focus, however, on the ways ubiquitylation controls the process of unperturbed DNA replication. We summarise the accumulated knowledge showing the leading role of ubiquitin driven protein degradation in setting up conditions favourable for replication origin licensing and S-phase entry. Importantly, we also present the emerging major role of ubiquitylation in coordination of the active DNA replication process: preventing re-replication, regulating the progression of DNA replication forks, chromatin re-establishment and disassembly of the replisome at the termination of replication forks. PMID:26121093

  16. Quarterly Progress Report on the Biological Monitoring Program for East Fork Poplar Creek

    SciTech Connect

    Adams, S.M.; Ashwood, T.L.; Cicerone, D.S.; Greeley, M.S. Jr.; Hill, W.R.; Kszos, L.A.

    1996-12-30

    In May 1985, a National Pollutant Discharge Elimination System (NPDES) permit was issued for the Oak Ridge Y-12 Plant. As a condition of the permit, a Biological Monitoring and Abatement Program ( BMAP) was developed to demonstrate that the effluent limitations established for the Y-12 Plant protect the classified uses of the receiving stream (East Fork Poplar Creek; EFPC), in particular, the growth and propagation of aquatic life (Lear et al. 1989). A second objective of the BMAP is to document the ecological effects resulting from the implementation of a water pollution control program designed to eliminate direct discharges of wastewaters to EFPC and to minimize the inadvertent release of pollutants to the environment. Because of the complex nature of the discharges to EFPC and the temporal and spatial variability in the composition of the discharges, a comprehensive, integrated approach to biological monitoring was developed. A new permit was issued to the Y-12 Plant on April 28, 1995 and became effective on July 1, 1995. Biological monitoring continues to be required under the new permit. The BMAP consists of four major tasks that reflect different but complementary approaches to evaluating the effects of the Y-12 Plant discharges on the aquatic integrity of EFPC. These tasks are (1) toxicity monitoring, (2) biological indicator studies, (3) bioaccumulation studies, and (4) ecological surveys of the periphyton, benthic macroinvertebrate, and fish communities.

  17. Quarterly Progress Report - Biological Monitoring Program for East Fork Poplar Creek

    SciTech Connect

    Adams, S. M.; Christensen, S. W.; Greeley, M.S. jr; McCracken, M.K.; Peterson, M.J.; Ryon, M.G.; Smith, J.G.; Southworth G. R.; Stewart, A. J.

    2001-01-19

    In May 1985, a National Pollutant Discharge Elimination System (NPDES) permit was issued for the Oak Ridge Y-12 National Security Complex (formerly the Oak Ridge Y-12 Plant). As a condition of the permit, a Biological Monitoring and Abatement Program (BMAP) was developed to demonstrate that the effluent limitations established for the Y-12 Complex protect the classified uses of the receiving stream (East Fork Poplar Creek; EFPC), in particular, the growth and propagation of aquatic life (Loar et al. 1989). A second objective of the BMAP is to document the ecological effects resulting from the implementation of a water pollution control program designed to eliminate direct discharges of wastewaters to EFPC and to minimize the inadvertent release of pollutants to the environment. Because of the complex nature of the discharges to EFPC and the temporal and spatial variability in the composition of the discharges, a comprehensive, integrated approach to biological monitoring was developed. A new permit was issued to the Y-12 Complex on April 28, 1995 and became effective on July 1, 1995. Biological monitoring continues to be required under the new permit. The BMAP consists of four major tasks that reflect different but complementary approaches to evaluating the effects of the Y-12 Complex discharges on the aquatic integrity of EFPC. These tasks are (1) toxicity monitoring, (2) biological indicator studies, (3) bioaccumulation studies, and (4) ecological surveys of the periphyton, benthic macroinvertebrate, and fish communities. Monitoring is currently being conducted at five primary EFPC sites, although sites may be excluded or added depending upon the specific objectives of the various tasks. Criteria used in selecting the sites include: (1) location of sampling sites used in other studies, (2) known or suspected sources of downstream impacts, (3) proximity to U.S. Department of Energy (DOE) Oak Ridge Reservation (ORR) boundaries, (4) concentration of mercury in the

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

    PubMed

    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

  19. Polyomavirus JC in the context of immunosuppression: a series of adaptive, DNA replication-driven recombination events in the development of progressive multifocal leukoencephalopathy.

    PubMed

    Johnson, Edward M; Wortman, Margaret J; Dagdanova, Ayuna V; Lundberg, Patric S; Daniel, Dianne C

    2013-01-01

    Polyomavirus JC (JCV) is the etiological agent of progressive multifocal leukoencephalopathy (PML), a demyelinating infection of oligodendrocytes in the brain. PML, a frequently fatal opportunistic infection in AIDS, has also emerged as a consequence of treatment with several new immunosuppressive therapeutic agents. Although nearly 80% of adults are seropositive, JCV attains an ability to infect glial cells in only a minority of people. Data suggest that JCV undergoes sequence alterations that accompany this ability, and these changes can be derived from an archetype strain by mutation, deletion, and duplication. While the introductory source and primary tissue reservoir of JCV remain unknown, lymphoid cells have been identified as potential intermediaries in progression of JCV to the brain. This review is focused on sequence changes in the noncoding control region (NCCR) of the virus. We propose an adaptive mechanism that involves a sequential series of DNA replication-driven NCCR recombination events involving stalled DNA replication forks at NCCR palindromic secondary structures. We shall describe how the NCCR sequence changes point to a model in which viral DNA replication drives NCCR recombination, allowing JCV adaptation to different cell types in its progression to neurovirulence. PMID:23690820

  20. RAD18, WRNIP1 and ATMIN promote ATM signalling in response to replication stress.

    PubMed

    Kanu, N; Zhang, T; Burrell, R A; Chakraborty, A; Cronshaw, J; DaCosta, C; Grönroos, E; Pemberton, H N; Anderton, E; Gonzalez, L; Sabbioneda, S; Ulrich, H D; Swanton, C; Behrens, A

    2016-07-28

    The DNA replication machinery invariably encounters obstacles that slow replication fork progression, and threaten to prevent complete replication and faithful segregation of sister chromatids. The resulting replication stress activates ATR, the major kinase involved in resolving impaired DNA replication. In addition, replication stress also activates the related kinase ATM, which is required to prevent mitotic segregation errors. However, the molecular mechanism of ATM activation by replication stress is not defined. Here, we show that monoubiquitinated Proliferating Cell Nuclear Antigen (PCNA), a marker of stalled replication forks, interacts with the ATM cofactor ATMIN via WRN-interacting protein 1 (WRNIP1). ATMIN, WRNIP1 and RAD18, the E3 ligase responsible for PCNA monoubiquitination, are specifically required for ATM signalling and 53BP1 focus formation induced by replication stress, not ionising radiation. Thus, WRNIP1 connects PCNA monoubiquitination with ATMIN/ATM to activate ATM signalling in response to replication stress and contribute to the maintenance of genomic stability. PMID:26549024

  1. A fork-clearing role for UvrD.

    PubMed

    Florés, Maria-José; Sanchez, Nicolas; Michel, Bénédicte

    2005-09-01

    The inactivation of a replication protein causes the disassembly of the replication machinery and creates a need for replication reactivation. In several replication mutants, restart occurs after the fork has been isomerized into a four-armed junction, a reaction called replication fork reversal. The repair helicase UvrD is essential for replication fork reversal upon inactivation of the polymerase (DnaE) or the beta-clamp (DnaN) subunits of the Escherichia coli polymerase III, and for the viability of dnaEts and dnaNts mutants at semi-permissive temperature. We show here that the inactivation of recA, recFOR, recJ or recQ recombination genes suppresses the requirement for UvrD for replication fork reversal and suppresses the lethality conferred by uvrD inactivation to Pol IIIts mutants at semi-permissive temperature. We propose that RecA binds inappropriately to blocked replication forks in the dnaEts and dnaNts mutants in a RecQ- RecJ- RecFOR-dependent way and that UvrD acts by removing RecA or a RecA-made structure, allowing replication fork reversal. This work thus reveals the existence of a futile reaction of RecA binding to blocked replication forks, that requires the action of UvrD for fork-clearing and proper replication restart. PMID:16135232

  2. [Mechanisms of inhibition of viral replication in plants]. Progress report

    SciTech Connect

    Not Available

    1992-09-01

    Progress is described concerning genetic mapping CMV movement genes for CMV coat protein in squash and ToMV gene in tomato. These gene products appear to be involved in resistance to squash and tomato mosaic viruses respectively.

  3. Cell Cycle Regulation of DNA Replication

    PubMed Central

    Sclafani, R. A.; Holzen, T. M.

    2008-01-01

    Eukaryotic DNA replication is regulated to ensure all chromosomes replicate once and only once per cell cycle. Replication begins at many origins scattered along each chromosome. Except for budding yeast, origins are not defined DNA sequences and probably are inherited by epigenetic mechanisms. Initiation at origins occurs throughout the S phase according to a temporal program that is important in regulating gene expression during development. Most replication proteins are conserved in evolution in eukaryotes and archaea, but not in bacteria. However, the mechanism of initiation is conserved and consists of origin recognition, assembly of pre-replication (pre-RC) initiative complexes, helicase activation, and replisome loading. Cell cycle regulation by protein phosphorylation ensures that pre-RC assembly can only occur in G1 phase, whereas helicase activation and loading can only occur in S phase. Checkpoint regulation maintains high fidelity by stabilizing replication forks and preventing cell cycle progression during replication stress or damage. PMID:17630848

  4. Repair of lesions and initiation of DNA replication in vertebrate cells. Progress report, 1981-1982

    SciTech Connect

    Taylor, J.H.

    1982-01-01

    Progress is reported in the following areas: (1) the search for sequences in Xenopus DNA that serve as origins for replication; (2) preparation of a partial library of Xenopus genomic DNA and search for other origins; and (3) base pair mismatch correction and DNA methylation. (ACR)

  5. Absence of MutSβ leads to the formation of slipped-DNA for CTG/CAG contractions at primate replication forks.

    PubMed

    Slean, Meghan M; Panigrahi, Gagan B; Castel, Arturo López; Pearson, August B; Tomkinson, Alan E; Pearson, Christopher E

    2016-06-01

    Typically disease-causing CAG/CTG repeats expand, but rare affected families can display high levels of contraction of the expanded repeat amongst offspring. Understanding instability is important since arresting expansions or enhancing contractions could be clinically beneficial. The MutSβ mismatch repair complex is required for CAG/CTG expansions in mice and patients. Oddly, by unknown mechanisms MutSβ-deficient mice incur contractions instead of expansions. Replication using CTG or CAG as the lagging strand template is known to cause contractions or expansions respectively; however, the interplay between replication and repair leading to this instability remains unclear. Towards understanding how repeat contractions may arise, we performed in vitro SV40-mediated replication of repeat-containing plasmids in the presence or absence of mismatch repair. Specifically, we separated repair from replication: Replication mediated by MutSβ- and MutSα-deficient human cells or cell extracts produced slipped-DNA heteroduplexes in the contraction- but not expansion-biased replication direction. Replication in the presence of MutSβ disfavoured the retention of replication products harbouring slipped-DNA heteroduplexes. Post-replication repair of slipped-DNAs by MutSβ-proficient extracts eliminated slipped-DNAs. Thus, a MutSβ-deficiency likely enhances repeat contractions because MutSβ protects against contractions by repairing template strand slip-outs. Replication deficient in LigaseI or PCNA-interaction mutant LigaseI revealed slipped-DNA formation at lagging strands. Our results reveal that distinct mechanisms lead to expansions or contractions and support inhibition of MutSβ as a therapeutic strategy to enhance the contraction of expanded repeats. PMID:27155933

  6. Inducing a Site Specific Replication Blockage in E. coli Using a Fluorescent Repressor Operator System.

    PubMed

    Mettrick, Karla A; Lawrence, Nikki; Mason, Claire; Weaver, Georgia M; Corocher, Tayla-Ann; Grainge, Ian

    2016-01-01

    Obstacles present on DNA, including tightly-bound proteins and various lesions, can severely inhibit the progression of the cell's replication machinery. The stalling of a replisome can lead to its dissociation from the chromosome, either in part or its entirety, leading to the collapse of the replication fork. The recovery from this collapse is a necessity for the cell to accurately complete chromosomal duplication and subsequently divide. Therefore, when the collapse occurs, the cell has evolved diverse mechanisms that take place to restore the DNA fork and allow replication to be completed with high fidelity. Previously, these replication repair pathways in bacteria have been studied using UV damage, which has the disadvantage of not being localized to a known site. This manuscript describes a system utilizing a Fluorescence Repressor Operator System (FROS) to create a site-specific protein block that can induce the stalling and collapse of replication forks in Escherichia coli. Protocols detail how the status of replication can be visualized in single living cells using fluorescence microscopy and DNA replication intermediates can be analyzed by 2-dimensional agarose gel electrophoresis. Temperature sensitive mutants of replisome components (e.g. DnaBts) can be incorporated into the system to induce a synchronous collapse of the replication forks. Furthermore, the roles of the recombination proteins and helicases that are involved in these processes can be studied using genetic knockouts within this system. PMID:27583408

  7. Isolation of the Cdc45/Mcm2-7/GINS (CMG) complex, a candidate for the eukaryotic DNA replication fork helicase.

    PubMed

    Moyer, Stephen E; Lewis, Peter W; Botchan, Michael R

    2006-07-01

    The protein Cdc45 plays a critical but poorly understood role in the initiation and elongation stages of eukaryotic DNA replication. To study Cdc45's function in DNA replication, we purified Cdc45 protein from Drosophila embryo extracts by a combination of traditional and immunoaffinity chromatography steps and found that the protein exists in a stable, high-molecular-weight complex with the Mcm2-7 hexamer and the GINS tetramer. The purified Cdc45/Mcm2-7/GINS complex is associated with an active ATP-dependent DNA helicase function. RNA interference knock-down experiments targeting the GINS and Cdc45 components establish that the proteins are required for the S phase transition in Drosophila cells. The data suggest that this complex forms the core helicase machinery for eukaryotic DNA replication. PMID:16798881

  8. Structural Biology of Replication Initiation Factor Mcm10

    PubMed Central

    Du, Wenyue; Stauffer, Melissa E.; Eichman, Brandt F.

    2016-01-01

    Minichromosome maintenance protein 10 (Mcm10) is a non-enzymatic replication factor required for proper assembly of the eukaryotic replication fork. Mcm10 interacts with single-stranded and double-stranded DNA, polymerase α, and Mcm2-7, and is important for activation of the pre-replicative complex and recruitment of subsequent proteins to the origin at the onset of S-phase. In addition, Mcm10 has recently been implicated in coordination of helicase and polymerase activities during replication fork progression. The nature of Mcm10's involvement in these activities, whether direct or indirect, remains unknown. However, recent biochemical and structural characterization of Mcm10 from multiple organisms has provided insights into how Mcm10 utilizes a modular architecture to act as a replisome scaffold, which helps to define possible roles in origin DNA melting, pol α recruitment and coordination of enzymatic activities during elongation. PMID:22918587

  9. The DNA repair endonuclease Mus81 facilitates fast DNA replication in the absence of exogenous damage

    PubMed Central

    Fu, Haiqing; Martin, Melvenia M.; Regairaz, Marie; Huang, Liang; You, Yang; Lin, Chi-Mei; Ryan, Michael; Kim, RyangGuk; Shimura, Tsutomu; Pommier, Yves; Aladjem, Mirit I.

    2015-01-01

    The Mus81 endonuclease resolves recombination intermediates and mediates cellular responses to exogenous replicative stress. Here, we show that Mus81 also regulates the rate of DNA replication during normal growth by promoting replication fork progression while reducing the frequency of replication initiation events. In the absence of Mus81 endonuclease activity, DNA synthesis is slowed and replication initiation events are more frequent. In addition, Mus81 deficient cells fail to recover from exposure to low doses of replication inhibitors and cell viability is dependent on the XPF endonuclease. Despite an increase in replication initiation frequency, cells lacking Mus81 use the same pool of replication origins as Mus81-expressing cells. Therefore, decelerated DNA replication in Mus81 deficient cells does not initiate from cryptic or latent origins not used during normal growth. These results indicate that Mus81 plays a key role in determining the rate of DNA replication without activating a novel group of replication origins. PMID:25879486

  10. 5-ASA Affects Cell Cycle Progression in Colorectal Cells by Reversibly Activating a Replication Checkpoint

    PubMed Central

    LUCIANI, M. GLORIA; CAMPREGHER, CHRISTOPH; FORTUNE, JOHN M.; KUNKEL, THOMAS A.; GASCHE, CHRISTOPH

    2007-01-01

    Background & Aims Individuals with inflammatory bowel disease are at risk of developing colorectal cancer (CRC). Epidemiologic, animal, and laboratory studies suggest that 5-amino-salicylic acid (5-ASA) protects from the development of CRC by altering cell cycle progression and by inducing apoptosis. Our previous results indicate that 5-ASA improves replication fidelity in colorectal cells, an effect that is active in reducing mutations. In this study, we hypothesized that 5-ASA restrains cell cycle progression by activating checkpoint pathways in colorectal cell lines, which would prevent tumor development and improve genomic stability. Methods CRC cells with different genetic backgrounds such as HT29, HCT116, HCT116p53−/−, HCT116+chr3, and LoVo were treated with 5-ASA for 2–96 hours. Cell cycle progression, phosphorylation, and DNA binding of cell cycle checkpoint proteins were analyzed. Results We found that 5-ASA at concentrations between 10 and 40 mmol/L affects cell cycle progression by inducing cells to accumulate in the S phase. This effect was independent of the hMLH1, hMSH2, and p53 status because it was observed to a similar extent in all cell lines under investigation. Moreover, wash-out experiments demonstrated reversibility within 48 hours. Although p53 did not have a causative role, p53 Ser15 was strongly phosphorylated. Proteins involved in the ATM-and-Rad3-related kinase (ATR)-dependent S-phase checkpoint response (Chk1 and Rad17) were also phosphorylated but not ataxia telengectasia mutated kinase. Conclusions Our data demonstrate that 5-ASA causes cells to reversibly accumulate in S phase and activate an ATR-dependent checkpoint. The activation of replication checkpoint may slow down DNA replication and improve DNA replication fidelity, which increases the maintenance of genomic stability and counteracts carcinogenesis. PMID:17241873

  11. Replication landscape of the human genome

    PubMed Central

    Petryk, Nataliya; Kahli, Malik; d'Aubenton-Carafa, Yves; Jaszczyszyn, Yan; Shen, Yimin; Silvain, Maud; Thermes, Claude; Chen, Chun-Long; Hyrien, Olivier

    2016-01-01

    Despite intense investigation, human replication origins and termini remain elusive. Existing data have shown strong discrepancies. Here we sequenced highly purified Okazaki fragments from two cell types and, for the first time, quantitated replication fork directionality and delineated initiation and termination zones genome-wide. Replication initiates stochastically, primarily within non-transcribed, broad (up to 150 kb) zones that often abut transcribed genes, and terminates dispersively between them. Replication fork progression is significantly co-oriented with the transcription. Initiation and termination zones are frequently contiguous, sometimes separated by regions of unidirectional replication. Initiation zones are enriched in open chromatin and enhancer marks, even when not flanked by genes, and often border ‘topologically associating domains' (TADs). Initiation zones are enriched in origin recognition complex (ORC)-binding sites and better align to origins previously mapped using bubble-trap than λ-exonuclease. This novel panorama of replication reveals how chromatin and transcription modulate the initiation process to create cell-type-specific replication programs. PMID:26751768

  12. ATR inhibitors VE-821 and VX-970 sensitize cancer cells to topoisomerase i inhibitors by disabling DNA replication initiation and fork elongation responses.

    PubMed

    Jossé, Rozenn; Martin, Scott E; Guha, Rajarshi; Ormanoglu, Pinar; Pfister, Thomas D; Reaper, Philip M; Barnes, Christopher S; Jones, Julie; Charlton, Peter; Pollard, John R; Morris, Joel; Doroshow, James H; Pommier, Yves

    2014-12-01

    Camptothecin and its derivatives, topotecan and irinotecan, are specific topoisomerase I (Top1) inhibitors and potent anticancer drugs killing cancer cells by producing replication-associated DNA double-strand breaks, and the indenoisoquinoline LMP-400 (indotecan) is a novel Top1 inhibitor in clinical trial. To develop novel drug combinations, we conducted a synthetic lethal siRNA screen using a library that targets nearly 7,000 human genes. Depletion of ATR, the main transducer of replication stress, came as a top candidate gene for camptothecin synthetic lethality. Validation studies using ATR siRNA and the ATR inhibitor VE-821 confirmed marked antiproliferative synergy with camptothecin and even greater synergy with LMP-400. Single-cell analyses and DNA fiber combing assays showed that VE-821 abrogates the S-phase replication elongation checkpoint and the replication origin-firing checkpoint induced by camptothecin and LMP-400. As expected, the combination of Top1 inhibitors with VE-821 inhibited the phosphorylation of ATR and Chk1; however, it strongly induced γH2AX. In cells treated with the combination, the γH2AX pattern changed over time from the well-defined Top1-induced damage foci to an intense peripheral and diffuse nuclear staining, which could be used as response biomarker. Finally, the clinical derivative of VE-821, VX-970, enhanced the in vivo tumor response to irinotecan without additional toxicity. A key implication of our work is the mechanistic rationale and proof of principle it provides to evaluate the combination of Top1 inhibitors with ATR inhibitors in clinical trials. PMID:25269479

  13. ATR inhibitors VE-821 and VX-970 sensitize cancer cells to topoisomerase I inhibitors by disabling DNA replication initiation and fork elongation responses

    PubMed Central

    Jossé, Rozenn; Martin, Scott E.; Guha, Rajarshi; Ormanoglu, Pinar; Pfister, Thomas D.; Reaper, Philip M.; Barnes, Christopher S.; Jones, Julie; Charlton, Peter; Pollard, John R.; Morris, Joel; Doroshow, James H.; Pommier, Yves

    2014-01-01

    Camptothecin and its derivatives, topotecan and irinotecan are specific topoisomerase I (Top1) inhibitors and potent anticancer drugs killing cancer cells by producing replication-associated DNA double-strand breaks, and the indenoisoquinoline LMP-400 (indotecan) is a novel Top1 inhibitor in clinical trial. To develop novel drug combinations, we conducted a synthetic lethal siRNA screen using a library that targets nearly 7,000 human genes. Depletion of ATR, the main transducer of replication stress came as a top candidate gene for camptothecin synthetic lethality. Validation studies using ATR siRNA and the ATR inhibitor VE-821, confirmed marked antiproliferative synergy with camptothecin, and even greater synergy with LMP-400. Single cell analyses and DNA fiber combing assays showed that VE-821 abrogates the S-phase replication elongation checkpoint and the replication origin-firing check point induced by camptothecin and LMP-400. As expected, the combination ofTop1 inhibitors with VE-821 inhibited the phosphorylation of ATR and Chk1; however, it strongly induced γH2AX. In cells treated with the combination, the γH2AX pattern changed overtime from the well-defined Top1-induced damage foci to an intense peripheral and diffuse nuclear staining, which could be used as response biomarker. Finally, the clinical derivative of VE-821, VX-970 enhanced the in vivo tumor response to irinotecan without additional toxicity. Akey implication of our work is the mechanistic rationale and proof-of-principle it provides to evaluate the combination of Top1 inhibitors with ATR inhibitors in clinical trials. PMID:25269479

  14. Phosphorylation of CMG helicase and Tof1 is required for programmed fork arrest.

    PubMed

    Bastia, Deepak; Srivastava, Pankaj; Zaman, Shamsu; Choudhury, Malay; Mohanty, Bidyut K; Bacal, Julien; Langston, Lance D; Pasero, Philippe; O'Donnell, Michael E

    2016-06-28

    Several important physiological transactions, including control of replicative life span (RLS), prevention of collision between replication and transcription, and cellular differentiation, require programmed replication fork arrest (PFA). However, a general mechanism of PFA has remained elusive. We previously showed that the Tof1-Csm3 fork protection complex is essential for PFA by antagonizing the Rrm3 helicase that displaces nonhistone protein barriers that impede fork progression. Here we show that mutations of Dbf4-dependent kinase (DDK) of Saccharomyces cerevisiae, but not other DNA replication factors, greatly reduced PFA at replication fork barriers in the spacer regions of the ribosomal DNA array. A key target of DDK is the mini chromosome maintenance (Mcm) 2-7 complex, which is known to require phosphorylation by DDK to form an active CMG [Cdc45 (cell division cycle gene 45), Mcm2-7, GINS (Go, Ichi, Ni, and San)] helicase. In vivo experiments showed that mutational inactivation of DDK caused release of Tof1 from the chromatin fractions. In vitro binding experiments confirmed that CMG and/or Mcm2-7 had to be phosphorylated for binding to phospho-Tof1-Csm3 but not to its dephosphorylated form. Suppressor mutations that bypass the requirement for Mcm2-7 phosphorylation by DDK restored PFA in the absence of the kinase. Retention of Tof1 in the chromatin fraction and PFA in vivo was promoted by the suppressor mcm5-bob1, which bypassed DDK requirement, indicating that under this condition a kinase other than DDK catalyzed the phosphorylation of Tof1. We propose that phosphorylation regulates the recruitment and retention of Tof1-Csm3 by the replisome and that this complex antagonizes the Rrm3 helicase, thereby promoting PFA, by preserving the integrity of the Fob1-Ter complex. PMID:27298353

  15. The Western progression of lyme disease: infectious and Nonclonal Borrelia burgdorferi Sensu Lato populations in Grand Forks County, North Dakota.

    PubMed

    Stone, Brandee L; Russart, Nathan M; Gaultney, Robert A; Floden, Angela M; Vaughan, Jefferson A; Brissette, Catherine A

    2015-01-01

    Scant attention has been paid to Lyme disease, Borrelia burgdorferi, Ixodes scapularis, or reservoirs in eastern North Dakota despite the fact that it borders high-risk counties in Minnesota. Recent reports of B. burgdorferi and I. scapularis in North Dakota, however, prompted a more detailed examination. Spirochetes cultured from the hearts of five rodents trapped in Grand Forks County, ND, were identified as B. burgdorferi sensu lato through sequence analyses of the 16S rRNA gene, the 16S rRNA gene-ileT intergenic spacer region, flaB, ospA, ospC, and p66. OspC typing revealed the presence of groups A, B, E, F, L, and I. Two rodents were concurrently carrying multiple OspC types. Multilocus sequence typing suggested the eastern North Dakota strains are most closely related to those found in neighboring regions of the upper Midwest and Canada. BALB/c mice were infected with B. burgdorferi isolate M3 (OspC group B) by needle inoculation or tick bite. Tibiotarsal joints and ear pinnae were culture positive, and B. burgdorferi M3 was detected by quantitative PCR (qPCR) in the tibiotarsal joints, hearts, and ear pinnae of infected mice. Uninfected larval I. scapularis ticks were able to acquire B. burgdorferi M3 from infected mice; M3 was maintained in I. scapularis during the molt from larva to nymph; and further, M3 was transmitted from infected I. scapularis nymphs to naive mice, as evidenced by cultures and qPCR analyses. These results demonstrate that isolate M3 is capable of disseminated infection by both artificial and natural routes of infection. This study confirms the presence of unique (nonclonal) and infectious B. burgdorferi populations in eastern North Dakota. PMID:25304515

  16. Chromatin Immunoprecipitation to Detect DNA Replication and Repair Factors

    PubMed Central

    Gadaleta, Mariana C.; Iwasaki, Osamu; Noguchi, Chiaki; Noma, Ken-Ichi; Noguchi, Eishi

    2015-01-01

    DNA replication is tightly coupled with DNA repair processes in order to preserve genomic integrity. During DNA replication, the replication fork encounters a variety of obstacles including DNA damage/adducts, secondary structures, and programmed fork-blocking sites, which are all difficult to replicate. The replication fork also collides with the transcription machinery, which shares the template DNA with the replisome complex. Under these conditions, replication forks stall, causing replication stress and/or fork collapse, ultimately leading to genomic instability. The mechanisms to overcome these replication problems remain elusive. Therefore, it is important to investigate how DNA repair and replication factors are recruited and coordinated at chromosomal regions that are difficult to replicate. In this chapter, we describe a chromatin immunoprecipitation method to locate proteins required for DNA repair during DNA replication in the fission yeast Schizosaccharomyces pombe. This method can also easily be adapted to study replisome components or chromatin-associated factors. PMID:25916713

  17. Single-Molecule Observation of Prokaryotic DNA Replication

    PubMed Central

    Tanner, Nathan A.; van Oijen, Antoine M.

    2010-01-01

    Recent advances in optical imaging and molecular manipulation techniques have made it possible to observe the activity of individual enzymes and study the dynamic properties of processes that are challenging to elucidate using ensemble-averaging techniques. The use of single-molecule approaches has proven to be particularly successful in the study of the dynamic interactions between the components at the replication fork. In this section, we describe the methods necessary for in vitro single-molecule studies of prokaryotic replication systems. Through these experiments, accurate information can be obtained on the rates and processivities of DNA unwinding and polymerization. The ability to monitor in real time the progress of a single replication fork allows for the detection of short-lived, intermediate states that would be difficult to visualize in bulk-phase assays. PMID:19563119

  18. Unwinding of forked DNA structures by UvrD.

    PubMed

    Cadman, Chris J; Matson, Steven W; McGlynn, Peter

    2006-09-01

    Many studies have demonstrated the need for processing of blocked replication forks to underpin genome duplication. UvrD helicase in Escherichia coli has been implicated in the processing of damaged replication forks, or the recombination intermediates formed from damaged forks. Here we show that UvrD can unwind forked DNA structures, in part due to the ability of UvrD to initiate unwinding from discontinuities within the phosphodiester backbone of DNA. UvrD does therefore have the capacity to target DNA intermediates of replication and recombination. Such an activity resulted in unwinding of what would be the parental duplex DNA ahead of either a stalled replication fork or a D-loop formed by recombination. However, UvrD had a substrate preference for fork structures having a nascent lagging strand at the branch point but no leading strand. Furthermore, at such structures the polarity of UvrD altered so that unwinding of the lagging strand predominated. This reaction is reminiscent of the PriC-Rep pathway of replication restart, suggesting that UvrD and Rep may have at least partially redundant functions. PMID:16890954

  19. Universal Temporal Profile of Replication Origin Activation in Eukaryotes

    NASA Astrophysics Data System (ADS)

    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.

  20. Replicative Nature of Indian Research, Essence of Scientific Temper, and Future of Scientific Progress*

    PubMed Central

    Singh, Ajai R.; Singh, Shakuntala A.

    2004-01-01

    A lot of Indian research is replicative in nature. This is because originality is at a premium here and mediocrity is in great demand. But replication has its merit as well because it helps in corroboration. And that is the bedrock on which many a fancied scientific hypothesis or theory stands, or falls. However, to go from replicative to original research will involve a massive effort to restructure the Indian psyche and an all round effort from numerous quarters. The second part of this paper deals with the essence of scientific temper,which need not have any basic friendship, or animosity, with religion, faith, superstition and other such entities. A true scientist follows two cardinal rules. He is never unwilling to accept the worth of evidence, howsoever damning to the most favourite of his theories. Second, and perhaps more important, for want of evidence, he withholds comment. He says neither yes nor no. Where will Science ultimately lead Man is the third part of this essay. One argument is that the conflict between Man and Science will continue tilleither of them is exhausted or wiped out. The other believes that it is Science which has to be harnessed for Man and not Man used for Science. And with the numerous checks and balances in place, Science will remain an effective tool for man's progress. The essential value-neutrality of Science will have to be supplemented by the values that man has upheld for centuries as fundamental, and which religious thought and moral philosophy have continuously professed. PMID:22815607

  1. The RdgC protein of Escherichia coli binds DNA and counters a toxic effect of RecFOR in strains lacking the replication restart protein PriA

    PubMed Central

    Moore, Timothy; McGlynn, Peter; Ngo, Hien-Ping; Sharples, Gary J.; Lloyd, Robert G.

    2003-01-01

    PriA protein provides a means to load the DnaB replicative helicase at DNA replication fork and D loop structures, and is therefore a key factor in the rescue of stalled or broken forks and subsequent replication restart. We show that the nucleoid-associated RdgC protein binds non-specifically to single-stranded (ss) DNA and double-stranded DNA. It is also essential for growth of a strain lacking PriA, indicating that it might affect replication fork progression or fork rescue. dnaC suppressors of priA overcome this inviability, especially when RecF, RecO or RecR is inactivated, indicating that RdgC avoids or counters a toxic effect of these proteins. Mutations modifying ssDNA-binding (SSB) protein also negate this toxic effect, suggesting that the toxicity reflects inappropriate loading of RecA on SSB-coated ssDNA, leading to excessive or untimely RecA activity. We suggest that binding of RdgC to DNA limits RecA loading, avoiding problems at replication forks that would otherwise require PriA to promote replication restart. Mutations in RNA polymerase also reduce the toxic effect of RecFOR, providing a further link between DNA replication, transcription and repair. PMID:12554673

  2. Single-Molecule Analysis of Replicating Yeast Chromosomes.

    PubMed

    Gallo, David; Wang, Gang; Yip, Christopher M; Brown, Grant W

    2016-02-01

    The faithful replication of eukaryotic chromosomal DNA occurs during S phase once per cell cycle. Replication is highly regulated and is initiated at special structures, termed origins, from which replication forks move out bidirectionally. A wide variety of techniques have been developed to study the features and kinetics of replication. Many of these, such as those based on flow cytometry and two-dimensional and pulsed-field gel electrophoresis, give a population-level view of replication. However, an alternative approach, DNA fiber analysis, which was originally developed more than 50 years ago, has the advantage of revealing features of replication at the level of individual DNA fibers. Initially based on autoradiography, this technique has been superseded by immunofluorescence-based detection of incorporated halogenated thymidine analogs. Furthermore, derivations of this technique have been developed to distribute and stretch the labeled DNA fibers uniformly on optically clear surfaces. As described here, one such technique-DNA combing, in which DNA is combed onto silanized coverslips-has been used successfully to monitor replication fork progression and origin usage in budding yeast. PMID:26832692

  3. 3. SOUTH FORK OF THE TULE RIVER MIDDLE FORK BRANCH ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    3. SOUTH FORK OF THE TULE RIVER MIDDLE FORK BRANCH FLUME AT THE NORTH FORK OF THE TULE RIVER MIDDLE FORK CROSSING SHOWING ORIGINAL DIMENSIONAL STONE PIER ON WEST BANK AT PHOTO CENTER, AND REMAINS OF ORIGINAL EAST BANK DIMENSIONAL STONE PIER AT PHOTO LEFT BELOW NEW (ca. 1931) EAST BANK PIER. VIEW TO SOUTHEAST. - Tule River Hydroelectric Project, Water Conveyance System, Middle Fork Tule River, Springville, Tulare County, CA

  4. A replicator-specific binding protein essential for site-specific initiation of DNA replication in mammalian cells.

    PubMed

    Zhang, Ya; Huang, Liang; Fu, Haiqing; Smith, Owen K; Lin, Chii Mei; Utani, Koichi; Rao, Mishal; Reinhold, William C; Redon, Christophe E; Ryan, Michael; Kim, RyangGuk; You, Yang; Hanna, Harlington; Boisclair, Yves; Long, Qiaoming; Aladjem, Mirit I

    2016-01-01

    Mammalian chromosome replication starts from distinct sites; however, the principles governing initiation site selection are unclear because proteins essential for DNA replication do not exhibit sequence-specific DNA binding. Here we identify a replication-initiation determinant (RepID) protein that binds a subset of replication-initiation sites. A large fraction of RepID-binding sites share a common G-rich motif and exhibit elevated replication initiation. RepID is required for initiation of DNA replication from RepID-bound replication origins, including the origin at the human beta-globin (HBB) locus. At HBB, RepID is involved in an interaction between the replication origin (Rep-P) and the locus control region. RepID-depleted murine embryonic fibroblasts exhibit abnormal replication fork progression and fewer replication-initiation events. These observations are consistent with a model, suggesting that RepID facilitates replication initiation at a distinct group of human replication origins. PMID:27272143

  5. A replicator-specific binding protein essential for site-specific initiation of DNA replication in mammalian cells

    PubMed Central

    Zhang, Ya; Huang, Liang; Fu, Haiqing; Smith, Owen K.; Lin, Chii Mei; Utani, Koichi; Rao, Mishal; Reinhold, William C.; Redon, Christophe E.; Ryan, Michael; Kim, RyangGuk; You, Yang; Hanna, Harlington; Boisclair, Yves; Long, Qiaoming; Aladjem, Mirit I.

    2016-01-01

    Mammalian chromosome replication starts from distinct sites; however, the principles governing initiation site selection are unclear because proteins essential for DNA replication do not exhibit sequence-specific DNA binding. Here we identify a replication-initiation determinant (RepID) protein that binds a subset of replication-initiation sites. A large fraction of RepID-binding sites share a common G-rich motif and exhibit elevated replication initiation. RepID is required for initiation of DNA replication from RepID-bound replication origins, including the origin at the human beta-globin (HBB) locus. At HBB, RepID is involved in an interaction between the replication origin (Rep-P) and the locus control region. RepID-depleted murine embryonic fibroblasts exhibit abnormal replication fork progression and fewer replication-initiation events. These observations are consistent with a model, suggesting that RepID facilitates replication initiation at a distinct group of human replication origins. PMID:27272143

  6. The paradox of simian immunodeficiency virus infection in sooty mangabeys: active viral replication without disease progression.

    PubMed

    Chakrabarti, Lisa A

    2004-01-01

    Simian immunodeficiency virus SIVsm causes an asymptomatic infection in its natural host, the sooty mangabey, but induces an immunodeficiency syndrome very similar to human AIDS when transferred to a new host species such as the rhesus macaque. Unexpectedly, SIVsm replication dynamics is comparable in the two species, with rapid accumulation of viral mutations and a high viral load detected in both mangabeys and macaques. In contrast, clear differences are observed in immune parameters. Pathogenic SIV infection in macaques is associated with decreased CD4+ T cell numbers and signs of generalized immune activation, such as increased numbers of cycling and apoptotic T cells, hyperplasic lymphoid tissues, and exacerbated immune responses. Mangabeys with asymptomatic SIV infection show normal T cell regeneration parameters and signs of a moderate immune response, appropriate in the setting of chronic viral infection. The comparative analysis of simian models thus suggests that viral load alone cannot account for progression to disease, and that the capacity of primate lentiviruses to induce abnormal immune activation underlies AIDS pathogenesis. PMID:14766388

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

    PubMed Central

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

    2016-01-01

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

  8. Force regulated dynamics of RPA on a DNA fork

    PubMed Central

    Kemmerich, Felix E.; Daldrop, Peter; Pinto, Cosimo; Levikova, Maryna; Cejka, Petr; Seidel, Ralf

    2016-01-01

    Replication protein A (RPA) is a single-stranded DNA binding protein, involved in most aspects of eukaryotic DNA metabolism. Here, we study the behavior of RPA on a DNA substrate that mimics a replication fork. Using magnetic tweezers we show that both yeast and human RPA can open forked DNA when sufficient external tension is applied. In contrast, at low force, RPA becomes rapidly displaced by the rehybridization of the DNA fork. This process appears to be governed by the binding or the release of an RPA microdomain (toehold) of only few base-pairs length. This gives rise to an extremely rapid exchange dynamics of RPA at the fork. Fork rezipping rates reach up to hundreds of base-pairs per second, being orders of magnitude faster than RPA dissociation from ssDNA alone. Additionally, we show that RPA undergoes diffusive motion on ssDNA, such that it can be pushed over long distances by a rezipping fork. Generally the behavior of both human and yeast RPA homologs is very similar. However, in contrast to yeast RPA, the dissociation of human RPA from ssDNA is greatly reduced at low Mg2+ concentrations, such that human RPA can melt DNA in absence of force. PMID:27016742

  9. The Fanconi Anemia Pathway Maintains Genome Stability by Coordinating Replication and Transcription.

    PubMed

    Schwab, Rebekka A; Nieminuszczy, Jadwiga; Shah, Fenil; Langton, Jamie; Lopez Martinez, David; Liang, Chih-Chao; Cohn, Martin A; Gibbons, Richard J; Deans, Andrew J; Niedzwiedz, Wojciech

    2015-11-01

    DNA replication stress can cause chromosomal instability and tumor progression. One key pathway that counteracts replication stress and promotes faithful DNA replication consists of the Fanconi anemia (FA) proteins. However, how these proteins limit replication stress remains largely elusive. Here we show that conflicts between replication and transcription activate the FA pathway. Inhibition of transcription or enzymatic degradation of transcription-associated R-loops (DNA:RNA hybrids) suppresses replication fork arrest and DNA damage occurring in the absence of a functional FA pathway. Furthermore, we show that simple aldehydes, known to cause leukemia in FA-deficient mice, induce DNA:RNA hybrids in FA-depleted cells. Finally, we demonstrate that the molecular mechanism by which the FA pathway limits R-loop accumulation requires FANCM translocase activity. Failure to activate a response to physiologically occurring DNA:RNA hybrids may critically contribute to the heightened cancer predisposition and bone marrow failure of individuals with mutated FA proteins. PMID:26593718

  10. Causes and Consequences of Replication Stress

    PubMed Central

    Zeman, Michelle K.; Cimprich, Karlene A.

    2015-01-01

    Replication stress is a complex phenomenon which has serious implications for genome stability, cell survival, and human disease. Generation of aberrant replication fork structures containing single-stranded DNA activates the replication stress response, primarily mediated by the kinase ATM- and Rad3-related (ATR). ATR and its downstream effectors stabilize and help to restart stalled replication forks, avoiding the generation of DNA damage and genome instability. Understanding these pathways may be key to diagnosis and treatment of human diseases caused by defective responses to replication stress. PMID:24366029

  11. DNA topoisomerase IIα controls replication origin cluster licensing and firing time in Xenopus egg extracts

    PubMed Central

    Gaggioli, Vincent; Le Viet, Barbara; Germe, Thomas; Hyrien, Olivier

    2013-01-01

    Sperm chromatin incubated in Xenopus egg extracts undergoes origin licensing and nuclear assembly before DNA replication. We found that depletion of DNA topoisomerase IIα (topo IIα), the sole topo II isozyme of eggs and its inhibition by ICRF-193, which clamps topo IIα around DNA have opposite effects on these processes. ICRF-193 slowed down replication origin cluster activation and fork progression in a checkpoint-independent manner, without altering replicon size. In contrast, topo IIα depletion accelerated origin cluster activation, and topo IIα add-back negated overinitiation. Therefore, topo IIα is not required for DNA replication, but topo IIα clamps slow replication, probably by forming roadblocks. ICRF-193 had no effect on DNA synthesis when added after nuclear assembly, confirming that topo IIα activity is dispensable for replication and revealing that topo IIα clamps formed on replicating DNA do not block replication, presumably because topo IIα acts behind and not in front of forks. Topo IIα depletion increased, and topo IIα addition reduced, chromatin loading of MCM2-7 replicative helicase, whereas ICRF-193 did not affect MCM2-7 loading. Therefore, topo IIα restrains MCM2-7 loading in an ICRF-193-resistant manner during origin licensing, suggesting a model for establishing the sequential firing of origin clusters. PMID:23757188

  12. Taking the thrombin "fork".

    PubMed

    Mann, Kenneth G

    2010-07-01

    The proverb that probably best exemplifies my career in research is attributable to Yogi Berra (http://www.yogiberra.com/), ie, "when you come to a fork in the road ... take it." My career is a consequence of chance interactions with great mentors and talented students and the opportunities provided by a succession of ground-breaking improvements in technology. PMID:20554951

  13. Replication domains are self-interacting structural chromatin units of human chromosomes

    NASA Astrophysics Data System (ADS)

    Arneodo, Alain

    2011-03-01

    In higher eukaryotes, the absence of specific sequence motifs marking the origins of replication has been a serious hindrance to the understanding of the mechanisms that regulate the initiation and the maintenance of the replication program in different cell types. In silico analysis of nucleotide compositional skew has predicted the existence, in the germline, of replication N-domains bordered by putative replication origins and where the skew decreases rather linearly as the signature of a progressive inversion of the average fork polarity. Here, from the demonstration that the average fork polarity can be directly extracted from the derivative of replication timing profiles, we develop a wavelet-based pattern recognition methodology to delineate replication U-domains where the replication timing profile is shaped as a U and its derivative as a N. Replication U-domains are robustly found in seven cell lines as covering a significant portion (40-50%) of the human genome where the replication timing data actually displays some plasticity between cell lines. The early replication initiation zones at U-domains borders are found to be hypersensitive to DNase I cleavage, to be associated with transcriptional activity and to present a significant enrichment in insular-binding proteins CTCF, the hallmark of an open chromatin structure. A comparative analysis of genome-wide chromatin interaction (HiC) data shows that replication-U domains correspond to self-interacting structural high order chromatin units of megabase characteristic size. Taken together, these findings provide evidence that the epigenetic compartmentalization of the human genome into autonomous replication U-domains comes along with an extensive remodelling of the threedimensional chromosome architecture during development or in specific diseases. The observed cell specific conservation of the replication timing between the human and mouse genomes strongly suggests that this chromosome organization into

  14. Physical Interaction between Replication Protein A (RPA) and MRN

    PubMed Central

    Oakley, Greg; Tillison, Kristin; Opiyo, Stephen; Glanzer, Jason; Horn, Jeffrey M.; Patrick, Steve M.

    2009-01-01

    Replication protein A (RPA) is a heterotrimeric protein consisting of RPA1, RPA2 and RPA3 subunits that binds to ssDNA with high affinity. The response to replication stress requires the recruitment of RPA and the MRE11/RAD50/NBS1 (MRN) complex. RPA bound to ssDNA stabilizes stalled replication forks by recruiting checkpoint proteins involved in fork stabilization. MRN can bind DNA structures encountered at stalled or collapsed replication forks, such as ssDNA-dsDNA junctions or breaks and promote the restart of DNA replication. Here, we demonstrate that RPA2 phosphorylation regulates the assembly of DNA damage-induced RPA and MRN foci. Using purified proteins, we observe a direct interaction between RPA with both NBS1 and MRE11. By utilizing RPA bound to ssDNA, we demonstrate that substituting RPA with phosphorylated RPA or a phosphomimetic decreases the interaction with the MRN complex. Also, the N-terminus of RPA1 is a critical component of the RPA-MRN protein-protein interaction. Deletion of the N-terminal oligonucleotide-oligosaccharide binding fold (OB-fold) of RPA1 abrogates RPA interactions with MRN and individual proteins of the MRN complex. Further identification of residues critical for MRN binding in the N-terminus of RPA1 show that substitution of Arg31 and Arg41 with alanines disrupts the RPA-MRN interaction and alters cell cycle progression in response to DNA damage. Thus, the N-terminus of RPA1 and phosphorylation of RPA2 regulate RPA-MRN interactions and are important in the response to DNA damage. PMID:19586055

  15. G-quadruplex-interacting compounds alter latent DNA replication and episomal persistence of KSHV

    PubMed Central

    Madireddy, Advaitha; Purushothaman, Pravinkumar; Loosbroock, Christopher P.; Robertson, Erle S.; Schildkraut, Carl L.; Verma, Subhash C.

    2016-01-01

    Kaposi's sarcoma associated herpesvirus (KSHV) establishes life-long latent infection by persisting as an extra-chromosomal episome in the infected cells and by maintaining its genome in dividing cells. KSHV achieves this by tethering its epigenome to the host chromosome by latency associated nuclear antigen (LANA), which binds in the terminal repeat (TR) region of the viral genome. Sequence analysis of the TR, a GC-rich DNA element, identified several potential Quadruplex G-Rich Sequences (QGRS). Since quadruplexes have the tendency to obstruct DNA replication, we used G-quadruplex stabilizing compounds to examine their effect on latent DNA replication and the persistence of viral episomes. Our results showed that these G-quadruplex stabilizing compounds led to the activation of dormant origins of DNA replication, with preferential bi-directional pausing of replications forks moving out of the TR region, implicating the role of the G-rich TR in the perturbation of episomal DNA replication. Over time, treatment with PhenDC3 showed a loss of viral episomes in the infected cells. Overall, these data show that G-quadruplex stabilizing compounds retard the progression of replication forks leading to a reduction in DNA replication and episomal maintenance. These results suggest a potential role for G-quadruplex stabilizers in the treatment of KSHV-associated diseases. PMID:26837574

  16. Slx5/Slx8 Promotes Replication Stress Tolerance by Facilitating Mitotic Progression.

    PubMed

    Thu, Yee Mon; Van Riper, Susan Kaye; Higgins, LeeAnn; Zhang, Tianji; Becker, Jordan Robert; Markowski, Todd William; Nguyen, Hai Dang; Griffin, Timothy Jon; Bielinsky, Anja Katrin

    2016-05-10

    Loss of minichromosome maintenance protein 10 (Mcm10) causes replication stress. We uncovered that S. cerevisiae mcm10-1 mutants rely on the E3 SUMO ligase Mms21 and the SUMO-targeted ubiquitin ligase complex Slx5/8 for survival. Using quantitative mass spectrometry, we identified changes in the SUMO proteome of mcm10-1 mutants and revealed candidates regulated by Slx5/8. Such candidates included subunits of the chromosome passenger complex (CPC), Bir1 and Sli15, known to facilitate spindle assembly checkpoint (SAC) activation. We show here that Slx5 counteracts SAC activation in mcm10-1 mutants under conditions of moderate replication stress. This coincides with the proteasomal degradation of sumoylated Bir1. Importantly, Slx5-dependent mitotic relief was triggered not only by Mcm10 deficiency but also by treatment with low doses of the alkylating drug methyl methanesulfonate. Based on these findings, we propose a model in which Slx5/8 allows for passage through mitosis when replication stress is tolerable. PMID:27134171

  17. Slx5/Slx8 promotes replication stress tolerance by facilitating mitotic progression

    PubMed Central

    Thu, Yee Mon; Van Riper, Susan Kaye; Higgins, LeeAnn; Zhang, Tianji; Becker, Jordan Robert; Markowski, Todd William; Nguyen, Hai Dang; Griffin, Timothy Jon; Bielinsky, Anja-Katrin

    2016-01-01

    SUMMARY Loss of minichromosome maintenance protein 10 (Mcm10) causes replication stress. We uncovered that S. cerevisiae mcm10-1 mutants rely on the E3 SUMO ligase Mms21 and the SUMO-targeted ubiquitin ligase complex Slx5/8 for survival. Using quantitative mass spectrometry, we identified changes in the SUMO proteome of mcm10-1 mutants and revealed candidates regulated by Slx5/8. Such candidates included subunits of the chromosome passenger complex (CPC), Bir1 and Sli15, known to facilitate spindle assembly checkpoint (SAC) activation. We show here that Slx5 counteracts SAC activation in mcm10-1 mutants under conditions of moderate replication stress. This coincides with the proteasomal degradation of sumoylated Bir1. Importantly, Slx5-dependent mitotic relief was not only triggered by Mcm10 deficiency but also by treatment with low doses of the alkylating drug methyl methanesulfonate. Based on these findings, we propose a model in which Slx5/8 allows for passage through mitosis when replication stress is tolerable. PMID:27134171

  18. Progress in the Pipeline: Replication of the Minority Training Program in Cancer Control Research

    PubMed Central

    Yancey, Antronette K.; Kagawa-Singer, Marjorie; Ratliff, Pamela; Valdez, Annalyn; Jiménez, Laura; Banks, Priscilla; Stewart, Susan; Roe, Kathleen M.; Pasick, Rena J.

    2013-01-01

    Background This paper evaluates the replicability of an NCI-funded didactic/experiential program to increase the diversity of doctorally-prepared cancer disparities investigators. Methods The program was developed and operated successfully for three years in Northern California when a replication was established at UCLA. Feasibility, process, impact, and outcome measures on UCSF and UCLA summer-institute participants were compared. Results Average participant rankings of the influence of the program on intention to apply for a doctorate were 9.1/10 (UCSF) and 8.6/10 (UCLA). A total of 22.5% of UCSF and 10% of UCLA participants have enrolled in, been accepted by, or completed doctoral programs. Among these alumni, 68% (21/31) of UCSF and 60% (3/5) of UCLA participants plan to conduct their doctoral research in cancer control. Conclusions This program has been successfully replicated and has met its objective to increase the pipeline of ethnically diverse doctoral-level public health researchers. Expansion of the program to other regions of the US is feasible and indicated. PMID:17542715

  19. Replication Stress in Mammalian Cells and Its Consequences for Mitosis

    PubMed Central

    Gelot, Camille; Magdalou, Indiana; Lopez, Bernard S.

    2015-01-01

    The faithful transmission of genetic information to daughter cells is central to maintaining genomic stability and relies on the accurate and complete duplication of genetic material during each cell cycle. However, the genome is routinely exposed to endogenous and exogenous stresses that can impede the progression of replication. Such replication stress can be an early cause of cancer or initiate senescence. Replication stress, which primarily occurs during S phase, results in consequences during mitosis, jeopardizing chromosome segregation and, in turn, genomic stability. The traces of replication stress can be detected in the daughter cells during G1 phase. Alterations in mitosis occur in two types: 1) local alterations that correspond to breaks, rearrangements, intertwined DNA molecules or non-separated sister chromatids that are confined to the region of the replication dysfunction; 2) genome-wide chromosome segregation resulting from centrosome amplification (although centrosomes do not contain DNA), which amplifies the local replication stress to the entire genome. Here, we discuss the endogenous causes of replication perturbations, the mechanisms of replication fork restart and the consequences for mitosis, chromosome segregation and genomic stability. PMID:26010955

  20. Intracellular Locations of Replication Proteins and the Origin of Replication during Chromosome Duplication in the Slowly Growing Human Pathogen Helicobacter pylori

    PubMed Central

    Sharma, Atul; Kamran, Mohammad; Verma, Vijay

    2014-01-01

    We followed the position of the replication complex in the pathogenic bacterium Helicobacter pylori using antibodies raised against the single-stranded DNA binding protein (HpSSB) and the replicative helicase (HpDnaB). The position of the replication origin, oriC, was also localized in growing cells by fluorescence in situ hybridization (FISH) with fluorescence-labeled DNA sequences adjacent to the origin. The replisome assembled at oriC near one of the cell poles, and the two forks moved together toward the cell center as replication progressed in the growing cell. Termination and resolution of the forks occurred near midcell, on one side of the septal membrane. The duplicated copies of oriC did not separate until late in elongation, when the daughter chromosomes segregated into bilobed nucleoids, suggesting sister chromatid cohesion at or near the oriC region. Components of the replication machinery, viz., HpDnaB and HpDnaG (DNA primase), were found associated with the cell membrane. A model for the assembly and location of the H. pylori replication machinery during chromosomal duplication is presented. PMID:24363345

  1. Discordant Impact of HLA on Viral Replicative Capacity and Disease Progression in Pediatric and Adult HIV Infection

    PubMed Central

    Adland, Emily; Paioni, Paolo; Thobakgale, Christina; Laker, Leana; Mori, Luisa; Muenchhoff, Maximilian; Csala, Anna; Clapson, Margaret; Flynn, Jacquie; Novelli, Vas; Hurst, Jacob; Naidoo, Vanessa; Shapiro, Roger; Huang, Kuan-Hsiang Gary; Frater, John; Prendergast, Andrew; Prado, Julia G.; Ndung’u, Thumbi; Walker, Bruce D.; Carrington, Mary; Jooste, Pieter; Goulder, Philip J. R.

    2015-01-01

    HLA class I polymorphism has a major influence on adult HIV disease progression. An important mechanism mediating this effect is the impact on viral replicative capacity (VRC) of the escape mutations selected in response to HLA-restricted CD8+ T-cell responses. Factors that contribute to slow progression in pediatric HIV infection are less well understood. We here investigate the relationship between VRC and disease progression in pediatric infection, and the effect of HLA on VRC and on disease outcome in adult and pediatric infection. Studying a South African cohort of >350 ART-naïve, HIV-infected children and their mothers, we first observed that pediatric disease progression is significantly correlated with VRC. As expected, VRCs in mother-child pairs were strongly correlated (p = 0.004). The impact of the protective HLA alleles, HLA-B*57, HLA-B*58:01 and HLA-B*81:01, resulted in significantly lower VRCs in adults (p<0.0001), but not in children. Similarly, in adults, but not in children, VRCs were significantly higher in subjects expressing the disease-susceptible alleles HLA-B*18:01/45:01/58:02 (p = 0.007). Irrespective of the subject, VRCs were strongly correlated with the number of Gag CD8+ T-cell escape mutants driven by HLA-B*57/58:01/81:01 present in each virus (p = 0.0002). In contrast to the impact of VRC common to progression in adults and children, the HLA effects on disease outcome, that are substantial in adults, are small and statistically insignificant in infected children. These data further highlight the important role that VRC plays both in adult and pediatric progression, and demonstrate that HLA-independent factors, yet to be fully defined, are predominantly responsible for pediatric non-progression. PMID:26076345

  2. Discordant Impact of HLA on Viral Replicative Capacity and Disease Progression in Pediatric and Adult HIV Infection.

    PubMed

    Adland, Emily; Paioni, Paolo; Thobakgale, Christina; Laker, Leana; Mori, Luisa; Muenchhoff, Maximilian; Csala, Anna; Clapson, Margaret; Flynn, Jacquie; Novelli, Vas; Hurst, Jacob; Naidoo, Vanessa; Shapiro, Roger; Huang, Kuan-Hsiang Gary; Frater, John; Prendergast, Andrew; Prado, Julia G; Ndung'u, Thumbi; Walker, Bruce D; Carrington, Mary; Jooste, Pieter; Goulder, Philip J R

    2015-06-01

    HLA class I polymorphism has a major influence on adult HIV disease progression. An important mechanism mediating this effect is the impact on viral replicative capacity (VRC) of the escape mutations selected in response to HLA-restricted CD8+ T-cell responses. Factors that contribute to slow progression in pediatric HIV infection are less well understood. We here investigate the relationship between VRC and disease progression in pediatric infection, and the effect of HLA on VRC and on disease outcome in adult and pediatric infection. Studying a South African cohort of >350 ART-naïve, HIV-infected children and their mothers, we first observed that pediatric disease progression is significantly correlated with VRC. As expected, VRCs in mother-child pairs were strongly correlated (p = 0.004). The impact of the protective HLA alleles, HLA-B*57, HLA-B*58:01 and HLA-B*81:01, resulted in significantly lower VRCs in adults (p<0.0001), but not in children. Similarly, in adults, but not in children, VRCs were significantly higher in subjects expressing the disease-susceptible alleles HLA-B*18:01/45:01/58:02 (p = 0.007). Irrespective of the subject, VRCs were strongly correlated with the number of Gag CD8+ T-cell escape mutants driven by HLA-B*57/58:01/81:01 present in each virus (p = 0.0002). In contrast to the impact of VRC common to progression in adults and children, the HLA effects on disease outcome, that are substantial in adults, are small and statistically insignificant in infected children. These data further highlight the important role that VRC plays both in adult and pediatric progression, and demonstrate that HLA-independent factors, yet to be fully defined, are predominantly responsible for pediatric non-progression. PMID:26076345

  3. Shaping the landscape of the Escherichia coli chromosome: replication-transcription encounters in cells with an ectopic replication origin

    PubMed Central

    Ivanova, Darja; Taylor, Toni; Smith, Sarah L.; Dimude, Juachi U.; Upton, Amy L.; Mehrjouy, Mana M.; Skovgaard, Ole; Sherratt, David J.; Retkute, Renata; Rudolph, Christian J.

    2015-01-01

    Each cell division requires the unwinding of millions of DNA base pairs to allow chromosome duplication and gene transcription. As DNA replication and transcription share the same template, conflicts between both processes are unavoidable and head-on collisions are thought to be particularly problematic. Surprisingly, a recent study reported unperturbed cell cycle progression in Escherichia coli cells with an ectopic replication origin in which highly transcribed rrn operons were forced to be replicated opposite to normal. In this study we have re-generated a similar strain and found the doubling time to be twice that of normal cells. Replication profiles of this background revealed significant deviations in comparison to wild-type profiles, particularly in highly transcribed regions and the termination area. These deviations were alleviated by mutations that either inactivate the termination area or destabilise RNA polymerase complexes and allow their easier displacement by replication forks. Our data demonstrate that head-on replication-transcription conflicts are highly problematic. Indeed, analysis of the replication profile of the previously published E. coli construct revealed a chromosomal rearrangement that alleviates replication-transcription conflicts in an intriguingly simple way. Our data support the idea that avoiding head-on collisions has significantly contributed to shaping the distinct architecture of bacterial chromosomes. PMID:26160884

  4. Shaping the landscape of the Escherichia coli chromosome: replication-transcription encounters in cells with an ectopic replication origin.

    PubMed

    Ivanova, Darja; Taylor, Toni; Smith, Sarah L; Dimude, Juachi U; Upton, Amy L; Mehrjouy, Mana M; Skovgaard, Ole; Sherratt, David J; Retkute, Renata; Rudolph, Christian J

    2015-09-18

    Each cell division requires the unwinding of millions of DNA base pairs to allow chromosome duplication and gene transcription. As DNA replication and transcription share the same template, conflicts between both processes are unavoidable and head-on collisions are thought to be particularly problematic. Surprisingly, a recent study reported unperturbed cell cycle progression in Escherichia coli cells with an ectopic replication origin in which highly transcribed rrn operons were forced to be replicated opposite to normal. In this study we have re-generated a similar strain and found the doubling time to be twice that of normal cells. Replication profiles of this background revealed significant deviations in comparison to wild-type profiles, particularly in highly transcribed regions and the termination area. These deviations were alleviated by mutations that either inactivate the termination area or destabilise RNA polymerase complexes and allow their easier displacement by replication forks. Our data demonstrate that head-on replication-transcription conflicts are highly problematic. Indeed, analysis of the replication profile of the previously published E. coli construct revealed a chromosomal rearrangement that alleviates replication-transcription conflicts in an intriguingly simple way. Our data support the idea that avoiding head-on collisions has significantly contributed to shaping the distinct architecture of bacterial chromosomes. PMID:26160884

  5. Cdc48 and a ubiquitin ligase drive disassembly of the CMG helicase at the end of DNA replication

    PubMed Central

    De Piccoli, Giacomo; Labib, Karim

    2015-01-01

    Chromosome replication is initiated by a universal mechanism in eukaryotic cells, involving the assembly and activation at replication origins of the CMG (Cdc45-MCM-GINS) DNA helicase, which is essential for the progression of replication forks. Disassembly of CMG is likely to be a key regulated step at the end of chromosome replication, but the mechanism was unknown until now. Here we show that the ubiquitin ligase known as SCFDia2 promotes ubiquitylation of CMG during the final stages of chromosome replication in Saccharomyces cerevisiae. The Cdc48/p97 segregase then associates with ubiquitylated CMG, leading rapidly to helicase disassembly. These findings indicate that the end of chromosome replication in eukaryotes is controlled in a similarly complex fashion to the much-better-characterized initiation step. PMID:25342810

  6. The 2008 National Assessment of Educational Progress (NAEP): A Visual Arts Replication Study

    ERIC Educational Resources Information Center

    Heid, Karen

    2016-01-01

    The 2008 National Assessment of Educational Progress (NAEP) Arts Assessment was administered to selected 8th grade students throughout the nation, and in 2009 the results from that administration were publicly reported. In the spring of 2010, building on the format and structure of the 2008 national assessment, the researcher administered a…

  7. 3. View of Clark Fork Vehicle Bridge facing southwest. Bridge ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    3. View of Clark Fork Vehicle Bridge facing southwest. Bridge from north shore of Clark Fork River. - Clark Fork Vehicle Bridge, Spanning Clark Fork River, serves Highway 200, Clark Fork, Bonner County, ID

  8. 4. View of Clark Fork Vehicle Bridge facing northeast. Bridge ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    4. View of Clark Fork Vehicle Bridge facing northeast. Bridge from south shoreof Clark Fork River showing 4 spans. - Clark Fork Vehicle Bridge, Spanning Clark Fork River, serves Highway 200, Clark Fork, Bonner County, ID

  9. 2. View of Clark Fork Vehicle Bridge facing northeast. Bridge ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    2. View of Clark Fork Vehicle Bridge facing northeast. Bridge from south shore of Clark Fork River showing 4 1/2 spans. - Clark Fork Vehicle Bridge, Spanning Clark Fork River, serves Highway 200, Clark Fork, Bonner County, ID

  10. 1. View of Clark Fork Vehicle Bridge facing west. Panorama ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    1. View of Clark Fork Vehicle Bridge facing west. Panorama showing the entire span of bridge from north shore of the Clark Fork River. - Clark Fork Vehicle Bridge, Spanning Clark Fork River, serves Highway 200, Clark Fork, Bonner County, ID

  11. 7. View of Clark Fork Vehicle Bridge facing northwest. Bridge ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    7. View of Clark Fork Vehicle Bridge facing northwest. Bridge from south shore of Clark Fork River showing 4 1/2 spans. - Clark Fork Vehicle Bridge, Spanning Clark Fork River, serves Highway 200, Clark Fork, Bonner County, ID

  12. DNA-damage accumulation and replicative arrest in Hutchinson-Gilford progeria syndrome.

    PubMed

    Musich, Phillip R; Zou, Yue

    2011-12-01

    A common feature of progeria syndromes is a premature aging phenotype and an enhanced accumulation of DNA damage arising from a compromised repair system. HGPS (Hutchinson-Gilford progeria syndrome) is a severe form of progeria in which patients accumulate progerin, a mutant lamin A protein derived from a splicing variant of the lamin A/C gene (LMNA). Progerin causes chromatin perturbations which result in the formation of DSBs (double-strand breaks) and abnormal DDR (DNA-damage response). In the present article, we review recent findings which resolve some mechanistic details of how progerin may disrupt DDR pathways in HGPS cells. We propose that progerin accumulation results in disruption of functions of some replication and repair factors, causing the mislocalization of XPA (xeroderma pigmentosum group A) protein to the replication forks, replication fork stalling and, subsequently, DNA DSBs. The binding of XPA to the stalled forks excludes normal binding by repair proteins, leading to DSB accumulation, which activates ATM (ataxia telangiectasia mutated) and ATR (ATM- and Rad3-related) checkpoints, and arresting cell-cycle progression. PMID:22103522

  13. Hyper-Acetylation of Histone H3K56 Limits Break-Induced Replication by Inhibiting Extensive Repair Synthesis

    PubMed Central

    Che, Jun; Smith, Stephanie; Kim, Yoo Jung; Shim, Eun Yong; Myung, Kyungjae; Lee, Sang Eun

    2015-01-01

    Break-induced replication (BIR) has been implicated in restoring eroded telomeres and collapsed replication forks via single-ended invasion and extensive DNA synthesis on the recipient chromosome. Unlike other recombination subtypes, DNA synthesis in BIR likely relies heavily on mechanisms enabling efficient fork progression such as chromatin modification. Herein we report that deletion of HST3 and HST4, two redundant de-acetylases of histone H3 Lysine 56 (H3K56), inhibits BIR, sensitizes checkpoint deficient cells to deoxyribonucleotide triphosphate pool depletion, and elevates translocation-type gross chromosomal rearrangements (GCR). The basis for deficiency in BIR and gene conversion with long gap synthesis in hst3Δ hst4Δ cells can be traced to a defect in extensive DNA synthesis. Distinct from other cellular defects associated with deletion of HST3 and HST4 including thermo-sensitivity and elevated spontaneous mutagenesis, the BIR defect in hst3Δ hst4Δ cannot be offset by the deletion of RAD17 or MMS22, but rather by the loss of RTT109 or ASF1, or in combination with the H3K56R mutation, which also restores tolerance to replication stress in mrc1 mutants. Our studies suggest that acetylation of H3K56 limits extensive repair synthesis and interferes with efficient fork progression in BIR. PMID:25705897

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

    PubMed

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

    2016-06-01

    A dividing cell has to duplicate its DNA precisely once during the cell cycle to preserve genome integrity avoiding the accumulation of genetic aberrations that promote diseases such as cancer. A large number of endogenous impacts can challenge DNA replication and cells harbor a battery of pathways to promote genome integrity during DNA replication. This includes suppressing new replication origin firing, stabilization of replicating forks, and the safe restart of forks to prevent any loss of genetic information. Here, we describe mechanisms by which oncogenes can interfere with DNA replication thereby causing DNA replication stress and genome instability. Further, we describe cellular and systemic responses to these insults with a focus on DNA replication restart pathways. Finally, we discuss the therapeutic potential of exploiting intrinsic replicative stress in cancer cells for targeted therapy. PMID:26805514

  15. Safeguarding genome stability: RASSF1A tumor suppressor regulates BRCA2 at stalled forks

    PubMed Central

    Pefani, Dafni Eleftheria; O'Neill, Eric

    2015-01-01

    While it has been widely established that defective fork restart after exposure to stress results in increased genomic instability, the importance of fork protection during stalling for safeguarding genomic integrity has recently been fully appreciated. BRCA2, Breast tumor suppressor, has dual functionality promoting not only DNA repair but also preventing DNA lesions at stalled forks. In response to replication stress, BRCA2 recruits RAD51 onto nascent DNA at stalled forks, protecting nascent DNA from nucleolitic cleavage. Phosphorylation of the BRCA2 C-terminal RAD51 binding site by CDK2 promotes RAD51 filament disassembly, leading to nucleolitic cleavage of newly synthesized DNA and compromised fork integrity. Recently we uncovered how the core Hippo pathway components RASSF1A, MST2 and LATS1 regulate CDK2 activity towards BRCA2, in response to fork stalling. In complex with LATS1, CDK2 exhibits reduced kinase activity which results in low levels of pBRCA2-S3291 and stable RAD51 filaments protecting nascent DNA from MRE11 cleavage. In the absence of the RASSF1A/MST2/LATS1/CDK2 pathway increased resection of newly synthesized DNA leads to chromosomal instability and malignant transformation. This function of RASSF1A in stalled replication fork protection adds to the role of RASSF1A as a tumor suppressor and builds up evidence for RASSF1A status and its prognostic and predictive value in cancer. PMID:25927241

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

    PubMed

    Yeo, Constance Qiao Xin; Alexander, Irina; Lin, Zhaoru; Lim, Shuhui; Aning, Obed Akwasi; Kumar, Ramesh; Sangthongpitag, Kanda; Pendharkar, Vishal; Ho, Vincent H B; Cheok, Chit Fang

    2016-04-01

    p53 tumor suppressor maintains genomic stability, typically acting through cell-cycle arrest, senescence, and apoptosis. We discovered a function of p53 in preventing conflicts between transcription and replication, independent of its canonical roles. p53 deficiency sensitizes cells to Topoisomerase (Topo) II inhibitors, resulting in DNA damage arising spontaneously during replication. Topoisomerase IIα (TOP2A)-DNA complexes preferentially accumulate in isogenic p53 mutant or knockout cells, reflecting an increased recruitment of TOP2A to regulate DNA topology. We propose that p53 acts to prevent DNA topological stress originating from transcription during the S phase and, therefore, promotes normal replication fork progression. Consequently, replication fork progression is impaired in the absence of p53, which is reversed by transcription inhibition. Pharmacologic inhibition of transcription also attenuates DNA damage and decreases Topo-II-DNA complexes, restoring cell viability in p53-deficient cells. Together, our results demonstrate a function of p53 that may underlie its role in tumor suppression. PMID:27052176

  17. The Eukaryotic Replication Machine.

    PubMed

    Zhang, D; O'Donnell, M

    2016-01-01

    The cellular replicating machine, or "replisome," is composed of numerous different proteins. The core replication proteins in all cell types include a helicase, primase, DNA polymerases, sliding clamp, clamp loader, and single-strand binding (SSB) protein. The core eukaryotic replisome proteins evolved independently from those of bacteria and thus have distinct architectures and mechanisms of action. The core replisome proteins of the eukaryote include: an 11-subunit CMG helicase, DNA polymerase alpha-primase, leading strand DNA polymerase epsilon, lagging strand DNA polymerase delta, PCNA clamp, RFC clamp loader, and the RPA SSB protein. There are numerous other proteins that travel with eukaryotic replication forks, some of which are known to be involved in checkpoint regulation or nucleosome handling, but most have unknown functions and no bacterial analogue. Recent studies have revealed many structural and functional insights into replisome action. Also, the first structure of a replisome from any cell type has been elucidated for a eukaryote, consisting of 20 distinct proteins, with quite unexpected results. This review summarizes the current state of knowledge of the eukaryotic core replisome proteins, their structure, individual functions, and how they are organized at the replication fork as a machine. PMID:27241931

  18. Replication-Associated Recombinational Repair: Lessons from Budding Yeast

    PubMed Central

    Bonner, Jaclyn N.; Zhao, Xiaolan

    2016-01-01

    Recombinational repair processes multiple types of DNA lesions. Though best understood in the repair of DNA breaks, recombinational repair is intimately linked to other situations encountered during replication. As DNA strands are decorated with many types of blocks that impede the replication machinery, a great number of genomic regions cannot be duplicated without the help of recombinational repair. This replication-associated recombinational repair employs both the core recombination proteins used for DNA break repair and the specialized factors that couple replication with repair. Studies from multiple organisms have provided insights into the roles of these specialized factors, with the findings in budding yeast being advanced through use of powerful genetics and methods for detecting DNA replication and repair intermediates. In this review, we summarize recent progress made in this organism, ranging from our understanding of the classical template switch mechanisms to gap filling and replication fork regression pathways. As many of the protein factors and biological principles uncovered in budding yeast are conserved in higher eukaryotes, these findings are crucial for stimulating studies in more complex organisms. PMID:27548223

  19. Tuning Forks and Monitor Screens.

    ERIC Educational Resources Information Center

    Harrison, M. A. T.

    2000-01-01

    Defines the vibrations of a tuning fork against a computer monitor screen as a pattern that can illustrate or explain physical concepts like wave vibrations, wave forms, and phase differences. Presents background information and demonstrates the experiment. (Author/YDS)

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

    PubMed Central

    Gemble, Simon; Ahuja, Akshay; Buhagiar-Labarchède, Géraldine; Onclercq-Delic, Rosine; Dairou, Julien; Biard, Denis S. F.; Lambert, Sarah; Lopes, Massimo; Amor-Guéret, Mounira

    2015-01-01

    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. PMID:26181065

  1. Midbrain-to-pons ratio in autopsy-confirmed progressive supranuclear palsy: replication in an independent cohort.

    PubMed

    Kaasinen, Valtteri; Kangassalo, Noora; Gardberg, Maria; Isotalo, Juuso; Karhu, Jari; Parkkola, Riitta; Sonninen, Pirkko

    2015-07-01

    Recent neuropathologically confirmed clinical data suggest that the midbrain-to-pons ratio, as calculated from conventional brain MRI, has high specificity and sensitivity for the diagnosis of progressive supranuclear palsy (PSP). Here, we aimed to replicate these findings in an independent autopsy-confirmed cohort of 6 PSP patients and 23 non-PSP patients. Patients with confirmed PSP had clearly lower midbrain-to-pons ratios compared to non-PSP patients (p < 0.0001). All non-PSP patients had midbrain-to-pons ratios higher than 0.50, whereas all but one PSP patient had a ratio lower than 0.50. The positive predictive value (PPV) of the ratio (<0.50) was 100% and the negative predictive value (NPV) was 95.8 %. The results of this second autopsy-confirmed sample confirm that midbrain-to-brain ratios constitute reliable and clinically useful estimates of diagnostic midbrain atrophy in relation to PSP pathology. PMID:25805708

  2. The Consequences of Replicating in the Wrong Orientation: Bacterial Chromosome Duplication without an Active Replication Origin

    PubMed Central

    Dimude, Juachi U.; Stockum, Anna; Midgley-Smith, Sarah L.; Upton, Amy L.; Foster, Helen A.; Khan, Arshad; Saunders, Nigel J.; Retkute, Renata

    2015-01-01

    ABSTRACT Chromosome replication is regulated in all organisms at the assembly stage of the replication machinery at specific origins. In Escherichia coli, the DnaA initiator protein regulates the assembly of replication forks at oriC. This regulation can be undermined by defects in nucleic acid metabolism. In cells lacking RNase HI, replication initiates independently of DnaA and oriC, presumably at persisting R-loops. A similar mechanism was assumed for origin-independent synthesis in cells lacking RecG. However, recently we suggested that this synthesis initiates at intermediates resulting from replication fork fusions. Here we present data suggesting that in cells lacking RecG or RNase HI, origin-independent synthesis arises by different mechanisms, indicative of these two proteins having different roles in vivo. Our data support the idea that RNase HI processes R-loops, while RecG is required to process replication fork fusion intermediates. However, regardless of how origin-independent synthesis is initiated, a fraction of forks will proceed in an orientation opposite to normal. We show that the resulting head-on encounters with transcription threaten cell viability, especially if taking place in highly transcribed areas. Thus, despite their different functions, RecG and RNase HI are both important factors for maintaining replication control and orientation. Their absence causes severe replication problems, highlighting the advantages of the normal chromosome arrangement, which exploits a single origin to control the number of forks and their orientation relative to transcription, and a defined termination area to contain fork fusions. Any changes to this arrangement endanger cell cycle control, chromosome dynamics, and, ultimately, cell viability. PMID:26530381

  3. 22. View of Clark Fork Vehicle Bridge facing downwest side. ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    22. View of Clark Fork Vehicle Bridge facing down-west side. Looking at road deck and vertical laced channel. - Clark Fork Vehicle Bridge, Spanning Clark Fork River, serves Highway 200, Clark Fork, Bonner County, ID

  4. 11. View of Clark Fork Vehicle Bridge facing northwest. Southernmost ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    11. View of Clark Fork Vehicle Bridge facing northwest. Southernmost span. Plaque was originally located where striped traffic sign is posted. - Clark Fork Vehicle Bridge, Spanning Clark Fork River, serves Highway 200, Clark Fork, Bonner County, ID

  5. 19. View of Clark Fork Vehicle Bridge facing north. Looking ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    19. View of Clark Fork Vehicle Bridge facing north. Looking at north abutment and underside of northernmost span. - Clark Fork Vehicle Bridge, Spanning Clark Fork River, serves Highway 200, Clark Fork, Bonner County, ID

  6. 20. View of Clark Fork Vehicle Bridge facing up. Looking ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    20. View of Clark Fork Vehicle Bridge facing up. Looking at understructure of northernmost span. - Clark Fork Vehicle Bridge, Spanning Clark Fork River, serves Highway 200, Clark Fork, Bonner County, ID

  7. 8. View of Clark Fork Vehicle Bridge facing southwest. Looking ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    8. View of Clark Fork Vehicle Bridge facing southwest. Looking at understructure of northernmost span. - Clark Fork Vehicle Bridge, Spanning Clark Fork River, serves Highway 200, Clark Fork, Bonner County, ID

  8. 12. View of Clark Fork Vehicle Bridge facing south. Approach ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    12. View of Clark Fork Vehicle Bridge facing south. Approach from the north road. Plaque was originally located where striped traffic sign is posted. - Clark Fork Vehicle Bridge, Spanning Clark Fork River, serves Highway 200, Clark Fork, Bonner County, ID

  9. 21. View of Clark Fork Vehicle Bridge facing west. Looking ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    21. View of Clark Fork Vehicle Bridge facing west. Looking at bridge deck, guard rail, juncture of two bridge spans. - Clark Fork Vehicle Bridge, Spanning Clark Fork River, serves Highway 200, Clark Fork, Bonner County, ID

  10. 18. View of Clark Fork Vehicle Bridge facing north. Looking ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    18. View of Clark Fork Vehicle Bridge facing north. Looking at north concrete abutment and timber stringers. - Clark Fork Vehicle Bridge, Spanning Clark Fork River, serves Highway 200, Clark Fork, Bonner County, ID

  11. High-resolution genomic assays provide insight into the division of labor between TLS and HDR in mammalian replication of damaged DNA.

    PubMed

    Livneh, Zvi; Cohen, Isadora S; Paz-Elizur, Tamar; Davidovsky, Dana; Carmi, Dalit; Swain, Umakanta; Mirlas-Neisberg, Nataly

    2016-08-01

    The multitude of DNA lesions that continuously form in DNA cannot all be detected and removed prior to replication. Thus, encounters of the replication fork with DNA damage become inevitable. Such encounters inhibit fork progression, leading to replication fork arrest or to replication re-priming downstream of the damage site. Either of these events will result in the formation of gap-lesion structures, in which a damaged base is located in a single stranded stretch of DNA, that is vulnerable to subsequent nicking. The double strand break that would ensue if ssDNA becomes nicked constitutes escalation of the damage from nucleotide(s)-specific to chromosomal scale. Cells employ two universal DNA damage tolerance (DDT) strategies to resolve these situations, by converting the gap-lesion structures into dsDNA without repairing the damage. The first is translesion DNA synthesis (TLS), in which a specialized low-fidelity DNA polymerase inserts a nucleotide opposite the damaged one. TLS is inherently mutagenic, due to the miscoding nature of most damaged nucleotides. The second strategy is homology-dependent repair (HDR), which relies on the presence of an identical intact sister chromatid. The molecular mechanisms that regulate the division of labor between these pathways are poorly understood. This review focuses on the balance between TLS and HDR in mammalian cells, discussing recent findings that were made possible thanks to newly developed high resolution genomic assays, and highlighting the role of the DNA lesion's properties in DDT pathway choice. PMID:27262613

  12. Inhibition of Histone Deacetylase 3 Causes Replication Stress in Cutaneous T Cell Lymphoma

    PubMed Central

    Wells, Christina E.; Bhaskara, Srividya; Stengel, Kristy R.; Zhao, Yue; Sirbu, Bianca; Chagot, Benjamin; Cortez, David; Khabele, Dineo; Chazin, Walter J.; Cooper, Andrew; Jacques, Vincent; Rusche, James; Eischen, Christine M.; McGirt, Laura Y.; Hiebert, Scott W.

    2013-01-01

    Given the fundamental roles of histone deacetylases (HDACs) in the regulation of DNA repair, replication, transcription and chromatin structure, it is fitting that therapies targeting HDAC activities are now being explored as anti-cancer agents. In fact, two histone deacetylase inhibitors (HDIs), SAHA and Depsipeptide, are FDA approved for single-agent treatment of refractory cutaneous T cell lymphoma (CTCL). An important target of these HDIs, histone deacetylase 3 (HDAC3), regulates processes such as DNA repair, metabolism, and tumorigenesis through the regulation of chromatin structure and gene expression. Here we show that HDAC3 inhibition using a first in class selective inhibitor, RGFP966, resulted in decreased cell growth in CTCL cell lines due to increased apoptosis that was associated with DNA damage and impaired S phase progression. Through isolation of proteins on nascent DNA (iPOND), we found that HDAC3 was associated with chromatin and is present at and around DNA replication forks. DNA fiber labeling analysis showed that inhibition of HDAC3 resulted in a significant reduction in DNA replication fork velocity within the first hour of drug treatment. These results suggest that selective inhibition of HDAC3 could be useful in treatment of CTCL by disrupting DNA replication of the rapidly cycling tumor cells, ultimately leading to cell death. PMID:23894374

  13. 21 CFR 882.1525 - Tuning fork.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Tuning fork. 882.1525 Section 882.1525 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES NEUROLOGICAL DEVICES Neurological Diagnostic Devices § 882.1525 Tuning fork. (a) Identification. A tuning fork is a mechanical device...

  14. Mechanism and Physiological Significance of Programmed Replication Termination

    PubMed Central

    Bastia, Deepak; Zaman, Shamsu

    2014-01-01

    Replication forks in both prokaryotic and eukaryotic systems pause at random sites due to depletion of dNTP pools, DNA damage, tight binding nonhistone proteins or unusual DNA sequences and/or structures, in a mostly non-polar fashion. However there is also physiologically programmed replication termination at sequence-specific authentic replication termini. Here, the structure and functions of programmed replication termini, their mechanism of action and their diverse physiological functions in prokaryotes and eukaryotes have been reviewed. PMID:24811316

  15. Long Range Interaction Between Protein Complexes in DNA Controls Replication and Cell Cycle Progression:. the Double Helix and Microtubules Behave like Elastically Braced Strings

    NASA Astrophysics Data System (ADS)

    Matsson, L.

    2001-09-01

    A nonstationary interaction model, that controls the gross behaviour of DNA replication and cell cycle progression, is derived in terms of manydody physics in a chemically open T cell. The model predicts a long range force F(φ) = - (κ/2) φ(1 - φ /N)(2 - φ /N) between the origin recognition complexes (ORCs) bound by DNA, φ being the number of ORCs, N the threshold for initiation, and κ the compressibility modulus in the lattice of ORCs which behaves like an elastically braced string. Initiation of DNA replication is induced by a switch of sign of F, from attraction (-) and assembly in the G1 phase (0 < φ < N), to repulsion (+) and partial disassembly in the S phase (N < φ < 2N), with release of licensing factors from pre-replication complexes (pre-RCs) and prevention of re-replication. Termination of replication is due to a vanishing of F at φ = 2N, when all primed replicons have been duplicated once, and F(0) = 0 corresponds to a resting cell in absence of a driving force at φ = 0. The switch of sign of F at φ = N also explains the dynamic instability in growing microtubules (MTs), as well as the switch in the interleukin-2 (IL2) interaction with its receptor in late G1, at the R-point, after which a T cell proceeds to replication without further exposure to IL2. Shape, slope and scale of the response curves derived agree well with data from dividing T cells and polymerizing MTs, the variable length of which is due to a nonlinear dependence on initial concentrations of guanosine-triphosphate (GTP) and tubulin dimers.

  16. Single-molecule analysis of DNA replication reveals novel features in the divergent eukaryotes Leishmania and Trypanosoma brucei versus mammalian cells

    PubMed Central

    Stanojcic, Slavica; Sollelis, Lauriane; Kuk, Nada; Crobu, Lucien; Balard, Yves; Schwob, Etienne; Bastien, Patrick; Pagès, Michel; Sterkers, Yvon

    2016-01-01

    Leishmania and Trypanosoma are unicellular parasites that possess markedly original biological features as compared to other eukaryotes. The Leishmania genome displays a constitutive ‘mosaic aneuploidy’, whereas in Trypanosoma brucei, the megabase-sized chromosomes are diploid. We accurately analysed DNA replication parameters in three Leishmania species and Trypanosoma brucei as well as mouse embryonic fibroblasts (MEF). Active replication origins were visualized at the single molecule level using DNA molecular combing. More than one active origin was found on most DNA fibres, showing that the chromosomes are replicated from multiple origins. Inter-origin distances (IODs) were measured and found very large in trypanosomatids: the mean IOD was 160 kb in T. brucei and 226 kb in L. mexicana. Moreover, the progression of replication forks was faster than in any other eukaryote analyzed so far (mean velocity 1.9 kb/min in T. brucei and 2.4–2.6 kb/min in Leishmania). The estimated total number of active DNA replication origins in trypanosomatids is ~170. Finally, 14.4% of unidirectional replication forks were observed in T. brucei, in contrast to 1.5–1.7% in Leishmania and 4% in MEF cells. The biological significance of these original features is discussed. PMID:26976742

  17. Do sister forks of bidirectionally growing replicons proceed at unequal rates

    SciTech Connect

    Dubey, D.D.; Raman, R.

    1987-02-01

    DNA fibre autoradiography in different tissues of the rodents Bandicota bengalensis and Nesokia indica reveals a high frequency of such bidirectionally replicating replicons whose sister hot tracks are of unequal size. These results suggest intrarepliconic difference in the rates of fork migration in the two directions.

  18. Structural basis for DNA binding by replication initiator Mcm10

    SciTech Connect

    Warren, Eric M.; Vaithiyalingam, Sivaraja; Haworth, Justin; Greer, Briana; Bielinsky, Anja-Katrin; Chazin, Walter J.; Eichman, Brandt F.

    2009-06-30

    Mcm10 is an essential eukaryotic DNA replication protein required for assembly and progression of the replication fork. The highly conserved internal domain (Mcm10-ID) has been shown to physically interact with single-stranded (ss) DNA, DNA polymerase alpha, and proliferating cell nuclear antigen (PCNA). The crystal structure of Xenopus laevis Mcm10-ID presented here reveals a DNA binding architecture composed of an oligonucleotide/oligosaccharide-fold followed in tandem by a variant and highly basic zinc finger. NMR chemical shift perturbation and mutational studies of DNA binding activity in vitro reveal how Mcm10 uses this unique surface to engage ssDNA. Corresponding mutations in Saccharomyces cerevisiae result in increased sensitivity to replication stress, demonstrating the functional importance of DNA binding by this region of Mcm10 to replication. In addition, mapping Mcm10 mutations known to disrupt PCNA, polymerase alpha, and DNA interactions onto the crystal structure provides insight into how Mcm10 might coordinate protein and DNA binding within the replisome.

  19. Timeless links replication termination to mitotic kinase activation.

    PubMed

    Dheekollu, Jayaraju; Wiedmer, Andreas; Hayden, James; Speicher, David; Gotter, Anthony L; Yen, Tim; Lieberman, Paul M

    2011-01-01

    The mechanisms that coordinate the termination of DNA replication with progression through mitosis are not completely understood. The human Timeless protein (Tim) associates with S phase replication checkpoint proteins Claspin and Tipin, and plays an important role in maintaining replication fork stability at physical barriers, like centromeres, telomeres and ribosomal DNA repeats, as well as at termination sites. We show here that human Tim can be isolated in a complex with mitotic entry kinases CDK1, Auroras A and B, and Polo-like kinase (Plk1). Plk1 bound Tim directly and colocalized with Tim at a subset of mitotic structures in M phase. Tim depletion caused multiple mitotic defects, including the loss of sister-chromatid cohesion, loss of mitotic spindle architecture, and a failure to exit mitosis. Tim depletion caused a delay in mitotic kinase activity in vivo and in vitro, as well as a reduction in global histone H3 S10 phosphorylation during G2/M phase. Tim was also required for the recruitment of Plk1 to centromeric DNA and formation of catenated DNA structures at human centromere alpha satellite repeats. Taken together, these findings suggest that Tim coordinates mitotic kinase activation with termination of DNA replication. PMID:21573113

  20. Perturbation of DNA replication and cell cycle progression by commonly used ( sup 3 H)thymidine labeling protocols

    SciTech Connect

    Hoy, C.A.; Lewis, E.D.; Schimke, R.T. )

    1990-04-01

    The effect of tritiated thymidine incorporation on DNA replication was studied in Chinese hamster ovary cells. Rapidly eluting (small) DNA from cells labeled with 2 microCi of ({sup 3}H)thymidine per ml (200 microCi/mmol) for 60 min matured to a large nonelutable size within approximately 2 to 4 h, as measured by the alkaline elution technique. However, DNA from cells exposed to 10 microCi of ({sup 3}H)thymidine per ml (66 microCi/mmol) was more rapidly eluting initially and did not mature to a nonelutable size during subsequent incubation. Semiconservative DNA replication measured by cesium chloride gradient analysis of bromodeoxyuridine-substituted DNA was also found to be affected by the final specific activity of the ({sup 3}H)thymidine used in the labeling protocol. Dramatic cell cycle perturbations accompanied these effects on DNA replication, suggesting that labeling protocols commonly used to study DNA metabolism produce aberrant DNA replication and subsequent cell cycle perturbations.

  1. 5. View of Clark Fork Vehicle Bridge facing east. Bridge ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    5. View of Clark Fork Vehicle Bridge facing east. Bridge from south shore of Clark Fork River-southernmost span. 1900-era Northern Pacific Railway Bridge in background. - Clark Fork Vehicle Bridge, Spanning Clark Fork River, serves Highway 200, Clark Fork, Bonner County, ID

  2. Ctf4 Links DNA Replication with Sister Chromatid Cohesion Establishment by Recruiting the Chl1 Helicase to the Replisome.

    PubMed

    Samora, Catarina P; Saksouk, Julie; Goswami, Panchali; Wade, Ben O; Singleton, Martin R; Bates, Paul A; Lengronne, Armelle; Costa, Alessandro; Uhlmann, Frank

    2016-08-01

    DNA replication during S phase is accompanied by establishment of sister chromatid cohesion to ensure faithful chromosome segregation. The Eco1 acetyltransferase, helped by factors including Ctf4 and Chl1, concomitantly acetylates the chromosomal cohesin complex to stabilize its cohesive links. Here we show that Ctf4 recruits the Chl1 helicase to the replisome via a conserved interaction motif that Chl1 shares with GINS and polymerase α. We visualize recruitment by EM analysis of a reconstituted Chl1-Ctf4-GINS assembly. The Chl1 helicase facilitates replication fork progression under conditions of nucleotide depletion, partly independently of Ctf4 interaction. Conversely, Ctf4 interaction, but not helicase activity, is required for Chl1's role in sister chromatid cohesion. A physical interaction between Chl1 and the cohesin complex during S phase suggests that Chl1 contacts cohesin to facilitate its acetylation. Our results reveal how Ctf4 forms a replisomal interaction hub that coordinates replication fork progression and sister chromatid cohesion establishment. PMID:27397686

  3. Crystallization and preliminary X-ray diffraction analysis of the Bacillus subtilis replication termination protein in complex with the 37-base-pair TerI-binding site

    SciTech Connect

    Vivian, J. P.; Porter, C.; Wilce, J. A.; Wilce, M. C. J.

    2006-11-01

    A preparation of replication terminator protein (RTP) of B. subtilis and a 37-base-pair TerI sequence (comprising two binding sites for RTP) has been purified and crystallized. The replication terminator protein (RTP) of Bacillus subtilis binds to specific DNA sequences that halt the progression of the replisome in a polar manner. These terminator complexes flank a defined region of the chromosome into which they allow replication forks to enter but not exit. Forcing the fusion of replication forks in a specific zone is thought to allow the coordination of post-replicative processes. The functional terminator complex comprises two homodimers each of 29 kDa bound to overlapping binding sites. A preparation of RTP and a 37-base-pair TerI sequence (comprising two binding sites for RTP) has been purified and crystallized. A data set to 3.9 Å resolution with 97.0% completeness and an R{sub sym} of 12% was collected from a single flash-cooled crystal using synchrotron radiation. The diffraction data are consistent with space group P622, with unit-cell parameters a = b = 118.8, c = 142.6 Å.

  4. The Fanconi Anemia Pathway Maintains Genome Stability by Coordinating Replication and Transcription

    PubMed Central

    Schwab, Rebekka A.; Nieminuszczy, Jadwiga; Shah, Fenil; Langton, Jamie; Lopez Martinez, David; Liang, Chih-Chao; Cohn, Martin A.; Gibbons, Richard J.; Deans, Andrew J.; Niedzwiedz, Wojciech

    2015-01-01

    Summary DNA replication stress can cause chromosomal instability and tumor progression. One key pathway that counteracts replication stress and promotes faithful DNA replication consists of the Fanconi anemia (FA) proteins. However, how these proteins limit replication stress remains largely elusive. Here we show that conflicts between replication and transcription activate the FA pathway. Inhibition of transcription or enzymatic degradation of transcription-associated R-loops (DNA:RNA hybrids) suppresses replication fork arrest and DNA damage occurring in the absence of a functional FA pathway. Furthermore, we show that simple aldehydes, known to cause leukemia in FA-deficient mice, induce DNA:RNA hybrids in FA-depleted cells. Finally, we demonstrate that the molecular mechanism by which the FA pathway limits R-loop accumulation requires FANCM translocase activity. Failure to activate a response to physiologically occurring DNA:RNA hybrids may critically contribute to the heightened cancer predisposition and bone marrow failure of individuals with mutated FA proteins. PMID:26593718

  5. Direct non transcriptional role of NF-Y in DNA replication.

    PubMed

    Benatti, Paolo; Belluti, Silvia; Miotto, Benoit; Neusiedler, Julia; Dolfini, Diletta; Drac, Marjorie; Basile, Valentina; Schwob, Etienne; Mantovani, Roberto; Blow, J Julian; Imbriano, Carol

    2016-04-01

    NF-Y is a heterotrimeric transcription factor, which plays a pioneer role in the transcriptional control of promoters containing the CCAAT-box, among which genes involved in cell cycle regulation, apoptosis and DNA damage response. The knock-down of the sequence-specific subunit NF-YA triggers defects in S-phase progression, which lead to apoptotic cell death. Here, we report that NF-Y has a critical function in DNA replication progression, independent from its transcriptional activity. NF-YA colocalizes with early DNA replication factories, its depletion affects the loading of replisome proteins to DNA, among which Cdc45, and delays the passage from early to middle-late S phase. Molecular combing experiments are consistent with a role for NF-Y in the control of fork progression. Finally, we unambiguously demonstrate a direct non-transcriptional role of NF-Y in the overall efficiency of DNA replication, specifically in the DNA elongation process, using a Xenopus cell-free system. Our findings broaden the activity of NF-Y on a DNA metabolism other than transcription, supporting the existence of specific TFs required for proper and efficient DNA replication. PMID:26732297

  6. 16 CFR 1512.13 - Requirements for front fork.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... REGULATIONS REQUIREMENTS FOR BICYCLES Regulations § 1512.13 Requirements for front fork. The front fork shall... fork test, § 1512.18(k)(1), without visible evidence of fracture. Sidewalk bicycles need not meet...

  7. 16 CFR 1512.13 - Requirements for front fork.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... REGULATIONS REQUIREMENTS FOR BICYCLES Regulations § 1512.13 Requirements for front fork. The front fork shall... fork test, § 1512.18(k)(1), without visible evidence of fracture. Sidewalk bicycles need not meet...

  8. 16 CFR 1512.13 - Requirements for front fork.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... REGULATIONS REQUIREMENTS FOR BICYCLES Regulations § 1512.13 Requirements for front fork. The front fork shall... fork test, § 1512.18(k)(1), without visible evidence of fracture. Sidewalk bicycles need not meet...

  9. 16 CFR 1512.13 - Requirements for front fork.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... REGULATIONS REQUIREMENTS FOR BICYCLES Regulations § 1512.13 Requirements for front fork. The front fork shall... fork test, § 1512.18(k)(1), without visible evidence of fracture. Sidewalk bicycles need not meet...

  10. 16 CFR 1512.13 - Requirements for front fork.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... REGULATIONS REQUIREMENTS FOR BICYCLES Regulations § 1512.13 Requirements for front fork. The front fork shall... fork test, § 1512.18(k)(1), without visible evidence of fracture. Sidewalk bicycles need not meet...

  11. Dicer in action at replication-transcription collisions

    PubMed Central

    Ren, Jie; Castel, Stephane E; Martienssen, Robert A

    2015-01-01

    Maintaining genome stability at sites of transcription and replication collision is a major challenge to cells. Recently, we have shown that in Schizosaccharomyces pombe Dicer promotes transcription termination at these sites, facilitating DNA replication and preventing replication fork restart that would otherwise occur via homologous recombination at the expense of genome stability. This novel role of Dicer could further explain its previously described role as a tumor suppressor. PMID:27308471

  12. Research reactor fork users manual

    SciTech Connect

    Hsue, S.T.; Menlove, H.O.; Bosler, G.E.; Dye, H.R.; Walton, G.; Halbig, J.K.; Siebelist, R.

    1993-11-01

    This manual describes the design features and operating characteristics of the research reactor fork. The system includes an ion chamber for gross gamma-ray counting, fission chambers for neutron counting, and a collimated high-resolution spectroscopy system for gamma-ray measurements. The neutron and ion chamber measurements are designed to be made underwater in spent-fuel cooling ponds. The neutron and gamma-ray detectors have been designed with high efficiencies to accommodate the relatively low emission rates of neutrons and gamma rays from low-burnup, research-type reactor fuel. This manual presents the design, performance, and test results for the system.

  13. Training Guidelines: Fork Lift Truck Driving.

    ERIC Educational Resources Information Center

    Ceramics, Glass, and Mineral Products Industry Training Board, Harrow (England).

    This manual of operative training guidelines for fork lift truck driving has been developed by the Ceramics, Glass and Mineral Products Industry Training Board (Great Britain) in consultation with a number of firms which manufacture fork lift trucks or which already have training--programs for their use. The purpose of the guidelines is to assist…

  14. Fifth-wheel fork truck adapter

    NASA Technical Reports Server (NTRS)

    Smith, P. L.

    1969-01-01

    Standard fifth wheel mounted on a rectangular steel structure adapted for use with a fork lift truck provides a fast, safe, and economical way of maneuvering semitrailers in close quarters at plants and warehouses. One operator can move and locate a semitrailer without dismounting from a fork lift truck.

  15. Dynamic look at DNA unwinding by a replicative helicase

    PubMed Central

    Lee, Seung-Jae; Syed, Salman; Enemark, Eric J.; Schuck, Stephen; Stenlund, Arne; Ha, Taekjip; Joshua-Tor, Leemor

    2014-01-01

    A prerequisite for DNA replication is the unwinding of duplex DNA catalyzed by a replicative hexameric helicase. Despite a growing body of research, key elements of helicase mechanism remain under substantial debate. In particular, the number of DNA strands encircled by the helicase ring during unwinding and the ring orientation at the replication fork completely contrast in contemporary mechanistic models. Here we use single-molecule and ensemble assays to address these questions for the papillomavirus E1 helicase. We find that E1 unwinds DNA with a strand-exclusion mechanism, with the N-terminal side of the helicase ring facing the replication fork. We show that E1 generates strikingly heterogeneous unwinding patterns stemming from varying degrees of repetitive movements, which is modulated by the DNA-binding domain. Together, our studies reveal previously unrecognized dynamic facets of replicative helicase unwinding mechanisms. PMID:24550505

  16. Dynamic look at DNA unwinding by a replicative helicase.

    PubMed

    Lee, Seung-Jae; Syed, Salman; Enemark, Eric J; Schuck, Stephen; Stenlund, Arne; Ha, Taekjip; Joshua-Tor, Leemor

    2014-03-01

    A prerequisite for DNA replication is the unwinding of duplex DNA catalyzed by a replicative hexameric helicase. Despite a growing body of research, key elements of helicase mechanism remain under substantial debate. In particular, the number of DNA strands encircled by the helicase ring during unwinding and the ring orientation at the replication fork completely contrast in contemporary mechanistic models. Here we use single-molecule and ensemble assays to address these questions for the papillomavirus E1 helicase. We find that E1 unwinds DNA with a strand-exclusion mechanism, with the N-terminal side of the helicase ring facing the replication fork. We show that E1 generates strikingly heterogeneous unwinding patterns stemming from varying degrees of repetitive movements, which is modulated by the DNA-binding domain. Together, our studies reveal previously unrecognized dynamic facets of replicative helicase unwinding mechanisms. PMID:24550505

  17. South Fork Holston River basin 1988 biomonitoring

    SciTech Connect

    Saylor, C.F.; Ahlstedt, S.A.

    1990-06-01

    There is concern over the effects of shifts in land use use practices on the aquatic fauna of streams in the South Fork Holston River basin in northwestern North Carolina and southwestern Virginia. Trout reproduction has noticeably declined in the Watauga River subbasin. The Watauga River and Elk River subbasins have been subjected to commercial and resort development. The Middle fork Holston River and the upper South Fork Holston River subbasins have been affected by agricultural and mining activities, respectively (Cox, 1986). To aid reclamation and management of the South Fork Holston basin, Tennessee Valley Authority (TVA) biologists conducted biomonitoring--including index of biotic integrity and macroinvertebrate sampling--on the Middle Fork Holston, South Fork Holston, Watauga, and Elk Rivers to assess cumulative impairment related to changes in habitat and pollutant loading in these subbasins. Biomonitoring can detect environmental degradation, help document problem areas, and assist in development of strategies for managing water quality. This report discusses the methods and materials and results of the biomonitoring of South Fork Holston River Basin. 13 refs., 5 figs., 12 tabs.

  18. 13. View of Clark Fork Vehicle Bridge facing south. Concrete ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    13. View of Clark Fork Vehicle Bridge facing south. Concrete barrier blocks access. Plaque was originally located where strioed traffic sign is posted at right. - Clark Fork Vehicle Bridge, Spanning Clark Fork River, serves Highway 200, Clark Fork, Bonner County, ID

  19. 14. View of Clark Fork Vehicle Bridge facing north. Approach ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    14. View of Clark Fork Vehicle Bridge facing north. Approach from the south. Concrete barrier blocks access. Plaque was originally located where striped traffic sign is posted at right. - Clark Fork Vehicle Bridge, Spanning Clark Fork River, serves Highway 200, Clark Fork, Bonner County, ID

  20. 24. View of one of the plaques from Clark Fork ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    24. View of one of the plaques from Clark Fork Vehicle Bridge. Presently located at the Bonner County Historical Museum in Sandpoint, Idaho. A plaque was attached at each end of the bridge. Only one remains. - Clark Fork Vehicle Bridge, Spanning Clark Fork River, serves Highway 200, Clark Fork, Bonner County, ID

  1. 23. View of Clark Fork Vehicle Bridge facing upwest side. ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    23. View of Clark Fork Vehicle Bridge facing up-west side. Looking at structural connection of top chord, vertical laced channel and diagonal bars. - Clark Fork Vehicle Bridge, Spanning Clark Fork River, serves Highway 200, Clark Fork, Bonner County, ID

  2. SMARCAL1 and replication stress

    PubMed Central

    Bansbach, Carol E; Cortez, David

    2010-01-01

    The SNF2 family of ATPases acts in the context of chromatin to regulate transcription, replication, repair and recombination. Defects in SNF2 genes cause many human diseases. For example, mutations in SMARCAL1 (also named HARP) cause Schimke immuno-osseous dysplasia (SIOD); a multi-system disorder characterized by growth defects, immune deficiencies, renal failure and other complex phenotypes. Several groups including ours recently identified SMARCAL1 as a replication stress response protein. Importantly, SMARCAL1 localizes to stalled replication forks and this localization of SMARCAL1 activity prevents DNA damage accumulation during DNA replication. We determined that SIOD-related SMARCAL1 mutants could not prevent replication-associated DNA damage in cells in which endogenous SMARCAL1 was silenced, establishing the first link between SIOD and a defect in a specific biological activity. Here, we also report that cells from patients with SIOD exhibit elevated levels of DNA damage that can be rescued by re-introduction of wild-type SMARCAL1. Our data suggest that loss of SMARCAL1 function in patients may cause DNA replication-associated genome instability that contributes to the pleiotropic phenotypes of SIOD. PMID:21327070

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

    NASA Astrophysics Data System (ADS)

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

    1994-07-01

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

  4. Mathematical modelling of eukaryotic DNA replication.

    PubMed

    Hyrien, Olivier; Goldar, Arach

    2010-01-01

    Eukaryotic DNA replication is a complex process. Replication starts at thousand origins that are activated at different times in S phase and terminates when converging replication forks meet. Potential origins are much more abundant than actually fire within a given S phase. The choice of replication origins and their time of activation is never exactly the same in any two cells. Individual origins show different efficiencies and different firing time probability distributions, conferring stochasticity to the DNA replication process. High-throughput microarray and sequencing techniques are providing increasingly huge datasets on the population-averaged spatiotemporal patterns of DNA replication in several organisms. On the other hand, single-molecule replication mapping techniques such as DNA combing provide unique information about cell-to-cell variability in DNA replication patterns. Mathematical modelling is required to fully comprehend the complexity of the chromosome replication process and to correctly interpret these data. Mathematical analysis and computer simulations have been recently used to model and interpret genome-wide replication data in the yeast Saccharomyces cerevisiae and Schizosaccharomyces pombe, in Xenopus egg extracts and in mammalian cells. These works reveal how stochasticity in origin usage confers robustness and reliability to the DNA replication process. PMID:20205354

  5. Maternal embryonic leucine zipper kinase (MELK) reduces replication stress in glioblastoma cells.

    PubMed

    Kig, Cenk; Beullens, Monique; Beke, Lijs; Van Eynde, Aleyde; Linders, Johannes T; Brehmer, Dirk; Bollen, Mathieu

    2013-08-16

    Maternal embryonic leucine zipper kinase (MELK) belongs to the subfamily of AMP-activated Ser/Thr protein kinases. The expression of MELK is very high in glioblastoma-type brain tumors, but it is not clear how this contributes to tumor growth. Here we show that the siRNA-mediated loss of MELK in U87 MG glioblastoma cells causes a G1/S phase cell cycle arrest accompanied by cell death or a senescence-like phenotype that can be rescued by the expression of siRNA-resistant MELK. This cell cycle arrest is mediated by an increased expression of p21(WAF1/CIP1), an inhibitor of cyclin-dependent kinases, and is associated with the hypophosphorylation of the retinoblastoma protein and the down-regulation of E2F target genes. The increased expression of p21 can be explained by the consecutive activation of ATM (ataxia telangiectasia mutated), Chk2, and p53. Intriguingly, the activation of p53 in MELK-deficient cells is not due to an increased stability of p53 but stems from the loss of MDMX (mouse double minute-X), an inhibitor of p53 transactivation. The activation of the ATM-Chk2 pathway in MELK-deficient cells is associated with the accumulation of DNA double-strand breaks during replication, as demonstrated by the appearance of γH2AX foci. Replication stress in these cells is also illustrated by an increased number of stalled replication forks and a reduced fork progression speed. Our data indicate that glioblastoma cells have elevated MELK protein levels to better cope with replication stress during unperturbed S phase. Hence, MELK inhibitors hold great potential for the treatment of glioblastomas as such or in combination with DNA-damaging therapies. PMID:23836907

  6. Chronic DNA Replication Stress Reduces Replicative Lifespan of Cells by TRP53-Dependent, microRNA-Assisted MCM2-7 Downregulation

    PubMed Central

    Bai, Gongshi; Smolka, Marcus B.; Schimenti, John C.

    2016-01-01

    Circumstances that compromise efficient DNA replication, such as disruptions to replication fork progression, cause a state known as DNA replication stress (RS). Whereas normally proliferating cells experience low levels of RS, excessive RS from intrinsic or extrinsic sources can trigger cell cycle arrest and senescence. Here, we report that a key driver of RS-induced senescence is active downregulation of the Minichromosome Maintenance 2–7 (MCM2-7) factors that are essential for replication origin licensing and which constitute the replicative helicase core. Proliferating cells produce high levels of MCM2-7 that enable formation of dormant origins that can be activated in response to acute, experimentally-induced RS. However, little is known about how physiological RS levels impact MCM2-7 regulation. We found that chronic exposure of primary mouse embryonic fibroblasts (MEFs) to either genetically-encoded or environmentally-induced RS triggered gradual MCM2-7 repression, followed by inhibition of replication and senescence that could be accelerated by MCM hemizygosity. The MCM2-7 reduction in response to RS is TRP53-dependent, and involves a group of Trp53-dependent miRNAs, including the miR-34 family, that repress MCM expression in replication-stressed cells before they undergo terminal cell cycle arrest. miR-34 ablation partially rescued MCM2-7 downregulation and genomic instability in mice with endogenous RS. Together, these data demonstrate that active MCM2-7 repression is a physiologically important mechanism for RS-induced cell cycle arrest and genome maintenance on an organismal level. PMID:26765334

  7. DNA replication meets genetic exchange: Chromosomal damage and its repair by homologous recombination

    PubMed Central

    Kuzminov, Andrei

    2001-01-01

    Proceedings of the National Academy of Sciences Colloquium on the roles of homologous recombination in DNA replication are summarized. Current findings in experimental systems ranging from bacteriophages to mammalian cell lines substantiate the idea that homologous recombination is a system supporting DNA replication when either the template DNA is damaged or the replication machinery malfunctions. There are several lines of supporting evidence: (i) DNA replication aggravates preexisting DNA damage, which then blocks subsequent replication; (ii) replication forks abandoned by malfunctioning replisomes become prone to breakage; (iii) mutants with malfunctioning replisomes or with elevated levels of DNA damage depend on homologous recombination; and (iv) homologous recombination primes DNA replication in vivo and can restore replication fork structures in vitro. The mechanisms of recombinational repair in bacteriophage T4, Escherichia coli, and Saccharomyces cerevisiae are compared. In vitro properties of the eukaryotic recombinases suggest a bigger role for single-strand annealing in the eukaryotic recombinational repair. PMID:11459990

  8. ATR inhibition rewires cellular signaling networks induced by replication stress.

    PubMed

    Wagner, Sebastian A; Oehler, Hannah; Voigt, Andrea; Dalic, Denis; Freiwald, Anja; Serve, Hubert; Beli, Petra

    2016-02-01

    The slowing down or stalling of replication forks is commonly known as replication stress and arises from multiple causes such as DNA lesions, nucleotide depletion, RNA-DNA hybrids, and oncogene activation. The ataxia telangiectasia and Rad3-related kinase (ATR) plays an essential role in the cellular response to replication stress and inhibition of ATR has emerged as therapeutic strategy for the treatment of cancers that exhibit high levels of replication stress. However, the cellular signaling induced by replication stress and the substrate spectrum of ATR has not been systematically investigated. In this study, we employed quantitative MS-based proteomics to define the cellular signaling after nucleotide depletion-induced replication stress and replication fork collapse following ATR inhibition. We demonstrate that replication stress results in increased phosphorylation of a subset of proteins, many of which are involved in RNA splicing and transcription and have previously not been associated with the cellular replication stress response. Furthermore, our data reveal the ATR-dependent phosphorylation following replication stress and discover novel putative ATR target sites on MCM6, TOPBP1, RAD51AP1, and PSMD4. We establish that ATR inhibition rewires cellular signaling networks induced by replication stress and leads to the activation of the ATM-driven double-strand break repair signaling. PMID:26572502

  9. SMARCAL1 maintains telomere integrity during DNA replication.

    PubMed

    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

  10. Cdc7 kinase mediates Claspin phosphorylation in DNA replication checkpoint.

    PubMed

    Kim, J M; Kakusho, N; Yamada, M; Kanoh, Y; Takemoto, N; Masai, H

    2008-05-29

    Cdc7 kinase is evolutionarily conserved and is involved in initiation and progression of DNA replication. However, roles of Cdc7 in checkpoint responses remain largely unknown. In this study, we show that deletion of the Cdc7 genes in mouse embryonic stem (ES) cells abrogates hydroxyurea (HU)- or UV-induced activation of Chk1. HU-induced Chk1 activation is also impaired in human cancer cell lines in which Cdc7 is depleted by siRNA, and Cdc7-depleted cells are more sensitive to HU treatment. In contrast, ATR and Rad17 are relocated to chromatin in these cells following HU treatment, indicating that stalled DNA replication forks are detected normally. Cdc7-depleted cells exhibit defects in chromatin association and phosphorylation of Claspin, suggesting that Cdc7 exerts its effect at least partially through Claspin. Consistent with this prediction, Cdc7 interacts with and phosphorylates Claspin. We propose that Cdc7 is required for activation of the ATR-Chk1 checkpoint pathway through regulation of Claspin. PMID:18084324

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

    PubMed

    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

  12. Claspin recruits Cdc7 kinase for initiation of DNA replication in human cells

    PubMed Central

    Yang, Chi-Chun; Suzuki, Masahiro; Yamakawa, Shiori; Uno, Syuzi; Ishii, Ai; Yamazaki, Satoshi; Fukatsu, Rino; Fujisawa, Ryo; Sakimura, Kenji; Tsurimoto, Toshiki; Masai, Hisao

    2016-01-01

    Claspin transmits replication stress signal from ATR to Chk1 effector kinase as a mediator. It also plays a role in efficient replication fork progression during normal growth. Here we have generated conditional knockout of Claspin and show that Claspin knockout mice are dead by E12.5 and Claspin knockout mouse embryonic fibroblast (MEF) cells show defect in S phase. Using the mutant cell lines, we report the crucial roles of the acidic patch (AP) near the C terminus of Claspin in initiation of DNA replication. Cdc7 kinase binds to AP and this binding is required for phosphorylation of Mcm. AP is involved also in intramolecular interaction with a N-terminal segment, masking the DNA-binding domain and a newly identified PIP motif, and Cdc7-mediated phosphorylation reduces the intramolecular interaction. Our results suggest a new role of Claspin in initiation of DNA replication during normal S phase through the recruitment of Cdc7 that facilitates phosphorylation of Mcm proteins. PMID:27401717

  13. Claspin recruits Cdc7 kinase for initiation of DNA replication in human cells.

    PubMed

    Yang, Chi-Chun; Suzuki, Masahiro; Yamakawa, Shiori; Uno, Syuzi; Ishii, Ai; Yamazaki, Satoshi; Fukatsu, Rino; Fujisawa, Ryo; Sakimura, Kenji; Tsurimoto, Toshiki; Masai, Hisao

    2016-01-01

    Claspin transmits replication stress signal from ATR to Chk1 effector kinase as a mediator. It also plays a role in efficient replication fork progression during normal growth. Here we have generated conditional knockout of Claspin and show that Claspin knockout mice are dead by E12.5 and Claspin knockout mouse embryonic fibroblast (MEF) cells show defect in S phase. Using the mutant cell lines, we report the crucial roles of the acidic patch (AP) near the C terminus of Claspin in initiation of DNA replication. Cdc7 kinase binds to AP and this binding is required for phosphorylation of Mcm. AP is involved also in intramolecular interaction with a N-terminal segment, masking the DNA-binding domain and a newly identified PIP motif, and Cdc7-mediated phosphorylation reduces the intramolecular interaction. Our results suggest a new role of Claspin in initiation of DNA replication during normal S phase through the recruitment of Cdc7 that facilitates phosphorylation of Mcm proteins. PMID:27401717

  14. FANCD2 Maintains Fork Stability in BRCA1/2-Deficient Tumors and Promotes Alternative End-Joining DNA Repair.

    PubMed

    Kais, Zeina; Rondinelli, Beatrice; Holmes, Amie; O'Leary, Colin; Kozono, David; D'Andrea, Alan D; Ceccaldi, Raphael

    2016-06-14

    BRCA1/2 proteins function in homologous recombination (HR)-mediated DNA repair and cooperate with Fanconi anemia (FA) proteins to maintain genomic integrity through replication fork stabilization. Loss of BRCA1/2 proteins results in DNA repair deficiency and replicative stress, leading to genomic instability and enhanced sensitivity to DNA-damaging agents. Recent studies have shown that BRCA1/2-deficient tumors upregulate Polθ-mediated alternative end-joining (alt-EJ) repair as a survival mechanism. Whether other mechanisms maintain genomic integrity upon loss of BRCA1/2 proteins is currently unknown. Here we show that BRCA1/2-deficient tumors also upregulate FANCD2 activity. FANCD2 is required for fork protection and fork restart in BRCA1/2-deficient tumors. Moreover, FANCD2 promotes Polθ recruitment at sites of damage and alt-EJ repair. Finally, loss of FANCD2 in BRCA1/2-deficient tumors enhances cell death. These results reveal a synthetic lethal relationship between FANCD2 and BRCA1/2, and they identify FANCD2 as a central player orchestrating DNA repair pathway choice at the replication fork. PMID:27264184

  15. Mutagenesis of ARS2 Domains To Assess Possible Roles in Cell Cycle Progression and MicroRNA and Replication-Dependent Histone mRNA Biogenesis

    PubMed Central

    O'Sullivan, Connor; Christie, Jennifer; Pienaar, Marcus; Gambling, Jake; Nickerson, Philip E. B.; Alford, Spencer C.; Chow, Robert L.

    2015-01-01

    ARS2 is a regulator of RNA polymerase II transcript processing through its role in the maturation of distinct nuclear cap-binding complex (CBC)-controlled RNA families. In this study, we examined ARS2 domain function in transcript processing. Structural modeling based on the plant ARS2 orthologue, SERRATE, revealed 2 previously uncharacterized domains in mammalian ARS2: an N-terminal domain of unknown function (DUF3546), which is also present in SERRATE, and an RNA recognition motif (RRM) that is present in metazoan ARS2 but not in plants. Both the DUF3546 and zinc finger domain (ZnF) were required for association with microRNA and replication-dependent histone mRNA. Mutations in the ZnF disrupted interaction with FLASH, a key component in histone pre-mRNA processing. Mutations targeting the Mid domain implicated it in DROSHA interaction and microRNA biogenesis. The unstructured C terminus was required for interaction with the CBC protein CBP20, while the RRM was required for cell cycle progression and for binding to FLASH. Together, our results support a bridging model in which ARS2 plays a central role in RNA recognition and processing through multiple protein and RNA interactions. PMID:26303529

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

    PubMed

    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

  17. Chromosome demise in the wake of ligase-deficient replication

    PubMed Central

    Kouzminova, Elena A.; Kuzminov, Andrei

    2012-01-01

    Summary Bacterial DNA ligases, NAD+-dependent enzymes, are distinct from eukaryotic ATP-dependent ligases, representing promising targets for broad-spectrum antimicrobials. Yet, the chromosomal consequences of ligase-deficient DNA replication, during which Okazaki fragments accumulate, are still unclear. Using ligA251(Ts), the strongest ligase mutant of Escherichia coli, we studied ligase-deficient DNA replication by genetic and physical approaches. Here we show that replication without ligase kills after a short resistance period. We found that double-strand break repair via RecA, RecBCD, RuvABC and RecG explains the transient resistance, whereas irreparable chromosomal fragmentation explains subsequent cell death. Remarkably, death is mostly prevented by elimination of linear DNA degradation activity of ExoV, suggesting that non-allelic double-strand breaks behind replication forks precipitate DNA degradation that enlarge them into allelic double-strand gaps. Marker frequency profiling of synchronized replication reveals stalling of ligase-deficient forks with subsequent degradation of the DNA synthesized without ligase. The mechanism that converts unsealed nicks behind replication forks first into repairable double-strand breaks and then into irreparable double-strand gaps may be behind lethality of any DNA damaging treatment. PMID:22582878

  18. Quantitative BrdU immunoprecipitation method demonstrates that Fkh1 and Fkh2 are rate-limiting activators of replication origins that reprogram replication timing in G1 phase.

    PubMed

    Peace, Jared M; Villwock, Sandra K; Zeytounian, John L; Gan, Yan; Aparicio, Oscar M

    2016-03-01

    The Saccharomyces cerevisiae Forkhead Box (FOX) proteins, Fkh1 and Fkh2, regulate diverse cellular processes including transcription, long-range DNA interactions during homologous recombination, and replication origin timing and long-range origin clustering. We hypothesized that, as stimulators of early origin activation, Fkh1 and Fkh2 abundance limits the rate of origin activation genome-wide. Existing methods, however, are not well-suited to quantitative, genome-wide measurements of origin firing between strains and conditions. To overcome this limitation, we developed qBrdU-seq, a quantitative method for BrdU incorporation analysis of replication dynamics, and applied it to show that overexpression of Fkh1 and Fkh2 advances the initiation timing of many origins throughout the genome resulting in a higher total level of origin initiations in early S phase. The higher initiation rate is accompanied by slower replication fork progression, thereby maintaining a normal length of S phase without causing detectable Rad53 checkpoint kinase activation. The advancement of origin firing time, including that of origins in heterochromatic domains, was established in late G1 phase, indicating that origin timing can be reset subsequently to origin licensing. These results provide novel insights into the mechanisms of origin timing regulation by identifying Fkh1 and Fkh2 as rate-limiting factors for origin firing that determine the ability of replication origins to accrue limiting factors and have the potential to reprogram replication timing late in G1 phase. PMID:26728715

  19. Replication Restart after Replication-Transcription Conflicts Requires RecA in Bacillus subtilis

    PubMed Central

    Million-Weaver, Samuel; Samadpour, Ariana Nakta

    2015-01-01

    ABSTRACT Efficient duplication of genomes depends on reactivation of replication forks outside the origin. Replication restart can be facilitated by recombination proteins, especially if single- or double-strand breaks form in the DNA. Each type of DNA break is processed by a distinct pathway, though both depend on the RecA protein. One common obstacle that can stall forks, potentially leading to breaks in the DNA, is transcription. Though replication stalling by transcription is prevalent, the nature of DNA breaks and the prerequisites for replication restart in response to these encounters remain unknown. Here, we used an engineered site-specific replication-transcription conflict to identify and dissect the pathways required for the resolution and restart of replication forks stalled by transcription in Bacillus subtilis. We found that RecA, its loader proteins RecO and AddAB, and the Holliday junction resolvase RecU are required for efficient survival and replication restart after conflicts with transcription. Genetic analyses showed that RecO and AddAB act in parallel to facilitate RecA loading at the site of the conflict but that they can each partially compensate for the other's absence. Finally, we found that RecA and either RecO or AddAB are required for the replication restart and helicase loader protein, DnaD, to associate with the engineered conflict region. These results suggest that conflicts can lead to both single-strand gaps and double-strand breaks in the DNA and that RecA loading and Holliday junction resolution are required for replication restart at regions of replication-transcription conflicts. IMPORTANCE Head-on conflicts between replication and transcription occur when a gene is expressed from the lagging strand. These encounters stall the replisome and potentially break the DNA. We investigated the necessary mechanisms for Bacillus subtilis cells to overcome a site-specific engineered conflict with transcription of a protein-coding gene

  20. Human Genome Replication Proceeds through Four Chromatin States

    PubMed Central

    Julienne, Hanna; Zoufir, Azedine; Audit, Benjamin; Arneodo, Alain

    2013-01-01

    Advances in genomic studies have led to significant progress in understanding the epigenetically controlled interplay between chromatin structure and nuclear functions. Epigenetic modifications were shown to play a key role in transcription regulation and genome activity during development and differentiation or in response to the environment. Paradoxically, the molecular mechanisms that regulate the initiation and the maintenance of the spatio-temporal replication program in higher eukaryotes, and in particular their links to epigenetic modifications, still remain elusive. By integrative analysis of the genome-wide distributions of thirteen epigenetic marks in the human cell line K562, at the 100 kb resolution of corresponding mean replication timing (MRT) data, we identify four major groups of chromatin marks with shared features. These states have different MRT, namely from early to late replicating, replication proceeds though a transcriptionally active euchromatin state (C1), a repressive type of chromatin (C2) associated with polycomb complexes, a silent state (C3) not enriched in any available marks, and a gene poor HP1-associated heterochromatin state (C4). When mapping these chromatin states inside the megabase-sized U-domains (U-shaped MRT profile) covering about 50% of the human genome, we reveal that the associated replication fork polarity gradient corresponds to a directional path across the four chromatin states, from C1 at U-domains borders followed by C2, C3 and C4 at centers. Analysis of the other genome half is consistent with early and late replication loci occurring in separate compartments, the former correspond to gene-rich, high-GC domains of intermingled chromatin states C1 and C2, whereas the latter correspond to gene-poor, low-GC domains of alternating chromatin states C3 and C4 or long C4 domains. This new segmentation sheds a new light on the epigenetic regulation of the spatio-temporal replication program in human and provides a

  1. Initial amplification of the HPV18 genome proceeds via two distinct replication mechanisms

    PubMed Central

    Orav, Marit; Geimanen, Jelizaveta; Sepp, Eva-Maria; Henno, Liisi; Ustav, Ene; Ustav, Mart

    2015-01-01

    Determining the mechanism of HPV18 replication is paramount for identifying possible drug targets against HPV infection. We used two-dimensional and three-dimensional gel electrophoresis techniques to identify replication intermediates arising during the initial amplification of HPV18 episomal genomes. We determined that the first rounds of HPV18 replication proceed via bidirectional theta structures; however, a notable accumulation of almost fully replicated HPV18 genomes indicates difficulties with the completion of theta replication. We also observed intermediates that were created by a second replication mechanism during the initial amplification of HPV18 genomes. The second replication mechanism does not utilize specific initiation or termination sequences and proceeds via a unidirectional replication fork. We suggest a significant role for the second replication mechanism during the initial replication of the HPV18 genome and propose that the second replication mechanism is recombination-dependent replication. PMID:26522968

  2. Initial amplification of the HPV18 genome proceeds via two distinct replication mechanisms.

    PubMed

    Orav, Marit; Geimanen, Jelizaveta; Sepp, Eva-Maria; Henno, Liisi; Ustav, Ene; Ustav, Mart

    2015-01-01

    Determining the mechanism of HPV18 replication is paramount for identifying possible drug targets against HPV infection. We used two-dimensional and three-dimensional gel electrophoresis techniques to identify replication intermediates arising during the initial amplification of HPV18 episomal genomes. We determined that the first rounds of HPV18 replication proceed via bidirectional theta structures; however, a notable accumulation of almost fully replicated HPV18 genomes indicates difficulties with the completion of theta replication. We also observed intermediates that were created by a second replication mechanism during the initial amplification of HPV18 genomes. The second replication mechanism does not utilize specific initiation or termination sequences and proceeds via a unidirectional replication fork. We suggest a significant role for the second replication mechanism during the initial replication of the HPV18 genome and propose that the second replication mechanism is recombination-dependent replication. PMID:26522968

  3. South Fork Latrine showing north and west sides, general view ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    South Fork Latrine showing north and west sides, general view to southeast - Fort McKinley, South Fork Latrine, West side of East Side Drive, approximately 225 feet south of Weymouth Way, Great Diamond Island, Portland, Cumberland County, ME

  4. South Fork Latrine, oblique view showing south and east sides; ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    South Fork Latrine, oblique view showing south and east sides; view northwest - Fort McKinley, South Fork Latrine, West side of East Side Drive, approximately 225 feet south of Weymouth Way, Great Diamond Island, Portland, Cumberland County, ME

  5. South Fork Latrine, east elevation showing structure in context, view ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    South Fork Latrine, east elevation showing structure in context, view west - Fort McKinley, South Fork Latrine, West side of East Side Drive, approximately 225 feet south of Weymouth Way, Great Diamond Island, Portland, Cumberland County, ME

  6. 6. Roaring Fork Motor Nature Trail, road view after stop ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    6. Roaring Fork Motor Nature Trail, road view after stop four. - Great Smoky Mountains National Park Roads & Bridges, Roaring Fork Motor Nature Trail, Between Cherokee Orchard Road & U.S. Route 321, Gatlinburg, Sevier County, TN

  7. 9. Roaring Fork Motor Nature Trail, Reagan House. Great ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    9. Roaring Fork Motor Nature Trail, Reagan House. - Great Smoky Mountains National Park Roads & Bridges, Roaring Fork Motor Nature Trail, Between Cherokee Orchard Road & U.S. Route 321, Gatlinburg, Sevier County, TN

  8. 8. Roaring Fork Motor Nature Trail, handbuilt rock pile. ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    8. Roaring Fork Motor Nature Trail, hand-built rock pile. - Great Smoky Mountains National Park Roads & Bridges, Roaring Fork Motor Nature Trail, Between Cherokee Orchard Road & U.S. Route 321, Gatlinburg, Sevier County, TN

  9. 5. Roaring Fork Motor Nature Trail, vista at stop three. ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    5. Roaring Fork Motor Nature Trail, vista at stop three. - Great Smoky Mountains National Park Roads & Bridges, Roaring Fork Motor Nature Trail, Between Cherokee Orchard Road & U.S. Route 321, Gatlinburg, Sevier County, TN

  10. 1. Roaring Fork Motor Nature Trail, entrance sign. Great ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    1. Roaring Fork Motor Nature Trail, entrance sign. - Great Smoky Mountains National Park Roads & Bridges, Roaring Fork Motor Nature Trail, Between Cherokee Orchard Road & U.S. Route 321, Gatlinburg, Sevier County, TN

  11. 3. Roaring Fork Motor Nature Trail, view between second and ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    3. Roaring Fork Motor Nature Trail, view between second and third stops - Great Smoky Mountains National Park Roads & Bridges, Roaring Fork Motor Nature Trail, Between Cherokee Orchard Road & U.S. Route 321, Gatlinburg, Sevier County, TN

  12. 2. Roaring Fork Motor Nature Trail, road view before first ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    2. Roaring Fork Motor Nature Trail, road view before first stop. - Great Smoky Mountains National Park Roads & Bridges, Roaring Fork Motor Nature Trail, Between Cherokee Orchard Road & U.S. Route 321, Gatlinburg, Sevier County, TN

  13. 7. Roaring Fork Motor Nature Trail, rocks along edge of ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    7. Roaring Fork Motor Nature Trail, rocks along edge of road. - Great Smoky Mountains National Park Roads & Bridges, Roaring Fork Motor Nature Trail, Between Cherokee Orchard Road & U.S. Route 321, Gatlinburg, Sevier County, TN

  14. Roaring Fork Motor Nature Trail, Title Sheet Great Smoky ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    Roaring Fork Motor Nature Trail, Title Sheet - Great Smoky Mountains National Park Roads & Bridges, Roaring Fork Motor Nature Trail, Between Cherokee Orchard Road & U.S. Route 321, Gatlinburg, Sevier County, TN

  15. 12. Roaring Fork Motor Nature Trail, place of a thousand ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    12. Roaring Fork Motor Nature Trail, place of a thousand drips, view from road. - Great Smoky Mountains National Park Roads & Bridges, Roaring Fork Motor Nature Trail, Between Cherokee Orchard Road & U.S. Route 321, Gatlinburg, Sevier County, TN

  16. 11. Roaring Fork Motor Nature Trail, boulders along road after ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    11. Roaring Fork Motor Nature Trail, boulders along road after stop 13. - Great Smoky Mountains National Park Roads & Bridges, Roaring Fork Motor Nature Trail, Between Cherokee Orchard Road & U.S. Route 321, Gatlinburg, Sevier County, TN

  17. 15. INSIDE VIEW OF FLUME, LOOKING DOWNSTREAM, LEFT FORK TO ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    15. INSIDE VIEW OF FLUME, LOOKING DOWNSTREAM, LEFT FORK TO SETTLING BASIN, SHOWING RIGHT FORK WITH GATE IN PLACE AND A FEW NEEDLES IN PLACE - Electron Hydroelectric Project, Along Puyallup River, Electron, Pierce County, WA

  18. Retained garden fork following cranial stab injury

    PubMed Central

    Gonya, Sonwabile; Mbatha, Andile; Moyeni, Nondabula; Enicker, Basil

    2016-01-01

    Retained garden fork is a rare complication of penetrating cranial trauma. Retained knife blade is the most commonly reported presentation. We report an unusual case of a 30-year-old male patient treated at our institution, who presented with a retained garden fork following a stab to the head, with no associated neurological deficits. Computerized tomographic scan of the brain was performed preoperatively to assess the trajectory of the weapon and parenchymal injury. A craniectomy was performed to facilitate removal of the weapon in the operating theatre under general anaesthesia. Intravenous prophylactic antibiotics were administered pre- and postoperatively to prevent septic complications. The patient recovered well and was discharged home. PMID:26747398

  19. 27 CFR 9.113 - North Fork of Long Island.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 27 Alcohol, Tobacco Products and Firearms 1 2013-04-01 2013-04-01 false North Fork of Long Island... North Fork of Long Island. (a) Name. The name of the viticultural area described in this section is “North Fork of Long Island.” (b) Approved maps. The appropriate maps for determining the boundaries...

  20. 27 CFR 9.113 - North Fork of Long Island.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 27 Alcohol, Tobacco Products and Firearms 1 2010-04-01 2010-04-01 false North Fork of Long Island... North Fork of Long Island. (a) Name. The name of the viticultural area described in this section is “North Fork of Long Island.” (b) Approved maps. The appropriate maps for determining the boundaries...

  1. 27 CFR 9.113 - North Fork of Long Island.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 27 Alcohol, Tobacco Products and Firearms 1 2011-04-01 2011-04-01 false North Fork of Long Island... North Fork of Long Island. (a) Name. The name of the viticultural area described in this section is “North Fork of Long Island.” (b) Approved maps. The appropriate maps for determining the boundaries...

  2. 27 CFR 9.113 - North Fork of Long Island.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 27 Alcohol, Tobacco Products and Firearms 1 2012-04-01 2012-04-01 false North Fork of Long Island... North Fork of Long Island. (a) Name. The name of the viticultural area described in this section is “North Fork of Long Island.” (b) Approved maps. The appropriate maps for determining the boundaries...

  3. 27 CFR 9.113 - North Fork of Long Island.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 27 Alcohol, Tobacco Products and Firearms 1 2014-04-01 2014-04-01 false North Fork of Long Island... North Fork of Long Island. (a) Name. The name of the viticultural area described in this section is “North Fork of Long Island.” (b) Approved maps. The appropriate maps for determining the boundaries...

  4. Genomics Analysis of Replicative Helicase DnaB Sequences in Proteobacteria

    PubMed Central

    Poggi, Silvana; Chandra, Sathees B.

    2014-01-01

    Replicative Helicase DnaB interacts with DnaA, DnaC, DnaG, and DNA polymerase III to commence replication, increase the movement rate of the replication fork, and to assemble part of the primosome. The formation of the replication fork is limited by the ability to load DnaB to the DNA, thus DnaB has shown to be vital to a large extent. In the absence of DnaB, the replication fork is not maintained and in a state of inactivity the replication fork degrades and collapses. To further understand importance of this enzyme from an evolutionary perspective, a genomic analysis DnaB protein sequences, chosen from five Proteobacteria subclasses was performed. Our analysis indicates that, DnaB replicative helicases of Alphaproteobacteria and Epsilonproteobacteria have diverged at an earlier stage from Betaproteobacteria, Deltaproteobacteria and Gammaproteobacteria as well as from one another. Our results were further supported, when we reanalyzed and reconstructed the phylogenetic tree after the inclusion of sequences from Actinobacteria and Firmicute phylum. In addition, Betaproteobacteria, Deltaproteobacteria, and Gammaproteobacteria appear to share a closer common ancestor than from the other two subclasses. The Dot-plot analysis indicated that, the region between amino acid residues 320 to 400 was strongly conserved among all five subclasses. PMID:25395727

  5. Single molecule analysis of Trypanosoma brucei DNA replication dynamics

    PubMed Central

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

    2015-01-01

    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

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

    PubMed

    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

  7. Replication stalling and heteroduplex formation within CAG/CTG trinucleotide repeats by mismatch repair.

    PubMed

    Viterbo, David; Michoud, Grégoire; Mosbach, Valentine; Dujon, Bernard; Richard, Guy-Franck

    2016-06-01

    Trinucleotide repeat expansions are responsible for at least two dozen neurological disorders. Mechanisms leading to these large expansions of repeated DNA are still poorly understood. It was proposed that transient stalling of the replication fork by the repeat tract might trigger slippage of the newly-synthesized strand over its template, leading to expansions or contractions of the triplet repeat. However, such mechanism was never formally proven. Here we show that replication fork pausing and CAG/CTG trinucleotide repeat instability are not linked, stable and unstable repeats exhibiting the same propensity to stall replication forks when integrated in a yeast natural chromosome. We found that replication fork stalling was dependent on the integrity of the mismatch-repair system, especially the Msh2p-Msh6p complex, suggesting that direct interaction of MMR proteins with secondary structures formed by trinucleotide repeats in vivo, triggers replication fork pauses. We also show by chromatin immunoprecipitation that Msh2p is enriched at trinucleotide repeat tracts, in both stable and unstable orientations, this enrichment being dependent on MSH3 and MSH6. Finally, we show that overexpressing MSH2 favors the formation of heteroduplex regions, leading to an increase in contractions and expansions of CAG/CTG repeat tracts during replication, these heteroduplexes being dependent on both MSH3 and MSH6. These heteroduplex regions were not detected when a mutant msh2-E768A gene in which the ATPase domain was mutated was overexpressed. Our results unravel two new roles for mismatch-repair proteins: stabilization of heteroduplex regions and transient blocking of replication forks passing through such repeats. Both roles may involve direct interactions between MMR proteins and secondary structures formed by trinucleotide repeat tracts, although indirect interactions may not be formally excluded. PMID:27045900

  8. 21 CFR 882.1525 - Tuning fork.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Tuning fork. 882.1525 Section 882.1525 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES... and to test for vibratory sense. (b) Classification. Class I (general controls). The device is...

  9. 21 CFR 882.1525 - Tuning fork.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Tuning fork. 882.1525 Section 882.1525 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES... is a mechanical device which resonates at a given frequency and is used to diagnose hearing...

  10. Development of the Pintle Release Fork Mechanism

    SciTech Connect

    BOGER, R.M.; DALE, R.

    1999-08-27

    An improved method of attachment of the pintle to the piston in the universal sampler is being developed. The mechanism utilizes a forked release disk which captures two balls in a cavity formed by a hole in the piston and a groove in the pintle rod.

  11. Monitoring S phase progression globally and locally using BrdU incorporation in TK+ yeast strains

    PubMed Central

    Lengronne, Armelle; Pasero, Philippe; Bensimon, Aaron; Schwob, Etienne

    2001-01-01

    Eukaryotic chromosome replication is initiated from numerous origins and its activation is temporally controlled by cell cycle and checkpoint mechanisms. Yeast has been very useful in defining the genetic elements required for initiation of DNA replication, but simple and precise tools to monitor S phase progression are lacking in this model organism. Here we describe a TK+ yeast strain and conditions that allow incorporation of exogenous BrdU into genomic DNA, along with protocols to detect the sites of DNA synthesis in yeast nuclei or on combed DNA molecules. S phase progression is monitored by quantification of BrdU in total yeast DNA or on individual chromosomes. Using these tools we show that yeast chromosomes replicate synchronously and that DNA synthesis occurs at discrete subnuclear foci. Analysis of BrdU signals along single DNA molecules from hydroxyurea-arrested cells reveals that replication forks stall 8–9 kb from origins that are placed 46 kb apart on average. Quantification of total BrdU incorporation suggests that 190 ‘early’ origins have fired in these cells and that late replicating territories might represent up to 40% of the yeast genome. More generally, the methods outlined here will help understand the kinetics of DNA replication in wild-type yeast and refine the phenotypes of several mutants. PMID:11266543

  12. Man-induced gradient adjustment of the South Fork Forked Deer River, west Tennessee

    USGS Publications Warehouse

    Simon, A.; Robbins, C.H.

    1987-01-01

    Channel modifications from 1968 to 1969 on the South Fork Forked Deer River in western Tennessee have caused upstream degradation, downstream aggradation, and bank failures along the altered channels, adjacent reaches, and tributaries. The result of these adjustments is a general decrease in gradient as the channel attempts to absorb the imposed increase in energy conditions created by channelization. Headward degradation at a rate of approximately 2.57 km/yr on the South Fork Forked Deer River caused from 1.52 m to about 3.14 m of incision over a 13.5 km reach from 1969 to 1981. As a consequence of substantially increased sediment supply, approximately 2.13 m of aggradation was induced downstream of this reach during the same period. This accumulation represents a 60% recovery of bed level at the downstream site since the completion of channel work in 1969. Gradient adjustment with time is described by exponential decay functions. The length of time required for adjustment to some new quasi-equilibrium condition is computed by these decay functions and is about 20 years from the completion of channel work. Adjusted slopes are less than predisturbed values, probably because straightened channels dissipate less energy by friction, allowing more energy for sediment transport. An equivalent sediment load, therefore, can be transported at a considerably gentler slope. The predisturbed slope exceeds the adjusted slope by an order of magnitude on the downstream reach of the South Fork Forked Deer River. ?? 1987 Springer-Verlag New York Inc.

  13. DNA replication stress in CHK1-depleted tumour cells triggers premature (S-phase) mitosis through inappropriate activation of Aurora kinase B

    PubMed Central

    Zuazua-Villar, P; Rodriguez, R; Gagou, M E; Eyers, P A; Meuth, M

    2014-01-01

    The disruption of DNA replication in cells triggers checkpoint responses that slow-down S-phase progression and protect replication fork integrity. These checkpoints are also determinants of cell fate and can help maintain cell viability or trigger cell death pathways. CHK1 has a pivotal role in such S-phase responses. It helps maintain fork integrity during replication stress and protects cells from several catastrophic fates including premature mitosis, premature chromosome condensation and apoptosis. Here we investigated the role of CHK1 in protecting cancer cells from premature mitosis and apoptosis. We show that premature mitosis (characterized by the induction of histone H3 phosphorylation, aberrant chromatin condensation, and persistent RPA foci in arrested S-phase cells) is induced in p53-deficient tumour cells depleted of CHK1 when DNA synthesis is disrupted. These events are accompanied by an activation of Aurora kinase B in S-phase cells that is essential for histone H3 Ser10 phosphorylation. Histone H3 phosphorylation precedes the induction of apoptosis in p53−/− tumour cell lines but does not appear to be required for this fate as an Aurora kinase inhibitor suppresses phosphorylation of both Aurora B and histone H3 but has little effect on cell death. In contrast, only a small fraction of p53+/+ tumour cells shows this premature mitotic response, although they undergo a more rapid and robust apoptotic response. Taken together, our results suggest a novel role for CHK1 in the control of Aurora B activation during DNA replication stress and support the idea that premature mitosis is a distinct cell fate triggered by the disruption of DNA replication when CHK1 function is suppressed. PMID:24853431

  14. Replication-guided nucleosome packing and nucleosome breathing expedite the formation of dense arrays

    PubMed Central

    Osberg, Brendan; Nuebler, Johannes; Korber, Philipp; Gerland, Ulrich

    2014-01-01

    The first level of genome packaging in eukaryotic cells involves the formation of dense nucleosome arrays, with DNA coverage near 90% in yeasts. How cells achieve such high coverage within a short time, e.g. after DNA replication, remains poorly understood. It is known that random sequential adsorption of impenetrable particles on a line reaches high density extremely slowly, due to a jamming phenomenon. The nucleosome-shifting action of remodeling enzymes has been proposed as a mechanism to resolve such jams. Here, we suggest two biophysical mechanisms which assist rapid filling of DNA with nucleosomes, and we quantitatively characterize these mechanisms within mathematical models. First, we show that the ‘softness’ of nucleosomes, due to nucleosome breathing and stepwise nucleosome assembly, significantly alters the filling behavior, speeding up the process relative to ‘hard’ particles with fixed, mutually exclusive DNA footprints. Second, we explore model scenarios in which the progression of the replication fork could eliminate nucleosome jamming, either by rapid filling in its wake or via memory of the parental nucleosome positions. Taken together, our results suggest that biophysical effects promote rapid nucleosome filling, making the reassembly of densely packed nucleosomes after DNA replication a simpler task for cells than was previously thought. PMID:25428353

  15. Replicating vaccines

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Early work on fish immunology and disease resistance demonstrated fish (like animals and humans) that survived infection were typically resistant to re-infection with the same pathogen. The concepts of resistance upon reinfection lead to the research and development of replicating (live) vaccines in...

  16. DNA replication origins in archaea

    PubMed Central

    Wu, Zhenfang; Liu, Jingfang; Yang, Haibo; Xiang, Hua

    2014-01-01

    DNA replication initiation, which starts at specific chromosomal site (known as replication origins), is the key regulatory stage of chromosome replication. Archaea, the third domain of life, use a single or multiple origin(s) to initiate replication of their circular chromosomes. The basic structure of replication origins is conserved among archaea, typically including an AT-rich unwinding region flanked by several conserved repeats (origin recognition box, ORB) that are located adjacent to a replication initiator gene. Both the ORB sequence and the adjacent initiator gene are considerably diverse among different replication origins, while in silico and genetic analyses have indicated the specificity between the initiator genes and their cognate origins. These replicator–initiator pairings are reminiscent of the oriC-dnaA system in bacteria, and a model for the negative regulation of origin activity by a downstream cluster of ORB elements has been recently proposed in haloarchaea. Moreover, comparative genomic analyses have revealed that the mosaics of replicator-initiator pairings in archaeal chromosomes originated from the integration of extrachromosomal elements. This review summarizes the research progress in understanding of archaeal replication origins with particular focus on the utilization, control and evolution of multiple replication origins in haloarchaea. PMID:24808892

  17. SMARCAL1 Resolves Replication Stress at ALT Telomeres.

    PubMed

    Cox, Kelli E; Maréchal, Alexandre; Flynn, Rachel Litman

    2016-02-01

    Cancer cells overcome replicative senescence by exploiting mechanisms of telomere elongation, a process often accomplished by reactivation of the enzyme telomerase. However, a subset of cancer cells lack telomerase activity and rely on the alternative lengthening of telomeres (ALT) pathway, a recombination-based mechanism of telomere elongation. Although the mechanisms regulating ALT are not fully defined, chronic replication stress at telomeres might prime these fragile regions for recombination. Here, we demonstrate that the replication stress response protein SMARCAL1 is a critical regulator of ALT activity. SMARCAL1 associates with ALT telomeres to resolve replication stress and ensure telomere stability. In the absence of SMARCAL1, persistently stalled replication forks at ALT telomeres deteriorate into DNA double-strand breaks promoting the formation of chromosome fusions. Our studies not only define a role for SMARCAL1 in ALT telomere maintenance, but also demonstrate that resolution of replication stress is a crucial step in the ALT mechanism. PMID:26832416

  18. The mammalian INO80 chromatin remodeling complex is required for replication stress recovery

    PubMed Central

    Vassileva, Ivelina; Yanakieva, Iskra; Peycheva, Michaela; Gospodinov, Anastas; Anachkova, Boyka

    2014-01-01

    A number of studies have implicated the yeast INO80 chromatin remodeling complex in DNA replication, but the function of the human INO80 complex during S phase remains poorly understood. Here, we have systematically investigated the involvement of the catalytic subunit of the human INO80 complex during unchallenged replication and under replication stress by following the effects of its depletion on cell survival, S-phase checkpoint activation, the fate of individual replication forks, and the consequences of fork collapse. We report that INO80 was specifically needed for efficient replication elongation, while it was not required for initiation of replication. In the absence of the Ino80 protein, cells became hypersensitive to hydroxyurea and displayed hyperactive ATR-Chk1 signaling. Using bulk and fiber labeling of DNA, we found that cells deficient for Ino80 and Arp8 had impaired replication restart after treatment with replication inhibitors and accumulated double-strand breaks as evidenced by the formation of γ-H2AX and Rad51 foci. These data indicate that under conditions of replication stress mammalian INO80 protects stalled forks from collapsing and allows their subsequent restart. PMID:25016522

  19. Replisome stall events have shaped the distribution of replication origins in the genomes of yeasts

    PubMed Central

    Newman, Timothy J.; Mamun, Mohammed A.; Nieduszynski, Conrad A.; Blow, J. Julian

    2013-01-01

    During S phase, the entire genome must be precisely duplicated, with no sections of DNA left unreplicated. Here, we develop a simple mathematical model to describe the probability of replication failing due to the irreversible stalling of replication forks. We show that the probability of complete genome replication is maximized if replication origins are evenly spaced, the largest inter-origin distances are minimized, and the end-most origins are positioned close to chromosome ends. We show that origin positions in the yeast Saccharomyces cerevisiae genome conform to all three predictions thereby maximizing the probability of complete replication if replication forks stall. Origin positions in four other yeasts—Kluyveromyces lactis, Lachancea kluyveri, Lachancea waltii and Schizosaccharomyces pombe—also conform to these predictions. Equating failure rates at chromosome ends with those in chromosome interiors gives a mean per nucleotide fork stall rate of ∼5 × 10−8, which is consistent with experimental estimates. Using this value in our theoretical predictions gives replication failure rates that are consistent with data from replication origin knockout experiments. Our theory also predicts that significantly larger genomes, such as those of mammals, will experience a much greater probability of replication failure genome-wide, and therefore will likely require additional compensatory mechanisms. PMID:23963700

  20. Cdc45 is limiting for replication initiation in humans

    PubMed Central

    Köhler, Carsten; Koalick, Dennis; Fabricius, Anja; Parplys, Ann Christin; Borgmann, Kerstin; Pospiech, Helmut; Grosse, Frank

    2016-01-01

    ABSTRACT Cdc45 is an essential protein that together with Mcm2-7 and GINS forms the eukaryotic replicative helicase CMG. Cdc45 seems to be rate limiting for the initial unwinding or firing of replication origins. In line with this view, Cdc45-overexpressing cells fired at least twice as many origins as control cells. However, these cells displayed an about 2-fold diminished fork elongation rate, a pronounced asymmetry of replication fork extension, and an early S phase arrest. This was accompanied by H2AX-phosphorylation and subsequent apoptosis. Unexpectedly, we did not observe increased ATR/Chk1 signaling but rather a mild ATM/Chk2 response. In addition, we detected accumulation of long stretches of single-stranded DNA, a hallmark of replication catastrophe. We conclude that increased origin firing by upregulated Cdc45 caused exhaustion of the single-strand binding protein RPA, which in consequence diminished the ATR/Chk1 response; the subsequently occurring fork breaks led to an ATM/Chk2 mediated phosphorylation of H2AX and eventually to apoptosis. PMID:26919204

  1. The B. subtilis Accessory Helicase PcrA Facilitates DNA Replication through Transcription Units

    PubMed Central

    Merrikh, Christopher N.; Brewer, Bonita J.; Merrikh, Houra

    2015-01-01

    In bacteria the concurrence of DNA replication and transcription leads to potentially deleterious encounters between the two machineries, which can occur in either the head-on (lagging strand genes) or co-directional (leading strand genes) orientations. These conflicts lead to replication fork stalling and can destabilize the genome. Both eukaryotic and prokaryotic cells possess resolution factors that reduce the severity of these encounters. Though Escherichia coli accessory helicases have been implicated in the mitigation of head-on conflicts, direct evidence of these proteins mitigating co-directional conflicts is lacking. Furthermore, the endogenous chromosomal regions where these helicases act, and the mechanism of recruitment, have not been identified. We show that the essential Bacillus subtilis accessory helicase PcrA aids replication progression through protein coding genes of both head-on and co-directional orientations, as well as rRNA and tRNA genes. ChIP-Seq experiments show that co-directional conflicts at highly transcribed rRNA, tRNA, and head-on protein coding genes are major targets of PcrA activity on the chromosome. Partial depletion of PcrA renders cells extremely sensitive to head-on conflicts, linking the essential function of PcrA to conflict resolution. Furthermore, ablating PcrA’s ATPase/helicase activity simultaneously increases its association with conflict regions, while incapacitating its ability to mitigate conflicts, and leads to cell death. In contrast, disruption of PcrA’s C-terminal RNA polymerase interaction domain does not impact its ability to mitigate conflicts between replication and transcription, its association with conflict regions, or cell survival. Altogether, this work establishes PcrA as an essential factor involved in mitigating transcription-replication conflicts and identifies chromosomal regions where it routinely acts. As both conflicts and accessory helicases are found in all domains of life, these results

  2. TRF1 negotiates TTAGGG repeat-associated replication problems by recruiting the BLM helicase and the TPP1/POT1 repressor of ATR signaling

    PubMed Central

    Zimmermann, Michal; Kibe, Tatsuya; Kabir, Shaheen

    2014-01-01

    The semiconservative replication of telomeres is facilitated by the shelterin component TRF1. Without TRF1, replication forks stall in the telomeric repeats, leading to ATR kinase signaling upon S-phase progression, fragile metaphase telomeres that resemble the common fragile sites (CFSs), and the association of sister telomeres. In contrast, TRF1 does not contribute significantly to the end protection functions of shelterin. We addressed the mechanism of TRF1 action using mouse conditional knockouts of BLM, TRF1, TPP1, and Rap1 in combination with expression of TRF1 and TIN2 mutants. The data establish that TRF1 binds BLM to facilitate lagging but not leading strand telomeric DNA synthesis. As the template for lagging strand telomeric DNA synthesis is the TTAGGG repeat strand, TRF1-bound BLM is likely required to remove secondary structures formed by these sequences. In addition, the data establish that TRF1 deploys TIN2 and the TPP1/POT1 heterodimers in shelterin to prevent ATR during telomere replication and repress the accompanying sister telomere associations. Thus, TRF1 uses two distinct mechanisms to promote replication of telomeric DNA and circumvent the consequences of replication stress. These data are relevant to the expression of CFSs and provide insights into TIN2, which is compromised in dyskeratosis congenita (DC) and related disorders. PMID:25344324

  3. RNAi-based screening identifies the Mms22L–Nfkbil2 complex as a novel regulator of DNA replication in human cells

    PubMed Central

    Piwko, Wojciech; Olma, Michael H; Held, Michael; Bianco, Julien N; Pedrioli, Patrick G A; Hofmann, Kay; Pasero, Philippe; Gerlich, Daniel W; Peter, Matthias

    2010-01-01

    Cullin 4 (Cul4)-based ubiquitin ligases emerged as critical regulators of DNA replication and repair. Over 50 Cul4-specific adaptors (DNA damage-binding 1 (Ddb1)–Cul4-associated factors; DCAFs) have been identified and are thought to assemble functionally distinct Cul4 complexes. Using a live-cell imaging-based RNAi screen, we analysed the function of DCAFs and Cul4-linked proteins, and identified specific subsets required for progression through G1 and S phase. We discovered C6orf167/Mms22-like protein (Mms22L) as a putative human orthologue of budding yeast Mms22, which, together with cullin Rtt101, regulates genome stability by promoting DNA replication through natural pause sites and damaged templates. Loss of Mms22L function in human cells results in S phase-dependent genomic instability characterised by spontaneous double-strand breaks and DNA damage checkpoint activation. Unlike yeast Mms22, human Mms22L does not stably bind to Cul4, but is degraded in a Cul4-dependent manner and upon replication stress. Mms22L physically and functionally interacts with the scaffold-like protein Nfkbil2 that co-purifies with histones, several chromatin remodelling and DNA replication/repair factors. Together, our results strongly suggest that the Mms22L–Nfkbil2 complex contributes to genome stability by regulating the chromatin state at stalled replication forks. PMID:21113133

  4. Quartz tuning fork based microwave impedance microscopy

    NASA Astrophysics Data System (ADS)

    Cui, Yong-Tao; Ma, Eric Yue; Shen, Zhi-Xun

    2016-06-01

    Microwave impedance microscopy (MIM), a near-field microwave scanning probe technique, has become a powerful tool to characterize local electrical responses in solid state samples. We present the design of a new type of MIM sensor based on quartz tuning fork and electrochemically etched thin metal wires. Due to a higher aspect ratio tip and integration with tuning fork, such design achieves comparable MIM performance and enables easy self-sensing topography feedback in situations where the conventional optical feedback mechanism is not available, thus is complementary to microfabricated shielded stripline-type probes. The new design also enables stable differential mode MIM detection and multiple-frequency MIM measurements with a single sensor.

  5. The mechanism of DNA replication termination in vertebrates

    PubMed Central

    Dewar, James M.; Budzowska, Magda; Walter, Johannes C.

    2015-01-01

    Eukaryotic DNA replication terminates when replisomes from adjacent replication origins converge. Termination involves local completion of DNA synthesis, decatenation of daughter molecules, and replisome disassembly. Termination has been difficult to study because termination events are generally asynchronous and sequence non-specific. To overcome these challenges, we paused converging replisomes with a site-specific barrier in Xenopus egg extracts. Upon removal of the barrier, forks underwent synchronous and site-specific termination, allowing mechanistic dissection of this process. We show that DNA synthesis does not slow detectably as forks approach each other and that leading strands pass each other unhindered before undergoing ligation to downstream lagging strands. Dissociation of CMG helicases occurs only after the final ligation step, and is not required for completion of DNA synthesis, strongly suggesting that converging CMGs pass one another and dissociate from double-stranded DNA. This termination mechanism allows rapid completion of DNA synthesis while avoiding premature replisome disassembly PMID:26322582

  6. RPA inhibition increases replication stress and suppresses tumor growth.

    PubMed

    Glanzer, Jason G; Liu, Shengqin; Wang, Ling; Mosel, Adam; Peng, Aimin; Oakley, Greg G

    2014-09-15

    The ATR/Chk1 pathway is a critical surveillance network that maintains genomic integrity during DNA replication by stabilizing the replication forks during normal replication to avoid replication stress. One of the many differences between normal cells and cancer cells is the amount of replication stress that occurs during replication. Cancer cells with activated oncogenes generate increased levels of replication stress. This creates an increased dependency on the ATR/Chk1 pathway in cancer cells and opens up an opportunity to preferentially kill cancer cells by inhibiting this pathway. In support of this idea, we have identified a small molecule termed HAMNO ((1Z)-1-[(2-hydroxyanilino)methylidene]naphthalen-2-one), a novel protein interaction inhibitor of replication protein A (RPA), a protein involved in the ATR/Chk1 pathway. HAMNO selectively binds the N-terminal domain of RPA70, effectively inhibiting critical RPA protein interactions that rely on this domain. HAMNO inhibits both ATR autophosphorylation and phosphorylation of RPA32 Ser33 by ATR. By itself, HAMNO treatment creates DNA replication stress in cancer cells that are already experiencing replication stress, but not in normal cells, and it acts synergistically with etoposide to kill cancer cells in vitro and slow tumor growth in vivo. Thus, HAMNO illustrates how RPA inhibitors represent candidate therapeutics for cancer treatment, providing disease selectivity in cancer cells by targeting their differential response to replication stress. Cancer Res; 74(18); 5165-72. ©2014 AACR. PMID:25070753

  7. RPA Inhibition increases Replication Stress and Suppresses Tumor Growth

    PubMed Central

    Glanzer, Jason G.; Liu, Shengqin; Wang, Ling; Mosel, Adam; Peng, Aimin; Oakley, Greg G.

    2014-01-01

    The ATR/Chk1 pathway is a critical surveillance network that maintains genomic integrity during DNA replication by stabilizing the replication forks during normal replication to avoid replication stress. One of the many differences between normal cells and cancer cells is the amount of replication stress that occurs during replication. Cancer cells with activated oncogenes generate increased levels of replication stress. This creates an increased dependency on the ATR/Chk1 pathway in cancer cells and opens up an opportunity to preferentially kill cancer cells by inhibiting this pathway. In support of this idea, we have identified a small molecule termed HAMNO ((1Z)-1-[(2-hydroxyanilino)methylidene]naphthalen-2-one), a novel protein interaction inhibitor of replication protein A (RPA), a protein involved in the ATR/Chk1 pathway. HAMNO selectively binds the N-terminal domain of RPA70, effectively inhibiting critical RPA protein interactions which rely on this domain. HAMNO inhibits both ATR autophosphorylation and phosphorylation of RPA32 Ser33 by ATR. By itself, HAMNO treatment creates DNA replication stress in cancer cells that are already experiencing replication stress, but not in normal cells, and it acts synergistically with etoposide to kill cancer cells in vitro and slow tumor growth in vivo. Thus, HAMNO illustrates how RPA inhibitors represent candidate therapeutics for cancer treatment, providing disease selectivity in cancer cells by targeting their differential response to replication stress. PMID:25070753

  8. BALD ROCK AND MIDDLE FORK FEATHER RIVER ROADLESS AREAS, CALIFORNIA.

    USGS Publications Warehouse

    Sorensen, Martin L.; Buehler, Alan R.

    1984-01-01

    The results of a mineral-resource assessment of the Bald Rock and Middle Fork Feather River Roadless Areas in California indicate several areas within the Middle Fork Feather River Roadless Area that have probable mineral-resource potential. A probable potential for placer gold exists at various localities, both in areas covered by Tertiary volcanic rocks and in small streams that drain into the Middle Fork of the Feather River. A probable potential for small deposits of chromite exists in tracts underlain by ultramafic rocks in the Melones fault zone. A probable potential for lead-silver deposits is recognized at the east end of the Middle Fork Feather River Roadless Area.

  9. Evolution of tail fork depth in genus Hirundo.

    PubMed

    Hasegawa, Masaru; Arai, Emi; Kutsukake, Nobuyuki

    2016-02-01

    A classic example of a sexually selected trait, the deep fork tail of the barn swallow Hirundo rustica is now claimed to have evolved and be maintained mainly via aerodynamic advantage rather than sexually selected advantage. However, this aerodynamic advantage hypothesis does not clarify which flight habits select for/against deep fork tails, causing diversity of tail fork depth in hirundines. Here, by focusing on the genus Hirundo, we investigated whether the large variation in tail fork depth could be explained by the differential flight habits. Using a phylogenetic comparative approach, we found that migrant species had deeper fork tails, but less colorful plumage, than the other species, indicating that migration favors a specific trait, deep fork tails. At the same time, tail fork depth but not plumage coloration decreased with increasing bill size - a proxy of prey size, suggesting that foraging on larger prey items favors shallower fork tails. Variation of tail fork depth in the genus Hirundo may be explained by differential flight habits, even without assuming sexual selection. PMID:26865972

  10. Connecting Replication and Repair: YoaA, a Helicase-Related Protein, Promotes Azidothymidine Tolerance through Association with Chi, an Accessory Clamp Loader Protein

    PubMed Central

    Brown, Laura T.; Sutera, Vincent A.; Zhou, Shen; Weitzel, Christopher S.; Cheng, Yisha; Lovett, Susan T.

    2015-01-01

    Elongating DNA polymerases frequently encounter lesions or structures that impede progress and require repair before DNA replication can be completed. Therefore, directing repair factors to a blocked fork, without interfering with normal replication, is important for proper cell function, and it is a process that is not well understood. To study this process, we have employed the chain-terminating nucleoside analog, 3’ azidothymidine (AZT) and the E. coli genetic system, for which replication and repair factors have been well-defined. By using high-expression suppressor screens, we identified yoaA, encoding a putative helicase, and holC, encoding the Chi component of the replication clamp loader, as genes that promoted tolerance to AZT. YoaA is a putative Fe-S helicase in the XPD/RAD3 family for which orthologs can be found in most bacterial genomes; E. coli has a paralog to YoaA, DinG, which possesses 5’ to 3’ helicase activity and an Fe-S cluster essential to its activity. Mutants in yoaA are sensitive to AZT exposure; dinG mutations cause mild sensitivity to AZT and exacerbate the sensitivity of yoaA mutant strains. Suppression of AZT sensitivity by holC or yoaA was mutually codependent and we provide evidence here that YoaA and Chi physically interact. Interactions of Chi with single-strand DNA binding protein (SSB) and with Psi were required to aid AZT tolerance, as was the proofreading 3’ exonuclease, DnaQ. Our studies suggest that repair is coupled to blocked replication through these interactions. We hypothesize that SSB, through Chi, recruits the YoaA helicase to replication gaps and that unwinding of the nascent strand promotes repair and AZT excision. This recruitment prevents the toxicity of helicase activity and aids the handoff of repair with replication factors, ensuring timely repair and resumption of replication. PMID:26544712

  11. DNA replication: damage tolerance at the assembly line.

    PubMed

    Blastyák, András

    2014-07-01

    Damage tolerance mechanisms ensure resumption of DNA synthesis at damage-replisome encounters. Replication fork reversal (RFR) is one such widely recognized mechanism that acts on replisomes where lagging strand synthesis continues upon leading strand synthesis block. The possibility to form such a structure is highly counter to our current understanding of the replisome dynamics of single replisomes. Here, I suggest a model that takes coupled bidirectional replisome organization into account to solve this apparent contradiction. PMID:24957737

  12. Tight Chk1 Levels Control Replication Cluster Activation in Xenopus

    PubMed Central

    Wiggins, Jennifer M.; Barbosa, Pedro; Libeau, Pierre; Priam, Pierre; Narassimprakash, Hemalatha; Grodzenski, Xenia; Marheineke, Kathrin

    2015-01-01

    DNA replication in higher eukaryotes initiates at thousands of origins according to a spatio-temporal program. The ATR/Chk1 dependent replication checkpoint inhibits the activation of later firing origins. In the Xenopus in vitro system initiations are not sequence dependent and 2-5 origins are grouped in clusters that fire at different times despite a very short S phase. We have shown that the temporal program is stochastic at the level of single origins and replication clusters. It is unclear how the replication checkpoint inhibits late origins but permits origin activation in early clusters. Here, we analyze the role of Chk1 in the replication program in sperm nuclei replicating in Xenopus egg extracts by a combination of experimental and modelling approaches. After Chk1 inhibition or immunodepletion, we observed an increase of the replication extent and fork density in the presence or absence of external stress. However, overexpression of Chk1 in the absence of external replication stress inhibited DNA replication by decreasing fork densities due to lower Cdk2 kinase activity. Thus, Chk1 levels need to be tightly controlled in order to properly regulate the replication program even during normal S phase. DNA combing experiments showed that Chk1 inhibits origins outside, but not inside, already active clusters. Numerical simulations of initiation frequencies in the absence and presence of Chk1 activity are consistent with a global inhibition of origins by Chk1 at the level of clusters but need to be combined with a local repression of Chk1 action close to activated origins to fit our data. PMID:26046346

  13. Preservation of Epigenetic Memory During DNA Replication

    PubMed Central

    Lowe, Matthew; Hostager, Reilly; Kikyo, Nobuaki

    2016-01-01

    Faithful duplication of a cell’s epigenetic state during DNA replication is essential for the maintenance of a cell’s lineage. One of the key mechanisms is the recruitment of several critical chromatin modifying enzymes to the replication fork by proliferating cell nuclear antigen (PCNA). Another mechanism is mediated by the dual function of some histone modifying enzymes as both “reader” and “writer” of the same modification. This capacity allows for parental histones to act as a seed to copy the modification onto nearby newly synthesized histones. In contrast to the vast quantity of research into the maintenance of epigenetic memory, little is known about how the recruitment of these maintenance enzymes changes during stem cell differentiation. This question is especially pertinent due to the recent emphasis on cell reprogramming for regenerative medicine. PMID:27158681

  14. Oxbow Conservation Area; Middle Fork John Day River, Annual Report 2001-2002.

    SciTech Connect

    Robertson, Shaun; Smith, Brent; Cochran, Brian

    2003-04-01

    In early 2001, the Confederated Tribes of Warm Springs, through their John Day Basin Office, concluded the acquisition of the Middle Fork Oxbow Ranch. Under a memorandum of agreement with the Bonneville Power Administration (BPA), the Tribes are required to provided BPA an 'annual written report generally describing the real property interests in the Project, HEP analyses undertaken or in progress, and management activities undertaken or in progress'. This report is to be provided to the BPA by 30 April of each year. This is the first annual report filed for the Oxbow Ranch property.

  15. The Npl3 hnRNP prevents R-loop-mediated transcription–replication conflicts and genome instability

    PubMed Central

    Santos-Pereira, José M.; Herrero, Ana B.; García-Rubio, María L.; Marín, Antonio; Moreno, Sergio; Aguilera, Andrés

    2013-01-01

    Transcription is a major obstacle for replication fork (RF) progression and a cause of genome instability. Part of this instability is mediated by cotranscriptional R loops, which are believed to increase by suboptimal assembly of the nascent messenger ribonucleoprotein particle (mRNP). However, no clear evidence exists that heterogeneous nuclear RNPs (hnRNPs), the basic mRNP components, prevent R-loop stabilization. Here we show that yeast Npl3, the most abundant RNA-binding hnRNP, prevents R-loop-mediated genome instability. npl3Δ cells show transcription-dependent and R-loop-dependent hyperrecombination and genome-wide replication obstacles as determined by accumulation of the Rrm3 helicase. Such obstacles preferentially occur at long and highly expressed genes, to which Npl3 is preferentially bound in wild-type cells, and are reduced by RNase H1 overexpression. The resulting replication stress confers hypersensitivity to double-strand break-inducing agents. Therefore, our work demonstrates that mRNP factors are critical for genome integrity and opens the option of using them as therapeutic targets in anti-cancer treatment. PMID:24240235

  16. 33 CFR 207.370 - Big Fork River, Minn.; logging.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 33 Navigation and Navigable Waters 3 2011-07-01 2011-07-01 false Big Fork River, Minn.; logging..., DEPARTMENT OF DEFENSE NAVIGATION REGULATIONS § 207.370 Big Fork River, Minn.; logging. (a) During the season... maintained at all times for the navigation of steamboats, flatboats, and other small craft. (b) A...

  17. 33 CFR 207.370 - Big Fork River, Minn.; logging.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 33 Navigation and Navigable Waters 3 2012-07-01 2012-07-01 false Big Fork River, Minn.; logging..., DEPARTMENT OF DEFENSE NAVIGATION REGULATIONS § 207.370 Big Fork River, Minn.; logging. (a) During the season... maintained at all times for the navigation of steamboats, flatboats, and other small craft. (b) A...

  18. 33 CFR 207.370 - Big Fork River, Minn.; logging.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 33 Navigation and Navigable Waters 3 2014-07-01 2014-07-01 false Big Fork River, Minn.; logging..., DEPARTMENT OF DEFENSE NAVIGATION REGULATIONS § 207.370 Big Fork River, Minn.; logging. (a) During the season... maintained at all times for the navigation of steamboats, flatboats, and other small craft. (b) A...

  19. 33 CFR 207.370 - Big Fork River, Minn.; logging.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 33 Navigation and Navigable Waters 3 2010-07-01 2010-07-01 false Big Fork River, Minn.; logging..., DEPARTMENT OF DEFENSE NAVIGATION REGULATIONS § 207.370 Big Fork River, Minn.; logging. (a) During the season... maintained at all times for the navigation of steamboats, flatboats, and other small craft. (b) A...

  20. 33 CFR 207.370 - Big Fork River, Minn.; logging.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 33 Navigation and Navigable Waters 3 2013-07-01 2013-07-01 false Big Fork River, Minn.; logging..., DEPARTMENT OF DEFENSE NAVIGATION REGULATIONS § 207.370 Big Fork River, Minn.; logging. (a) During the season... maintained at all times for the navigation of steamboats, flatboats, and other small craft. (b) A...

  1. South Fork Telephone Switchboard Building, general view in setting showing ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    South Fork Telephone Switchboard Building, general view in setting showing (N) side; view (S) - Fort McKinley, South Fork Telephone Switchboard Building, South side of Weymouth Way, approximately 100 feet west of East Side Drive, Great Diamond Island, Portland, Cumberland County, ME

  2. South Fork Latrine, interior showing head with steel tank mounted ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    South Fork Latrine, interior showing head with steel tank mounted to wall; view south - Fort McKinley, South Fork Latrine, West side of East Side Drive, approximately 225 feet south of Weymouth Way, Great Diamond Island, Portland, Cumberland County, ME

  3. South Fork Telephone Switchboard Building, interior west room showing hardwood ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    South Fork Telephone Switchboard Building, interior west room showing hardwood floor; view south - Fort McKinley, South Fork Telephone Switchboard Building, South side of Weymouth Way, approximately 100 feet west of East Side Drive, Great Diamond Island, Portland, Cumberland County, ME

  4. South Fork Telephone Switchboard Building, oblique view of (W) and ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    South Fork Telephone Switchboard Building, oblique view of (W) and (S) sides, view to northeast - Fort McKinley, South Fork Telephone Switchboard Building, South side of Weymouth Way, approximately 100 feet west of East Side Drive, Great Diamond Island, Portland, Cumberland County, ME

  5. South Fork Telephone Switchboard Building, oblique view of east side; ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    South Fork Telephone Switchboard Building, oblique view of east side; view northwest - Fort McKinley, South Fork Telephone Switchboard Building, South side of Weymouth Way, approximately 100 feet west of East Side Drive, Great Diamond Island, Portland, Cumberland County, ME

  6. 14. Roaring Fork Motor Nature Trail, Place of a thousand ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    14. Roaring Fork Motor Nature Trail, Place of a thousand drips, view with three culvert pipes. - Great Smoky Mountains National Park Roads & Bridges, Roaring Fork Motor Nature Trail, Between Cherokee Orchard Road & U.S. Route 321, Gatlinburg, Sevier County, TN

  7. 27 CFR 9.65 - North Fork of Roanoke.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 27 Alcohol, Tobacco Products and Firearms 1 2014-04-01 2014-04-01 false North Fork of Roanoke. 9.65 Section 9.65 Alcohol, Tobacco Products and Firearms ALCOHOL AND TOBACCO TAX AND TRADE BUREAU, DEPARTMENT OF THE TREASURY ALCOHOL AMERICAN VITICULTURAL AREAS Approved American Viticultural Areas § 9.65 North Fork of Roanoke. (a) Name. The name...

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

    PubMed Central

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

    2016-01-01

    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. PMID:26935043

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

    NASA Astrophysics Data System (ADS)

    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.

  10. The PriA Replication Restart Protein Blocks Replicase Access Prior to Helicase Assembly and Directs Template Specificity through Its ATPase Activity*

    PubMed Central

    Manhart, Carol M.; McHenry, Charles S.

    2013-01-01

    The PriA protein serves as an initiator for the restart of DNA replication on stalled replication forks and as a checkpoint protein that prevents the replicase from advancing in a strand displacement reaction on forks that do not contain a functional replicative helicase. We have developed a primosomal protein-dependent fluorescence resonance energy transfer (FRET) assay using a minimal fork substrate composed of synthetic oligonucleotides. We demonstrate that a self-loading reaction, which proceeds at high helicase concentrations, occurs by threading of a preassembled helicase over free 5′-ends, an event that can be blocked by attaching a steric block to the 5′-end or coating DNA with single-stranded DNA binding protein. The specificity of PriA for replication forks is regulated by its intrinsic ATPase. ATPase-defective PriA K230R shows a strong preference for substrates that contain no gap between the leading strand and the duplex portion of the fork, as demonstrated previously. Wild-type PriA prefers substrates with larger gaps, showing maximal activity on substrates on which PriA K230R is inactive. We demonstrate that PriA blocks replicase function on forks by blocking its binding. PMID:23264623

  11. Molecular replication

    NASA Technical Reports Server (NTRS)

    Orgel, L. E.

    1986-01-01

    The object of our research program is to understand how polynucleotide replication originated on the primitive Earth. This is a central issue in studies of the origins of life, since a process similar to modern DNA and RNA synthesis is likely to have formed the basis for the most primitive system of genetic information transfer. The major conclusion of studies so far is that a preformed polynucleotide template under many different experimental conditions will facilitate the synthesis of a new oligonucleotide with a sequence complementary to that of the template. It has been shown, for example, that poly(C) facilitates the synthesis of long oligo(G)s and that the short template CCGCC facilities the synthesis of its complement GGCGG. Very recently we have shown that template-directed synthesis is not limited to the standard oligonucleotide substrates. Nucleic acid-like molecules with a pyrophosphate group replacing the phosphate of the standard nucleic acid backbone are readily synthesized from deoxynucleotide 3'-5'-diphosphates on appropriate templates.

  12. Design of a tuning-fork gyro made of quartz

    NASA Astrophysics Data System (ADS)

    Jia, Yubin; Sun, Yunan; Qin, BingKun; Cui, Fang; Chen, Gang

    1998-08-01

    Based on piezoelectric effect of quartz, a design of tuning- fork gyroscopes made of quartz was presented in this paper. The gyroscope is a kind of micro-machined quartz angular rate sensor. Its structure is similar to a tuning fork in quartz watch. In the gyroscope, the piezoelectric effect in quartz is used both to excite a reference vibration in the plane of tuning fork and to detect a vibration normal to this plane due to an externally applied rotation. The amplitude of the second vibration is directly proportional to the angular velocity of the applied rotation. In contrast to ordinary types of tuning fork gyroscopes, this gyroscope uses a single piece of quartz, the sensor element is of a design in which the only vibrationally, active areas are the tines of the tuning fork.

  13. The tight linkage between DNA replication and double-strand break repair in bacteriophage T4

    PubMed Central

    George, James W.; Stohr, Bradley A.; Tomso, Daniel J.; Kreuzer, Kenneth N.

    2001-01-01

    Double-strand break (DSB) repair and DNA replication are tightly linked in the life cycle of bacteriophage T4. Indeed, the major mode of phage DNA replication depends on recombination proteins and can be stimulated by DSBs. DSB-stimulated DNA replication is dramatically demonstrated when T4 infects cells carrying two plasmids that share homology. A DSB on one plasmid triggered extensive replication of the second plasmid, providing a useful model for T4 recombination-dependent replication (RDR). This system also provides a view of DSB repair in T4-infected cells and revealed that the DSB repair products had been replicated in their entirety by the T4 replication machinery. We analyzed the detailed structure of these products, which do not fit the simple predictions of any of three models for DSB repair. We also present evidence that the T4 RDR system functions to restart stalled or inactivated replication forks. First, we review experiments involving antitumor drug-stabilized topoisomerase cleavage complexes. The results suggest that forks blocked at cleavage complexes are resolved by recombinational repair, likely involving RDR. Second, we show here that the presence of a T4 replication origin on one plasmid substantially stimulated recombination events between it and a homologous second plasmid that did not contain a T4 origin. Furthermore, replication of the second plasmid was increased when the first plasmid contained the T4 origin. Our interpretation is that origin-initiated forks become inactivated at some frequency during replication of the first plasmid and are then restarted via RDR on the second plasmid. PMID:11459966

  14. A Hand-Off Mechanism for Primosome Assembly in Replication Restart

    SciTech Connect

    Lopper, Matthew; Boonsombat, Ruethairat; Sandler, Steven J.; Keck, James L.

    2008-11-12

    Collapsed DNA replication forks must be reactivated through origin-independent reloading of the replication machinery (replisome) to ensure complete duplication of cellular genomes. In E. coli, the PriA-dependent pathway is the major replication restart mechanism and requires primosome proteins PriA, PriB, and DnaT for replisome reloading. However, the molecular mechanisms that regulate origin-independent replisome loading are not fully understood. Here, we demonstrate that assembly of primosome protein complexes represents a key regulatory mechanism, as inherently weak PriA-PriB and PriB-DnaT interactions are strongly stimulated by single-stranded DNA. Furthermore, the binding site on PriB for single-stranded DNA partially overlaps the binding sites for PriA and DnaT, suggesting a dynamic primosome assembly process in which single-stranded DNA is handed off from one primosome protein to another as a repaired replication fork is reactivated. This model helps explain how origin-independent initiation of DNA replication is restricted to repaired replication forks, preventing overreplication of the genome.

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

    PubMed Central

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

    2015-01-01

    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

  16. The dual role of FOXF2 in regulation of DNA replication and the epithelial-mesenchymal transition in breast cancer progression.

    PubMed

    Lo, Pang-Kuo; Lee, Ji Shin; Liang, Xiaohui; Sukumar, Saraswati

    2016-10-01

    Dysregulation of Forkhead-box (FOX) transcription factors is linked to cancers of numerous tissue types. Here, we report that FOXF2 is frequently silenced in luminal-type and HER2-positive breast cancers, but is overexpressed in basal-like breast cancers; thus, FOXF2 appears to play distinct roles in different breast cancer subtypes. Inactivation of FOXF2 in luminal-type and HER2-positive breast cancers is attributable to epigenetic silencing. Silencing of FOXF2 is associated with poor prognosis in luminal-type breast cancer. Ectopic expression of FOXF2 in luminal and HER2-positive breast cancer cells suppresses their tumorigenic properties in vitro and in vivo via inhibition of the CDK2-RB-E2F cascade. The in vivo function of FOXF2 is to maintain the stringency of DNA replication, and its loss triggers dysregulation of DNA replication, which in turn activates the p53 checkpoint pathway. Besides its role in cell cycle regulation, FOXF2 is functionally required for mobility and epithelial-to-mesenchymal transition (EMT) of normal breast epithelial cells. In basal-like breast cancer cells, the cell-cycle function of FOXF2 is impaired. However, the EMT function of FOXF2 is still required for mobility, invasiveness and anchorage-independent growth of basal-like breast cancer cells. Our gene expression profiling studies demonstrate that FOXF2 regulates the expression of genes implicated in cell cycle and EMT regulation. Moreover, FOXF2 is highly co-expressed with basal- and metastasis-related genes in breast cancer. These findings suggest that FOXF2 has a dual role in breast tumorigenesis and functions as either a tumor suppressor or an oncogene depending on the breast tumor subtype. PMID:27377963

  17. Fork gratings based on ferroelectric liquid crystals.

    PubMed

    Ma, Y; Wei, B Y; Shi, L Y; Srivastava, A K; Chigrinov, V G; Kwok, H-S; Hu, W; Lu, Y Q

    2016-03-21

    In this article, we disclose a fork grating (FG) based on the photo-aligned ferroelectric liquid crystal (FLC). The Digital Micro-mirror Device based system is used as a dynamic photomask to generated different holograms. Because of controlled anchoring energy, the photo alignment process offers optimal conditions for the multi-domain FLC alignment. Two different electro-optical modes namely DIFF/TRANS and DIFF/OFF switchable modes have been proposed where the diffraction can be switched either to no diffraction or to a completely black state, respectively. The FLC FG shows high diffraction efficiency and fast response time of 50µs that is relatively faster than existing technologies. Thus, the FLC FG may pave a good foundation toward optical vertices generation and manipulation that could find applications in a variety of devices. PMID:27136779

  18. Chemical and biological sensing using tuning forks

    DOEpatents

    Tao, Nongjian; Boussaad, Salah

    2012-07-10

    A device for sensing a chemical analyte is disclosed. The device is comprised of a vibrating structure having first and second surfaces and having an associated resonant frequency and a wire coupled between the first and second surfaces of the vibrating structure, wherein the analyte interacts with the wire and causes a change in the resonant frequency of the vibrating structure. The vibrating structure can include a tuning fork. The vibrating structure can be comprised of quartz. The wire can be comprised of polymer. A plurality of vibrating structures are arranged in an array to increase confidence by promoting a redundancy of measurement or to detect a plurality of chemical analytes. A method of making a device for sensing a chemical analyte is also disclosed.

  19. Wireless tuning fork gyroscope for biomedical applications

    NASA Astrophysics Data System (ADS)

    Abraham, Jose K.; Varadan, Vijay K.; Whitchurch, Ashwin K.; Sarukesi, K.

    2003-07-01

    This paper presents the development of a Bluetooth enabled wireless tuning fork gyroscope for the biomedical applications, including gait phase detection system, human motion analysis and physical therapy. This gyroscope is capable of measuring rotation rates between -90 and 90 and it can read the rotation information using a computer. Currently, the information from a gyroscope can trigger automobile airbag deployment during rollover, improve the accuracy and reliability of GPS navigation systems and stabilize moving platforms such as automobiles, airplanes, robots, antennas, and industrial equipment. Adding wireless capability to the existing gyroscope could help to expand its applications in many areas particularly in biomedical applications, where a continuous patient monitoring is quite difficult. This wireless system provides information on several aspects of activities of patients for real-time monitoring in hospitals.

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

    PubMed Central

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

    2015-01-01

    DNA polymerase beta (Pol β) is a key enzymefor the protection against oxidative DNA lesions via itsrole 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

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

    PubMed

    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

  2. NORTH FORK JOHN DAY RIVER ROADLESS AREA, OREGON.

    USGS Publications Warehouse

    Evans, James G.; Conyac, Martin D.

    1984-01-01

    A mineral survey of the North Fork John Day River Roadless Area in Oregon indicates that a narrow belt along the river has a substantiated resource potential for placer gold, and several other drainages tributary to the North Fork a probable resource potential for placer or lode gold. Further study of the roadless area may reveal other areas with a potential for gold, and could help to delineate bedrock or additional placer resources, especially in drainages tributary to the North Fork. This work could also point to other mineral deposits near the roadless boundary.

  3. Insulin-like growth factor-I extends in vitro replicative life span of skeletal muscle satellite cells by enhancing G1/S cell cycle progression via the activation of phosphatidylinositol 3'-kinase/Akt signaling pathway

    NASA Technical Reports Server (NTRS)

    Chakravarthy, M. V.; Abraha, T. W.; Schwartz, R. J.; Fiorotto, M. L.; Booth, F. W.

    2000-01-01

    Interest is growing in methods to extend replicative life span of non-immortalized stem cells. Using the insulin-like growth factor I (IGF-I) transgenic mouse in which the IGF-I transgene is expressed during skeletal muscle development and maturation prior to isolation and during culture of satellite cells (the myogenic stem cells of mature skeletal muscle fibers) as a model system, we elucidated the underlying molecular mechanisms of IGF-I-mediated enhancement of proliferative potential of these cells. Satellite cells from IGF-I transgenic muscles achieved at least five additional population doublings above the maximum that was attained by wild type satellite cells. This IGF-I-induced increase in proliferative potential was mediated via activation of the phosphatidylinositol 3'-kinase/Akt pathway, independent of mitogen-activated protein kinase activity, facilitating G(1)/S cell cycle progression via a down-regulation of p27(Kip1). Adenovirally mediated ectopic overexpression of p27(Kip1) in exponentially growing IGF-I transgenic satellite cells reversed the increase in cyclin E-cdk2 kinase activity, pRb phosphorylation, and cyclin A protein abundance, thereby implicating an important role for p27(Kip1) in promoting satellite cell senescence. These observations provide a more complete dissection of molecular events by which increased local expression of a growth factor in mature skeletal muscle fibers extends replicative life span of primary stem cells than previously known.

  4. Gas Phase Photoacoustic Spectroscopy in the long-wave IR using Quartz Tuning Forks and Amplitude Modulated Quantum Cascade Lasers

    SciTech Connect

    Wojcik, Michael D.; Phillips, Mark C.; Cannon, Bret D.

    2006-12-31

    A paper to accompany a 20 minute talk about the progress of a DARPA funded project called LPAS. ABSTRACT: We demonstrate the performance of a novel long-wave infrared photoacoustic laser absorbance spectrometer for gas-phase species using an amplitude modulated (AM) quantum cascade (QC) laser and a quartz tuning fork microphone. Photoacoustic signal was generated by focusing the output of a Fabry-Perot QC laser operating at 8.41 micron between the legs of a quartz tuning fork which served as a transducer for the transient acoustic pressure wave. The QC laser was modulated at the resonant frequency of the tuning fork (32.8 kHz). This sensor was calibrated using the infrared absorber Freon-134a by performing a simultanious absorption measurement using a 35 cm absorption cell. The NEAS of this instrument was determined to be 2 x 10^-8 W cm^-1 /Hz^1/2 and the fundamental sensitivity of this technique is limited by the noise floor of the tuning fork itself.

  5. Translesion DNA polymerases remodel the replisome and alter the speed of the replicative helicase.

    PubMed

    Indiani, Chiara; Langston, Lance D; Yurieva, Olga; Goodman, Myron F; O'Donnell, Mike

    2009-04-14

    All cells contain specialized translesion DNA polymerases that replicate past sites of DNA damage. We find that Escherichia coli translesion DNA polymerase II (Pol II) and polymerase IV (Pol IV) function with DnaB helicase and regulate its rate of unwinding, slowing it to as little as 1 bp/s. Furthermore, Pol II and Pol IV freely exchange with the polymerase III (Pol III) replicase on the beta-clamp and function with DnaB helicase to form alternative replisomes, even before Pol III stalls at a lesion. DNA damage-induced levels of Pol II and Pol IV dominate the clamp, slowing the helicase and stably maintaining the architecture of the replication machinery while keeping the fork moving. We propose that these dynamic actions provide additional time for normal excision repair of lesions before the replication fork reaches them and also enable the appropriate translesion polymerase to sample each lesion as it is encountered. PMID:19279203

  6. Translesion DNA polymerases remodel the replisome and alter the speed of the replicative helicase

    PubMed Central

    Indiani, Chiara; Langston, Lance D.; Yurieva, Olga; Goodman, Myron F.; O'Donnell, Mike

    2009-01-01

    All cells contain specialized translesion DNA polymerases that replicate past sites of DNA damage. We find that Escherichia coli translesion DNA polymerase II (Pol II) and polymerase IV (Pol IV) function with DnaB helicase and regulate its rate of unwinding, slowing it to as little as 1 bp/s. Furthermore, Pol II and Pol IV freely exchange with the polymerase III (Pol III) replicase on the β-clamp and function with DnaB helicase to form alternative replisomes, even before Pol III stalls at a lesion. DNA damage-induced levels of Pol II and Pol IV dominate the clamp, slowing the helicase and stably maintaining the architecture of the replication machinery while keeping the fork moving. We propose that these dynamic actions provide additional time for normal excision repair of lesions before the replication fork reaches them and also enable the appropriate translesion polymerase to sample each lesion as it is encountered. PMID:19279203

  7. Telomerase is essential to alleviate pif1-induced replication stress at telomeres.

    PubMed

    Chang, Michael; Luke, Brian; Kraft, Claudine; Li, Zhijian; Peter, Matthias; Lingner, Joachim; Rothstein, Rodney

    2009-11-01

    Pif1, an evolutionarily conserved helicase, negatively regulates telomere length by removing telomerase from chromosome ends. Pif1 has also been implicated in DNA replication processes such as Okazaki fragment maturation and replication fork pausing. We find that overexpression of Saccharomyces cervisiae PIF1 results in dose-dependent growth inhibition. Strong overexpression causes relocalization of the DNA damage response factors Rfa1 and Mre11 into nuclear foci and activation of the Rad53 DNA damage checkpoint kinase, indicating that the toxicity is caused by accumulation of DNA damage. We screened the complete set of approximately 4800 haploid gene deletion mutants and found that moderate overexpression of PIF1, which is only mildly toxic on its own, causes growth defects in strains with mutations in genes involved in DNA replication and the DNA damage response. Interestingly, we find that telomerase-deficient strains are also sensitive to PIF1 overexpression. Our data are consistent with a model whereby increased levels of Pif1 interfere with DNA replication, causing collapsed replication forks. At chromosome ends, collapsed forks result in truncated telomeres that must be rapidly elongated by telomerase to maintain viability. PMID:19704012

  8. 117. Laurel Fork Viaduct. Elevation view of this 545 1939 ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    117. Laurel Fork Viaduct. Elevation view of this 545 1939 steel girder viaduct. Example of structure with plain reinforced concrete arches. Looking northwest. - Blue Ridge Parkway, Between Shenandoah National Park & Great Smoky Mountains, Asheville, Buncombe County, NC

  9. 35. CHARGING DOOR OF CUPOLA FORM LOFT, WITH FORKS FOR ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    35. CHARGING DOOR OF CUPOLA FORM LOFT, WITH FORKS FOR FEEDING COKE, FOUNDRY BELOW-LOOKING NORTH. - W. A. Young & Sons Foundry & Machine Shop, On Water Street along Monongahela River, Rices Landing, Greene County, PA

  10. 8. EEL RIVER SOUTH FORK BRIDGE, OLD HIGHWAY 101. NORTH ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    8. EEL RIVER SOUTH FORK BRIDGE, OLD HIGHWAY 101. NORTH OF LEGGETT, HUMBOLDT COUNTY, CALIFORNIA. LOOKING N. - Redwood National & State Parks Roads, California coast from Crescent City to Trinidad, Crescent City, Del Norte County, CA

  11. 9. EEL RIVER SOUTH FORK BRIDGE, OLD HIGHWAY 101. NORTH ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    9. EEL RIVER SOUTH FORK BRIDGE, OLD HIGHWAY 101. NORTH OF LEGGETT, HUMBOLDT COUNTY, CALIFORNIA. LOOKING W. - Redwood National & State Parks Roads, California coast from Crescent City to Trinidad, Crescent City, Del Norte County, CA

  12. Little Known Facts about the Common Tuning Fork.

    ERIC Educational Resources Information Center

    Ong, P. P.

    2002-01-01

    Explains the physical principles of the tuning fork which has a common use in teaching laboratories. Includes information on its vibration, frequency of vibration, elasticity, and reasons for having two prongs. (YDS)

  13. 17. DETAIL VIEW OF WHAT APPEARS TO BE STIRRING FORK ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    17. DETAIL VIEW OF WHAT APPEARS TO BE STIRRING FORK THAT MIXED COFFEE BEANS AS THEY WERE HUSKED - Hacienda Cafetalera Santa Clara, Coffee Mill, KM 19, PR Route 372, Hacienda La Juanita, Yauco Municipio, PR

  14. HOMESTEAD, LAKE FORK, AND LICK CREEK ROADLESS AREAS, OREGON.

    USGS Publications Warehouse

    Evans, James G.; Conyac, Martin D.

    1984-01-01

    A mineral survey concluded that the Homestead, Lake Fork and Lick Creek Roadless Area, Oregon offer little promise for the occurrence of mineral or energy resources in the bedrock. Probable mineral-resource potential is assigned to the west and north parts of the Lake Fork Roadless Area, where gold resources may occur in glacial deposits and alluvium transported into this area from sources outside the roadless area to the west.

  15. Extensive RPA2 hyperphosphorylation promotes apoptosis in response to DNA replication stress in CHK1 inhibited cells.

    PubMed

    Zuazua-Villar, Pedro; Ganesh, Anil; Phear, Geraldine; Gagou, Mary E; Meuth, Mark

    2015-11-16

    The replication protein A (RPA)-ssDNA complex formed at arrested replication forks recruits key proteins to activate the ATR-CHK1 signalling cascade. When CHK1 is inhibited during DNA replication stress, RPA2 is extensively hyperphosphorylated. Here, we investigated the role of RPA2 hyperphosphorylation in the fate of cells when CHK1 is inhibited. We show that proteins normally involved in DNA repair (RAD51) or control of RPA phosphorylation (the PP4 protein phosphatase complex) are not recruited to the genome after treatment with CHK1 and DNA synthesis inhibitors. This is not due to RPA2 hyperphosphorylation as suppression of this response does not restore loading suggesting that recruitment requires active CHK1. To determine whether RPA2 hyperphosphorylation protects stalled forks from collapse or induction of apoptosis in CHK1 inhibited cells during replication stress, cells expressing RPA2 genes mutated at key phosphorylation sites were characterized. Mutant RPA2 rescued cells from RPA2 depletion and reduced the level of apoptosis induced by treatment with CHK1 and replication inhibitors however the incidence of double strand breaks was not affected. Our data indicate that RPA2 hyperphosphorylation promotes cell death during replication stress when CHK1 function is compromised but does not appear to be essential for replication fork integrity. PMID:26271993

  16. TRAIP is a PCNA-binding ubiquitin ligase that protects genome stability after replication stress

    PubMed Central

    Hoffmann, Saskia; Smedegaard, Stine; Nakamura, Kyosuke; Mortuza, Gulnahar B.; Räschle, Markus; Ibañez de Opakua, Alain; Oka, Yasuyoshi; Feng, Yunpeng; Blanco, Francisco J.; Mann, Matthias; Montoya, Guillermo; Groth, Anja; Bekker-Jensen, Simon

    2016-01-01

    Cellular genomes are highly vulnerable to perturbations to chromosomal DNA replication. Proliferating cell nuclear antigen (PCNA), the processivity factor for DNA replication, plays a central role as a platform for recruitment of genome surveillance and DNA repair factors to replication forks, allowing cells to mitigate the threats to genome stability posed by replication stress. We identify the E3 ubiquitin ligase TRAIP as a new factor at active and stressed replication forks that directly interacts with PCNA via a conserved PCNA-interacting peptide (PIP) box motif. We show that TRAIP promotes ATR-dependent checkpoint signaling in human cells by facilitating the generation of RPA-bound single-stranded DNA regions upon replication stress in a manner that critically requires its E3 ligase activity and is potentiated by the PIP box. Consequently, loss of TRAIP function leads to enhanced chromosomal instability and decreased cell survival after replication stress. These findings establish TRAIP as a PCNA-binding ubiquitin ligase with an important role in protecting genome integrity after obstacles to DNA replication. PMID:26711499

  17. Ubiquitin-family modifications in the replication of DNA damage.

    PubMed

    Lehmann, Alan R

    2011-09-16

    The cell uses specialised Y-family DNA polymerases or damage avoidance mechanisms to replicate past damaged sites in DNA. These processes are under complex regulatory systems, which employ different types of post-translational modification. All the Y-family polymerases have ubiquitin binding domains that bind to mono-ubiquitinated PCNA to effect the switching from replicative to Y-family polymerase. Ubiquitination and de-ubiquitination of PCNA are tightly regulated. There is also evidence for another as yet unidentified ubiquitinated protein being involved in recruitment of Y-family polymerases to chromatin. Poly-ubiquitination of PCNA stimulates damage avoidance, and, at least in yeast, PCNA is SUMOylated to prevent unwanted recombination events at the replication fork. The Y-family polymerases themselves can be ubiquitinated and, in the case of DNA polymerase η, this results in the polymerase being excluded from chromatin. PMID:21704031

  18. The Budding Yeast Ubiquitin Protease Ubp7 Is a Novel Component Involved in S Phase Progression.

    PubMed

    Böhm, Stefanie; Szakal, Barnabas; Herken, Benjamin W; Sullivan, Meghan R; Mihalevic, Michael J; Kabbinavar, Faiz F; Branzei, Dana; Clark, Nathan L; Bernstein, Kara A

    2016-02-26

    DNA damage must be repaired in an accurate and timely fashion to preserve genome stability. Cellular mechanisms preventing genome instability are crucial to human health because genome instability is considered a hallmark of cancer. Collectively referred to as the DNA damage response, conserved pathways ensure proper DNA damage recognition and repair. The function of numerous DNA damage response components is fine-tuned by posttranslational modifications, including ubiquitination. This not only involves the enzyme cascade responsible for conjugating ubiquitin to substrates but also requires enzymes that mediate directed removal of ubiquitin. Deubiquitinases remove ubiquitin from substrates to prevent degradation or to mediate signaling functions. The Saccharomyces cerevisiae deubiquitinase Ubp7 has been characterized previously as an endocytic factor. However, here we identify Ubp7 as a novel factor affecting S phase progression after hydroxyurea treatment and demonstrate an evolutionary and genetic interaction of Ubp7 with DNA damage repair pathways of homologous recombination and nucleotide excision repair. We find that deletion of UBP7 sensitizes cells to hydroxyurea and cisplatin and demonstrate that factors that stabilize replication forks are critical under these conditions. Furthermore, ubp7Δ cells exhibit an S phase progression defect upon checkpoint activation by hydroxyurea treatment. ubp7Δ mutants are epistatic to factors involved in histone maintenance and modification, and we find that a subset of Ubp7 is chromatin-associated. In summary, our results suggest that Ubp7 contributes to S phase progression by affecting the chromatin state at replication forks, and we propose histone H2B ubiquitination as a potential substrate of Ubp7. PMID:26740628

  19. A nuclease-hypersensitive region forms de novo after chromosome replication.

    PubMed

    Solomon, M J; Varshavsky, A

    1987-10-01

    Regular nucleosome arrays in eucaryotic chromosomes are punctuated at specific locations, such as active promoters and replication origins, by apparently nucleosome-free sites, also called nuclease-hypersensitive, or exposed, regions. The -400-base pair-exposed region within simian virus 40 (SV40) chromosomes is present in approximately 20% of the chromosomes in lytically infected cells and encompasses the replication origin, transcriptional enhancer, and both late and early SV40 promoters. We report that nearly all SV40 chromosomes lacked the exposed region during replication and that newly formed chromosomes acquired the exposed region of the same degree as did bulk SV40 chromosomes within 1 h after replication. Furthermore, a much lower but significant level of exposure was detectable in late SV40 replication intermediates, indicating that formation of the exposed region could start within minutes after passage of the replication fork. PMID:2824998

  20. Local regulation of the Srs2 helicase by the SUMO-like domain protein Esc2 promotes recombination at sites of stalled replication

    PubMed Central

    Urulangodi, Madhusoodanan; Sebesta, Marek; Menolfi, Demis; Szakal, Barnabas; Sollier, Julie; Sisakova, Alexandra; Krejci, Lumir; Branzei, Dana

    2015-01-01

    Accurate completion of replication relies on the ability of cells to activate error-free recombination-mediated DNA damage bypass at sites of perturbed replication. However, as anti-recombinase activities are also recruited to replication forks, how recombination-mediated damage bypass is enabled at replication stress sites remained puzzling. Here we uncovered that the conserved SUMO-like domain-containing Saccharomyces cerevisiae protein Esc2 facilitates recombination-mediated DNA damage tolerance by allowing optimal recruitment of the Rad51 recombinase specifically at sites of perturbed replication. Mechanistically, Esc2 binds stalled replication forks and counteracts the anti-recombinase Srs2 helicase via a two-faceted mechanism involving chromatin recruitment and turnover of Srs2. Importantly, point mutations in the SUMO-like domains of Esc2 that reduce its interaction with Srs2 cause suboptimal levels of Rad51 recruitment at damaged replication forks. In conclusion, our results reveal how recombination-mediated DNA damage tolerance is locally enabled at sites of replication stress and globally prevented at undamaged replicating chromosomes. PMID:26443850

  1. Bacteriophage replication modules.

    PubMed

    Weigel, Christoph; Seitz, Harald

    2006-05-01

    Bacteriophages (prokaryotic viruses) are favourite model systems to study DNA replication in prokaryotes, and provide examples for every theoretically possible replication mechanism. In addition, the elucidation of the intricate interplay of phage-encoded replication factors with 'host' factors has always advanced the understanding of DNA replication in general. Here we review bacteriophage replication based on the long-standing observation that in most known phage genomes the replication genes are arranged as modules. This allows us to discuss established model systems--f1/fd, phiX174, P2, P4, lambda, SPP1, N15, phi29, T7 and T4--along with those numerous phages that have been sequenced but not studied experimentally. The review of bacteriophage replication mechanisms and modules is accompanied by a compendium of replication origins and replication/recombination proteins (available as supplementary material online). PMID:16594962

  2. Replication of Tobamovirus RNA.

    PubMed

    Ishibashi, Kazuhiro; Ishikawa, Masayuki

    2016-08-01

    Tobacco mosaic virus and other tobamoviruses have served as models for studying the mechanisms of viral RNA replication. In tobamoviruses, genomic RNA replication occurs via several steps: (a) synthesis of viral replication proteins by translation of the genomic RNA; (b) translation-coupled binding of the replication proteins to a 5'-terminal region of the genomic RNA; (c) recruitment of the genomic RNA by replication proteins onto membranes and formation of a complex with host proteins TOM1 and ARL8; (d) synthesis of complementary (negative-strand) RNA in the complex; and (e) synthesis of progeny genomic RNA. This article reviews current knowledge on tobamovirus RNA replication, particularly regarding how the genomic RNA is specifically selected as a replication template and how the replication proteins are activated. We also focus on the roles of the replication proteins in evading or suppressing host defense systems. PMID:27296148

  3. Incomplete replication generates somatic DNA alterations within Drosophila polytene salivary gland cells

    PubMed Central

    Yarosh, Will

    2014-01-01

    DNA replication remains unfinished in many Drosophila polyploid cells, which harbor disproportionately fewer copies of late-replicating chromosomal regions. By analyzing paired-end high-throughput sequence data from polytene larval salivary gland cells, we define 112 underreplicated (UR) euchromatic regions 60–480 kb in size. To determine the effects of underreplication on genome integrity, we analyzed anomalous read pairs and breakpoint reads throughout the euchromatic genome. Each UR euchromatic region contains many different deletions 10–500 kb in size, while very few deletions are present in fully replicated chromosome regions or UR zones from embryo DNA. Thus, during endocycles, stalled forks within UR regions break and undergo local repair instead of remaining stable and generating nested forks. As a result, each salivary gland cell contains hundreds of unique deletions that account for their copy number reductions. Similar UR regions and deletions were observed in ovarian DNA, suggesting that incomplete replication, fork breakage, and repair occur widely in polytene cells. UR regions are enriched in genes encoding immunoglobulin superfamily proteins and contain many neurally expressed and homeotic genes. We suggest that the extensive somatic DNA instability described here underlies position effect variegation, molds the structure of polytene chromosomes, and should be investigated for possible functions. PMID:25128500

  4. The Drosophila Werner exonuclease participates in an exonuclease-independent response to replication stress.

    PubMed

    Bolterstein, Elyse; Rivero, Rachel; Marquez, Melissa; McVey, Mitch

    2014-06-01

    Members of the RecQ family of helicases are known for their roles in DNA repair, replication, and recombination. Mutations in the human RecQ helicases, WRN and BLM, cause Werner and Bloom syndromes, which are diseases characterized by genome instability and an increased risk of cancer. While WRN contains both a helicase and an exonuclease domain, the Drosophila melanogaster homolog, WRNexo, contains only the exonuclease domain. Therefore the Drosophila model system provides a unique opportunity to study the exonuclease functions of WRN separate from the helicase. We created a null allele of WRNexo via imprecise P-element excision. The null WRNexo mutants are not sensitive to double-strand break-inducing reagents, suggesting that the exonuclease does not play a key role in homologous recombination-mediated repair of DSBs. However, WRNexo mutant embryos have a reduced hatching frequency and larvae are sensitive to the replication fork-stalling reagent, hydroxyurea (HU), suggesting that WRNexo is important in responding to replication stress. The role of WRNexo in the HU-induced stress response is independent of Rad51. Interestingly, the hatching defect and HU sensitivity of WRNexo mutants do not occur in flies containing an exonuclease-dead copy of WRNexo, suggesting that the role of WRNexo in replication is independent of exonuclease activity. Additionally, WRNexo and Blm mutants exhibit similar sensitivity to HU and synthetic lethality in combination with mutations in structure-selective endonucleases. We propose that WRNexo and BLM interact to promote fork reversal following replication fork stalling and in their absence regressed forks are restarted through a Rad51-mediated process. PMID:24709634

  5. Archaeal DNA replication.

    PubMed

    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

  6. A Checkpoint-Related Function of the MCM Replicative Helicase Is Required to Avert Accumulation of RNA:DNA Hybrids during S-phase and Ensuing DSBs during G2/M.

    PubMed

    Vijayraghavan, Sriram; Tsai, Feng-Ling; Schwacha, Anthony

    2016-08-01

    The Mcm2-7 complex is the catalytic core of the eukaryotic replicative helicase. Here, we identify a new role for this complex in maintaining genome integrity. Using both genetic and cytological approaches, we find that a specific mcm allele (mcm2DENQ) causes elevated genome instability that correlates with the appearance of numerous DNA-damage associated foci of γH2AX and Rad52. We further find that the triggering events for this genome instability are elevated levels of RNA:DNA hybrids and an altered DNA topological state, as over-expression of either RNaseH (an enzyme specific for degradation of RNA in RNA:DNA hybrids) or Topoisomerase 1 (an enzyme that relieves DNA supercoiling) can suppress the mcm2DENQ DNA-damage phenotype. Moreover, the observed DNA damage has several additional unusual properties, in that DNA damage foci appear only after S-phase, in G2/M, and are dependent upon progression into metaphase. In addition, we show that the resultant DNA damage is not due to spontaneous S-phase fork collapse. In total, these unusual mcm2DENQ phenotypes are markedly similar to those of a special previously-studied allele of the checkpoint sensor kinase ATR/MEC1, suggesting a possible regulatory interplay between Mcm2-7 and ATR during unchallenged growth. As RNA:DNA hybrids primarily result from transcription perturbations, we suggest that surveillance-mediated modulation of the Mcm2-7 activity plays an important role in preventing catastrophic conflicts between replication forks and transcription complexes. Possible relationships among these effects and the recently discovered role of Mcm2-7 in the DNA replication checkpoint induced by HU treatment are discussed. PMID:27556397

  7. A Checkpoint-Related Function of the MCM Replicative Helicase Is Required to Avert Accumulation of RNA:DNA Hybrids during S-phase and Ensuing DSBs during G2/M

    PubMed Central

    2016-01-01

    The Mcm2-7 complex is the catalytic core of the eukaryotic replicative helicase. Here, we identify a new role for this complex in maintaining genome integrity. Using both genetic and cytological approaches, we find that a specific mcm allele (mcm2DENQ) causes elevated genome instability that correlates with the appearance of numerous DNA-damage associated foci of γH2AX and Rad52. We further find that the triggering events for this genome instability are elevated levels of RNA:DNA hybrids and an altered DNA topological state, as over-expression of either RNaseH (an enzyme specific for degradation of RNA in RNA:DNA hybrids) or Topoisomerase 1 (an enzyme that relieves DNA supercoiling) can suppress the mcm2DENQ DNA-damage phenotype. Moreover, the observed DNA damage has several additional unusual properties, in that DNA damage foci appear only after S-phase, in G2/M, and are dependent upon progression into metaphase. In addition, we show that the resultant DNA damage is not due to spontaneous S-phase fork collapse. In total, these unusual mcm2DENQ phenotypes are markedly similar to those of a special previously-studied allele of the checkpoint sensor kinase ATR/MEC1, suggesting a possible regulatory interplay between Mcm2-7 and ATR during unchallenged growth. As RNA:DNA hybrids primarily result from transcription perturbations, we suggest that surveillance-mediated modulation of the Mcm2-7 activity plays an important role in preventing catastrophic conflicts between replication forks and transcription complexes. Possible relationships among these effects and the recently discovered role of Mcm2-7 in the DNA replication checkpoint induced by HU treatment are discussed. PMID:27556397

  8. Replicating repetitive DNA.

    PubMed

    Tognetti, Silvia; Speck, Christian

    2016-05-27

    The function and regulation of repetitive DNA, the 'dark matter' of the genome, is still only rudimentarily understood. Now a study investigating DNA replication of repetitive centromeric chromosome segments has started to expose a fascinating replication program that involves suppression of ATR signalling, in particular during replication stress. PMID:27230530

  9. Replication of Structured DNA and its implication in epigenetic stability

    PubMed Central

    Cea, Valentina; Cipolla, Lina; Sabbioneda, Simone

    2015-01-01

    DNA replication is an extremely risky process that cells have to endure in order to correctly duplicate and segregate their genome. This task is particularly sensitive to DNA damage and multiple mechanisms have evolved to protect DNA replication as a block to the replication fork could lead to genomic instability and possibly cell death. The DNA in the genome folds, for the most part, into the canonical B-form but in some instances can form complex secondary structures such as G-quadruplexes (G4). These G rich regions are thermodynamically stable and can constitute an obstacle to DNA and RNA metabolism. The human genome contains more than 350,000 sequences potentially capable to form G-quadruplexes and these structures are involved in a variety of cellular processes such as initiation of DNA replication, telomere maintenance and control of gene expression. Only recently, we started to understand how G4 DNA poses a problem to DNA replication and how its successful bypass requires the coordinated activity of ssDNA binding proteins, helicases and specialized DNA polymerases. Their role in the resolution and replication of structured DNA crucially prevents both genetic and epigenetic instability across the genome. PMID:26136769

  10. A Multi-Fork Z-Axis Quartz Micromachined Gyroscope

    PubMed Central

    Feng, Lihui; Zhao, Ke; Sun, Yunan; Cui, Jianmin; Cui, Fang; Yang, Aiying

    2013-01-01

    A novel multi-fork z-axis gyroscope is presented in this paper. Different from traditional quartz gyroscopes, the lateral electrodes of the sense beam can be arranged in simple patterns; as a result, the fabrication is simplified. High sensitivity is achieved by the multi-fork design. The working principles are introduced, while the finite element method (FEM) is used to simulate the modal and sensitivity. A quartz fork is fabricated, and a prototype is assembled. Impedance testing shows that the drive frequency and sense frequency are similar to the simulations, and the quality factor is approximately 10,000 in air. The scale factor is measured to be 18.134 mV/(°/s) and the nonlinearity is 0.40% in a full-scale input range of ±250 °/s. PMID:24048339

  11. DNA replication is altered in Immunodeficiency Centromeric instability Facial anomalies (ICF) cells carrying DNMT3B mutations

    PubMed Central

    Lana, Erica; Mégarbané, André; Tourrière, Hélène; Sarda, Pierre; Lefranc, Gérard; Claustres, Mireille; De Sario, Albertina

    2012-01-01

    ICF syndrome is a rare autosomal recessive disorder that is characterized by Immunodeficiency, Centromeric instability, and Facial anomalies. In all, 60% of ICF patients have mutations in the DNMT3B (DNA methyltransferase 3B) gene, encoding a de novo DNA methyltransferase. In ICF cells, constitutive heterochromatin is hypomethylated and decondensed, metaphase chromosomes undergo rearrangements (mainly involving juxtacentromeric regions), and more than 700 genes are aberrantly expressed. This work shows that DNA replication is also altered in ICF cells: (i) heterochromatic genes replicate earlier in the S-phase; (ii) global replication fork speed is higher; and (iii) S-phase is shorter. These replication defects may result from chromatin changes that modify DNA accessibility to the replication machinery and/or from changes in the expression level of genes involved in DNA replication. This work highlights the interest of using ICF cells as a model to investigate how DNA methylation regulates DNA replication in humans. PMID:22378288

  12. The Escherichia coli UvrD helicase is essential for Tus removal during recombination-dependent replication restart from Ter sites.

    PubMed

    Bidnenko, Vladimir; Lestini, Roxane; Michel, Bénédicte

    2006-10-01

    Blocking replication forks in the Escherichia coli chromosome by ectopic Ter sites renders the RecBCD pathway of homologous recombination and SOS induction essential for viability. In this work, we show that the E. coli helicase II (UvrD) is also essential for the growth of cells where replication forks are arrested at ectopic Ter sites. We propose that UvrD is required for Tus removal from Ter sites. The viability of a SOS non-inducible Ter-blocked strain is fully restored by the expression of the two SOS-induced proteins UvrD and RecA at high level, indicating that these are the only two SOS-induced proteins required for replication across Ter/Tus complexes. Several observations suggest that UvrD acts in concert with homologous recombination and we propose that UvrD is associated with recombination-initiated replication forks and that it removes Tus when a PriA-dependent, restarted replication fork goes across the Ter/Tus complex. Finally, expression of the UvrD homologue from Bacilus subtilis PcrA restores the growth of uvrD-deficient Ter-blocked cells, indicating that the capacity to dislodge Tus is conserved in this distant bacterial species. PMID:17020578

  13. Single-molecule analysis of the E. coli replisome and use of clamps to bypass replication barriers

    PubMed Central

    Georgescu, Roxana E.; Yao, Nina Y.; O’Donnell, Mike

    2010-01-01

    The process of chromosome duplication faces many obstacles. One way to circumvent blocks is to hop over them by placing a new clamp on a downstream primer. This resembles lagging strand synthesis, where the tight grip of polymerase to the clamp and DNA must be overcome upon completing each Okazaki fragment so it can transfer to new primed sites. This review focuses on recent single-molecule studies showing that E. coli Pol III can hop from one clamp to another without leaving the replication fork. This capability provides a means to circumvent obstacles like transcription or DNA lesions without fork collapse. PMID:20388515

  14. Bank stability and channel width adjustment, East Fork River, Wyoming.

    USGS Publications Warehouse

    Andrews, E.D.

    1982-01-01

    Frequent surveys of eight cross sections located in self-formed reaches of the East Fork River, Wyoming, during the 1974 snowmelt flood showed a close relation between channel morphology and scour and fill. Those cross sections narrower than the mean reach width filled at discharges less than bankfull and scoured at discharges greater than bankfull. Those cross sections wider than the mean reach width scoured at discharges less than bankfull and filled at discharges greater than bankfull. Bank stability, and to some extent the adjustment of stream channel width, in the East Fork River study reach appears to be controlled by the processes of scour and fill. -from Author

  15. 78 FR 13660 - Middle Fork American River Project; Notice of Availability of the Final Environmental Impact...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-02-28

    ... Energy Regulatory Commission Middle Fork American River Project; Notice of Availability of the Final Environmental Impact Statement for the Middle Fork American River Hydrolectric Project In accordance with the... Projects has reviewed the application for license for the Middle Fork American River Hydroelectric...

  16. 76 FR 35909 - Temporary Concession Contract for Big South Fork National Recreation Area, TN/KY

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-06-20

    ... contract for Big South Fork National Recreation Area, TN/KY. SUMMARY: Pursuant to 36 CFR 51.24, public... National Park Service Temporary Concession Contract for Big South Fork National Recreation Area, TN/KY... contract for the conduct of certain visitor services within Big South Fork National Recreation...

  17. Mec1, INO80, and the PAF1 complex cooperate to limit transcription replication conflicts through RNAPII removal during replication stress

    PubMed Central

    Poli, Jérôme; Gerhold, Christian-Benedikt; Tosi, Alessandro; Hustedt, Nicole; Seeber, Andrew; Sack, Ragna; Herzog, Franz; Pasero, Philippe; Shimada, Kenji; Hopfner, Karl-Peter; Gasser, Susan M.

    2016-01-01

    Little is known about how cells ensure DNA replication in the face of RNA polymerase II (RNAPII)-mediated transcription, especially under conditions of replicative stress. Here we present genetic and proteomic analyses from budding yeast that uncover links between the DNA replication checkpoint sensor Mec1–Ddc2 (ATR–ATRIP), the chromatin remodeling complex INO80C (INO80 complex), and the transcription complex PAF1C (PAF1 complex). We found that a subset of chromatin-bound RNAPII is degraded in a manner dependent on Mec1, INO80, and PAF1 complexes in cells exposed to hydroxyurea (HU). On HU, Mec1 triggers the efficient removal of PAF1C and RNAPII from transcribed genes near early firing origins. Failure to evict RNAPII correlates inversely with recovery from replication stress: paf1Δ cells, like ino80 and mec1 mutants, fail to restart forks efficiently after stalling. Our data reveal unexpected synergies between INO80C, Mec1, and PAF1C in the maintenance of genome integrity and suggest a mechanism of RNAPII degradation that reduces transcription–replication fork collision. PMID:26798134

  18. SV40 DNA replication: From the A gene to a nanomachine

    PubMed Central

    Fanning, Ellen; Zhao, Kun

    2009-01-01

    Duplication of the simian virus 40 (SV40) genome is the best understood eukaryotic DNA replication process to date. Like most prokaryotic genomes, the SV40 genome is a circular duplex DNA organized in a single replicon. This small viral genome, its association with host histones in nucleosomes, and its dependence on the host cell milieu for replication factors and precursors led to its adoption as a simple and powerful model. The steps in replication, the viral initiator, the host proteins, and their mechanisms of action were initially defined using a cell-free SV40 replication reaction. Although our understanding of the vastly more complex host replication fork is advancing, no eukaryotic replisome has yet been reconstituted and the SV40 paradigm remains a point of reference. This article reviews some of the milestones in the development of this paradigm and speculates on its potential utility to address unsolved questions in eukaryotic genome maintenance. PMID:19101707

  19. 4D Visualization of replication foci in mammalian cells corresponding to individual replicons

    PubMed Central

    Chagin, V. O.; Casas-Delucchi, C. S.; Reinhart, M.; Schermelleh, L.; Markaki, Y.; Maiser, A.; Bolius, J. J.; Bensimon, A.; Fillies, M.; Domaing, P.; Rozanov, Y. M.; Leonhardt, H.; Cardoso, M. C.

    2016-01-01

    Since the pioneering proposal of the replicon model of DNA replication 50 years ago, the predicted replicons have not been identified and quantified at the cellular level. Here, we combine conventional and super-resolution microscopy of replication sites in live and fixed cells with computational image analysis. We complement these data with genome size measurements, comprehensive analysis of S-phase dynamics and quantification of replication fork speed and replicon size in human and mouse cells. These multidimensional analyses demonstrate that replication foci (RFi) in three-dimensional (3D) preserved somatic mammalian cells can be optically resolved down to single replicons throughout S-phase. This challenges the conventional interpretation of nuclear RFi as replication factories, that is, the complex entities that process multiple clustered replicons. Accordingly, 3D genome organization and duplication can be now followed within the chromatin context at the level of individual replicons. PMID:27052570

  20. 4D Visualization of replication foci in mammalian cells corresponding to individual replicons.

    PubMed

    Chagin, V O; Casas-Delucchi, C S; Reinhart, M; Schermelleh, L; Markaki, Y; Maiser, A; Bolius, J J; Bensimon, A; Fillies, M; Domaing, P; Rozanov, Y M; Leonhardt, H; Cardoso, M C

    2016-01-01

    Since the pioneering proposal of the replicon model of DNA replication 50 years ago, the predicted replicons have not been identified and quantified at the cellular level. Here, we combine conventional and super-resolution microscopy of replication sites in live and fixed cells with computational image analysis. We complement these data with genome size measurements, comprehensive analysis of S-phase dynamics and quantification of replication fork speed and replicon size in human and mouse cells. These multidimensional analyses demonstrate that replication foci (RFi) in three-dimensional (3D) preserved somatic mammalian cells can be optically resolved down to single replicons throughout S-phase. This challenges the conventional interpretation of nuclear RFi as replication factories, that is, the complex entities that process multiple clustered replicons. Accordingly, 3D genome organization and duplication can be now followed within the chromatin context at the level of individual replicons. PMID:27052570

  1. Replication stress caused by low MCM expression limits fetal erythropoiesis and hematopoietic stem cell functionality

    PubMed Central

    Alvarez, Silvia; Díaz, Marcos; Flach, Johanna; Rodriguez-Acebes, Sara; López-Contreras, Andrés J.; Martínez, Dolores; Cañamero, Marta; Fernández-Capetillo, Oscar; Isern, Joan; Passegué, Emmanuelle; Méndez, Juan

    2015-01-01

    Replicative stress during embryonic development influences ageing and predisposition to disease in adults. A protective mechanism against replicative stress is provided by the licensing of thousands of origins in G1 that are not necessarily activated in the subsequent S-phase. These ‘dormant' origins provide a backup in the presence of stalled forks and may confer flexibility to the replication program in specific cell types during differentiation, a role that has remained unexplored. Here we show, using a mouse strain with hypomorphic expression of the origin licensing factor mini-chromosome maintenance (MCM)3 that limiting origin licensing in vivo affects the functionality of hematopoietic stem cells and the differentiation of rapidly-dividing erythrocyte precursors. Mcm3-deficient erythroblasts display aberrant DNA replication patterns and fail to complete maturation, causing lethal anemia. Our results indicate that hematopoietic progenitors are particularly sensitive to replication stress, and full origin licensing ensures their correct differentiation and functionality. PMID:26456157

  2. The Balance between Recombination Enzymes and Accessory Replicative Helicases in Facilitating Genome Duplication.

    PubMed

    Syeda, Aisha H; Atkinson, John; Lloyd, Robert G; McGlynn, Peter

    2016-01-01

    Accessory replicative helicases aid the primary replicative helicase in duplicating protein-bound DNA, especially transcribed DNA. Recombination enzymes also aid genome duplication by facilitating the repair of DNA lesions via strand exchange and also processing of blocked fork DNA to generate structures onto which the replisome can be reloaded. There is significant interplay between accessory helicases and recombination enzymes in both bacteria and lower eukaryotes but how these replication repair systems interact to ensure efficient genome duplication remains unclear. Here, we demonstrate that the DNA content defects of Escherichia coli cells lacking the strand exchange protein RecA are driven primarily by conflicts between replication and transcription, as is the case in cells lacking the accessory helicase Rep. However, in contrast to Rep, neither RecA nor RecBCD, the helicase/exonuclease that loads RecA onto dsDNA ends, is important for maintaining rapid chromosome duplication. Furthermore, RecA and RecBCD together can sustain viability in the absence of accessory replicative helicases but only when transcriptional barriers to replication are suppressed by an RNA polymerase mutation. Our data indicate that the minimisation of replisome pausing by accessory helicases has a more significant impact on successful completion of chromosome duplication than recombination-directed fork repair. PMID:27483323

  3. The Balance between Recombination Enzymes and Accessory Replicative Helicases in Facilitating Genome Duplication

    PubMed Central

    Syeda, Aisha H.; Atkinson, John; Lloyd, Robert G.; McGlynn, Peter

    2016-01-01

    Accessory replicative helicases aid the primary replicative helicase in duplicating protein-bound DNA, especially transcribed DNA. Recombination enzymes also aid genome duplication by facilitating the repair of DNA lesions via strand exchange and also processing of blocked fork DNA to generate structures onto which the replisome can be reloaded. There is significant interplay between accessory helicases and recombination enzymes in both bacteria and lower eukaryotes but how these replication repair systems interact to ensure efficient genome duplication remains unclear. Here, we demonstrate that the DNA content defects of Escherichia coli cells lacking the strand exchange protein RecA are driven primarily by conflicts between replication and transcription, as is the case in cells lacking the accessory helicase Rep. However, in contrast to Rep, neither RecA nor RecBCD, the helicase/exonuclease that loads RecA onto dsDNA ends, is important for maintaining rapid chromosome duplication. Furthermore, RecA and RecBCD together can sustain viability in the absence of accessory replicative helicases but only when transcriptional barriers to replication are suppressed by an RNA polymerase mutation. Our data indicate that the minimisation of replisome pausing by accessory helicases has a more significant impact on successful completion of chromosome duplication than recombination-directed fork repair. PMID:27483323

  4. A tale of two HSV-1 helicases: roles of phage and animal virus helicases in DNA replication and recombination.

    PubMed

    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

  5. Dynamic Escherichia coli SeqA complexes organize the newly replicated DNA at a considerable distance from the replisome

    PubMed Central

    Helgesen, Emily; Fossum-Raunehaug, Solveig; Sætre, Frank; Schink, Kay Oliver; Skarstad, Kirsten

    2015-01-01

    The Escherichia coli SeqA protein binds to newly replicated, hemimethylated DNA behind replication forks and forms structures consisting of several hundred SeqA molecules bound to about 100 kb of DNA. It has been suggested that SeqA structures either direct the new sister DNA molecules away from each other or constitute a spacer that keeps the sisters together. We have developed an image analysis script that automatically measures the distance between neighboring foci in cells. Using this tool as well as direct stochastic optical reconstruction microscopy (dSTORM) we find that in cells with fluorescently tagged SeqA and replisome the sister SeqA structures were situated close together (less than about 30 nm apart) and relatively far from the replisome (on average 200–300 nm). The results support the idea that newly replicated sister molecules are kept together behind the fork and suggest the existence of a stretch of DNA between the replisome and SeqA which enjoys added stabilization. This could be important in facilitating DNA transactions such as recombination, mismatch repair and topoisomerase activity. In slowly growing cells without ongoing replication forks the SeqA protein was found to reside at the fully methylated origins prior to initiation of replication. PMID:25722374

  6. The DNA-Binding Domain of S. pombe Mrc1 (Claspin) Acts to Enhance Stalling at Replication Barriers

    PubMed Central

    Zech, Juergen; Godfrey, Emma Louise; Masai, Hisao; Hartsuiker, Edgar; Dalgaard, Jacob Zeuthen

    2015-01-01

    During S-phase replication forks can stall at specific genetic loci. At some loci, the stalling events depend on the replisome components Schizosaccharomyces pombe Swi1 (Saccharomyces cerevisiae Tof1) and Swi3 (S. cerevisiae Csm3) as well as factors that bind DNA in a site-specific manner. Using a new genetic screen we identified Mrc1 (S. cerevisiae Mrc1/metazoan Claspin) as a replisome component involved in replication stalling. Mrc1 is known to form a sub-complex with Swi1 and Swi3 within the replisome and is required for the intra-S phase checkpoint activation. This discovery is surprising as several studies show that S. cerevisiae Mrc1 is not required for replication barrier activity. In contrast, we show that deletion of S. pombe mrc1 leads to an approximately three-fold reduction in barrier activity at several barriers and that Mrc1’s role in replication fork stalling is independent of its role in checkpoint activation. Instead, S. pombe Mrc1 mediated fork stalling requires the presence of a functional copy of its phylogenetically conserved DNA binding domain. Interestingly, this domain is on the sequence level absent from S. cerevisiae Mrc1. Our study indicates that direct interactions between the eukaryotic replisome and the DNA are important for site-specific replication stalling. PMID:26201080

  7. NEK8 links the ATR-regulated replication stress response and S phase CDK activity to renal ciliopathies.

    PubMed

    Choi, Hyo Jei Claudia; Lin, Jia-Ren; Vannier, Jean-Baptiste; Slaats, Gisela G; Kile, Andrew C; Paulsen, Renee D; Manning, Danielle K; Beier, David R; Giles, Rachel H; Boulton, Simon J; Cimprich, Karlene A

    2013-08-22

    Renal ciliopathies are a leading cause of kidney failure, but their exact etiology is poorly understood. NEK8/NPHP9 is a ciliary kinase associated with two renal ciliopathies in humans and mice, nephronophthisis (NPHP) and polycystic kidney disease. Here, we identify NEK8 as a key effector of the ATR-mediated replication stress response. Cells lacking NEK8 form spontaneous DNA double-strand breaks (DSBs) that further accumulate when replication forks stall, and they exhibit reduced fork rates, unscheduled origin firing, and increased replication fork collapse. NEK8 suppresses DSB formation by limiting cyclin A-associated CDK activity. Strikingly, a mutation in NEK8 that is associated with renal ciliopathies affects its genome maintenance functions. Moreover, kidneys of NEK8 mutant mice accumulate DNA damage, and loss of NEK8 or replication stress similarly disrupts renal cell architecture in a 3D-culture system. Thus, NEK8 is a critical component of the DNA damage response that links replication stress with cystic kidney disorders. PMID:23973373

  8. The evolution of replicators.

    PubMed Central

    Szathmáry, E

    2000-01-01

    Replicators of interest in chemistry, biology and culture are briefly surveyed from a conceptual point of view. Systems with limited heredity have only a limited evolutionary potential because the number of available types is too low. Chemical cycles, such as the formose reaction, are holistic replicators since replication is not based on the successive addition of modules. Replicator networks consisting of catalytic molecules (such as reflexively autocatalytic sets of proteins, or reproducing lipid vesicles) are hypothetical ensemble replicators, and their functioning rests on attractors of their dynamics. Ensemble replicators suffer from the paradox of specificity: while their abstract feasibility seems to require a high number of molecular types, the harmful effect of side reactions calls for a small system size. No satisfactory solution to this problem is known. Phenotypic replicators do not pass on their genotypes, only some aspects of the phenotype are transmitted. Phenotypic replicators with limited heredity include genetic membranes, prions and simple memetic systems. Memes in human culture are unlimited hereditary, phenotypic replicators, based on language. The typical path of evolution goes from limited to unlimited heredity, and from attractor-based to modular (digital) replicators. PMID:11127914

  9. SETD2 loss-of-function promotes renal cancer branched evolution through replication stress and impaired DNA repair

    PubMed Central

    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

    2015-01-01

    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

  10. SETD2 loss-of-function promotes renal cancer branched evolution through replication stress and impaired DNA repair.

    PubMed

    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

  11. Search for additional replication terminators in the Bacillus subtilis 168 chromosome.

    PubMed

    Griffiths, A A; Wake, R G

    1997-05-01

    The Bacillus subtilis 168 chromosome is known to contain at least six DNA replication terminators in the terminus region of the chromosome. By using a degenerate DNA probe for the consensus terminator sequence and low-stringency hybridization conditions, several additional minor hybridizing bands were identified. DNA corresponding to the most intense of these bands was cloned and characterized. Although localized in the terminus region, it could not bind RTP and possibly represents a degenerate terminator. A search of the SubtiList database identified an additional terminator sequence in the terminus region, near glnA. It was shown to bind RTP and to function in blocking replication fork movement in a polar manner. Its orientation conformed to the replication fork trap arrangement of the other terminators. The low-stringency hybridization experiments failed to identify any terminus region-type terminators in the region of the chromosome where postinitiation control sequences (STer sites) are known to reside. The two most likely terminators in STer site regions, in terms of sequence similarity to terminus region terminators, were identified through sequence searching. They were synthesized and were found not to bind RTP under conditions that allowed binding to terminus region terminators. Neither did they elicit fork arrest, when present in a plasmid, under stringent conditions. It is concluded that the STer site terminators, at least the first two to the left of oriC, do not have the typical consensus A+B site makeup of terminus region terminators. PMID:9150236

  12. Structural basis for DNA strand separation by a hexameric replicative helicase

    PubMed Central

    Chaban, Yuriy; Stead, Jonathan A.; Ryzhenkova, Ksenia; Whelan, Fiona; Lamber, Ekaterina P.; Antson, Alfred; Sanders, Cyril M.; Orlova, Elena V.

    2015-01-01

    Hexameric helicases are processive DNA unwinding machines but how they engage with a replication fork during unwinding is unknown. Using electron microscopy and single particle analysis we determined structures of the intact hexameric helicase E1 from papillomavirus and two complexes of E1 bound to a DNA replication fork end-labelled with protein tags. By labelling a DNA replication fork with streptavidin (dsDNA end) and Fab (5′ ssDNA) we located the positions of these labels on the helicase surface, showing that at least 10 bp of dsDNA enter the E1 helicase via a side tunnel. In the currently accepted ‘steric exclusion’ model for dsDNA unwinding, the active 3′ ssDNA strand is pulled through a central tunnel of the helicase motor domain as the dsDNA strands are wedged apart outside the protein assembly. Our structural observations together with nuclease footprinting assays indicate otherwise: strand separation is taking place inside E1 in a chamber above the helicase domain and the 5′ passive ssDNA strands exits the assembly through a separate tunnel opposite to the dsDNA entry point. Our data therefore suggest an alternative to the current general model for DNA unwinding by hexameric helicases. PMID:26240379

  13. Two-dimensional gel analysis of rolling circle replication in the presence and absence of bacteriophage T4 primase.

    PubMed Central

    Belanger, K G; Mirzayan, C; Kreuzer, H E; Alberts, B M; Kreuzer, K N

    1996-01-01

    The rolling circle DNA replication structures generated by the in vitro phage T4 replication system were analyzed using two-dimensional agarose gels. Replication structures were generated in the presence or absence of T4 primase (gp61), permitting the analysis of replication forks with either duplex or single-stranded tails. A characteristic arc shape was visualized when forks with single-stranded tails were cleaved by a restriction enzyme with the help of an oligonucleotide that anneals to restriction sites in the single-stranded tail. After calibrating the gel system with this well-studied rolling circle replication reaction, we then analyzed the in vivo replication directed by a T4 replication origin cloned within a plasmid. DNA samples were generated from infections with either wild-type or primase-deletion mutant phage. The only replicative arc that could be detected in the wild-type sample corresponded to duplex Y forms, consistent with very efficient lagging strand synthesis. Surprisingly, we obtained evidence for both duplex and single-stranded DNA tails in the samples from the primase-deficient infection. We conclude that a relatively inefficient mechanism primes lagging strand DNA synthesis in vivo when gp61 is absent. PMID:8668550

  14. Ten Things You Should Do with a Tuning Fork

    ERIC Educational Resources Information Center

    Lincoln, James

    2013-01-01

    Tuning forks are wonderful tools for teaching physics. Every physics classroom should have several and every physics student should be taught how to use them. In this article, I highlight 10 enriching demonstrations that most teachers might not know, as well as provide tips to enhance the demonstrations teachers might already be doing. Some of…

  15. 80. Laurel Fork Creek Bridge #2. Example of a concrete ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    80. Laurel Fork Creek Bridge #2. Example of a concrete slab bridge with T beams. It was built in 1937 and the wing walls were faced with stone to blend with its surroundings. Looking northeast. - Blue Ridge Parkway, Between Shenandoah National Park & Great Smoky Mountains, Asheville, Buncombe County, NC

  16. 132. FORKS DIVERSION, HIGH LINE AND LOW LINE CANALS, TWIN ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    132. FORKS DIVERSION, HIGH LINE AND LOW LINE CANALS, TWIN FALLS COUNTY, SOUTH OF HANSEN, IDAHO; POWER GATES FOR HYDRO-ELECTRIC. - Milner Dam & Main Canal: Twin Falls Canal Company, On Snake River, 11 miles West of city of Burley, Idaho, Twin Falls, Twin Falls County, ID

  17. Self-replicating systems.

    PubMed

    Clixby, Gregory; Twyman, Lance

    2016-05-01

    Over the past 25 years, there has been a surge of development in research towards self-replication and self-replicating systems. The interest in these systems relates to one of the most fundamental questions posed in all fields of science - How did life on earth begin? Investigating how the self-replication process evolved may hold the key to understanding the emergence and evolution of living systems and, ultimately, gain a clear insight into the origin of life on earth. This introductory review aims to highlight the fundamental prerequisites of self-replication along with the important research that has been conducted over the past few decades. PMID:27086507

  18. USP7 is a SUMO deubiquitinase essential for DNA replication

    PubMed Central

    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

  19. Asynchronous DNA replication within the human. beta. -globin gene locus

    SciTech Connect

    Epner, E.; Forrester, W.C.; Groudine, M. )

    1988-11-01

    The timing of DNA replication of the human {beta}-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 {beta}-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-{gamma}-globin gene region and approximately 20 kilobases 5' to the {epsilon}-globin gene and 20 kilobases 3' to the {beta}-globin gene, replicate later and throughout S phase. A similar area is also present in the {alpha}-globin gene region in K562 cells. They suggest that these regions may represent sites of termination of replication forks.

  20. Proficient Replication of the Yeast Genome by a Viral DNA Polymerase.

    PubMed

    Stodola, Joseph L; Stith, Carrie M; Burgers, Peter M

    2016-05-27

    DNA replication in eukaryotic cells requires minimally three B-family DNA polymerases: Pol α, Pol δ, and Pol ϵ. Pol δ replicates and matures Okazaki fragments on the lagging strand of the replication fork. Saccharomyces cerevisiae Pol δ is a three-subunit enzyme (Pol3-Pol31-Pol32). A small C-terminal domain of the catalytic subunit Pol3 carries both iron-sulfur cluster and zinc-binding motifs, which mediate interactions with Pol31, and processive replication with the replication clamp proliferating cell nuclear antigen (PCNA), respectively. We show that the entire N-terminal domain of Pol3, containing polymerase and proofreading activities, could be effectively replaced by those from bacteriophage RB69, and could carry out chromosomal DNA replication in yeast with remarkable high fidelity, provided that adaptive mutations in the replication clamp PCNA were introduced. This result is consistent with the model that all essential interactions for DNA replication in yeast are mediated through the small C-terminal domain of Pol3. The chimeric polymerase carries out processive replication with PCNA in vitro; however, in yeast, it requires an increased involvement of the mutagenic translesion DNA polymerase ζ during DNA replication. PMID:27072134

  1. Oak Grove Fork Habitat Improvement Project, 1988 Annual Report.

    SciTech Connect

    Bettin, Scott

    1989-04-01

    The Lower Oak Grove Fork of the Clackamas River is a fifth-order tributary of the Clackamas River drainage supporting depressed runs of coho and chinook salmon, and summer and winter steelhead. Habitat condition rating for the Lower Oak Grove is good, but smelt production estimates are below the average for Clackamas River tributaries. Limiting factors in the 3.8 miles of the Lower Oak Grove supporting anadromous fish include an overall lack of quality spawning and rearing habitat. Beginning in 1986. measures to improve fish habitat in the Lower Oak Grove were developed in coordination with the Oregon Department of Fish and Wildlife (ODF&W) and Portland General Electric (PGE) fisheries biologists. Prior to 1986, no measures had been applied to the stream to mitigate for PGE's storage and regulation of flows in the Oak Grove Fork (Timothy Lake, Harriet Lake). Catchable rainbow trout are stocked by ODF&W two or three times a year during the trout fishing season in the lowermost portion of the Oak Grove Fork near two Forest Service campgrounds (Ripplebrook and Rainbow). The 1987 field season marked the third year of efforts to improve fish habitat of the Lower Oak Grove Fork and restore anadromous fish production. The efforts included the development of an implementation plan for habitat improvement activities in the Lower Oak Grove Fork. post-project monitoring. and maintenance of the 1986 improvement structures. No new structures were constructed or placed in 1987. Fiscal year 1988 brought a multitude of changes which delayed implementation of plans developed in 1987. The most prominent change was the withdrawal of the proposed Spotted Owl Habitat Area (SOHA) which overlapped the Oak Grove project implementation area. Another was the change in the Forest Service biologist responsible for implementation and design of this project.

  2. HOWARD FORK ACID ROCK DRAINAGE SOURCE INTERCEPTION STUDY; HOWARD FORK OF THE SAN MIGUEL RIVER NEAR OPHIR, COLORADO

    EPA Science Inventory

    This project proposes to analyze regional hydrogeology as it relates to mine workings which discharge significant heavy metals into the Howard Fork of the San Miguel River and recommend strategies to intercept and divert water away from mineralized zones. The study also includes...

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

    PubMed

    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

  4. Who Needs Replication?

    ERIC Educational Resources Information Center

    Porte, Graeme

    2013-01-01

    In this paper, the editor of a recent Cambridge University Press book on research methods discusses replicating previous key studies to throw more light on their reliability and generalizability. Replication research is presented as an accepted method of validating previous research by providing comparability between the original and replicated…

  5. The oligomeric Rep protein of Mungbean yellow mosaic India virus (MYMIV) is a likely replicative helicase

    PubMed Central

    Choudhury, Nirupam Roy; Malik, Punjab Singh; Singh, Dharmendra Kumar; Islam, Mohammad Nurul; Kaliappan, Kosalai; Mukherjee, Sunil Kumar

    2006-01-01

    Geminiviruses replicate by rolling circle mode of replication (RCR) and the viral Rep protein initiates RCR by the site-specific nicking at a conserved nonamer (TAATATT↓ AC) sequence. The mechanism of subsequent steps of the replication process, e.g. helicase activity to drive fork-elongation, etc. has largely remained obscure. Here we show that Rep of a geminivirus, namely, Mungbean yellow mosaic India virus (MYMIV), acts as a replicative helicase. The Rep-helicase, requiring ≥6 nt space for its efficient activity, translocates in the 3′→5′ direction, and the presence of forked junction in the substrate does not influence the activity to any great extent. Rep forms a large oligomeric complex and the helicase activity is dependent on the oligomeric conformation (∼24mer). The role of Rep as a replicative helicase has been demonstrated through ex vivo studies in Saccharomyces cerevisiae and in planta analyses in Nicotiana tabacum. We also establish that such helicase activity is not confined to the MYMIV system alone, but is also true with at least two other begomoviruses, viz., Mungbean yellow mosaic virus (MYMV) and Indian cassava mosaic virus (ICMV). PMID:17142233

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

    PubMed Central

    Graindorge, Dany; Martineau, Sylvain; Machon, Christelle; Arnoux, Philippe; Guitton, Jérôme; Francesconi, Stefania; Frochot, Céline; Sage, Evelyne; Girard, Pierre-Marie

    2015-01-01

    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. PMID:26485711

  7. Helicases that underpin replication of protein-bound DNA in Escherichia coli.

    PubMed

    McGlynn, Peter

    2011-04-01

    A pre-requisite for successful cell division in any organism is synthesis of an accurate copy of the genetic information needed for survival. This copying process is a mammoth task, given the amount of DNA that must be duplicated, but potential blocks to replication fork movement also pose a challenge for genome duplication. Damage to the template inhibits the replication machinery but proteins bound to the template such as RNA polymerases also present barriers to replication. This review discusses recent results from Escherichia coli that shed light on the roles of helicases in overcoming protein-DNA barriers to replication and that may illustrate fundamental aspects of how duplication of protein-bound DNA is underpinned in all organisms. PMID:21428948

  8. Analysis of fork-join program response times on multiprocessors

    SciTech Connect

    Towsley, D.; Stankovic, J.A. . Dept. of Computer and Information Science); Rommel, C.G. )

    1990-07-01

    In this paper, the authors develop analytic models for a shared memory multiprocessor that executes fork-join parallel programs. Here a fork-join program is one that consists of a set of n {ge} 1 parallel tasks. All of the tasks of a program arrive simultaneously to the system and the job is assumed to complete when the last task completes. They develop and analyze models for two processor sharing policies, called task scheduling processor sharing and job scheduling processor sharing. The first policy schedules tasks independently of each other and allows parallel execution of an individual program, whereas the second policy schedules each job as a unit and thereby does not allow parallel execution of an individual program.

  9. Optimal information provision for maximizing flow in a forked lattice

    NASA Astrophysics Data System (ADS)

    Imai, Takeaki; Nishinari, Katsuhiro

    2015-06-01

    In a forked road, the provision of inappropriate information to car drivers sometimes leads to undesirable situations such as one-sided congestion, which is called the hunting phenomenon in real traffic. To address such problems, we propose a forked exclusion model and investigate the behavior of traffic flow in two routes, providing various types of information to a limited number of traveling particles according to the share rate of information. To analytically understand the phenomena, we develop a coarse-grained representation of the model. By analyzing the model, we find the most effective types of information to minimize particles' travel time and the existence of an optimal share rate according to route conditions.

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

    PubMed

    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

  11. Ten Things You Should Do with a Tuning Fork

    NASA Astrophysics Data System (ADS)

    Lincoln, James

    2013-03-01

    Tuning forks are wonderful tools for teaching physics. Every physics classroom should have several and every physics student should be taught how to use them. In this article, I highlight 10 enriching demonstrations that most teachers might not know, as well as provide tips to enhance the demonstrations teachers might already be doing. Some of these demonstrations have appeared in literature before, but this paper will serve as a collection of ideas that will be helpful for both the beginner and master teacher.

  12. NORTH FORK OF THE AMERICAN RIVER WILDERNESS STUDY AREA, CALIFORNIA.

    USGS Publications Warehouse

    Harwood, David S.; Federspiel, Francis E.

    1984-01-01

    Mineral-resource surveys of the North Fork of the American River Wilderness study area, California have identified a zone of substantiated resource potential for gold and silver. Zones of probable gold and silver potential occur in the eastern part of the area between the Wubbena and La Trinidad mines and locally around the Marrs mine. A zone with probable chromium potential occurs in the serpentinite belt along the western border of the area. No energy resources were identified in this study.

  13. South Fork Clearwater River Habitat Enhancement, Nez Perce National Forest.

    SciTech Connect

    Siddall, Phoebe

    1992-04-01

    In 1984, the Nez Perce National forest and the Bonneville Power Administration entered into a contractual agreement which provided for improvement of spring chinook salmon and summer steelhead trout habitat in south Fork Clearwater River tributaries. Project work was completed in seven main locations: Crooked River, Red River, Meadow Creek Haysfork Gloryhole, Cal-Idaho Gloryhole, Fisher Placer and Leggett Placer. This report describes restoration activities at each of these sites.

  14. Enhanced Deletion Formation by Aberrant DNA Replication in Escherichia Coli

    PubMed Central

    Saveson, C. J.; Lovett, S. T.

    1997-01-01

    Repeated genes and sequences are prone to genetic rearrangements including deletions. We have investigated deletion formation in Escherichia coli strains mutant for various replication functions. Deletion was selected between 787 base pair tandem repeats carried either on a ColE1-derived plasmid or on the E. coli chromosome. Only mutations in functions associated with DNA Polymerase III elevated deletion rates in our assays. Especially large increases were observed in strains mutant in dnaQ, the ε editing subunit of Pol III, and dnaB, the replication fork helicase. Mutations in several other functions also altered deletion formation: the α polymerase (dnaE), the γ clamp loader complex (holC, dnaX), and the β clamp (dnaN) subunits of Pol III and the primosomal proteins, dnaC and priA. Aberrant replication stimulated deletions through several pathways. Whereas the elevation in dnaB strains was mostly recA- and lexA-dependent, that in dnaQ strains was mostly recA- and lexA-independent. Deletion product analysis suggested that slipped mispairing, producing monomeric replicon products, may be preferentially increased in a dnaQ mutant and sister-strand exchange, producing dimeric replicon products, may be elevated in dnaE mutants. We conclude that aberrant Polymerase III replication can stimulate deletion events through several mechanisms of deletion and via both recA-dependent and independent pathways. PMID:9177997

  15. Replication of Low Density Electroformed Normal Incidence Optics

    NASA Technical Reports Server (NTRS)

    Ritter, Joseph M.; Burdine, Robert (Technical Monitor)

    2001-01-01

    Replicated electroformed light-weight nickel alloy mirrors can have high strength, low areal density (less than 3kg/m2), smooth finish, and controllable alloy composition. Progress at NASA MSFC SOMTC in developing normal incidence replicated Nickel mirrors will be reported.

  16. Replication of Low Density Electroformed Normal Incidence Optics

    NASA Technical Reports Server (NTRS)

    Ritter, Joseph M.

    2000-01-01

    Replicated electroformed light-weight nickel alloy mirrors can have high strength, low areal density (<3kg/m2), smooth finish, and controllable alloy composition. Progress at NASA MSFC SOMTC in developing normal incidence replicated Nickel mirrors will be reported.

  17. Modeling DNA Replication.

    ERIC Educational Resources Information Center

    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)

  18. DNA Replication Origins

    PubMed Central

    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 environment in origin selection and the mechanisms used by initiators to recognize replication origins. Close examination of bacterial and archaeal replication origins reveals an array of DNA sequence motifs that position individual initiator protein molecules and promote initiator oligomerization on origin DNA. Conversely, the need for specific recognition sequences in eukaryotic replication origins is relaxed. In fact, the primary rule for origin selection appears to be flexibility, a feature that is modulated either by structural elements or by epigenetic mechanisms at least partly linked to the organization of the genome for gene expression. PMID:23838439

  19. Replication of lightweight mirrors

    NASA Astrophysics Data System (ADS)

    Chen, Ming Y.; Matson, Lawrence E.; Lee, Heedong; Chen, Chenggang

    2009-08-01

    The fabrication of lightweight mirror assemblages via a replication technique offers great potential for eliminating the high cost and schedule associated with the grinding and polishing steps needed for conventional glass or SiC mirrors. A replication mandrel is polished to an inverse figure shape and to the desired finish quality. It is then, coated with a release layer, the appropriate reflective layer, and followed by a laminate for coefficient of thermal expansion (CTE) tailorability and strength. This optical membrane is adhered to a mirror structural substrate with a low shrinkage, CTE tailored adhesive. Afterwards, the whole assembly is separated from the mandrel. The mandrel is then cleaned and reused for the next replication run. The ultimate goal of replication is to preserve the surface finish and figure of the optical membrane upon its release from the mandrel. Successful replication requires a minimization of the residual stresses within the optical coating stack, the curing stresses from the adhesive and the thermal stress resulting from CTE mismatch between the structural substrate, the adhesive, and the optical membrane. In this paper, the results on replicated trials using both metal/metal and ceramic/ceramic laminates adhered to light weighted structural substrates made from syntactic foams (both inorganic and organic) will be discussed.

  20. phiX174 cistron A protein is a multifunctional enzyme in DNA replication.

    PubMed

    Eisenberg, S; Griffith, J; Kornberg, A

    1977-08-01

    The cistron A protein induced by phage varphiX174 nicks (produces a single-strand break in) the viral strand of the superhelical varphiX duplex DNA, thereby forming a complex with the DNA. The protein, seen bound to the DNA in the electron microscope, was located in the restriction endonuclease fragment between nucleotides 4290 and 4330 on the varphiX map [Sanger, F., Air, G. M., Barrel, B. G., Brown, N. L., Coulson, A. R., Fiddes, J. C., Hutchison, C. A., III, Slocomb, P. M. Y. & Smith, M. (1977) Nature 265, 687-695]. Replication also was initiated at this point, thus identifying the site of cistron A protein nicking and binding as the origin of replication. The cisA-DNA complex (separated from free cistron A protein), upon the addition of Escherichia coli rep protein, ATP, and DNA binding protein, is unwound to generate a single-stranded linear [presumably the nicked (+) strand] and a circular [presumably the (-) strand] molecule. The cisA-DNA complex, upon the further addition of DNA polymerase III holoenzyme and deoxynucleoside triphosphates, supports replication to generate viral, single-stranded circles, as many as 15 circles per cisA-DNA complex. The replicating intermediates seen in the electron microscope are a novel form of "rolling circle" [Gilbert, W. & Dressler, D. H. (1969) Cold Spring Harbor Symp. Quant. Biol. 33, 473-485]. The 5' end (presumably with the cistron A protein bound to it) is locked in the replication fork and loops back to accompany the strand-separation and replication fork around the template [(-) strand] circle. Thus, the multiple functions of cistron A protein include: (i) nicking the viral strand at the origin of replication to initiate a round of replication, (ii) participating in a complex which supports fork movement in strand separation and replication, (iii) nicking again at the regenerated origin to produce a unit-length DNA, and (iv) ligating the newly generated 3'-OH end to the 5'-phosphate-complexed end to form a circular

  1. Many replications do not causal inferences make: the need for critical replications to test competing explanations of nonrandomized studies.

    PubMed

    Larzelere, Robert E; Cox, Ronald B; Swindle, Taren M

    2015-05-01

    Although direct replications are ideal for randomized studies, areas of psychological science that lack randomized studies should incorporate Rosenbaum's (2001) distinction between trivial and nontrivial replications, relabeled herein as exact and critical replications. If exact replications merely repeat systematic biases, they cannot enhance cumulative progress in psychological science. In contrast, critical replications distinguish between competing explanations by using crucial tests to clarify the underlying causal influences. We illustrate this potential with examples from research on corrective actions by professionals (e.g., psychotherapy, Ritalin) and parents (e.g., spanking, homework assistance), where critical replications are needed to overcome the inherent selection bias due to corrective actions being triggered by children's symptoms. Purported causal effects must first prove to be replicable after plausible confounds such as selection bias are eliminated. Subsequent critical replications can then compare plausible alternative explanations of the average unbiased causal effect and of individual differences in those effects. We conclude that this type of systematic sequencing of critical replications has more potential for making the kinds of discriminations typical of cumulative progress in science than do exact replications alone, especially in areas where randomized studies are unavailable. PMID:25987516

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

    PubMed

    Kubota, Takashi; Katou, Yuki; Nakato, Ryuichiro; Shirahige, Katsuhiko; Donaldson, Anne D

    2015-08-01

    The sliding clamp PCNA is a crucial component of the DNA replication machinery. Timely PCNA loading and unloading are central for genome integrity and must be strictly coordinated with other DNA processing steps during replication. Here, we show that the S. cerevisiae Elg1 replication factor C-like complex (Elg1-RLC) unloads PCNA genome-wide following Okazaki fragment ligation. In the absence of Elg1, PCNA is retained on chromosomes in the wake of replication forks, rather than at specific sites. Degradation of the Okazaki fragment ligase Cdc9 leads to PCNA accumulation on chromatin, similar to the accumulation caused by lack of Elg1. We demonstrate that Okazaki fragment ligation is the critical prerequisite for PCNA unloading, since Chlorella virus DNA ligase can substitute for Cdc9 in yeast and simultaneously promotes PCNA unloading. Our results suggest that Elg1-RLC acts as a general PCNA unloader and is dependent upon DNA ligation during chromosome replication. PMID:26212319

  3. The crystal structure of Neisseria gonorrhoeae PriB reveals mechanistic differences among bacterial DNA replication restart pathways

    SciTech Connect

    Dong, Jinlan; George, Nicholas P.; Duckett, Katrina L.; DeBeer, Madeleine A.P.; Lopper, Matthew E.

    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.

  4. Replication stress induced site-specific phosphorylation targets WRN to the ubiquitin-proteasome pathway

    PubMed Central

    Su, Fengtao; Bhattacharya, Souparno; Abdisalaam, Salim; Mukherjee, Shibani; Yajima, Hirohiko; Yang, Yanyong; Mishra, Ritu; Srinivasan, Kalayarasan; Ghose, Subroto; Chen, David J.; Yannone, Steven M.; Asaithamby, Aroumougame

    2016-01-01

    Faithful and complete genome replication in human cells is essential for preventing the accumulation of cancer-promoting mutations. WRN, the protein defective in Werner syndrome, plays critical roles in preventing replication stress, chromosome instability, and tumorigenesis. Herein, we report that ATR-mediated WRN phosphorylation is needed for DNA replication and repair upon replication stress. A serine residue, S1141, in WRN is phosphorylated in vivo by the ATR kinase in response to replication stress. ATR-mediated WRN S1141 phosphorylation leads to ubiquitination of WRN, facilitating the reversible interaction of WRN with perturbed replication forks and subsequent degradation of WRN. The dynamic interaction between WRN and DNA is required for the suppression of new origin firing and Rad51-dependent double-stranded DNA break repair. Significantly, ATR-mediated WRN phosphorylation is critical for the suppression of chromosome breakage during replication stress. These findings reveal a unique role for WRN as a modulator of DNA repair, replication, and recombination, and link ATR-WRN signaling to the maintenance of genome stability. PMID:26695548

  5. RECQ5 helicase promotes resolution of conflicts between replication and transcription in human cells.

    PubMed

    Urban, Vaclav; Dobrovolna, Jana; Hühn, Daniela; Fryzelkova, Jana; Bartek, Jiri; Janscak, Pavel

    2016-08-15

    Collisions between replication and transcription machineries represent a significant source of genomic instability. RECQ5 DNA helicase binds to RNA-polymerase (RNAP) II during transcription elongation and suppresses transcription-associated genomic instability. Here, we show that RECQ5 also associates with RNAPI and enforces the stability of ribosomal DNA arrays. We demonstrate that RECQ5 associates with transcription complexes in DNA replication foci and counteracts replication fork stalling in RNAPI- and RNAPII-transcribed genes, suggesting that RECQ5 exerts its genome-stabilizing effect by acting at sites of replication-transcription collisions. Moreover, RECQ5-deficient cells accumulate RAD18 foci and BRCA1-dependent RAD51 foci that are both formed at sites of interference between replication and transcription and likely represent unresolved replication intermediates. Finally, we provide evidence for a novel mechanism of resolution of replication-transcription collisions wherein the interaction between RECQ5 and proliferating cell nuclear antigen (PCNA) promotes RAD18-dependent PCNA ubiquitination and the helicase activity of RECQ5 promotes the processing of replication intermediates. PMID:27502483

  6. ATR-mediated phosphorylation of FANCI regulates dormant origin firing in response to replication stress

    PubMed Central

    Chen, Yu-Hung; Jones, Mathew J. K.; Yin, Yandong; Crist, Sarah B.; Colnaghi, Luca; Sims, Robert J.; Rothenberg, Eli; Jallepalli, Prasad V.; Huang, Tony T.

    2015-01-01

    SUMMARY Excess dormant origins bound by the minichromosome maintenance (MCM) replicative helicase complex play a critical role in preventing replication stress, chromosome instability and tumorigenesis. In response to DNA damage, replicating cells must coordinate DNA repair and dormant origin firing to ensure complete and timely replication of the genome; how cells regulate this process remains elusive. Herein, we identify a member of the Fanconi Anemia (FA) DNA repair pathway, FANCI, as a key effector of dormant origin firing in response to replication stress. Cells lacking FANCI have reduced number of origins, increased inter-origin distances and slowed proliferation rates. Intriguingly, ATR-mediated FANCI phosphorylation inhibits dormant origin firing while promoting replication fork restart/DNA repair. Using super-resolution microscopy, we show that FANCI co-localizes with MCM-bound chromatin in response to replication stress. These data reveal a unique role for FANCI as a modulator of dormant origin firing and links timely genome replication to DNA repair. PMID:25843623

  7. Mitochondrial DNA replication proceeds via a ‘bootlace’ mechanism involving the incorporation of processed transcripts

    PubMed Central

    Reyes, Aurelio; Kazak, Lawrence; Wood, Stuart R.; Yasukawa, Takehiro; Jacobs, Howard T.; Holt, Ian J.

    2013-01-01

    The observation that long tracts of RNA are associated with replicating molecules of mitochondrial DNA (mtDNA) suggests that the mitochondrial genome of mammals is copied by an unorthodox mechanism. Here we show that these RNA-containing species are present in living cells and tissue, based on interstrand cross-linking. Using DNA synthesis in organello, we demonstrate that isolated mitochondria incorporate radiolabeled RNA precursors, as well as DNA precursors, into replicating DNA molecules. RNA-containing replication intermediates are chased into mature mtDNA, to which they are thus in precursor–product relationship. While a DNA chain terminator rapidly blocks the labeling of mitochondrial replication intermediates, an RNA chain terminator does not. Furthermore, processed L-strand transcripts can be recovered from gel-extracted mtDNA replication intermediates. Therefore, instead of concurrent DNA and RNA synthesis, respectively, on the leading and lagging strands, preformed processed RNA is incorporated as a provisional lagging strand during mtDNA replication. These findings indicate that RITOLS is a physiological mechanism of mtDNA replication, and that it involves a ‘bootlace' mechanism, in which processed transcripts are successively hybridized to the lagging-strand template, as the replication fork advances. PMID:23595151

  8. Uracil DNA Glycosylase BKRF3 Contributes to Epstein-Barr Virus DNA Replication through Physical Interactions with Proteins in Viral DNA Replication Complex

    PubMed Central

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

    2014-01-01

    activity executes at the right time and the right place in DNA replication forks, complex formation with other components in the DNA replication machinery provides an important regulation for UDG function. In this study, we provide the mechanism for EBV UDG BKRF3 nuclear targeting and the interacting domains of BKRF3 with viral DNA replication proteins. Through knockout and complementation approaches, we further demonstrate that in addition to UDG activity, the interaction of BKRF3 with viral proteins in the replication compartment is crucial for efficient viral DNA replication. PMID:24872582

  9. E. coli minichromosome replication in vitro and in vivo: comparative analyses of replication intermediates.

    PubMed

    Munson, B R; Hucul, J A; Maier, P G; Krajewski, C A; Helmstetter, C E

    1987-10-01

    The process of replication of Escherichia coli minichromosomes was examined by following the intermediates formed in vitro and in vivo. Replication initiated on a supercoiled closed circular (CC) monomer, proceeded rapidly to a late but incomplete stage in polymerization (the LC form) in both systems, passed more slowly through a series of open and closed circular catenated dimers with varying extents of intertwining between the monomer units, and then yielded, after decatenation, the supercoiled CC monomer. The replication patterns of two different minichromosomes were similar, although the LC form and the multiply intertwined dimers were much more evident in the smaller pAL4 than in pAL2. The same basic replication scheme was seen in vitro and in vivo but completion of polymerization and processing of the dimers were slower in vitro. Some radioactivity was detected in OC monomer early during replication, consistent with occasional decatenation of LC structures to produce OC molecules which then completed replication to form CC molecules. However, progression to CC catenated dimers prior to formation of CC monomers represented the major replication pathway. PMID:3307923

  10. Challenges in Replicating Interventions

    PubMed Central

    Bell, Stephanie G; Newcomer, Susan F; Bachrach, Christine; Borawski, Elaine; Jemmott, John L; Morrison, Diane; Stanton, Bonita; Tortolero, Susan; Zimmerman, Richard

    2007-01-01

    Purpose This paper describes and reflects on an effort to document, through a set of six interventions, the process of adapting effective youth risk behavior interventions for new settings. It provides insights into how this might best be accomplished. It discusses six studies funded by NIH starting in 1999. The studies were funded in response to a Request for Applications [RFA] to replicate HIV prevention interventions for youth. Researchers were to select an HIV risk reduction intervention program shown to be effective in one adolescent population and to replicate it in a new community or different adolescent population. This was to be done while systematically documenting those processes and aspects of the intervention hypothesized to be critical to the development of community-based, culturally sensitive programs. The replication was to assess the variations necessary to gain cooperation, implement a locally feasible and meaningful intervention, and evaluate the outcomes in the new setting. Methods This paper lays out the rationale for this initiative and describes the goals and the approaches to adaptation of the funded researchers. Results The paper discusses issues relevant to all interventions, those unique to replication and to these replications in particular. It then reflects on the processes and the consequences of the adaptations. It does not address the further challenges in taking a successful intervention “to scale.” Conclusions Replications of effective interventions face all of the challenges of implementation design plus additional challenges of balancing fidelity to the original intervention and sensitivity to the needs of new populations. PMID:17531757

  11. Replicated Composite Optics Development

    NASA Technical Reports Server (NTRS)

    Engelhaupt, Darell

    1997-01-01

    Advanced optical systems for applications such as grazing incidence Wolter I x-ray mirror assemblies require extraordinary mirror surfaces in ten-ns of fine surface finish and figure. The impeccable mirror surface is on the inside of the rotational mirror form. One practical method of producing devices with these requirements is to first fabricate an exterior surface for the optical device then replicate that surface to have the inverse component with lightweight characteristics. The replicate optic is not better than the master or mandrel from which it is made. This task is a continuance of previous studies to identify methods and materials for forming these extremely low roughness optical components.

  12. Interaction force microscopy based on quartz tuning fork force sensor

    NASA Astrophysics Data System (ADS)

    Qin, Yexian

    The ability to sense small changes in the interaction force between a scanning probe microscope (SPM) tip and a substrate requires cantilevers with a sharp mechanical resonance. A typical commercially available cantilever in air is characterized by a resonance with a Q factor of 100 ˜ 300. The low Q factor can be attributed to imperfections in the cantilever itself as well as damping effects of the surrounding air. To substantially increase the Q factor, novel concepts are required. For this reason, we have performed a systematic study of quartz tuning fork resonators for possible use with SPMs. We find that tuning fork resonators operating in air are characterized by Q factors in the order of 104, thereby greatly improving the SPM's ability to measure small shifts in the interaction force. By carefully attaching commercially available SPM tips to the tuning fork, it is possible to obtain SPM images using non-contact imaging techniques and analyze the tip-sample interactions. The assembly of uniform molecular monolayers on atomically flat substrates for molecular electronics applications has received widespread attention during the past ten years. Scanning probe techniques are often used to assess substrate topography, molecular ordering and electronic properties, yet little is known about the fundamental tip-molecule interaction. To address this issue we have built an Interaction Force Microscope using a quartz tuning fork to probe tip-molecular monolayer interactions using scanning probe microscopy. The high quality factor and stable resonant frequency of a quartz tuning fork allows accurate measurement of small shifts in the resonant frequency as the tip interacts with the substrate. To permit an accurate measure of surface interaction forces, the electrical and piezomechanical properties of a tuning fork have been calibrated using a fiber optical interferometer. In prior work [1], we have studied molecular layers formed from either 4-Trifluoro

  13. Regulation of DNA Replication Timing on Human Chromosome by a Cell-Type Specific DNA Binding Protein SATB1

    PubMed Central

    Oda, Masako; Kanoh, Yutaka; Watanabe, Yoshihisa; Masai, Hisao

    2012-01-01

    Background Replication timing of metazoan DNA during S-phase may be determined by many factors including chromosome structures, nuclear positioning, patterns of histone modifications, and transcriptional activity. It may be determined by Mb-domain structures, termed as “replication domains”, and recent findings indicate that replication timing is under developmental and cell type-specific regulation. Methodology/Principal Findings We examined replication timing on the human 5q23/31 3.5-Mb segment in T cells and non-T cells. We used two independent methods to determine replication timing. One is quantification of nascent replicating DNA in cell cycle-fractionated stage-specific S phase populations. The other is FISH analyses of replication foci. Although the locations of early- and late-replicating domains were common between the two cell lines, the timing transition region (TTR) between early and late domains were offset by 200-kb. We show that Special AT-rich sequence Binding protein 1 (SATB1), specifically expressed in T-cells, binds to the early domain immediately adjacent to TTR and delays the replication timing of the TTR. Measurement of the chromosome copy number along the TTR during synchronized S phase suggests that the fork movement may be slowed down by SATB1. Conclusions Our results reveal a novel role of SATB1 in cell type-specific regulation of replication timing along the chromosome. PMID:22879953

  14. BLACK FORK MOUNTAIN ROADLESS AREA, ARAKANSAS AND OKLAHOMA.

    USGS Publications Warehouse

    Miller, Mary H.

    1984-01-01

    Black Fork Mountain Roadless Area covers about 21 sq mi in the Ouachita National Forest in Polk County, Arkansas and LeFlore County, Oklahoma. On the basis of a mineral survey the area has little promise for the occurrence of metallic mineral resources. Stone and sand and gravel suitable for construction purposes occur in the Jackfork Sandstone and the Stanley Shale which also occur outside the roadless area. Although the potential for gas and oil is unknown and no resource potential was identified, some investigators believe that there is a possibility for the occurrence of gas and oil in the roadless area.

  15. Glue-free tuning fork shear-force microscope

    NASA Astrophysics Data System (ADS)

    Mühlschlegel, P.; Toquant, J.; Pohl, D. W.; Hecht, B.

    2006-01-01

    A scanning near-field optical microscope without any glued parts is described. Key elements are the optical fiber probe/tuning fork junction and the piezotube scanner assembly. In both cases, fixation is achieved by means of controlled pressure and elastic deformation. The avoidance of glued connections was found to improve the Q factor of the shear-force sensor as well as to facilitate the replacement of the fiber probe and other parts of the scanner head. We present approach curves and shear-force images that demonstrate the performance and stability of the system.

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

    PubMed Central

    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

  17. Oxbow Conservation Area; Middle Fork John Day River, Annual Report 2003-2004.

    SciTech Connect

    Cochran, Brian

    2004-02-01

    In early 2001, the Confederated Tribes of Warm Springs, through their John Day Basin Office, concluded the acquisition of the Oxbow Ranch, now know as the Oxbow Conservation Area (OCA). Under a memorandum of agreement with the Bonneville Power Administration (BPA), the Tribes are required to provided BPA an 'annual written report generally describing the real property interests in the Project, HEP analyses undertaken or in progress, and management activities undertaken or in progress'. The project during 2003 was crippled due to the aftermath of the BPA budget crisis. Some objectives were not completed during the first half of this contract because of limited funds in the 2003 fiscal year. The success of this property purchase can be seen on a daily basis. Water rights were utilized only in the early, high water season and only from diversion points with functional fish screens. After July 1, all of the OCA water rights were put instream. Riparian fences on the river, Ruby and Granite Boulder creeks continued to promote important vegetation to provide shade and bank stabilization. Hundreds of willow, dogwood, Douglas-fir, and cottonwood were planted along the Middle Fork John Day River. Livestock grazing on the property was carefully managed to ensure the protection of fish and wildlife habitat, while promoting meadow vigor and producing revenue for property taxes. Monitoring of property populations, resources, and management activities continued in 2003 to build a database for future management of this and other properties in the region.

  18. 77 FR 39675 - Wallowa-Whitman National Forest, Baker County, OR; North Fork Burnt River Mining

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-07-05

    ... River Mining AGENCY: Forest Service, USDA. ACTION: Correction--Notice of intent to prepare a supplement... changed to the Whitman District Ranger. This 2012 North Fork Burnt River Mining Record of Decision will replace and supercede the 2004 North Fork Burnt River Mining Record of Decision only where necessary...

  19. 76 FR 6114 - Lincoln National Forest, New Mexico, North Fork Eagle Creek Wells Special Use Authorization

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-02-03

    ... North Fork well field (Village of Ruidoso 2006). The Village of Ruidoso drilled four production wells on... National Forest System land; and (2) Minimizing impacts of groundwater drawdown from this well field to... approximately 31 percent of its water supply from the North Fork well field. During drought conditions prior...

  20. Origin & Evolution of the Grand Forks Human Nutrition Research Center, 1970-90

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In the early 1960s William E Cornatzer, MD, PhD, suggested the need for increased USDA research concerning human nutrition, and creation of the Grand Forks Human Nutrition Laboratory (Grand Forks Human Nutrition Research Center). He shared ideas with Senator Milton R. Young of North Dakota, who requ...

  1. 16 CFR Figure 1 to Part 1512 - Bicycle Front Fork Cantilever Bending Test Rig

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 16 Commercial Practices 2 2011-01-01 2011-01-01 false Bicycle Front Fork Cantilever Bending Test Rig 1 Figure 1 to Part 1512 Commercial Practices CONSUMER PRODUCT SAFETY COMMISSION FEDERAL HAZARDOUS... Fork Cantilever Bending Test Rig EC03OC91.070...

  2. 16 CFR Figure 1 to Part 1512 - Bicycle Front Fork Cantilever Bending Test Rig

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 16 Commercial Practices 2 2010-01-01 2010-01-01 false Bicycle Front Fork Cantilever Bending Test Rig 1 Figure 1 to Part 1512 Commercial Practices CONSUMER PRODUCT SAFETY COMMISSION FEDERAL HAZARDOUS... Fork Cantilever Bending Test Rig EC03OC91.070...

  3. 16 CFR Figure 1 to Part 1512 - Bicycle Front Fork Cantilever Bending Test Rig

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 16 Commercial Practices 2 2014-01-01 2014-01-01 false Bicycle Front Fork Cantilever Bending Test Rig 1 Figure 1 to Part 1512 Commercial Practices CONSUMER PRODUCT SAFETY COMMISSION FEDERAL HAZARDOUS... Fork Cantilever Bending Test Rig EC03OC91.070...

  4. 16 CFR Figure 1 to Part 1512 - Bicycle Front Fork Cantilever Bending Test Rig

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 16 Commercial Practices 2 2013-01-01 2013-01-01 false Bicycle Front Fork Cantilever Bending Test Rig 1 Figure 1 to Part 1512 Commercial Practices CONSUMER PRODUCT SAFETY COMMISSION FEDERAL HAZARDOUS... Fork Cantilever Bending Test Rig EC03OC91.070...

  5. 16 CFR Figure 1 to Part 1512 - Bicycle Front Fork Cantilever Bending Test Rig

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 16 Commercial Practices 2 2012-01-01 2012-01-01 false Bicycle Front Fork Cantilever Bending Test Rig 1 Figure 1 to Part 1512 Commercial Practices CONSUMER PRODUCT SAFETY COMMISSION FEDERAL HAZARDOUS... Fork Cantilever Bending Test Rig EC03OC91.070...

  6. 77 FR 45597 - Middle Fork American River Project; Notice of Availability of the Draft Environmental Impact...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-08-01

    ... Energy Regulatory Commission Middle Fork American River Project; Notice of Availability of the Draft Environmental Impact Statement for the Middle Fork American River Hydrolectric Project and Intention To Hold... CFR part 380 ), the Office of Energy Projects has reviewed the application for license for the...

  7. 33 CFR 208.26 - Altus Dam and Reservoir, North Fork Red River, Okla.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 33 Navigation and Navigable Waters 3 2013-07-01 2013-07-01 false Altus Dam and Reservoir, North Fork Red River, Okla. 208.26 Section 208.26 Navigation and Navigable Waters CORPS OF ENGINEERS, DEPARTMENT OF THE ARMY, DEPARTMENT OF DEFENSE FLOOD CONTROL REGULATIONS § 208.26 Altus Dam and Reservoir, North Fork Red River, Okla. The Bureau...

  8. 33 CFR 208.33 - Cheney Dam and Reservoir, North Fork of Ninnescah River, Kans.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 33 Navigation and Navigable Waters 3 2012-07-01 2012-07-01 false Cheney Dam and Reservoir, North Fork of Ninnescah River, Kans. 208.33 Section 208.33 Navigation and Navigable Waters CORPS OF ENGINEERS, DEPARTMENT OF THE ARMY, DEPARTMENT OF DEFENSE FLOOD CONTROL REGULATIONS § 208.33 Cheney Dam and Reservoir, North Fork of Ninnescah River,...

  9. 33 CFR 208.26 - Altus Dam and Reservoir, North Fork Red River, Okla.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 33 Navigation and Navigable Waters 3 2012-07-01 2012-07-01 false Altus Dam and Reservoir, North Fork Red River, Okla. 208.26 Section 208.26 Navigation and Navigable Waters CORPS OF ENGINEERS, DEPARTMENT OF THE ARMY, DEPARTMENT OF DEFENSE FLOOD CONTROL REGULATIONS § 208.26 Altus Dam and Reservoir, North Fork Red River, Okla. The Bureau...

  10. 33 CFR 208.33 - Cheney Dam and Reservoir, North Fork of Ninnescah River, Kans.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 33 Navigation and Navigable Waters 3 2013-07-01 2013-07-01 false Cheney Dam and Reservoir, North Fork of Ninnescah River, Kans. 208.33 Section 208.33 Navigation and Navigable Waters CORPS OF ENGINEERS, DEPARTMENT OF THE ARMY, DEPARTMENT OF DEFENSE FLOOD CONTROL REGULATIONS § 208.33 Cheney Dam and Reservoir, North Fork of Ninnescah River,...

  11. 16 CFR 1512.14 - Requirements for fork and frame assembly.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 16 Commercial Practices 2 2010-01-01 2010-01-01 false Requirements for fork and frame assembly. 1512.14 Section 1512.14 Commercial Practices CONSUMER PRODUCT SAFETY COMMISSION FEDERAL HAZARDOUS... assembly. The fork and frame assembly shall be tested for strength by application of a load of 890 N...

  12. EVALUATING THE EFFECTIVENESS OF NUTRIENT MANAGEMENT IN THE CLARK FORK-PEND OREILLE WATERSHED

    EPA Science Inventory

    (1) Analyze the nutrient concentration and algal standing crop trend in the Clark Fork River portion of the watershed; (2) Provide a web-based data access system for reporting Clark Fork River monitoring information; and (3) Evaluate the effectiveness of current nutrient reductio...

  13. Mutual interactions of oscillating quartz tuning forks in superfluid 4He

    NASA Astrophysics Data System (ADS)

    Sheshin, G.; Gritsenko, I.; Schmoranzer, D.; Skrbek, L.

    2013-10-01

    The quartz tuning fork has recently become a popular experimental tool for investigations of both classical and quantum turbulence in cryogenic helium. Its increased use in low-temperature experiments and a number of puzzling results obtained in the past have led to many questions concerning the interaction of multiple tuning forks or the interaction of tuning forks with other oscillators. We report measurements performed in He II at low temperatures around 360 mK, on the mutual interaction of tuning forks placed in the same volume of fluid, and examine the responsible mechanisms in an effort to discriminate between acoustic coupling and interaction via quantized vortices. To this end, the interaction of two tuning forks is investigated by analyzing their recorded resonance curves, looking for any nonelectrical crosstalk. Further, the force-velocity characteristics of a detector tuning fork are measured for different operating velocities of a generator tuning fork. As a complementary measurement, the intensity of sound waves is recorded using a set of miniature receivers. We confirm the current knowledge on acoustic emission by tuning forks in He II and verify properties of their radiation patterns. We conclude that in our experiment the interaction is almost entirely mediated by sound waves.

  14. 76 FR 62038 - Boundary Establishment for North Fork Crooked National Wild and Scenic River, Ochoco National...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-10-06

    ... Forest Service Boundary Establishment for North Fork Crooked National Wild and Scenic River, Ochoco National Forest, Crook County, Oregon AGENCY: Forest Service, USDA. ACTION: Notice of availability. SUMMARY..., Washington Office, is transmitting the final boundary of the North Fork Crooked National Wild and...

  15. First report of anther smut caused by Microbotryum violaceum on forked catchfly in Turkey

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Forked catchfly (Silene dichotoma Ehrh.), family Caryophyllaceae, is a common and native plant in rangelands and pastures in Turkey. It is also an introduced plant that is widely distributed in North America. In May, 2007 about 20 forked catchfly plants on the campus of Ondokuz Mayis University, i...

  16. ATM and KAT5 safeguard replicating chromatin against formaldehyde damage

    PubMed Central

    Ortega-Atienza, Sara; Wong, Victor C.; DeLoughery, Zachary; Luczak, Michal W.; Zhitkovich, Anatoly

    2016-01-01

    Many carcinogens damage both DNA and protein constituents of chromatin, and it is unclear how cells respond to this compound injury. We examined activation of the main DNA damage-responsive kinase ATM and formation of DNA double-strand breaks (DSB) by formaldehyde (FA) that forms histone adducts and replication-blocking DNA-protein crosslinks (DPC). We found that low FA doses caused a strong and rapid activation of ATM signaling in human cells, which was ATR-independent and restricted to S-phase. High FA doses inactivated ATM via its covalent dimerization and formation of larger crosslinks. FA-induced ATM signaling showed higher CHK2 phosphorylation but much lower phospho-KAP1 relative to DSB inducers. Replication blockage by DPC did not produce damaged forks or detectable amounts of DSB during the main wave of ATM activation, which did not require MRE11. Chromatin-monitoring KAT5 (Tip60) acetyltransferase was responsible for acetylation and activation of ATM by FA. KAT5 and ATM were equally important for triggering of intra-S-phase checkpoint and ATM signaling promoted recovery of normal human cells after low-dose FA. Our results revealed a major role of the KAT5-ATM axis in protection of replicating chromatin against damage by the endogenous carcinogen FA. PMID:26420831

  17. Replicating viruses for gynecologic cancer therapy.

    PubMed

    Park, J W; Kim, M

    2016-01-01

    Despite advanced therapeutic treatments, gynecologic malignancies such as cervical and ovarian cancers are still the top ten leading cause of cancer death among women in South Korea. Thus a novel and innovative approach is urgently needed. Naturally occurring viruses are live, replication-proficient viruses that specifically infect human cancer cells while sparing normal cell counterparts. Since the serendipitous discovery of the naturally oncotropic virus targeting gynecologic cancer in 1920s, various replicating viruses have shown various degrees of safety and efficacy in preclinical or clinical applications for gynecologic cancer therapy. Cellular oncogenes and tumor suppressor genes, which are frequently dysregulated in gynecologic malignancies, play an important role in determining viral oncotropism. Published articles describing replicating, oncolytic viruses for gynecologic cancers are thoroughly reviewed. This review outlines the discovery of replication-proficient virus strains for targeting gynecologic malignancies, recent progresses elucidating molecular connections between oncogene/tumor suppressor gene abnormalities and viral oncotropism, and the associated preclinical/clinical implications. The authors would also like to propose future directions in the utility of the replicating viruses for gynecologic cancer therapy. PMID:27352554

  18. Replicative DNA polymerases.

    PubMed

    Johansson, Erik; Dixon, Nicholas

    2013-06-01

    In 1959, Arthur Kornberg was awarded the Nobel Prize for his work on the principles by which DNA is duplicated by DNA polymerases. Since then, it has been confirmed in all branches of life that replicative DNA polymerases require a single-stranded template to build a complementary strand, but they cannot start a new DNA strand de novo. Thus, they also depend on a primase, which generally assembles a short RNA primer to provide a 3'-OH that can be extended by the replicative DNA polymerase. The general principles that (1) a helicase unwinds the double-stranded DNA, (2) single-stranded DNA-binding proteins stabilize the single-stranded DNA, (3) a primase builds a short RNA primer, and (4) a clamp loader loads a clamp to (5) facilitate the loading and processivity of the replicative polymerase, are well conserved among all species. Replication of the genome is remarkably robust and is performed with high fidelity even in extreme environments. Work over the last decade or so has confirmed (6) that a common two-metal ion-promoted mechanism exists for the nucleotidyltransferase reaction that builds DNA strands, and (7) that the replicative DNA polymerases always act as a key component of larger multiprotein assemblies, termed replisomes. Furthermore (8), the integrity of replisomes is maintained by multiple protein-protein and protein-DNA interactions, many of which are inherently weak. This enables large conformational changes to occur without dissociation of replisome components, and also means that in general replisomes cannot be isolated intact. PMID:23732474

  19. Role of DNA damage response pathways in preventing carcinogenesis caused by intrinsic replication stress.

    PubMed

    Wallace, M D; Southard, T L; Schimenti, K J; Schimenti, J C

    2014-07-10

    Defective DNA replication can result in genomic instability, cancer and developmental defects. To understand the roles of DNA damage response (DDR) genes on carcinogenesis in mutants defective for core DNA replication components, we utilized the Mcm4(Chaos3/Chaos3) ('Chaos3') mouse model that, by virtue of an amino-acid alteration in MCM4 that destabilizes the MCM2-7 DNA replicative helicase, has fewer dormant replication origins and an increased number of stalled replication forks. This leads to genomic instability and cancer in most Chaos3 mice. We found that animals doubly mutant for Chaos3 and components of the ataxia telangiectasia-mutated (ATM) double-strand break response pathway (Atm, p21/Cdkn1a and Chk2/Chek2) had decreased tumor latency and/or increased tumor susceptibility. Tumor latency and susceptibility differed between genetic backgrounds and genders, with females demonstrating an overall greater cancer susceptibility to Atm and p21 deficiency than males. Atm deficiency was semilethal in the Chaos3 background and impaired embryonic fibroblast proliferation, suggesting that ATM drug inhibitors might be useful against tumors with DNA replication defects. Hypomorphism for the 9-1-1 component Hus1 did not affect tumor latency or susceptibility in Chaos3 animals, and tumors in these mice did not exhibit impaired ATR pathway signaling. These and other data indicate that under conditions of systemic replication stress, the ATM pathway is particularly important both for cancer suppression and viability during development. PMID:23975433

  20. 76 FR 35009 - Draft Oil and Gas Management Plan/Environmental Impact Statement for Big South Fork National...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-06-15

    ... National Park Service Draft Oil and Gas Management Plan/Environmental Impact Statement for Big South Fork... Big South Fork National River and Recreation Area and Obed Wild and Scenic River. SUMMARY: Pursuant to.../DEIS) for the proposed Big South Fork National River and Recreation Area (BISO) and Obed Wild...

  1. Environment of deposition of Clear Fork Formation: Yoakum County, Texas

    SciTech Connect

    Moore, B.K.

    1987-05-01

    The Clear Fork Formation is Permian (Leonardian) in age and constitutes a major oil-bearing unit in the Permian basin of west Texas. In Yoakum County, west Texas, the upper Clear Fork carbonates record a subtidal upward-shoaling sequence of deposition. A small bryozoan-algal patch reef is situated within these carbonates near the southern edge of the North Basin platform. The reef is completely dolomitized, but paramorphic replacement has facilitated a study of the paleoecology, lateral variations, and community succession within this buildup. Build-ups of this type are scarcely known in strata of Permian age. The reef was apparently founded on a coquina horizon at the base of the buildup. The reef apparently had a low-relief, dome-shaped morphology. The trapping and binding of sediment by bryozoa appear to have been the main constructional process. A significant role was also played by encrusting forams and the early precipitation of submarine cements, both of which added rigidity to the structure. The reef also contains a low-diversity community of other invertebrates. Algal constituents predominate at the basinward edge of the buildup. The reef was formed entirely subaqueously on a broad, relatively shallow tropical marine carbonate shelf environment. An understanding of the lithofacies distribution and paragenesis within this sequence will provide information on porosity variations and the nature and distribution of permeability barriers. Such information is useful in reservoir modeling studies and for secondary recovery techniques in shelf-edge carbonate reservoirs of this type.

  2. Telomere replication: poised but puzzling

    PubMed Central

    Sampathi, Shilpa; Chai, Weihang

    2011-01-01

    Abstract Faithful replication of chromosomes is essential for maintaining genome stability. Telomeres, the chromosomal termini, pose quite a challenge to replication machinery due to the complexity in their structures and sequences. Efficient and complete replication of chromosomes is critical to prevent aberrant telomeres as well as to avoid unnecessary loss of telomere DNA. Compelling evidence supports the emerging picture of synergistic actions between DNA replication proteins and telomere protective components in telomere synthesis. This review discusses the actions of various replication and telomere-specific binding proteins that ensure accurate telomere replication and their roles in telomere maintenance and protection. PMID:21122064

  3. Hepatitis D Virus Replication.

    PubMed

    Taylor, John M

    2015-11-01

    This work reviews specific related aspects of hepatitis delta virus (HDV) reproduction, including virion structure, the RNA genome, the mode of genome replication, the delta antigens, and the assembly of HDV using the envelope proteins of its helper virus, hepatitis B virus (HBV). These topics are considered with perspectives ranging from a history of discovery through to still-unsolved problems. HDV evolution, virus entry, and associated pathogenic potential and treatment of infections are considered in other articles in this collection. PMID:26525452

  4. The KRAB Zinc Finger Protein Roma/Zfp157 Is a Critical Regulator of Cell-Cycle Progression and Genomic Stability

    PubMed Central

    Ho, Teresa L.F.; Guilbaud, Guillaume; Blow, J. Julian; Sale, Julian E.; Watson, Christine J.

    2016-01-01

    Summary Regulation of DNA replication and cell division is essential for tissue growth and maintenance of genomic integrity and is particularly important in tissues that undergo continuous regeneration such as mammary glands. We have previously shown that disruption of the KRAB-domain zinc finger protein Roma/Zfp157 results in hyperproliferation of mammary epithelial cells (MECs) during pregnancy. Here, we delineate the mechanism by which Roma engenders this phenotype. Ablation of Roma in MECs leads to unscheduled proliferation, replication stress, DNA damage, and genomic instability. Furthermore, mouse embryonic fibroblasts (MEFs) depleted for Roma exhibit downregulation of p21Cip1 and geminin and have accelerated replication fork velocities, which is accompanied by a high rate of mitotic errors and polyploidy. In contrast, overexpression of Roma in MECs halts cell-cycle progression, whereas siRNA-mediated p21Cip1 knockdown ameliorates, in part, this phenotype. Thus, Roma is an essential regulator of the cell cycle and is required to maintain genomic stability. PMID:27149840

  5. Mapping of a replication origin within the promoter region of two unlinked, abundantly transcribed actin genes of Physarum polycephalum.

    PubMed

    Bénard, M; Lagnel, C; Pallotta, D; Pierron, G

    1996-03-01

    We analyzed the replication of two unlinked actin genes, ardB and ardC , which are abundantly transcribed in the naturally synchronous plasmodium of the slime mold Physarum polycephalum. Detection and size measurements of single-stranded nascent replication intermediates (RIs) demonstrate that these two genes are concomitantly replicated at the onset of the 3-h S phase and tightly linked to replication origins. Appearance of RIs on neutral-neutral two-dimensional gels at specific time points in early S phase and analysis of their structure confirmed these results and further established that, in both cases, an efficient, site-specific, bidirectional origin of replication is localized within the promoter region of the gene. We also determined similar elongation rates for the divergent replication forks of the ardC gene replicon. Finally, taking advantage of a restriction fragment length polymorphism, we studied allelic replicons and demonstrate similar localizations and a simultaneous firing of allelic replication origins. Computer search revealed a low level of homology between the promoters of ardB and ardC and, most notably, the absence of DNA sequences similar to the yeast autonomously replicating sequence consensus sequence in these Physarum origin regions. Our results with the ardB and ardC actin genes support the model of early replicating origins located within the promoter regions of abundantly transcribed genes in P. polycephalum. PMID:8622700

  6. Grand Forks - East Grand Forks Urban Water Resources Study. Energy conservation and recreation appendix, public involvement appendix. Final report

    SciTech Connect

    Not Available

    1981-07-01

    The goal of the Corps of Engineers Urban Study Program is to provide planning assistance to local interests in a variety of water resource areas. The St. Paul District conducted the Grand Forks-East Grand Forks (GF/EGF) Urban Water Resources Study which was a cooperative effort among local, state and federal agencies. Primary attention was given to flood control, water supply and wastewater management; supporting investigations addressed recreation and energy conservation. The recreation investigation consists of the leisure time analysis conducted in stage 2 of the urban study by the Heritage Conservation and Recreation Service, Department of the Interior. The leisure time analysis compared the study area's recreational needs to the available and planned facilities and identified unsatisfied needs. The thermography investigation was conducted in spring 1978 in response to the public's growing awareness of energy conservation and the Corps' desire to make the public aware of the urban study in a meaningful, useful fashion. The investigation consisted of aerial infrared photography, public displays of photographs, and distribution of information on energy saving practices. This investigation was a one-time effort with no plans for follow-up.

  7. Replicating Damaged DNA in Eukaryotes

    PubMed Central

    Chatterjee, Nimrat; Siede, Wolfram

    2013-01-01

    DNA damage is one of many possible perturbations that challenge the mechanisms that preserve genetic stability during the copying of the eukaryotic genome in S phase. This short review provides, in the first part, a general introduction to the topic and an overview of checkpoint responses. In the second part, the mechanisms of error-free tolerance in response to fork-arresting DNA damage will be discussed in some detail. PMID:24296172

  8. Therapeutic targeting of replicative immortality

    PubMed Central

    Yaswen, Paul; MacKenzie, Karen L.; Keith, W. Nicol; Hentosh, Patricia; Rodier, Francis; Zhu, Jiyue; Firestone, Gary L.; Matheu, Ander; Carnero, Amancio; Bilsland, Alan; Sundin, Tabetha; Honoki, Kanya; Fujii, Hiromasa; Georgakilas, Alexandros G.; Amedei, Amedeo; Amin, Amr; Helferich, Bill; Boosani, Chandra S.; Guha, Gunjan; Ciriolo, Maria Rosa; Chen, Sophie; Mohammed, Sulma I.; Azmi, Asfar S.; Bhakta, Dipita; Halicka, Dorota; Niccolai, Elena; Aquilano, Katia; Ashraf, S. Salman; Nowsheen, Somaira; Yang, Xujuan

    2015-01-01

    One of the hallmarks of malignant cell populations is the ability to undergo continuous proliferation. This property allows clonal lineages to acquire sequential aberrations that can fuel increasingly autonomous growth, invasiveness, and therapeutic resistance. Innate cellular mechanisms have evolved to regulate replicative potential as a hedge against malignant progression. When activated in the absence of normal terminal differentiation cues, these mechanisms can result in a state of persistent cytostasis. This state, termed “senescence,” can be triggered by intrinsic cellular processes such as telomere dysfunction and oncogene expression, and by exogenous factors such as DNA damaging agents or oxidative environments. Despite differences in upstream signaling, senescence often involves convergent interdependent activation of tumor suppressors p53 and p16/pRB, but can be induced, albeit with reduced sensitivity, when these suppressors are compromised. Doses of conventional genotoxic drugs required to achieve cancer cell senescence are often much lower than doses required to achieve outright cell death. Additional therapies, such as those targeting cyclin dependent kinases or components of the PI3K signaling pathway, may induce senescence specifically in cancer cells by circumventing defects in tumor suppressor pathways or exploiting cancer cells’ heightened requirements for telomerase. Such treatments sufficient to induce cancer cell senescence could provide increased patient survival with fewer and less severe side effects than conventional cytotoxic regimens. This positive aspect is countered by important caveats regarding senescence reversibility, genomic instability, and paracrine effects that may increase heterogeneity and adaptive resistance of surviving cancer cells. Nevertheless, agents that effectively disrupt replicative immortality will likely be valuable components of new combinatorial approaches to cancer therapy. PMID:25869441

  9. Therapeutic targeting of replicative immortality.

    PubMed

    Yaswen, Paul; MacKenzie, Karen L; Keith, W Nicol; Hentosh, Patricia; Rodier, Francis; Zhu, Jiyue; Firestone, Gary L; Matheu, Ander; Carnero, Amancio; Bilsland, Alan; Sundin, Tabetha; Honoki, Kanya; Fujii, Hiromasa; Georgakilas, Alexandros G; Amedei, Amedeo; Amin, Amr; Helferich, Bill; Boosani, Chandra S; Guha, Gunjan; Ciriolo, Maria Rosa; Chen, Sophie; Mohammed, Sulma I; Azmi, Asfar S; Bhakta, Dipita; Halicka, Dorota; Niccolai, Elena; Aquilano, Katia; Ashraf, S Salman; Nowsheen, Somaira; Yang, Xujuan

    2015-12-01

    One of the hallmarks of malignant cell populations is the ability to undergo continuous proliferation. This property allows clonal lineages to acquire sequential aberrations that can fuel increasingly autonomous growth, invasiveness, and therapeutic resistance. Innate cellular mechanisms have evolved to regulate replicative potential as a hedge against malignant progression. When activated in the absence of normal terminal differentiation cues, these mechanisms can result in a state of persistent cytostasis. This state, termed "senescence," can be triggered by intrinsic cellular processes such as telomere dysfunction and oncogene expression, and by exogenous factors such as DNA damaging agents or oxidative environments. Despite differences in upstream signaling, senescence often involves convergent interdependent activation of tumor suppressors p53 and p16/pRB, but can be induced, albeit with reduced sensitivity, when these suppressors are compromised. Doses of conventional genotoxic drugs required to achieve cancer cell senescence are often much lower than doses required to achieve outright cell death. Additional therapies, such as those targeting cyclin dependent kinases or components of the PI3K signaling pathway, may induce senescence specifically in cancer cells by circumventing defects in tumor suppressor pathways or exploiting cancer cells' heightened requirements for telomerase. Such treatments sufficient to induce cancer cell senescence could provide increased patient survival with fewer and less severe side effects than conventional cytotoxic regimens. This positive aspect is countered by important caveats regarding senescence reversibility, genomic instability, and paracrine effects that may increase heterogeneity and adaptive resistance of surviving cancer cells. Nevertheless, agents that effectively disrupt replicative immortality will likely be valuable components of new combinatorial approaches to cancer therapy. PMID:25869441

  10. Functional association of poly(ADP-ribose) polymerase with DNA polymerase alpha-primase complex: a link between DNA strand break detection and DNA replication.

    PubMed Central

    Dantzer, F; Nasheuer, H P; Vonesch, J L; de Murcia, G; Ménissier-de Murcia, J

    1998-01-01

    Poly(ADP-ribose) polymerase (PARP) is an element of the DNA damage surveillance network evolved by eukaryotic cells to cope with numerous environmental and endogenous genotoxic agents. PARP has been found to be involved in vivo in both cell proliferation and base excision repair of DNA. In this study the interaction between PARP and the DNA polymerase alpha-primase tetramer has been examined. We provide evidence that in proliferating cells: (i) PARP is physically associated with the catalytic subunit of the DNA polymerase alpha-primase tetramer, an association confirmed by confocal microscopy, demonstrating that both enzymes are co-localized at the nuclear periphery of HeLa cells; (ii) this interaction requires the integrity of the second zinc finger of PARP and is maximal during the S and G2/M phases of the cell cycle; (iii) PARP-deficient cells derived from PARP knock-out mice exhibited reduced DNA polymerase activity, compared with the parental cells, a reduction accentuated following exposure to sublethal doses of methylmethanesulfonate. Altogether, the present results strongly suggest that PARP participates in a DNA damage survey mechanism implying its nick-sensor function as part of the control of replication fork progression when breaks are present in the template. PMID:9518481

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

    PubMed Central

    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. Replication Research and Special Education

    ERIC Educational Resources Information Center

    Travers, Jason C.; Cook, Bryan G.; Therrien, William J.; Coyne, Michael D.

    2016-01-01

    Replicating previously reported empirical research is a necessary aspect of an evidence-based field of special education, but little formal investigation into the prevalence of replication research in the special education research literature has been conducted. Various factors may explain the lack of attention to replication of special education…

  13. Geomorphic Characterization of the Middle Fork Saline River: Garland, Perry, and Saline Counties, Arkansas

    USGS Publications Warehouse

    Pugh, Aaron L.; Garday, Thomas J.; Redman, Ronald

    2008-01-01

    This report was prepared to help address concerns raised by local residents, State, and Federal agencies about the current geomorphic conditions of the Middle Fork Saline River. Over the past 30 years the Middle Fork Saline River Basin has experienced a marked increase in urbanization. The report summarizes the Middle Fork?s current (2003) channel characteristics at nine stream reaches in the upper 91 square miles of the basin. Assessments at each study reach included comparing measured stream geometry dimensions (cross-sectional area, top width, and mean depth) at bankfull stage to regional hydraulic geometry curves for the Ouachita Mountains Physiographic Province of Arkansas and Oklahoma, evaluations of streambed materials and sinuosity, and classification of individual stream reach types. When compared to the Ouachita Mountains? regional hydraulic geometry curves for natural, stable, stream reaches, five of the nine study reaches had slightly smaller crosssectional areas, longer top widths, and shallower depths. Streambed material analysis indicates that the Middle Fork is a bedrock influenced, gravel dominated stream with lesser amounts of sand and cobbles. Slight increases in sinuosity from 1992 to 2002 at seven of the nine study reaches indicate a slight decrease in stream channel slope. Analyses of the Middle Fork?s hydraulic geometry and sinuosity indicate that the Middle Fork is currently overly wide and shallow, but is slowly adjusting towards a deeper, narrower hydraulic geometry. Using the Rosgen system of channel classification, the two upstream study reaches classified as B4c/1 stream types; which were moderately entrenched, riffle dominated channels, with infrequently spaced pools. The downstream seven study reaches classified as C4/1 stream types; which were slightly entrenched, meandering, gravel-dominated, riffle/ pool channels with well developed flood plains. Analyses of stream reach types suggest that the downstream reaches of the Middle Fork

  14. Replication stress is a potent driver of functional decline in ageing haematopoietic stem cells

    PubMed Central

    Flach, Johanna; Bakker, Sietske T.; Mohrin, Mary; Conroy, Pauline C.; Pietras, Eric M.; Reynaud, Damien; Alvarez, Silvia; Diolaiti, Morgan E.; Ugarte, Fernando; Forsberg, E. Camilla; Le Beau, Michelle M.; Stohr, Bradley A.; Méndez, Juan; Morrison, Ciaran G.; Passegué, Emmanuelle

    2015-01-01

    Haematopoietic stem cells (HSCs) self-renew for life, thereby making them one of the few blood cells that truly age1,2. Paradoxically, although HSCs numerically expand with age, their functional activity declines over time, resulting in degraded blood production and impaired engraftment following transplantation2. While many drivers of HSC ageing have been proposed2–5, the reason why HSC function degrades with age remains unknown. Here we show that cycling old HSCs in mice have heightened levels of replication stress associated with cell cycle defects and chromosome gaps or breaks, which are due to decreased expression of mini-chromosome maintenance (MCM) helicase components and altered dynamics of DNA replication forks. Nonetheless, old HSCs survive replication unless confronted with a strong replication challenge, such as transplantation. Moreover, once old HSCs re-establish quiescence, residual replication stress on ribosomal DNA (rDNA) genes leads to the formation of nucleolar-associated γH2AX signals, which persist owing to ineffective H2AX dephosphorylation by mislocalized PP4c phosphatase rather than ongoing DNA damage. Persistent nucleolar γH2AX also acts as a histone modification marking the transcriptional silencing of rDNA genes and decreased ribosome biogenesis in quiescent old HSCs. Our results identify replication stress as a potent driver of functional decline in old HSCs, and highlight the MCM DNA helicase as a potential molecular target for rejuvenation therapies. PMID:25079315

  15. LY2606368 Causes Replication Catastrophe and Antitumor Effects through CHK1-Dependent Mechanisms.

    PubMed

    King, Constance; Diaz, H Bruce; McNeely, Samuel; Barnard, Darlene; Dempsey, Jack; Blosser, Wayne; Beckmann, Richard; Barda, David; Marshall, Mark S

    2015-09-01

    CHK1 is a multifunctional protein kinase integral to both the cellular response to DNA damage and control of the number of active replication forks. CHK1 inhibitors are currently under investigation as chemopotentiating agents due to CHK1's role in establishing DNA damage checkpoints in the cell cycle. Here, we describe the characterization of a novel CHK1 inhibitor, LY2606368, which as a single agent causes double-stranded DNA breakage while simultaneously removing the protection of the DNA damage checkpoints. The action of LY2606368 is dependent upon inhibition of CHK1 and the corresponding increase in CDC25A activation of CDK2, which increases the number of replication forks while reducing their stability. Treatment of cells with LY2606368 results in the rapid appearance of TUNEL and pH2AX-positive double-stranded DNA breaks in the S-phase cell population. Loss of the CHK1-dependent DNA damage checkpoints permits cells with damaged DNA to proceed into early mitosis and die. The majority of treated mitotic nuclei consist of extensively fragmented chromosomes. Inhibition of apoptosis by the caspase inhibitor Z-VAD-FMK had no effect on chromosome fragmentation, indicating that LY2606368 causes replication catastrophe. Changes in the ratio of RPA2 to phosphorylated H2AX following LY2606368 treatment further support replication catastrophe as the mechanism of DNA damage. LY2606368 shows similar activity in xenograft tumor models, which results in significant tumor growth inhibition. LY2606368 is a potent representative of a novel class of drugs for the treatment of cancer that acts through replication catastrophe. PMID:26141948

  16. DNA replication stress and cancer: cause or cure?

    PubMed

    Taylor, Elaine M; Lindsay, Howard D

    2016-01-01

    There is an extensive and growing body of evidence that DNA replication stress is a major driver in the development and progression of many cancers, and that these cancers rely heavily on replication stress response pathways for their continued proliferation. This raises the possibility that the pathways that ordinarily protect cells from the accumulation of cancer-causing mutations may actually prove to be effective therapeutic targets for a wide range of malignancies. In this review, we explore the mechanisms by which sustained proliferation can lead to replication stress and genome instability, and discuss how the pattern of mutations observed in human cancers is supportive of this oncogene-induced replication stress model. Finally, we go on to consider the implications of replication stress both as a prognostic indicator and, more encouragingly, as a potential target in cancer treatment. PMID:26616915

  17. Replication Origin Selection Regulates the Distribution of Meiotic Recombination

    PubMed Central

    Wu, Pei-Yun Jenny; Nurse, Paul

    2014-01-01

    Summary The program of DNA replication, defined by the temporal and spatial pattern of origin activation, is altered during development and in cancers. However, whether changes in origin usage play a role in regulating specific biological processes remains unknown. We investigated the consequences of modifying origin selection on meiosis in fission yeast. Genome-wide changes in the replication program of premeiotic S phase do not affect meiotic progression, indicating that meiosis neither activates nor requires a particular origin pattern. In contrast, local changes in origin efficiencies between different replication programs lead to changes in Rad51 recombination factor binding and recombination frequencies in these domains. We observed similar results for Rad51 when changes in efficiencies were generated by directly targeting expression of the Cdc45 replication factor. We conclude that origin selection is a key determinant for organizing meiotic recombination, providing evidence that genome-wide modifications in replication program can modulate cellular physiology. PMID:24560273

  18. Rad18 is required for functional interactions between FANCD2, BRCA2, and Rad51 to repair DNA topoisomerase 1-poisons induced lesions and promote fork recovery

    PubMed Central

    Tripathi, Kaushlendra; Mani, Chinnadurai; Clark, David W; Palle, Komaraiah

    2016-01-01

    Camptothecin (CPT) and its analogues are chemotherapeutic agents that covalently and reversibly link DNA Topoisomerase I to its nicked DNA intermediate eliciting the formation of DNA double strand breaks (DSB) during replication. The repair of these DSB involves multiple DNA damage response and repair proteins. Here we demonstrate that CPT-induced DNA damage promotes functional interactions between BRCA2, FANCD2, Rad18, and Rad51 to repair the replication-associated DSB through homologous recombination (HR). Loss of any of these proteins leads to equal disruption of HR repair, causes chromosomal aberrations and sensitizes cells to CPT. Rad18 appears to function upstream in this repair pathway as its downregulation prevents activation of FANCD2, diminishes BRCA2 and Rad51 protein levels, formation of nuclear foci of all three proteins and recovery of stalled or collapsed replication forks in response to CPT. Taken together this work further elucidates the complex interplay of DNA repair proteins in the repair of replication-associated DSB. PMID:26871286

  19. Improved Tuning Fork for Terahertz Quartz-Enhanced Photoacoustic Spectroscopy.

    PubMed

    Sampaolo, Angelo; Patimisco, Pietro; Giglio, Marilena; Vitiello, Miriam S; Beere, Harvey E; Ritchie, David A; Scamarcio, Gaetano; Tittel, Frank K; Spagnolo, Vincenzo

    2016-01-01

    We report on a quartz-enhanced photoacoustic (QEPAS) sensor for methanol (CH₃OH) detection employing a novel quartz tuning fork (QTF), specifically designed to enhance the QEPAS sensing performance in the terahertz (THz) spectral range. A discussion of the QTF properties in terms of resonance frequency, quality factor and acousto-electric transduction efficiency as a function of prong sizes and spacing between the QTF prongs is presented. The QTF was employed in a QEPAS sensor system using a 3.93 THz quantum cascade laser as the excitation source in resonance with a CH₃OH rotational absorption line located at 131.054 cm(-1). A minimum detection limit of 160 ppb in 30 s integration time, corresponding to a normalized noise equivalent absorption NNEA = 3.75 × 10(-11) cm(-1)W/Hz(½), was achieved, representing a nearly one-order-of-magnitude improvement with respect to previous reports. PMID:27023552

  20. Improved Tuning Fork for Terahertz Quartz-Enhanced Photoacoustic Spectroscopy

    PubMed Central

    Sampaolo, Angelo; Patimisco, Pietro; Giglio, Marilena; Vitiello, Miriam S.; Beere, Harvey E.; Ritchie, David A.; Scamarcio, Gaetano; Tittel, Frank K.; Spagnolo, Vincenzo

    2016-01-01

    We report on a quartz-enhanced photoacoustic (QEPAS) sensor for methanol (CH3OH) detection employing a novel quartz tuning fork (QTF), specifically designed to enhance the QEPAS sensing performance in the terahertz (THz) spectral range. A discussion of the QTF properties in terms of resonance frequency, quality factor and acousto-electric transduction efficiency as a function of prong sizes and spacing between the QTF prongs is presented. The QTF was employed in a QEPAS sensor system using a 3.93 THz quantum cascade laser as the excitation source in resonance with a CH3OH rotational absorption line located at 131.054 cm−1. A minimum detection limit of 160 ppb in 30 s integration time, corresponding to a normalized noise equivalent absorption NNEA = 3.75 × 10−11 cm−1W/Hz½, was achieved, representing a nearly one-order-of-magnitude improvement with respect to previous reports. PMID:27023552

  1. Geochemical map of the North Fork John Day River Roadless Area, Grant County, Oregon

    USGS Publications Warehouse

    Evans, James G.

    1986-01-01

    The North Fork John Day River Roadless Area comprised 21,210 acres in the Umatilla and Wallowa-Whitman National Forests, Grant County, Oregon, about 30 miles northwest of Baker, Oregon. The irregularly shaped area extends for about 1 mile on both sides of a 25-mile segment of the North Fork John Day River from Big Creek on the west to North Fork John Day Campground on the east. Most of the roadless area is in the northern half of the Desolation Butte 15-minute quadrangle. The eastern end of the area is in parts of the Granite and Trout Meadows 7½-minute quadrangles.

  2. Q-factor optimization of a tuning-fork/fiber sensor for shear-force detection

    NASA Astrophysics Data System (ADS)

    Morville, Jérôme; Liu, Jinquan; Callegari, Andrea; Chergui, Majed

    2005-02-01

    We present the results of an experimental and theoretical study on the optimum design of shear-force sensors, used in scanning probe microscopes. We have optimized a configuration consisting of a tuning-fork/fiber-tip assembly, achieving quality factors (Q) exceeding 8000, and have presented a theoretical analysis of the design wherein the force holding the fiber and fork in contact is provided solely by elastic mechanical deformation, which allows full control of the performance of the system. On this basis, we constructed a high-quality-factor configuration with the fiber glued onto the tuning fork.

  3. On cavitation in liquid helium in a flow due to a vibrating quartz fork

    NASA Astrophysics Data System (ADS)

    Blažková, M.; Schmoranzer, D.; Skrbek, L.

    2008-04-01

    Cavitation in normal and superfluid liquid He4 at saturated vapor pressure and slightly elevated pressures has been experimentally studied in a flow due to quartz forks vibrating at high amplitudes. Above the temperature- and pressure-dependent critical velocity, heterogeneous cavitation is observed both visually and electrically, as a breakdown of the resonance response of the fork. We compare our results with available experimental and discuss them using existing theoretical models. In particular, we show that thermal effects leading to local overheating of the vicinity of the fork have to be taken into account, especially in normal liquid He4.

  4. A tuning fork gyroscope with compensated imbalance signal

    NASA Astrophysics Data System (ADS)

    Arnold, E.; Nuscheler, F.

    2007-05-01

    This paper is presenting a robust gyroscope sensor with an electrical and mechanical self-test option and the ability to suppress the quadrature error. The presented sensor is based on a tuning-fork working principle. The mechanical part is assembled in bulk-technology produced with a wet etching process. The two detection elements are manufactured with a standard CMOS-process and the material of the two thin-film actuators is AlN (aluminium-nitrid). The two actuators can be controlled independently from each other. Two electronic PCB's were developed for actuating and measurement. One is including the analogue signal path; the second PCB is the digital electronics consisting of a FPGA and other peripherals. The tuning fork is actuated in a primary oscillation mode also called drive mode. For keeping the oscillation in resonance, a digital PLL is used in a forced feedback loop. To have a constant energy in the drive mode an Amplitude-Gain-Control (AGC) is implemented. An appearing angular rate causes the corriolis-force which is actuating secondary oscillation, also called detection mode. The amplitude of this oscillation is proportional to the angular rate. The signal has a component resulting from the mechanical imbalance. To separate these two signal parts from each other a synchronous demodulator followed by a digital filter chain has been developed. To achieve the maximum suppression of the imbalance signal a control-loop is used to shift the phases of the two actuation signals. This creates an additional force that compensates the movement as a result of the mechanical imbalance. With the implementation of this control loop the performance of the sensor was increased. An enhanced temperature stability over operation was achieved with the means of this compensation.

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

    PubMed

    Bilousova, Ganna; Marusyk, Andriy; Porter, Christopher C; Cardiff, Robert D; DeGregori, James

    2005-12-01

    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. PMID:16277552

  6. Chromatin and DNA replication.

    PubMed

    MacAlpine, David M; Almouzni, Geneviève

    2013-08-01

    The size of a eukaryotic genome presents a unique challenge to the cell: package and organize the DNA to fit within the confines of the nucleus while at the same time ensuring sufficient dynamics to allow access to specific sequences and features such as genes and regulatory elements. This is achieved via the dynamic nucleoprotein organization of eukaryotic DNA into chromatin. The basic unit of chromatin, the nucleosome, comprises a core particle with 147 bp of DNA wrapped 1.7 times around an octamer of histones. The nucleosome is a highly versatile and modular structure, both in its composition, with the existence of various histone variants, and through the addition of a series of posttranslational modifications on the histones. This versatility allows for both short-term regulatory responses to external signaling, as well as the long-term and multigenerational definition of large functional chromosomal domains within the nucleus, such as the centromere. Chromatin organization and its dynamics participate in essentially all DNA-templated processes, including transcription, replication, recombination, and repair. Here we will focus mainly on nucleosomal organization and describe the pathways and mechanisms that contribute to assembly of this organization and the role of chromatin in regulating the DNA replication program. PMID:23751185

  7. Bed-material entrainment potential, Roaring Fork River at Basalt, Colorado

    USGS Publications Warehouse

    Elliott, John G.

    2002-01-01

    The Roaring Fork River at Basalt, Colorado, has a frequently mobile streambed composed of gravel, cobbles, and boulders. Recent urban and highway development on the flood plain, earlier attempts to realign and confine the channel, and flow obstructions such as bridge openings and piers have altered the hydrology, hydraulics, sediment transport, and sediment deposition areas of the Roaring Fork. Entrainment and deposition of coarse sediment on the streambed and in large alluvial bars have reduced the flood-conveying capacity of the river. Previous engineering studies have identified flood-prone areas and hazards related to inundation and high streamflow velocity, but those studies have not evaluated the potential response of the channel to discharges that entrain the coarse streambed. This study builds upon the results of earlier flood studies and identifies some potential areas of concern associated with bed-material entrainment. Cross-section surveys and simulated water-surface elevations from a previously run HEC?RAS model were used to calculate the boundary shear stress on the mean streambed, in the thalweg, and on the tops of adjacent alluvial bars for four reference streamflows. Sediment-size characteristics were determined for surficial material on the streambed, on large alluvial bars, and on a streambank. The median particle size (d50) for the streambed samples was 165 millimeters and for the alluvial bars and bank samples was 107 millimeters. Shear stresses generated by the 10-, 50-, and 100-year floods, and by a more common flow that just inundated most of the alluvial bars in the study reach were calculated at 14 of the cross sections used in the Roaring Fork River HEC?RAS model. The Shields equation was used with a Shields parameter of 0.030 to estimate the critical shear stress for entrainment of the median sediment particle size on the mean streambed, in the thalweg, and on adjacent alluvial bar surfaces at the 14 cross sections. Sediment

  8. The hexameric structure of the human mitochondrial replicative helicase Twinkle

    PubMed Central

    Fernández-Millán, Pablo; Lázaro, Melisa; Cansız-Arda, Şirin; Gerhold, Joachim M.; Rajala, Nina; Schmitz, Claus-A.; Silva-Espiña, Cristina; Gil, David; Bernadó, Pau; Valle, Mikel; Spelbrink, Johannes N.; Solà, Maria

    2015-01-01

    The mitochondrial replicative helicase Twinkle is involved in strand separation at the replication fork of mitochondrial DNA (mtDNA). Twinkle malfunction is associated with rare diseases that include late onset mitochondrial myopathies, neuromuscular disorders and fatal infantile mtDNA depletion syndrome. We examined its 3D structure by electron microscopy (EM) and small angle X-ray scattering (SAXS) and built the corresponding atomic models, which gave insight into the first molecular architecture of a full-length SF4 helicase that includes an N-terminal zinc-binding domain (ZBD), an intermediate RNA polymerase domain (RPD) and a RecA-like hexamerization C-terminal domain (CTD). The EM model of Twinkle reveals a hexameric two-layered ring comprising the ZBDs and RPDs in one layer and the CTDs in another. In the hexamer, contacts in trans with adjacent subunits occur between ZBDs and RPDs, and between RPDs and CTDs. The ZBDs show important structural heterogeneity. In solution, the scattering data are compatible with a mixture of extended hexa- and heptameric models in variable conformations. Overall, our structural data show a complex network of dynamic interactions that reconciles with the structural flexibility required for helicase activity. PMID:25824949

  9. Replication stress and cancer: it takes two to tango

    PubMed Central

    Lecona, Emilio; Fernández-Capetillo, Oscar

    2016-01-01

    Problems arising during DNA replication require the activation of the ATR-CHK1 pathway to ensure the stabilization and repair of the forks, and to prevent the entry into mitosis with unreplicated genomes. Whereas the pathway is essential at the cellular level, limiting its activity is particularly detrimental for some cancer cells. Here we review the links between replication stress (RS) and cancer, which provide a rationale for the use of ATR and Chk1 inhibitors in chemotherapy. First, we describe how the activation of oncogene-induced RS promotes genome rearrangements and chromosome instability, both of which could potentially fuel carcinogenesis. Next, we review the various pathways that contribute to the suppression of RS, and how mutations in these components lead to increased cancer incidence and/or accelerated ageing. Finally, we summarize the evidence showing that tumours with high levels of RS are dependent on a proficient RS-response, and therefore vulnerable to ATR or Chk1 inhibitors. PMID:25257608

  10. Strand-biased cytosine deamination at the replication fork causes cytosine to thymine mutations in Escherichia coli

    PubMed Central

    Bhagwat, Ashok S.; Hao, Weilong; Townes, Jesse P.; Lee, Heewook; Tang, Haixu; Foster, Patricia L.

    2016-01-01

    The rate of cytosine deamination is much higher in single-stranded DNA (ssDNA) than in double-stranded DNA, and copying the resulting uracils causes C to T mutations. To study this phenomenon, the catalytic domain of APOBEC3G (A3G-CTD), an ssDNA-specific cytosine deaminase, was expressed in an Escherichia coli strain defective in uracil repair (ung mutant), and the mutations that accumulated over thousands of generations were determined by whole-genome sequencing. C:G to T:A transitions dominated, with significantly more cytosines mutated to thymine in the lagging-strand template (LGST) than in the leading-strand template (LDST). This strand bias was present in both repair-defective and repair-proficient cells and was strongest and highly significant in cells expressing A3G-CTD. These results show that the LGST is accessible to cellular cytosine deaminating agents, explains the well-known GC skew in microbial genomes, and suggests the APOBEC3 family of mutators may target the LGST in the human genome. PMID:26839411

  11. HENRY'S FORK AND SNAKE RIVER BASIN, IDAHO - WATER QUALITY REPORT, 1973

    EPA Science Inventory

    Reported problems in the Henrys Fork and Snake River Basin (17040202, 17040203, 17040201) include bacteria levels exceeding water quality standards, dissolved oxygen standards violations, and excessive algal blooms resulting in aesthetic problems and contributing to DO depression...

  12. ADAPTATION OPPORTUNITIES FOR SURFACE WATER RESOURCES IN THE ROARING FORK WATERSHED, COLORADO.

    EPA Science Inventory

    This project will assess climate-related impacts to the Roring Fork River (near Aspen, Colorado) and identify adaptive opportunities for surface water users, to support a larger global change assessment by the city of Aspen, CO (the Canary Initiative).

  13. 16 CFR 1512.14 - Requirements for fork and frame assembly.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... SUBSTANCES ACT REGULATIONS REQUIREMENTS FOR BICYCLES Regulations § 1512.14 Requirements for fork and frame... significantly limits the steering angle over which the wheel can be turned. Sidewalk bicycles are exempt...

  14. 16 CFR 1512.14 - Requirements for fork and frame assembly.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... SUBSTANCES ACT REGULATIONS REQUIREMENTS FOR BICYCLES Regulations § 1512.14 Requirements for fork and frame... significantly limits the steering angle over which the wheel can be turned. Sidewalk bicycles are exempt...

  15. 16 CFR 1512.14 - Requirements for fork and frame assembly.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... SUBSTANCES ACT REGULATIONS REQUIREMENTS FOR BICYCLES Regulations § 1512.14 Requirements for fork and frame... significantly limits the steering angle over which the wheel can be turned. Sidewalk bicycles are exempt...

  16. 16 CFR 1512.14 - Requirements for fork and frame assembly.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... SUBSTANCES ACT REGULATIONS REQUIREMENTS FOR BICYCLES Regulations § 1512.14 Requirements for fork and frame... significantly limits the steering angle over which the wheel can be turned. Sidewalk bicycles are exempt...

  17. The Cell Cycle Timing of Human Papillomavirus DNA Replication

    PubMed Central

    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

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

    PubMed

    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

  19. Probing Liquid ^4He with Quartz Tuning Forks Using a Novel Multifrequency Lock-in Technique

    NASA Astrophysics Data System (ADS)

    Bradley, D. I.; Haley, R. P.; Kafanov, S.; Noble, M. T.; Pickett, G. R.; Tsepelin, V.; Vonka, J.; Wilcox, T.

    2016-09-01

    We report on a novel technique to measure quartz tuning forks, and possibly other vibrating objects, in a quantum fluid using a multifrequency lock-in amplifier. The multifrequency technique allows to measure the resonance curve of a vibrating object much faster than a conventional single frequency lock-in amplifier technique. Forks with resonance frequencies of 12 kHz and 16 kHz were excited and measured electro-mechanically either at a single frequency or at up to 40 different frequencies simultaneously around the same mechanical mode. The response of each fork was identical for both methods and validates the use of the multifrequency lock-in technique to probe properties of liquid helium at low fork velocities. Using both methods we measured the resonance frequency and drag of two 25-μ m-wide quartz tuning forks immersed in liquid ^4He in the temperature range from 4.2 K to 1.5 K at saturated vapour pressure. The damping and shift of resonance frequency experienced by both tuning forks at low velocities are well described by hydrodynamic contributions in the framework of the two-fluid model. The sensitivity of the 25-μ m-wide tuning forks is larger compared to similar 75-μ m-wide forks and in combination with the faster multifrequency lock-in technique could be used to improve thermometry in liquid ^4He. The multifrequency technique could also be used for studies of the onset of non-linear phenomena such as quantum turbulence and cavitation in superfluids.

  20. Probing Liquid ^4 He with Quartz Tuning Forks Using a Novel Multifrequency Lock-in Technique

    NASA Astrophysics Data System (ADS)

    Bradley, D. I.; Haley, R. P.; Kafanov, S.; Noble, M. T.; Pickett, G. R.; Tsepelin, V.; Vonka, J.; Wilcox, T.

    2016-06-01

    We report on a novel technique to measure quartz tuning forks, and possibly other vibrating objects, in a quantum fluid using a multifrequency lock-in amplifier. The multifrequency technique allows to measure the resonance curve of a vibrating object much faster than a conventional single frequency lock-in amplifier technique. Forks with resonance frequencies of 12 kHz and 16 kHz were excited and measured electro-mechanically either at a single frequency or at up to 40 different frequencies simultaneously around the same mechanical mode. The response of each fork was identical for both methods and validates the use of the multifrequency lock-in technique to probe properties of liquid helium at low fork velocities. Using both methods we measured the resonance frequency and drag of two 25-μ m-wide quartz tuning forks immersed in liquid ^4 He in the temperature range from 4.2 K to 1.5 K at saturated vapour pressure. The damping and shift of resonance frequency experienced by both tuning forks at low velocities are well described by hydrodynamic contributions in the framework of the two-fluid model. The sensitivity of the 25-μ m-wide tuning forks is larger compared to similar 75-μ m-wide forks and in combination with the faster multifrequency lock-in technique could be used to improve thermometry in liquid ^4 He. The multifrequency technique could also be used for studies of the onset of non-linear phenomena such as quantum turbulence and cavitation in superfluids.