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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. ATR-like kinase Mec1 facilitates both chromatin accessibility at DNA replication forks and replication fork progression during replication stress.

    PubMed

    Rodriguez, Jairo; Tsukiyama, Toshio

    2013-01-01

    Faithful DNA replication is essential for normal cell division and differentiation. In eukaryotic cells, DNA replication takes place on chromatin. This poses the critical question as to how DNA replication can progress through chromatin, which is inhibitory to all DNA-dependent processes. Here, we developed a novel genome-wide method to measure chromatin accessibility to micrococcal nuclease (MNase) that is normalized for nucleosome density, the NCAM (normalized chromatin accessibility to MNase) assay. This method enabled us to discover that chromatin accessibility increases specifically at and ahead of DNA replication forks in normal S phase and during replication stress. We further found that Mec1, a key regulatory ATR-like kinase in the S-phase checkpoint, is required for both normal chromatin accessibility around replication forks and replication fork rate during replication stress, revealing novel functions for the kinase in replication stress response. These results suggest a possibility that Mec1 may facilitate DNA replication fork progression during replication stress by increasing chromatin accessibility around replication forks.

  3. Chk1 requirement for high global rates of replication fork progression during normal vertebrate S phase.

    PubMed

    Petermann, Eva; Maya-Mendoza, Apolinar; Zachos, George; Gillespie, David A F; Jackson, Dean A; Caldecott, Keith W

    2006-04-01

    Chk1 protein kinase maintains replication fork stability in metazoan cells in response to DNA damage and DNA replication inhibitors. Here, we have employed DNA fiber labeling to quantify, for the first time, the extent to which Chk1 maintains global replication fork rates during normal vertebrate S phase. We report that replication fork rates in Chk1(-/-) chicken DT40 cells are on average half of those observed with wild-type cells. Similar results were observed if Chk1 was inhibited or depleted in wild-type DT40 cells or HeLa cells by incubation with Chk1 inhibitor or small interfering RNA. In addition, reduced rates of fork extension were observed with permeabilized Chk1(-/-) cells in vitro. The requirement for Chk1 for high fork rates during normal S phase was not to suppress promiscuous homologous recombination at replication forks, because inhibition of Chk1 similarly slowed fork progression in XRCC3(-/-) DT40 cells. Rather, we observed an increased number of replication fibers in Chk1(-/-) cells in which the nascent strand is single-stranded, supporting the idea that slow global fork rates in unperturbed Chk1(-/-) cells are associated with the accumulation of aberrant replication fork structures.

  4. 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

  5. Low rate of replication fork progression lengthens the replication timing of a locus containing an early firing origin

    PubMed Central

    Bénard, Marianne; Maric, Chrystelle; Pierron, Gérard

    2007-01-01

    Invariance of temporal order of genome replication in eukaryotic cells and its correlation with gene activity has been well-documented. However, recent data suggest a relax control of replication timing. To evaluate replication schedule accuracy, we detailed the replicational organization of the developmentally regulated php locus that we previously found to be lately replicated, even though php gene is highly transcribed in naturally synchronous plasmodia of Physarum. Unexpectedly, bi-dimensional agarose gel electrophoreses of DNA samples prepared at specific time points of S phase showed that replication of the locus actually begins at the onset of S phase but it proceeds through the first half of S phase, so that complete replication of php-containing DNA fragments occurs in late S phase. Origin mapping located replication initiation upstream php coding region. This proximity and rapid fork progression through the coding region result in an early replication of php gene. We demonstrated that afterwards an unusually low fork rate and unidirectional fork pausing prolong complete replication of php locus, and we excluded random replication timing. Importantly, we evidenced that the origin linked to php gene in plasmodium is not fired in amoebae when php expression dramatically reduced, further illustrating replication-transcription coupling in Physarum. PMID:17717000

  6. Low rate of replication fork progression lengthens the replication timing of a locus containing an early firing origin.

    PubMed

    Bénard, Marianne; Maric, Chrystelle; Pierron, Gérard

    2007-01-01

    Invariance of temporal order of genome replication in eukaryotic cells and its correlation with gene activity has been well-documented. However, recent data suggest a relax control of replication timing. To evaluate replication schedule accuracy, we detailed the replicational organization of the developmentally regulated php locus that we previously found to be lately replicated, even though php gene is highly transcribed in naturally synchronous plasmodia of Physarum. Unexpectedly, bi-dimensional agarose gel electrophoreses of DNA samples prepared at specific time points of S phase showed that replication of the locus actually begins at the onset of S phase but it proceeds through the first half of S phase, so that complete replication of php-containing DNA fragments occurs in late S phase. Origin mapping located replication initiation upstream php coding region. This proximity and rapid fork progression through the coding region result in an early replication of php gene. We demonstrated that afterwards an unusually low fork rate and unidirectional fork pausing prolong complete replication of php locus, and we excluded random replication timing. Importantly, we evidenced that the origin linked to php gene in plasmodium is not fired in amoebae when php expression dramatically reduced, further illustrating replication-transcription coupling in Physarum.

  7. Spontaneous slow replication fork progression elicits mitosis alterations in homologous recombination-deficient mammalian cells.

    PubMed

    Wilhelm, Therese; Magdalou, Indiana; Barascu, Aurélia; Técher, Hervé; Debatisse, Michelle; Lopez, Bernard S

    2014-01-14

    Homologous recombination deficient (HR(-)) mammalian cells spontaneously display reduced replication fork (RF) movement and mitotic extra centrosomes. We show here that these cells present a complex mitotic phenotype, including prolonged metaphase arrest, anaphase bridges, and multipolar segregations. We then asked whether the replication and the mitotic phenotypes are interdependent. First, we determined low doses of hydroxyurea that did not affect the cell cycle distribution or activate CHK1 phosphorylation but did slow the replication fork movement of wild-type cells to the same level than in HR(-) cells. Remarkably, these low hydroxyurea doses generated the same mitotic defects (and to the same extent) in wild-type cells as observed in unchallenged HR(-) cells. Reciprocally, supplying nucleotide precursors to HR(-) cells suppressed both their replication deceleration and mitotic extra centrosome phenotypes. Therefore, subtle replication stress that escapes to surveillance pathways and, thus, fails to prevent cells from entering mitosis alters metaphase progression and centrosome number, resulting in multipolar mitosis. Importantly, multipolar mitosis results in global unbalanced chromosome segregation involving the whole genome, even fully replicated chromosomes. These data highlight the cross-talk between chromosome replication and segregation, and the importance of HR at the interface of these two processes for protection against general genome instability.

  8. Highly transcribed RNA polymerase II genes are impediments to replication fork progression in Saccharomyces cerevisiae

    PubMed Central

    Azvolinsky, Anna; Giresi, Paul G.; Lieb, Jason D.; Zakian, Virginia A.

    2009-01-01

    SUMMARY Replication forks face multiple obstacles that slow their progression. By two-dimensional gel analysis, yeast forks pause at stable DNA protein complexes, and this pausing is greatly increased in the absence of the Rrm3 helicase. We used a genome wide approach to identify 96 sites of very high DNA polymerase binding in wild type cells. Most of these binding sites were not previously identified pause sites. Rather, the most highly represented genomic category among high DNA polymerase binding sites was the open reading frames (ORFs) of highly transcribed RNA polymerase II genes. Twice as many pause sites were identified in rrm3 compared to wild type cells as pausing in this strain occurred at both highly transcribed RNA polymerase II genes and the previously identified protein DNA complexes. ORFs of highly transcribed RNA polymerase II genes are the first class of natural pause sites that are not exacerbated in rrm3 cells. PMID:19560424

  9. Developmental control of gene copy number by repression of replication initiation and fork progression.

    PubMed

    Sher, Noa; Bell, George W; Li, Sharon; Nordman, Jared; Eng, Thomas; Eaton, Matthew L; Macalpine, David M; Orr-Weaver, Terry L

    2012-01-01

    Precise DNA replication is crucial for genome maintenance, yet this process has been inherently difficult to study on a genome-wide level in untransformed differentiated metazoan cells. To determine how metazoan DNA replication can be repressed, we examined regions selectively under-replicated in Drosophila polytene salivary glands, and found they are transcriptionally silent and enriched for the repressive H3K27me3 mark. In the first genome-wide analysis of binding of the origin recognition complex (ORC) in a differentiated metazoan tissue, we find that ORC binding is dramatically reduced within these large domains, suggesting reduced initiation as one mechanism leading to under-replication. Inhibition of replication fork progression by the chromatin protein SUUR is an additional repression mechanism to reduce copy number. Although repressive histone marks are removed when SUUR is mutated and copy number restored, neither transcription nor ORC binding is reinstated. Tethering of the SUUR protein to a specific site is insufficient to block replication, however. These results establish that developmental control of DNA replication, at both the initiation and elongation stages, is a mechanism to change gene copy number during differentiation.

  10. Replication fork collapse at replication terminator sequences.

    PubMed

    Bidnenko, Vladimir; Ehrlich, S Dusko; Michel, Bénédicte

    2002-07-15

    Replication fork arrest is a source of genome re arrangements, and the recombinogenic properties of blocked forks are likely to depend on the cause of blockage. Here we study the fate of replication forks blocked at natural replication arrest sites. For this purpose, Escherichia coli replication terminator sequences Ter were placed at ectopic positions on the bacterial chromosome. The resulting strain requires recombinational repair for viability, but replication forks blocked at Ter are not broken. Linear DNA molecules are formed upon arrival of a second round of replication forks that copy the DNA strands of the first blocked forks to the end. A model that accounts for the requirement for homologous recombination for viability in spite of the lack of chromosome breakage is proposed. This work shows that natural and accidental replication arrests sites are processed differently.

  11. Hydroxyurea-stalled replication forks become progressively inactivated and require two different RAD51-mediated pathways for restart and repair.

    PubMed

    Petermann, Eva; Orta, Manuel Luís; Issaeva, Natalia; Schultz, Niklas; Helleday, Thomas

    2010-02-26

    Faithful DNA replication is essential to all life. Hydroxyurea (HU) depletes the cells of dNTPs, which initially results in stalled replication forks that, after prolonged treatment, collapse into DSBs. Here, we report that stalled replication forks are efficiently restarted in a RAD51-dependent process that does not trigger homologous recombination (HR). The XRCC3 protein, which is required for RAD51 foci formation, is also required for replication restart of HU-stalled forks, suggesting that RAD51-mediated strand invasion supports fork restart. In contrast, replication forks collapsed by prolonged replication blocks do not restart, and global replication is rescued by new origin firing. We find that RAD51-dependent HR is triggered for repair of collapsed replication forks, without apparent restart. In conclusion, our data suggest that restart of stalled replication forks and HR repair of collapsed replication forks require two distinct RAD51-mediated pathways.

  12. 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.

  13. 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

  14. 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

  15. 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.

  16. R-loop-mediated genomic instability is caused by impairment of replication fork progression.

    PubMed

    Gan, Wenjian; Guan, Zhishuang; Liu, Jie; Gui, Ting; Shen, Keng; Manley, James L; Li, Xialu

    2011-10-01

    Transcriptional R loops are anomalous RNA:DNA hybrids that have been detected in organisms from bacteria to humans. These structures have been shown in eukaryotes to result in DNA damage and rearrangements; however, the mechanisms underlying these effects have remained largely unknown. To investigate this, we first show that R-loop formation induces chromosomal DNA rearrangements and recombination in Escherichia coli, just as it does in eukaryotes. More importantly, we then show that R-loop formation causes DNA replication fork stalling, and that this in fact underlies the effects of R loops on genomic stability. Strikingly, we found that attenuation of replication strongly suppresses R-loop-mediated DNA rearrangements in both E. coli and HeLa cells. Our findings thus provide a direct demonstration that R-loop formation impairs DNA replication and that this is responsible for the deleterious effects of R loops on genome stability from bacteria to humans.

  17. Pyrimidine dimers block simian virus 40 replication forks

    SciTech Connect

    Berger, C.A.; Edenberg, H.J.

    1986-10-01

    UV light produces lesions, predominantly pyrimidine dimers, which inhibit DNA replication in mammalian cells. The mechanism of inhibition is controversial: is synthesis of a daughter strand halted at a lesion while the replication fork moves on and reinitiates downstream, or is fork progression itself blocked for some time at the site of a lesion. We directly addressed this question by using electron microscopy to examine the distances of replication forks from the origin in unirradiated and UV-irradiated simian virus 40 chromosomes. If UV lesions block replication fork progression, the forks should be asymmetrically located in a large fraction of the irradiated molecules; if replication forks move rapidly past lesions, the forks should be symmetrically located. A large fraction of the simian virus 40 replication forks in irradiated molecules were asymmetrically located, demonstrating that UV lesions present at the frequency of pyrimidine dimers block replication forks. As a mechanism for this fork blockage, we propose that polymerization of the leading strand makes a significant contribution to the energetics of fork movement, so any lesion in the template for the leading strand which blocks polymerization should also block fork movement.

  18. 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

    2016-02-04

    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.

  19. 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

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

    PubMed Central

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

    2016-01-01

    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

  1. 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

  2. Impediments to replication fork movement: stabilisation, reactivation and genome instability.

    PubMed

    Lambert, Sarah; Carr, Antony M

    2013-03-01

    Maintaining genome stability is essential for the accurate transmission of genetic material. Genetic instability is associated with human genome disorders and is a near-universal hallmark of cancer cells. Genetic variation is also the driving force of evolution, and a genome must therefore display adequate plasticity to evolve while remaining sufficiently stable to prevent mutations and chromosome rearrangements leading to a fitness disadvantage. A primary source of genome instability are errors that occur during chromosome replication. More specifically, obstacles to the movement of replication forks are known to underlie many of the gross chromosomal rearrangements seen both in human cells and in model organisms. Obstacles to replication fork progression destabilize the replisome (replication protein complex) and impact on the integrity of forked DNA structures. Therefore, to ensure the successful progression of a replication fork along with its associated replisome, several distinct strategies have evolved. First, there are well-orchestrated mechanisms that promote continued movement of forks through potential obstacles. Second, dedicated replisome and fork DNA stabilization pathways prevent the dysfunction of the replisome if its progress is halted. Third, should stabilisation fail, there are mechanisms to ensure damaged forks are accurately fused with a converging fork or, when necessary, re-associated with the replication proteins to continue replication. Here, we review what is known about potential barriers to replication fork progression, how these are tolerated and their impact on genome instability.

  3. 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.

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

    PubMed

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

    2013-11-20

    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.

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

    PubMed

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

    2015-08-13

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

  6. Assembly of Slx4 signaling complexes behind DNA replication forks

    PubMed Central

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

    2015-01-01

    Obstructions to replication fork progression, referred to collectively as DNA replication stress, challenge genome stability. In Saccharomyces cerevisiae, cells lacking RTT107 or SLX4 show genome instability and sensitivity to DNA replication stress and are defective in the completion of DNA replication during recovery from replication stress. We demonstrate that Slx4 is recruited to chromatin behind stressed replication forks, in a region that is spatially distinct from that occupied by the replication machinery. Slx4 complex formation is nucleated by Mec1 phosphorylation of histone H2A, which is recognized by the constitutive Slx4 binding partner Rtt107. Slx4 is essential for recruiting the Mec1 activator Dpb11 behind stressed replication forks, and Slx4 complexes are important for full activity of Mec1. We propose that Slx4 complexes promote robust checkpoint signaling by Mec1 by stably recruiting Dpb11 within a discrete domain behind the replication fork, during DNA replication stress. PMID:26113155

  7. Checkpoint regulation of replication forks: global or local?

    PubMed

    Iyer, Divya Ramalingam; Rhind, Nicholas

    2013-12-01

    Cell-cycle checkpoints are generally global in nature: one unattached kinetochore prevents the segregation of all chromosomes; stalled replication forks inhibit late origin firing throughout the genome. A potential exception to this rule is the regulation of replication fork progression by the S-phase DNA damage checkpoint. In this case, it is possible that the checkpoint is global, and it slows all replication forks in the genome. However, it is also possible that the checkpoint acts locally at sites of DNA damage, and only slows those forks that encounter DNA damage. Whether the checkpoint regulates forks globally or locally has important mechanistic implications for how replication forks deal with damaged DNA during S-phase.

  8. Preventing Replication Fork Collapse to Maintain Genome Integrity

    PubMed Central

    Cortez, David

    2015-01-01

    Billions of base pairs of DNA must be replicated trillions of times in a human lifetime. Complete and accurate replication once and only once per cell division cycle is essential to maintain genome integrity and prevent disease. Impediments to replication fork progression including difficult to replicate DNA sequences, conflicts with transcription, and DNA damage further add to the genome maintenance challenge. These obstacles frequently cause fork stalling, but only rarely cause a failure to complete replication. Robust mechanisms ensure that stalled forks remain stable and capable of either resuming DNA synthesis or being rescued by converging forks. However, when failures do happen the fork collapses leading to genome rearrangements, cell death and disease. Despite intense interest, the mechanisms to repair damaged replication forks, stabilize them, and ensure successful replication remain only partly understood. Different models of fork collapse have been proposed with varying descriptions of what happens to the DNA and replisome. Here, I will define fork collapse and describe what is known about how the replication checkpoint prevents it to maintain genome stability. PMID:25957489

  9. Arrest of replication fork progression at sites of topoisomerase II-mediated DNA cleavage in human leukemia CEM cells incubated with VM-26.

    PubMed

    Catapano, C V; Carbone, G M; Pisani, F; Qiu, J; Fernandes, D J

    1997-05-13

    Recent studies have shown that the anticancer drugs VM-26 and mitoxantrone stabilize preferentially the binding of topoisomerase IIalpha to replicating compared to nonreplicating DNA. To further understand the mechanisms by which cleavable complex-forming topoisomerase II inhibitors interfere with DNA replication, we examined the effects of VM-26 on this process in human leukemia CEM cells. Both the inhibition of DNA synthesis and cell survival were directly related to the total amount of drug-stabilized cleavable complexes formed in VM-26-treated cells. DNA chain elongation was also inhibited in a concentration-dependent fashion in these cells, which suggested that VM-26-stabilized cleavable complexes interfered with the movement of DNA replication forks. To test this hypothesis directly, we monitored replication fork progression at a specific site of VM-26-induced DNA cleavage. A topoisomerase II-mediated cleavage site was detected in the first exon of the c-myc gene in VM-26-treated cells. This cleavage site was downstream of a putative replication origin located in the 5' flanking region of the gene. Replication forks, which moved through this region of the c-myc gene in the 5' to 3' direction, were specifically arrested at this site in VM-26-treated cells, but not in untreated or aphidicolin-treated cells. These studies provide the first direct evidence that a VM-26-stabilized topoisomerase II-DNA cleavable complex acts as a replication fork barrier at a specific genomic site in mammalian cells. Furthermore, the data support the hypothesis that the replication fork arrest induced by cleavable complex-forming topoisomerase II inhibitors leads to the generation of irreversible DNA damage and cytotoxicity in proliferating cells.

  10. Topological locking restrains replication fork reversal

    PubMed Central

    Fierro-Fernández, Marta; Hernández, Pablo; Krimer, Dora B.; Stasiak, Andrzej; Schvartzman, Jorge B.

    2007-01-01

    Two-dimensional agarose gel electrophoresis, psoralen cross-linking, and electron microscopy were used to study the effects of positive supercoiling on fork reversal in isolated replication intermediates of bacterial DNA plasmids. The results obtained demonstrate that the formation of Holliday-like junctions at both forks of a replication bubble creates a topological constraint that prevents further regression of the forks. We propose that this topological locking of replication intermediates provides a biological safety mechanism that protects DNA molecules against extensive fork reversals. PMID:17242356

  11. Claspin promotes normal replication fork rates in human cells.

    PubMed

    Petermann, Eva; Helleday, Thomas; Caldecott, Keith W

    2008-06-01

    The S phase-specific adaptor protein Claspin mediates the checkpoint response to replication stress by facilitating phosphorylation of Chk1 by ataxia-telangiectasia and Rad3-related (ATR). Evidence suggests that these components of the ATR pathway also play a critical role during physiological S phase. Chk1 is required for high rates of global replication fork progression, and Claspin interacts with the replication machinery and might therefore monitor normal DNA replication. Here, we have used DNA fiber labeling to investigate, for the first time, whether human Claspin is required for high rates of replication fork progression during normal S phase. We report that Claspin-depleted HeLa and HCT116 cells display levels of replication fork slowing similar to those observed in Chk1-depleted cells. This was also true in primary human 1BR3 fibroblasts, albeit to a lesser extent, suggesting that Claspin is a universal requirement for high replication fork rates in human cells. Interestingly, Claspin-depleted cells retained significant levels of Chk1 phosphorylation at both Ser317 and Ser345, raising the possibility that Claspin function during normal fork progression may extend beyond facilitating phosphorylation of either individual residue. Consistent with this possibility, depletion of Chk1 and Claspin together doubled the percentage of very slow forks, compared with depletion of either protein alone.

  12. Replication forks reverse at high frequency upon replication stress in Physarum polycephalum.

    PubMed

    Maric, Chrystelle; Bénard, Marianne

    2014-12-01

    The addition of hydroxyurea after the onset of S phase allows replication to start and permits the successive detecting of replication-dependent joint DNA molecules and chicken foot structures in the synchronous nuclei of Physarum polycephalum. We find evidence for a very high frequency of reversed replication forks upon replication stress. The formation of these reversed forks is dependent on the presence of joint DNA molecules, the impediment of the replication fork progression by hydroxyurea, and likely on the propensity of some replication origins to reinitiate replication to counteract the action of this compound. As hydroxyurea treatment enables us to successively detect the appearance of joint DNA molecules and then of reversed replication forks, we propose that chicken foot structures are formed both from the regression of hydroxyurea-frozen joint DNA molecules and from hydroxyurea-stalled replication forks. These experiments underscore the transient nature of replication fork regression, which becomes detectable due to the hydroxyurea-induced slowing down of replication fork progression.

  13. 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.

  14. 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.

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

    PubMed Central

    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

  16. 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.

  17. Replication Fork Velocities at Adjacent Replication Origins Are Coordinately Modified during DNA Replication in Human Cells

    PubMed Central

    Conti, Chiara; Saccà, Barbara; Herrick, John; Lalou, Claude; Pommier, Yves

    2007-01-01

    The spatial organization of replicons into clusters is believed to be of critical importance for genome duplication in higher eukaryotes, but its functional organization still remains to be fully clarified. The coordinated activation of origins is insufficient on its own to account for a timely completion of genome duplication when interorigin distances vary significantly and fork velocities are constant. Mechanisms coordinating origin distribution with fork progression are still poorly elucidated, because of technical difficulties of visualizing the process. Taking advantage of a single molecule approach, we delineated and compared the DNA replication kinetics at the genome level in human normal primary and malignant cells. Our results show that replication forks moving from one origin, as well as from neighboring origins, tend to exhibit the same velocity, although the plasticity of the replication program allows for their adaptation to variable interorigin distances. We also found that forks that emanated from closely spaced origins tended to move slower than those associated with long replicons. Taken together, our results indicate a functional role for origin clustering in the dynamic regulation of genome duplication. PMID:17522385

  18. Supercoiling, knotting and replication fork reversal in partially replicated plasmids

    PubMed Central

    Olavarrieta, L.; Martínez-Robles, M. L.; Sogo, J. M.; Stasiak, A.; Hernández, P.; Krimer, D. B.; Schvartzman, J. B.

    2002-01-01

    To study the structure of partially replicated plasmids, we cloned the Escherichia coli polar replication terminator TerE in its active orientation at different locations in the ColE1 vector pBR18. The resulting plasmids, pBR18-TerE@StyI and pBR18-TerE@EcoRI, were analyzed by neutral/neutral two-dimensional agarose gel electrophoresis and electron microscopy. Replication forks stop at the Ter–TUS complex, leading to the accumulation of specific replication intermediates with a mass 1.26 times the mass of non-replicating plasmids for pBR18-TerE@StyI and 1.57 times for pBR18-TerE@EcoRI. The number of knotted bubbles detected after digestion with ScaI and the number and electrophoretic mobility of undigested partially replicated topoisomers reflect the changes in plasmid topology that occur in DNA molecules replicated to different extents. Exposure to increasing concentrations of chloroquine or ethidium bromide revealed that partially replicated topoisomers (CCCRIs) do not sustain positive supercoiling as efficiently as their non-replicating counterparts. It was suggested that this occurs because in partially replicated plasmids a positive ΔLk is absorbed by regression of the replication fork. Indeed, we showed by electron microscopy that, at least in the presence of chloroquine, some of the CCCRIs of pBR18-Ter@StyI formed Holliday-like junction structures characteristic of reversed forks. However, not all the positive supercoiling was absorbed by fork reversal in the presence of high concentrations of ethidium bromide. PMID:11809877

  19. Gaps and forks in DNA replication: Rediscovering old models.

    PubMed

    Lehmann, Alan R; Fuchs, Robert P

    2006-12-01

    Most current models for replication past damaged lesions envisage that translesion synthesis occurs at the replication fork. However older models suggested that gaps were left opposite lesions to allow the replication fork to proceed, and these gaps were subsequently sealed behind the replication fork. Two recent articles lend support to the idea that bypass of the damage occurs behind the fork. In the first paper, electron micrographs of DNA replicated in UV-irradiated yeast cells show regions of single-stranded DNA both at the replication forks and behind the fork, the latter being consistent with the presence of gaps in the daughter-strands opposite lesions. The second paper describes an in vitro DNA replication system reconstituted from purified bacterial proteins. Repriming of synthesis downstream from a blocked fork occurred not only on the lagging strand as expected, but also on the leading strand, demonstrating that contrary to widely accepted beliefs, leading strand synthesis does not need to be continuous.

  20. New histone supply regulates replication fork speed and PCNA unloading

    PubMed Central

    Mejlvang, Jakob; Feng, Yunpeng; Alabert, Constance; Neelsen, Kai J.; Jasencakova, Zuzana; Zhao, Xiaobei; Lees, Michael; Sandelin, Albin; Pasero, Philippe; Lopes, Massimo

    2014-01-01

    Correct duplication of DNA sequence and its organization into chromatin is central to genome function and stability. However, it remains unclear how cells coordinate DNA synthesis with provision of new histones for chromatin assembly to ensure chromosomal stability. In this paper, we show that replication fork speed is dependent on new histone supply and efficient nucleosome assembly. Inhibition of canonical histone biosynthesis impaired replication fork progression and reduced nucleosome occupancy on newly synthesized DNA. Replication forks initially remained stable without activation of conventional checkpoints, although prolonged histone deficiency generated DNA damage. PCNA accumulated on newly synthesized DNA in cells lacking new histones, possibly to maintain opportunity for CAF-1 recruitment and nucleosome assembly. Consistent with this, in vitro and in vivo analysis showed that PCNA unloading is delayed in the absence of nucleosome assembly. We propose that coupling of fork speed and PCNA unloading to nucleosome assembly provides a simple mechanism to adjust DNA replication and maintain chromatin integrity during transient histone shortage. PMID:24379417

  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. Developmental regulation of DNA replication: replication fork barriers and programmed gene amplification in Tetrahymena thermophila.

    PubMed

    Zhang, Z; Macalpine, D M; Kapler, G M

    1997-10-01

    The palindromic Tetrahymena ribosomal DNA (rDNA) minichromosome is amplified 10,000-fold during development. Subsequent vegetative replication is cell cycle regulated. rDNA replication differs fundamentally in cycling vegetative and nondividing amplifying cells. Using two-dimensional gel electrophoresis, we show for the first time that replication origins that direct gene amplification also function in normal dividing cells. Two classes of amplification intermediates were identified. The first class is indistinguishable from vegetative rDNA, initiating in just one of the two 5' nontranscribed spacer (NTS) copies in the rDNA palindrome at either of two closely spaced origins. Thus, these origins are active throughout the life cycle and their regulation changes at different developmental stages. The second, novel class of amplification intermediates is generated by multiple initiation events. Intermediates with mass greater than fully replicated DNA were observed, suggesting that onionskin replication occurs at this stage. Unlike amplified rDNA in Xenopus laevis, the novel Tetrahymena species are not produced by random initiation; replication also initiates in the 5' NTS. Surprisingly, a replication fork barrier which is activated only in these amplifying molecules blocks the progression of forks near the center of the palindrome. Whereas barriers have been previously described, this is the first instance in which programmed regulation of replication fork progression has been demonstrated in a eukaryote.

  3. 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.

  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. RECQ1 is required for cellular resistance to replication stress and catalyzes strand exchange on stalled replication fork structures.

    PubMed

    Popuri, Venkateswarlu; Croteau, Deborah L; Brosh, Robert M; Bohr, Vilhelm A

    2012-11-15

    RECQ1 is the most abundant of the five human RecQ helicases, but little is known about its biological significance. Recent studies indicate that RECQ1 is associated with origins of replication, suggesting a possible role in DNA replication. However, the functional role of RECQ1 at damaged or stalled replication forks is still unknown. Here, for the first time, we show that RECQ1 promotes strand exchange on synthetic stalled replication fork-mimicking structures and comparatively analyze RECQ1 with the other human RecQ helicases. RECQ1 actively unwinds the leading strand of the fork, similar to WRN, while RECQ4 and RECQ5β can only unwind the lagging strand of the replication fork. Human replication protein A modulates the strand exchange activity of RECQ1 and shifts the equilibrium more to the unwinding mode, an effect also observed for WRN. Stable depletion of RECQ1 affects cell proliferation and renders human cells sensitive to various DNA damaging agents that directly or indirectly block DNA replication fork progression. Consequently, loss of RECQ1 activates DNA damage response signaling, leads to hyper-phosphorylation of RPA32 and activation of CHK1, indicating replication stress. Furthermore, depletion of RECQ1 leads to chromosomal condensation defects and accumulation of under-condensed chromosomes. Collectively, our observations provide a new insight into the role of RECQ1 in replication fork stabilization and its role in the DNA damage response to maintain genomic stability.

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

    PubMed

    Waldminghaus, Torsten; Weigel, Christoph; Skarstad, Kirsten

    2012-07-01

    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.

  7. The MCM helicase: linking checkpoints to the replication fork.

    PubMed

    Forsburg, Susan L

    2008-02-01

    The MCM (minichromosome maintenance) complex is a helicase which is essential for DNA replication. Recent results suggest that the MCM helicase is important for replication fork integrity, and may function as a target of the replication checkpoint. Interactions between MCM proteins, checkpoint kinases, and repair and recovery proteins suggest that MCMs are proximal effectors of replication fork stability in the cell and are likely to play an important role in maintaining genome integrity. PMID:18208397

  8. PTEN regulates RPA1 and protects DNA replication forks.

    PubMed

    Wang, Guangxi; Li, Yang; Wang, Pan; Liang, Hui; Cui, Ming; Zhu, Minglu; Guo, Limei; Su, Qian; Sun, Yujie; McNutt, Michael A; Yin, Yuxin

    2015-11-01

    Tumor suppressor PTEN regulates cellular activities and controls genome stability through multiple mechanisms. In this study, we report that PTEN is necessary for the protection of DNA replication forks against replication stress. We show that deletion of PTEN leads to replication fork collapse and chromosomal instability upon fork stalling following nucleotide depletion induced by hydroxyurea. PTEN is physically associated with replication protein A 1 (RPA1) via the RPA1 C-terminal domain. STORM and iPOND reveal that PTEN is localized at replication sites and promotes RPA1 accumulation on replication forks. PTEN recruits the deubiquitinase OTUB1 to mediate RPA1 deubiquitination. RPA1 deletion confers a phenotype like that observed in PTEN knockout cells with stalling of replication forks. Expression of PTEN and RPA1 shows strong correlation in colorectal cancer. Heterozygous disruption of RPA1 promotes tumorigenesis in mice. These results demonstrate that PTEN is essential for DNA replication fork protection. We propose that RPA1 is a target of PTEN function in fork protection and that PTEN maintains genome stability through regulation of DNA replication.

  9. 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.

  10. 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

  11. Nascent DNA Proteomics Reveals a Chromatin Remodeler Required for Topoisomerase I Loading at Replication Forks.

    PubMed

    Ribeyre, Cyril; Zellweger, Ralph; Chauvin, Maeva; Bec, Nicole; Larroque, Christian; Lopes, Massimo; Constantinou, Angelos

    2016-04-12

    During transcription and DNA replication, the DNA template is overwound ahead of RNA and DNA polymerases and relaxed by DNA topoisomerases. Inhibitors of topoisomerases are potent anti-cancer agents. Camptothecin traps topoisomerase I on DNA and exerts preferential cytotoxicity toward cancer cells by way of its interference with the progression of replication forks. Starting with an unbiased proteomic analysis, we find that the chromatin remodeling complex BAZ1B-SMARCA5 accumulates near replication forks in camptothecin-exposed cells. We report that BAZ1B associates with topoisomerase I and facilitates its access to replication forks. Single-molecule analyses of replication structures show that BAZ1B contributes to replication interference by camptothecin. A lack of BAZ1B confers increased cellular tolerance of camptothecin. These findings reveal BAZ1B as a key facilitator of topoisomerase I function during DNA replication that affects the response of cancer cells to topoisomerase I inhibitors.

  12. Metnase promotes restart and repair of stalled and collapsed replication forks.

    PubMed

    De Haro, Leyma P; Wray, Justin; Williamson, Elizabeth A; Durant, Stephen T; Corwin, Lori; Gentry, Amanda C; Osheroff, Neil; Lee, Suk-Hee; Hromas, Robert; Nickoloff, Jac A

    2010-09-01

    Metnase is a human protein with methylase (SET) and nuclease domains that is widely expressed, especially in proliferating tissues. Metnase promotes non-homologous end-joining (NHEJ), and knockdown causes mild hypersensitivity to ionizing radiation. Metnase also promotes plasmid and viral DNA integration, and topoisomerase IIα (TopoIIα)-dependent chromosome decatenation. NHEJ factors have been implicated in the replication stress response, and TopoIIα has been proposed to relax positive supercoils in front of replication forks. Here we show that Metnase promotes cell proliferation, but it does not alter cell cycle distributions, or replication fork progression. However, Metnase knockdown sensitizes cells to replication stress and confers a marked defect in restart of stalled replication forks. Metnase promotes resolution of phosphorylated histone H2AX, a marker of DNA double-strand breaks at collapsed forks, and it co-immunoprecipitates with PCNA and RAD9, a member of the PCNA-like RAD9-HUS1-RAD1 intra-S checkpoint complex. Metnase also promotes TopoIIα-mediated relaxation of positively supercoiled DNA. Metnase is not required for RAD51 focus formation after replication stress, but Metnase knockdown cells show increased RAD51 foci in the presence or absence of replication stress. These results establish Metnase as a key factor that promotes restart of stalled replication forks, and implicate Metnase in the repair of collapsed forks.

  13. FEN1 ensures telomere stability by facilitating replication fork re-initiation.

    PubMed

    Saharia, Abhishek; Teasley, Daniel C; Duxin, Julien P; Dao, Benjamin; Chiappinelli, Katherine B; Stewart, Sheila A

    2010-08-27

    Telomeres are terminal repetitive DNA sequences whose stability requires the coordinated actions of telomere-binding proteins and the DNA replication and repair machinery. Recently, we demonstrated that the DNA replication and repair protein Flap endonuclease 1 (FEN1) is required for replication of lagging strand telomeres. Here, we demonstrate for the first time that FEN1 is required for efficient re-initiation of stalled replication forks. At the telomere, we find that FEN1 depletion results in replicative stress as evidenced by fragile telomere expression and sister telomere loss. We show that FEN1 participation in Okazaki fragment processing is not required for efficient telomere replication. Instead we find that FEN1 gap endonuclease activity, which processes DNA structures resembling stalled replication forks, and the FEN1 interaction with the RecQ helicases are vital for telomere stability. Finally, we find that FEN1 depletion neither impacts cell cycle progression nor in vitro DNA replication through non-telomeric sequences. Our finding that FEN1 is required for efficient replication fork re-initiation strongly suggests that the fragile telomere expression and sister telomere losses observed upon FEN1 depletion are the direct result of replication fork collapse. Together, these findings suggest that other nucleases compensate for FEN1 loss throughout the genome during DNA replication but fail to do so at the telomere. We propose that FEN1 maintains stable telomeres by facilitating replication through the G-rich lagging strand telomere, thereby ensuring high fidelity telomere replication.

  14. Anticipating chromosomal replication fork arrest: SSB targets repair DNA helicases to active forks.

    PubMed

    Lecointe, François; Sérèna, Céline; Velten, Marion; Costes, Audrey; McGovern, Stephen; Meile, Jean-Christophe; Errington, Jeffrey; Ehrlich, S Dusko; Noirot, Philippe; Polard, Patrice

    2007-10-01

    In bacteria, several salvage responses to DNA replication arrest culminate in reassembly of the replisome on inactivated forks to resume replication. The PriA DNA helicase is a prominent trigger of this replication restart process, preceded in many cases by a repair and/or remodeling of the arrested fork, which can be performed by many specific proteins. The mechanisms that target these rescue effectors to damaged forks in the cell are unknown. We report that the single-stranded DNA binding (SSB) protein is the key factor that links PriA to active chromosomal replication forks in vivo. This targeting mechanism determines the efficiency by which PriA reaches its specific DNA-binding site in vitro and directs replication restart in vivo. The RecG and RecQ DNA helicases, which are involved in intricate replication reactivation pathways, also associate with the chromosomal replication forks by similarly interacting with SSB. These results identify SSB as a platform for linking a 'repair toolbox' with active replication forks, providing a first line of rescue responses to accidental arrest.

  15. Directionality of replication fork movement determined by two-dimensional native-native DNA agarose gel electrophoresis.

    PubMed

    Ivessa, Andreas S

    2013-01-01

    The analysis of replication intermediates by the neutral-neutral two-dimensional agarose gel technique allows determining the chromosomal positions where DNA replication initiates, whether replication forks pause or stall at specific sites, or whether two DNA molecules undergo DNA recombination events. This technique does not, however, immediately tell in which direction replication forks migrate through the DNA region under investigation. Here, we describe the procedure to determine the direction of replication fork progression by carrying out a restriction enzyme digest of DNA imbedded in agarose after the completion of the first dimension of a 2D gel.

  16. Maintaining replication fork integrity in UV-irradiated Escherichia coli cells.

    PubMed

    Rudolph, Christian J; Upton, Amy L; Lloyd, Robert G

    2008-09-01

    In dividing cells, the stalling of replication fork complexes by impediments to DNA unwinding or by template imperfections that block synthesis by the polymerase subunits is a serious threat to genomic integrity and cell viability. What happens to stalled forks depends on the nature of the offending obstacle. In UV-irradiated Escherichia coli cells DNA synthesis is delayed for a considerable period, during which forks undergo extensive processing before replication can resume. Thus, restart depends on factors needed to load the replicative helicase, indicating that the replisome may have dissociated. It also requires the RecFOR proteins, which are known to load RecA recombinase on single-stranded DNA, implying that template strands are exposed. To gain a further understanding of how UV irradiation affects replication and how replication resumes after a block, we used fluorescence microscopy and BrdU or radioisotope labelling to examine chromosome replication and cell cycle progression. Our studies confirm that RecFOR promote efficient reactivation of stalled forks and demonstrate that they are also needed for productive replication initiated at the origin, or triggered elsewhere by damage to the DNA. Although delayed, all modes of replication do recover in the absence of these proteins, but nascent DNA strands are degraded more extensively by RecJ exonuclease. However, these strands are also degraded in the presence of RecFOR when restart is blocked by other means, indicating that RecA loading is not sufficient to stabilise and protect the fork. This is consistent with the idea that RecA actively promotes restart. Thus, in contrast to eukaryotic cells, there may be no factor in bacterial cells acting specifically to stabilise stalled forks. Instead, nascent strands may be protected by the simple expedient of promoting restart. We also report that the efficiency of fork reactivation is not affected in polB mutants. PMID:18644471

  17. Molecular anatomy and regulation of a stable replisome at a paused eukaryotic DNA replication fork.

    PubMed

    Calzada, Arturo; Hodgson, Ben; Kanemaki, Masato; Bueno, Avelino; Labib, Karim

    2005-08-15

    Eukaryotic cells regulate the progression and integrity of DNA replication forks to maintain genomic stability and couple DNA synthesis to other processes. The budding yeast proteins Mrc1 and Tof1 associate with the putative MCM-Cdc45 helicase and limit progression of the replisome when nucleotides are depleted, and the checkpoint kinases Mec1 and Rad53 stabilize such stalled forks and prevent disassembly of the replisome. Forks also pause transiently during unperturbed chromosome replication, at sites where nonnucleosomal proteins bind DNA tightly. We describe a method for inducing prolonged pausing of forks at protein barriers assembled at unique sites on a yeast chromosome, allowing us to examine for the first time the effects of pausing upon replisome integrity. We show that paused forks maintain an intact replisome that contains Mrc1, Tof1, MCM-Cdc45, GINS, and DNA polymerases alpha and epsilon and that recruits the Rrm3 helicase. Surprisingly, pausing does not require Mrc1, although Tof1 and Csm3 are both important. In addition, the integrity of the paused forks does not require Mec1, Rad53, or recombination. We also show that paused forks at analogous barriers in the rDNA are regulated similarly. These data indicate that paused and stalled eukaryotic replisomes resemble each other but are regulated differently.

  18. Triplex structures induce DNA double strand breaks via replication fork collapse in NER deficient cells

    PubMed Central

    Kaushik Tiwari, Meetu; Adaku, Nneoma; Peart, Natoya; Rogers, Faye A.

    2016-01-01

    Structural alterations in DNA can serve as natural impediments to replication fork stability and progression, resulting in DNA damage and genomic instability. Naturally occurring polypurine mirror repeat sequences in the human genome can create endogenous triplex structures evoking a robust DNA damage response. Failures to recognize or adequately process these genomic lesions can result in loss of genomic integrity. Nucleotide excision repair (NER) proteins have been found to play a prominent role in the recognition and repair of triplex structures. We demonstrate using triplex-forming oligonucleotides that chromosomal triplexes perturb DNA replication fork progression, eventually resulting in fork collapse and the induction of double strand breaks (DSBs). We find that cells deficient in the NER damage recognition proteins, XPA and XPC, accumulate more DSBs in response to chromosomal triplex formation than NER-proficient cells. Furthermore, we demonstrate that XPC-deficient cells are particularly prone to replication-associated DSBs in the presence of triplexes. In the absence of XPA or XPC, deleterious consequences of triplex-induced genomic instability may be averted by activating apoptosis via dual phosphorylation of the H2AX protein. Our results reveal that damage recognition by XPC and XPA is critical to maintaining replication fork integrity and preventing replication fork collapse in the presence of triplex structures. PMID:27298253

  19. Single strand transposition at the host replication fork

    PubMed Central

    Lavatine, Laure; He, Susu; Caumont-Sarcos, Anne; Guynet, Catherine; Marty, Brigitte; Chandler, Mick; Ton-Hoang, Bao

    2016-01-01

    Members of the IS200/IS605 insertion sequence family differ fundamentally from classical IS essentially by their specific single-strand (ss) transposition mechanism, orchestrated by the Y1 transposase, TnpA, a small HuH enzyme which recognizes and processes ss DNA substrates. Transposition occurs by the ‘peel and paste’ pathway composed of two steps: precise excision of the top strand as a circular ss DNA intermediate; and subsequent integration into a specific ssDNA target. Transposition of family members was experimentally shown or suggested by in silico high-throughput analysis to be intimately coupled to the lagging strand template of the replication fork. In this study, we investigated factors involved in replication fork targeting and analysed DNA-binding properties of the transposase which can assist localization of ss DNA substrates on the replication fork. We showed that TnpA interacts with the β sliding clamp, DnaN and recognizes DNA which mimics replication fork structures. We also showed that dsDNA can facilitate TnpA targeting ssDNA substrates. We analysed the effect of Ssb and RecA proteins on TnpA activity in vitro and showed that while RecA does not show a notable effect, Ssb inhibits integration. Finally we discuss the way(s) in which integration may be directed into ssDNA at the replication fork. PMID:27466393

  20. Stabilization and targeting of INO80 to replication forks by BAP1 during normal DNA synthesis.

    PubMed

    Lee, Han-Sae; Lee, Shin-Ai; Hur, Shin-Kyoung; Seo, Jeong-Wook; Kwon, Jongbum

    2014-10-06

    The INO80 chromatin-remodelling complex has been implicated in DNA replication during stress in yeast. However, its role in normal DNA replication and its underlying mechanisms remain unclear. Here, we show that INO80 binds to replication forks and promotes fork progression in human cells under unperturbed, normal conditions. We find that Ino80, which encodes the catalytic ATPase of INO80, is essential for mouse embryonic DNA replication and development. Ino80 is recruited to replication forks through interaction with ubiquitinated H2A--aided by BRCA1-associated protein-1 (BAP1), a tumour suppressor and nuclear de-ubiquitinating enzyme that also functions to stabilize Ino80. Importantly, Ino80 is downregulated in BAP1-defective cancer cells due to the lack of an Ino80 stabilization mechanism via BAP1. Our results establish a role for INO80 in normal DNA replication and uncover a mechanism by which this remodeler is targeted to replication forks, suggesting a molecular basis for the tumour-suppressing function of BAP1.

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

    PubMed

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

    2016-07-01

    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

  2. Termination of DNA replication forks: "Breaking up is hard to do".

    PubMed

    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.

  3. 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

  4. 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.

  5. 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

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

    PubMed

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

    2016-07-25

    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.

  7. 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

  8. Replication fork arrest and termination of chromosome replication in Bacillus subtilis.

    PubMed

    Wake, R G

    1997-08-15

    Sporulation in Bacillus subtilis provided the first evidence for the presence of sequence-specific replication fork arrest (Ter) sites in the terminus region of the bacterial chromosome. These sites, when complexed with the replication terminator protein (RTP), block movement of a replication fork in a polar manner. The Ter sites are organized into two opposed groups which force the approaching forks to meet and fuse within a restricted terminus region. While the precise advantage provided to the cell through the presence of the so-called replication fork trap is not patently obvious, the same situation appears to have evolved independently in Escherichia coli. The molecular mechanism by which the RTP-Ter complex of B. subtilis (or the analogous but apparently unrelated complex in E. coli) functions is currently unresolved and subject to intense investigation. Replication fork arrest in B. subtilis, requiring RTP, also occurs under conditions of the stringent response at so-called STer sites that lie close to and on both sides of oriC. These sites are yet to be identified and characterized. How they are induced to function under stringent conditions is of considerable interest, and could provide vital clues about the mechanism of fork arrest by RTP-terminator complexes in general.

  9. 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.

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

    PubMed

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

    2016-03-27

    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 associated

  11. 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

  12. Mechanism of chromosomal DNA replication initiation and replication fork stabilization in eukaryotes.

    PubMed

    Wu, LiHong; Liu, Yang; Kong, DaoChun

    2014-05-01

    Chromosomal DNA replication is one of the central biological events occurring inside cells. Due to its large size, the replication of genomic DNA in eukaryotes initiates at hundreds to tens of thousands of sites called DNA origins so that the replication could be completed in a limited time. Further, eukaryotic DNA replication is sophisticatedly regulated, and this regulation guarantees that each origin fires once per S phase and each segment of DNA gets duplication also once per cell cycle. The first step of replication initiation is the assembly of pre-replication complex (pre-RC). Since 1973, four proteins, Cdc6/Cdc18, MCM, ORC and Cdt1, have been extensively studied and proved to be pre-RC components. Recently, a novel pre-RC component called Sap1/Girdin was identified. Sap1/Girdin is required for loading Cdc18/Cdc6 to origins for pre-RC assembly in the fission yeast and human cells, respectively. At the transition of G1 to S phase, pre-RC is activated by the two kinases, cyclindependent kinase (CDK) and Dbf4-dependent kinase (DDK), and subsequently, RPA, primase-polα, PCNA, topoisomerase, Cdc45, polδ, and polɛ are recruited to DNA origins for creating two bi-directional replication forks and initiating DNA replication. As replication forks move along chromatin DNA, they frequently stall due to the presence of a great number of replication barriers on chromatin DNA, such as secondary DNA structures, protein/DNA complexes, DNA lesions, gene transcription. Stalled forks must require checkpoint regulation for their stabilization. Otherwise, stalled forks will collapse, which results in incomplete DNA replication and genomic instability. This short review gives a concise introduction regarding the current understanding of replication initiation and replication fork stabilization.

  13. 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

  14. 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

    2016-05-12

    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

  15. 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

  16. Formation of a DNA loop at the replication fork generated by bacteriophage T7 replication proteins.

    PubMed

    Park, K; Debyser, Z; Tabor, S; Richardson, C C; Griffith, J D

    1998-02-27

    Intermediates in the replication of circular and linear M13 double-stranded DNA by bacteriophage T7 proteins have been examined by electron microscopy. Synthesis generated double-stranded DNA molecules containing a single replication fork with a linear duplex tail. A complex presumably consisting of T7 DNA polymerase and gene 4 helicase/primase molecules was present at the fork together with a variable amount of single-stranded DNA sequestered by gene 2.5 single-stranded DNA binding protein. Analysis of the length distribution of Okazaki fragments formed at different helicase/primase concentrations was consistent with coupling of leading and lagging strand replication. Fifteen to forty percent of the templates engaged in replication have a DNA loop at the replication fork. The loops are fully double-stranded with an average length of approximately 1 kilobase. Labeling with biotinylated dCTP showed that the loops consist of newly synthesized DNA, and synchronization experiments using a linear template with a G-less cassette demonstrated that the loops are formed by active displacement of the lagging strand. A long standing feature of models for coupled leading/lagging strand replication has been the presence of a DNA loop at the replication fork. This study provides the first direct demonstration of such loops.

  17. Recombination at DNA replication fork barriers is not universal and is differentially regulated by Swi1

    PubMed Central

    Pryce, David W.; Ramayah, Soshila; Jaendling, Alessa; McFarlane, Ramsay J.

    2009-01-01

    DNA replication stress has been implicated in the etiology of genetic diseases, including cancers. It has been proposed that genomic sites that inhibit or slow DNA replication fork progression possess recombination hotspot activity and can form potential fragile sites. Here we used the fission yeast, Schizosaccharomyces pombe, to demonstrate that hotspot activity is not a universal feature of replication fork barriers (RFBs), and we propose that most sites within the genome that form RFBs do not have recombination hotspot activity under nonstressed conditions. We further demonstrate that Swi1, the TIMELESS homologue, differentially controls the recombination potential of RFBs, switching between being a suppressor and an activator of recombination in a site-specific fashion. PMID:19273851

  18. Fork it over: the cohesion establishment factor Ctf7p and DNA replication.

    PubMed

    Skibbens, Robert V; Maradeo, Marie; Eastman, Laura

    2007-08-01

    To produce viable progeny, cells must identify the products of chromosome replication as sister chromatids, pair them together and then maintain this cohesion until chromosome segregation. It is well established that cohesin ring-like structures maintain sister chromatid cohesion, but the molecular mechanism by which only sisters become paired (termed establishment) is highly controversial. One of the first establishment models posited in the literature suggested that cohesin complexes associated with each sister become tethered together through an active process that is intimately coupled to progression of the DNA replication fork. A subsequent model posited that the replication fork simply passes through pre-loaded cohesin rings, entrapping within both sister chromatids. The recent findings that the establishment factor Ctf7p/Eco1p is recruited to DNA and binds both a DNA polymerase processivity factor (PCNA) and the cohesin regulator Pds5p test current models of sister chromatid pairing.

  19. BOD1L Is Required to Suppress Deleterious Resection of Stressed Replication Forks.

    PubMed

    Higgs, Martin R; Reynolds, John J; Winczura, Alicja; Blackford, Andrew N; Borel, Valérie; Miller, Edward S; Zlatanou, Anastasia; Nieminuszczy, Jadwiga; Ryan, Ellis L; Davies, Nicholas J; Stankovic, Tatjana; Boulton, Simon J; Niedzwiedz, Wojciech; Stewart, Grant S

    2015-08-01

    Recognition and repair of damaged replication forks are essential to maintain genome stability and are coordinated by the combined action of the Fanconi anemia and homologous recombination pathways. These pathways are vital to protect stalled replication forks from uncontrolled nucleolytic activity, which otherwise causes irreparable genomic damage. Here, we identify BOD1L as a component of this fork protection pathway, which safeguards genome stability after replication stress. Loss of BOD1L confers exquisite cellular sensitivity to replication stress and uncontrolled resection of damaged replication forks, due to a failure to stabilize RAD51 at these forks. Blocking DNA2-dependent resection, or downregulation of the helicases BLM and FBH1, suppresses both catastrophic fork processing and the accumulation of chromosomal damage in BOD1L-deficient cells. Thus, our work implicates BOD1L as a critical regulator of genome integrity that restrains nucleolytic degradation of damaged replication forks.

  20. The stability of nucleosomes at the replication fork.

    PubMed

    Gasser, R; Koller, T; Sogo, J M

    1996-05-01

    Purified simian virus (SV40) minichromosomes were photoreacted with psoralen under various conditions that moderately destabilize nucleosomes. This assay allows indirect distinction between stable nucleosomes, partially unravelled nucleosomes and nucleosomes containing (or lacking) histone H1. In replicating molecules the passage of the replication machinery destabilizes the nucleosomal organization of the chromatin fiber over a distance of 650 to 1100 bp. In front of the fork, an average of two nucleosomes are destabilized presumably by the dissociation of histone H1 and the advancing replication machinery. On daughter strands, the first nucleosome is detected at a distance of about 260 nucleotides from the elongation point. This nucleosome is interpreted to contain no histone H1, while no stepwise association of (H3-H4)2 tetramers with H2A/H2B dimers on nascent DNA can be detected in vivo. The second nucleosome after the replication fork appears to contain histone H1. The prolonged nuclease sensitivity of newly replicated chromatin described in the literature therefore may not be due to a slow reassociation of histone H1.

  1. Telomere-bound TRF1 and TRF2 stall the replication fork at telomeric repeats

    PubMed Central

    Ohki, Rieko; Ishikawa, Fuyuki

    2004-01-01

    Vertebrate telomeres consist of tandem repeats of T2AG3 and associated proteins including the telomeric DNA-binding proteins, TRF1 and TRF2. It has been proposed that telomeres assume two interswitchable states, the open state that is accessible to various trans-acting factors and the closed state that excludes those factors. TRF1 and TRF2 are believed to promote the formation of the closed state. However, little is known about how those two states influence DNA replication. We analyzed the effects of TRF1 and TRF2 on telomeric replication both in vitro and in vivo. By exploiting the in vitro replication system of linear SV40 DNA, we found that telomeric repeats are a poor replication template. Moreover, the addition of recombinant TRF1 and TRF2 significantly stalled the replication fork progression at telomeric repeats. When TRF1 was overexpressed in HeLa cells, cells with 4N DNA content were accumulated. Furthermore, cytological analyses revealed that the replication focus overlapped with telomere signals at a significantly higher frequency in TRF1-overexpressing cells than in control cells. The results suggest that TRF1 and TRF2 exert inhibitory effects on replication fork progression. PMID:15007108

  2. Coordinated protein and DNA remodeling by human HLTF on stalled replication fork.

    PubMed

    Achar, Yathish Jagadheesh; Balogh, David; Haracska, Lajos

    2011-08-23

    Human helicase-like transcription factor (HLTF) exhibits ubiquitin ligase activity for proliferating cell nuclear antigen (PCNA) polyubiquitylation as well as double-stranded DNA translocase activity for remodeling stalled replication fork by fork reversal, which can support damage bypass by template switching. However, a stalled replication fork is surrounded by various DNA-binding proteins which can inhibit the access of damage bypass players, and it is unknown how these proteins become displaced. Here we reveal that HLTF has an ATP hydrolysis-dependent protein remodeling activity, by which it can remove proteins bound to the replication fork. Moreover, we demonstrate that HLTF can displace a broad spectrum of proteins such as replication protein A (RPA), PCNA, and replication factor C (RFC), thereby providing the first example for a protein clearing activity at the stalled replication fork. Our findings clarify how remodeling of a stalled replication fork can occur if it is engaged in interactions with masses of proteins.

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

    PubMed

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

    2015-10-15

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

  4. Protection or resection: BOD1L as a novel replication fork protection factor.

    PubMed

    Higgs, Martin R; Stewart, Grant S

    2016-01-01

    Replication stress, defined as the slowing or stalling of cellular DNA replication forks, represents a serious threat to genome stability. Numerous cellular pathways protect against replication stress and maintain genomic integrity. Among these, the Fanconi Anemia/homologous recombination pathways are critical for recognizing and repairing stalled replication forks. Members of these pathways play a vital role in protecting damaged forks from uncontrolled attack from cellular nucleases, which would otherwise render these irreparable. Recent studies have begun to shed light on the protective factors necessary to suppress nucleolytic over-processing of nascent DNA, and on the different cellular nucleases involved. Here, we review our recent identification of a novel fork protection factor, BOD1L, and discuss its role in preventing the processing of stalled replication forks within the context of current knowledge of the replication fork 'protectosome'.

  5. Architecture of the replication fork stalled at the 3' end of yeast ribosomal genes.

    PubMed

    Gruber, M; Wellinger, R E; Sogo, J M

    2000-08-01

    Every unit of the rRNA gene cluster of Saccharomyces cerevisiae contains a unique site, termed the replication fork barrier (RFB), where progressing replication forks are stalled in a polar manner. In this work, we determined the positions of the nascent strands at the RFB at nucleotide resolution. Within an HpaI-HindIII fragment essential for the RFB, a major and two closely spaced minor arrest sites were found. In the majority of molecules, the stalled lagging strand was completely processed and the discontinuously synthesized nascent lagging strand was extended three bases farther than the continuously synthesized leading strand. A model explaining these findings is presented. Our analysis included for the first time the use of T4 endonuclease VII, an enzyme recognizing branched DNA molecules. This enzyme cleaved predominantly in the newly synthesized homologous arms, thereby specifically releasing the leading arm.

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

    PubMed

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

    2015-11-16

    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.

  7. Replication forks blocked by protein-DNA complexes have limited stability in vitro.

    PubMed

    McGlynn, Peter; Guy, Colin P

    2008-08-29

    There are many barriers that replication forks must overcome in order to duplicate a genome in vivo. These barriers include damage to the template DNA and proteins bound to this template. If replication is halted by such a block, then the block must be either removed or bypassed for replication to continue. If continuation of replication employs the original fork, avoiding the need to reload the replication apparatus, then the blocked replisome must retain functionality. In vivo studies of Escherichia coli replication forks suggest that replication forks blocked by protein-DNA complexes retain the ability to resume replication upon removal of the block for several hours. Here we tested the functional stability of replication forks reconstituted in vitro and blocked by lac repressor-operator complexes. Once a fork comes to a halt at such a block, it cannot continue subsequently to translocate through the block until addition of IPTG induces repressor dissociation. However, the ability to resume replication is retained only for 4-6 min regardless of the topological state of the template DNA. Comparison of our in vitro data with previous in vivo data suggests that either accessory factors that stabilise blocked forks are present in vivo or the apparent stability of blocked forks in vivo is due to continual reloading of the replication apparatus at the site of the block.

  8. 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

  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. 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; Di Perna, Roberta; 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.

  11. Continued DNA synthesis in replication checkpoint mutants leads to fork collapse.

    PubMed

    Sabatinos, Sarah A; Green, Marc D; Forsburg, Susan L

    2012-12-01

    Hydroxyurea (HU) treatment activates the intra-S phase checkpoint proteins Cds1 and Mrc1 to prevent replication fork collapse. We found that prolonged DNA synthesis occurs in cds1Δ and mrc1Δ checkpoint mutants in the presence of HU and continues after release. This is coincident with increased DNA damage measured by phosphorylated histone H2A in whole cells during release. High-resolution live-cell imaging shows that mutants first accumulate extensive replication protein A (RPA) foci, followed by increased Rad52. Both DNA synthesis and RPA accumulation require the MCM helicase. We propose that a replication fork "collapse point" in HU-treated cells describes the point at which accumulated DNA damage and instability at individual forks prevent further replication. After this point, cds1Δ and mrc1Δ forks cannot complete genome replication. These observations establish replication fork collapse as a dynamic process that continues after release from HU block.

  12. DNA damage-induced replication fork regression and processing in Escherichia coli.

    PubMed

    Courcelle, Justin; Donaldson, Janet R; Chow, Kin-Hoe; Courcelle, Charmain T

    2003-02-14

    DNA lesions that block replication are a primary cause of rearrangements, mutations, and lethality in all cells. After ultraviolet (UV)-induced DNA damage in Escherichia coli, replication recovery requires RecA and several other recF pathway proteins. To characterize the mechanism by which lesion-blocked replication forks recover, we used two-dimensional agarose gel electrophoresis to show that replication-blocking DNA lesions induce a transient reversal of the replication fork in vivo. The reversed replication fork intermediate is stabilized by RecA and RecF and is degraded by the RecQ-RecJ helicase-nuclease when these proteins are absent. We propose that fork regression allows repair enzymes to gain access to the replication-blocking lesion, allowing processive replication to resume once the blocking lesion is removed. PMID:12543983

  13. UV stalled replication forks restart by re-priming in human fibroblasts.

    PubMed

    Elvers, Ingegerd; Johansson, Fredrik; Groth, Petra; Erixon, Klaus; Helleday, Thomas

    2011-09-01

    Restarting stalled replication forks is vital to avoid fatal replication errors. Previously, it was demonstrated that hydroxyurea-stalled replication forks rescue replication either by an active restart mechanism or by new origin firing. To our surprise, using the DNA fibre assay, we only detect a slightly reduced fork speed on a UV-damaged template during the first hour after UV exposure, and no evidence for persistent replication fork arrest. Interestingly, no evidence for persistent UV-induced fork stalling was observed even in translesion synthesis defective, Polη(mut) cells. In contrast, using an assay to measure DNA molecule elongation at the fork, we observe that continuous DNA elongation is severely blocked by UV irradiation, particularly in UV-damaged Polη(mut) cells. In conclusion, our data suggest that UV-blocked replication forks restart effectively through re-priming past the lesion, leaving only a small gap opposite the lesion. This allows continuation of replication on damaged DNA. If left unfilled, the gaps may collapse into DNA double-strand breaks that are repaired by a recombination pathway, similar to the fate of replication forks collapsed after hydroxyurea treatment.

  14. UV stalled replication forks restart by re-priming in human fibroblasts

    PubMed Central

    Elvers, Ingegerd; Johansson, Fredrik; Groth, Petra; Erixon, Klaus; Helleday, Thomas

    2011-01-01

    Restarting stalled replication forks is vital to avoid fatal replication errors. Previously, it was demonstrated that hydroxyurea-stalled replication forks rescue replication either by an active restart mechanism or by new origin firing. To our surprise, using the DNA fibre assay, we only detect a slightly reduced fork speed on a UV-damaged template during the first hour after UV exposure, and no evidence for persistent replication fork arrest. Interestingly, no evidence for persistent UV-induced fork stalling was observed even in translesion synthesis defective, Polηmut cells. In contrast, using an assay to measure DNA molecule elongation at the fork, we observe that continuous DNA elongation is severely blocked by UV irradiation, particularly in UV-damaged Polηmut cells. In conclusion, our data suggest that UV-blocked replication forks restart effectively through re-priming past the lesion, leaving only a small gap opposite the lesion. This allows continuation of replication on damaged DNA. If left unfilled, the gaps may collapse into DNA double-strand breaks that are repaired by a recombination pathway, similar to the fate of replication forks collapsed after hydroxyurea treatment. PMID:21646340

  15. FANCJ couples replication past natural fork barriers with maintenance of chromatin structure

    PubMed Central

    Schwab, Rebekka A.; Nieminuszczy, Jadwiga; Shin-ya, Kazuo

    2013-01-01

    Defective DNA repair causes Fanconi anemia (FA), a rare childhood cancer–predisposing syndrome. At least 15 genes are known to be mutated in FA; however, their role in DNA repair remains unclear. Here, we show that the FANCJ helicase promotes DNA replication in trans by counteracting fork stalling on replication barriers, such as G4 quadruplex structures. Accordingly, stabilization of G4 quadruplexes in ΔFANCJ cells restricts fork movements, uncouples leading- and lagging-strand synthesis and generates small single-stranded DNA gaps behind the fork. Unexpectedly, we also discovered that FANCJ suppresses heterochromatin spreading by coupling fork movement through replication barriers with maintenance of chromatin structure. We propose that FANCJ plays an essential role in counteracting chromatin compaction associated with unscheduled replication fork stalling and restart, and suppresses tumorigenesis, at least partially, in this replication-specific manner. PMID:23530069

  16. 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.

  17. FANCD2 influences replication fork processes and genome stability in response to clustered DSBs

    PubMed Central

    Zhu, Jiayun; Su, Fengtao; Mukherjee, Shibani; Mori, Eiichiro; Hu, Burong; Asaithamby, Aroumougame

    2015-01-01

    Fanconi Anemia (FA) is a cancer predisposition syndrome and the factors defective in FA are involved in DNA replication, DNA damage repair and tumor suppression. Here, we show that FANCD2 is critical for genome stability maintenance in response to high-linear energy transfer (LET) radiation. We found that FANCD2 is monoubiquitinated and recruited to the sites of clustered DNA double-stranded breaks (DSBs) specifically in S/G2 cells after high-LET radiation. Further, FANCD2 facilitated the repair of clustered DSBs in S/G2 cells and proper progression of S-phase. Furthermore, lack of FANCD2 led to a reduced rate of replication fork progression and elevated levels of both replication fork stalling and new origin firing in response to high-LET radiation. Mechanistically, FANCD2 is required for correct recruitment of RPA2 and Rad51 to the sites of clustered DSBs and that is critical for proper processing of clustered DSBs. Significantly, FANCD2-decifient cells exhibited defective chromosome segregation, elevated levels of chromosomal aberrations, and anchorage-independent growth in response to high-LET radiation. These findings establish FANCD2 as a key factor in genome stability maintenance in response to high-LET radiation and as a promising target to improve cancer therapy. PMID:26083937

  18. 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.

  19. CDK1 phosphorylates WRN at collapsed replication forks

    PubMed Central

    Palermo, Valentina; Rinalducci, Sara; Sanchez, Massimo; Grillini, Francesca; Sommers, Joshua A.; Brosh, Robert M.; Zolla, Lello; Franchitto, Annapaola; Pichierri, Pietro

    2016-01-01

    Regulation of end-processing is critical for accurate repair and to switch between homologous recombination (HR) and non-homologous end joining (NHEJ). End resection is a two-stage process but very little is known about regulation of the long-range resection, especially in humans. WRN participates in one of the two alternative long-range resection pathways mediated by DNA2 or EXO1. Here we demonstrate that phosphorylation of WRN by CDK1 is essential to perform DNA2-dependent end resection at replication-related DSBs, promoting HR, replication recovery and chromosome stability. Mechanistically, S1133 phosphorylation of WRN is dispensable for relocalization in foci but is involved in the interaction with the MRE11 complex. Loss of WRN phosphorylation negatively affects MRE11 foci formation and acts in a dominant negative manner to prevent long-range resection altogether, thereby licensing NHEJ at collapsed forks. Collectively, we unveil a CDK1-dependent regulation of the WRN-DNA2-mediated resection and identify an undescribed function of WRN as a DSB repair pathway switch. PMID:27634057

  20. CDK1 phosphorylates WRN at collapsed replication forks.

    PubMed

    Palermo, Valentina; Rinalducci, Sara; Sanchez, Massimo; Grillini, Francesca; Sommers, Joshua A; Brosh, Robert M; Zolla, Lello; Franchitto, Annapaola; Pichierri, Pietro

    2016-01-01

    Regulation of end-processing is critical for accurate repair and to switch between homologous recombination (HR) and non-homologous end joining (NHEJ). End resection is a two-stage process but very little is known about regulation of the long-range resection, especially in humans. WRN participates in one of the two alternative long-range resection pathways mediated by DNA2 or EXO1. Here we demonstrate that phosphorylation of WRN by CDK1 is essential to perform DNA2-dependent end resection at replication-related DSBs, promoting HR, replication recovery and chromosome stability. Mechanistically, S1133 phosphorylation of WRN is dispensable for relocalization in foci but is involved in the interaction with the MRE11 complex. Loss of WRN phosphorylation negatively affects MRE11 foci formation and acts in a dominant negative manner to prevent long-range resection altogether, thereby licensing NHEJ at collapsed forks. Collectively, we unveil a CDK1-dependent regulation of the WRN-DNA2-mediated resection and identify an undescribed function of WRN as a DSB repair pathway switch. PMID:27634057

  1. Complex of Escherichia coli primary replicative helicase DnaB protein with a replication fork: recognition and structure.

    PubMed

    Jezewska, M J; Rajendran, S; Bujalowski, W

    1998-03-01

    Interactions of the Escherichia coli replicative helicase DnaB protein, with DNA replication fork substrates, have been studied using rigorous fluorescence titration, fluorescence energy transfer, and analytical ultracentrifugation methods. DnaB binds the 5' single-arm fork, the 3' single-arm fork, and the two-arm fork with stoichiometries of 1, 1, and 2 DnaB hexamers per fork, independent of the length of the duplex part of the fork. Within the structurally heterogeneous binding site, the helicase accesses most of the 20 nucleotide residues of an arm. The dsDNA of the fork does not contribute to the affinity; however, it affects the positioning of the enzyme on the 5' or 3' arm. Fluorescence energy transfer experiments provide direct evidence that the DnaB helicase binds the 5' arm of the fork in a single orientation, with respect to the duplex part of the fork. The 33-kDa domains of the hexamer face the dsDNA, while the small 12-kDa domains face the 5' end of the arm. In the complex with the 3' arm, the helicase is bound in an opposite orientation when compared to the 5' arm. This is the first determination of the strict, single orientation of a helicase in the complex with a replication fork. The 3' arm accommodates a DnaB hexamer, while another hexamer is associated with the 5' arm. The complex of two DnaB hexamers bound in opposite orientations with each arm of the fork may play an important role during bidirectional replication of the E. coli DNA.

  2. RecA protein promotes the regression of stalled replication forks in vitro.

    PubMed

    Robu, M E; Inman, R B; Cox, M M

    2001-07-17

    Replication forks are halted by many types of DNA damage. At the site of a leading-strand DNA lesion, forks may stall and leave the lesion in a single-strand gap. Fork regression is the first step in several proposed pathways that permit repair without generating a double-strand break. Using model DNA substrates designed to mimic one of the known structures of a fork stalled at a leading-strand lesion, we show here that RecA protein of Escherichia coli will promote a fork regression reaction in vitro. The regression process exhibits an absolute requirement for ATP hydrolysis and is enhanced when dATP replaces ATP. The reaction is not affected by the inclusion of the RecO and R proteins. We present this reaction as one of several potential RecA protein roles in the repair of stalled and/or collapsed replication forks in bacteria. PMID:11459955

  3. The intra-S phase checkpoint targets Dna2 to prevent stalled replication forks from reversing.

    PubMed

    Hu, Jiazhi; Sun, Lei; Shen, Fenfen; Chen, Yufei; Hua, Yu; Liu, Yang; Zhang, Mian; Hu, Yiren; Wang, Qingsong; Xu, Wei; Sun, Fei; Ji, Jianguo; Murray, Johanne M; Carr, Antony M; Kong, Daochun

    2012-06-01

    When replication forks stall at damaged bases or upon nucleotide depletion, the intra-S phase checkpoint ensures they are stabilized and can restart. In intra-S checkpoint-deficient budding yeast, stalling forks collapse, and ∼10% form pathogenic chicken foot structures, contributing to incomplete replication and cell death (Lopes et al., 2001; Sogo et al., 2002; Tercero and Diffley, 2001). Using fission yeast, we report that the Cds1(Chk2) effector kinase targets Dna2 on S220 to regulate, both in vivo and in vitro, Dna2 association with stalled replication forks in chromatin. We demonstrate that Dna2-S220 phosphorylation and the nuclease activity of Dna2 are required to prevent fork reversal. Consistent with this, Dna2 can efficiently cleave obligate precursors of fork regression-regressed leading or lagging strands-on model replication forks. We propose that Dna2 cleavage of regressed nascent strands prevents fork reversal and thus stabilizes stalled forks to maintain genome stability during replication stress.

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

    PubMed Central

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

    2014-01-01

    Replication fork stalling can promote genomic instability, predisposing to cancer and other diseases1–3. Stalled replication forks may be processed by sister chromatid recombination (SCR), generating error-free or error-prone homologous recombination (HR) outcomes4–8. 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 forks9. Although BRCA1 and BRCA2 affect replication fork processing10–12, direct evidence that BRCA genes regulate HR at stalled chromosomal replication forks is lacking due to a dearth of tools for studying this process. We report that the Escherichia coli Tus/Ter complex13–16 can be engineered to induce site-specific replication fork stalling and chromosomal HR/SCR in mammalian cells. Tus/Ter-induced HR entails processing of bidirectionally arrested forks. We find that the BRCA1 C-terminal tandem BRCT repeat and regions of BRCA1 encoded by exon 11—two BRCA1 elements implicated in tumor suppression—control Tus/Ter-induced HR. 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 restriction endonuclease-mediated chromosomal double strand break (DSB). Therefore, HR at stalled forks is regulated differently from HR at DSBs arising independently of a fork. We propose that aberrant long-tract HR at stalled replication forks contributes to genomic instability and breast/ovarian cancer predisposition in BRCA mutant cells. PMID:24776801

  5. Replication initiation and elongation fork rates within a differentially expressed human multicopy locus in early S phase.

    PubMed Central

    Scott, R S; Truong, K Y; Vos, J M

    1997-01-01

    Replication of the 400 copies of the 43 kb human ribosomal RNA (rDNA) locus spans most of the S phase. To examine the basis for the unusual pattern of rDNA replication, a sensitive strategy was developed to map origins of DNA replication and measure apparent rates of fork progression within a chromosomal locus. This technique, termed differential intragenomic replication timing, revealed that initiation within the actively transcribed rDNA occurred in early S within a 10.7 kb region spanning the promoter and 5' external transcribed spacer. Forks emanating from this early bidirectional origin progressed at an apparent slow rate with the sense and anti-sense forks moving at 0.32 and 0.23 kb/min. Using a photochemical-based technique, the chromatin status of the rDNA repeats was assayed throughout the S phase. Approximately 85% of the rDNA repeats were in a transcriptionally active chromatin structure at the start of S phase. A progressive decrease in the transcription state of the rDNA loci was observed, reaching a minimum between 3 and 6 h in mid S phase. Altogether, the data suggest a link between RNA polymerase I mediated transcription and site-specific initiation of DNA replication within the rDNA multicopy locus. PMID:9358159

  6. Topoisomerase I poisoning results in PARP-mediated replication fork reversal.

    PubMed

    Ray Chaudhuri, Arnab; Hashimoto, Yoshitami; Herrador, Raquel; Neelsen, Kai J; Fachinetti, Daniele; Bermejo, Rodrigo; Cocito, Andrea; Costanzo, Vincenzo; Lopes, Massimo

    2012-04-01

    Topoisomerase I (Top1) releases torsional stress during DNA replication and transcription and is inhibited by camptothecin and camptothecin-derived cancer chemotherapeutics. Top1 inhibitor cytotoxicity is frequently linked to double-strand break (DSB) formation as a result of Top1 being trapped on a nicked DNA intermediate in replicating cells. Here we use yeast, mammalian cell lines and Xenopus laevis egg extracts to show that Top1 poisons rapidly induce replication-fork slowing and reversal, which can be uncoupled from DSB formation at sublethal inhibitor doses. Poly(ADP-ribose) polymerase activity, but not single-stranded break repair in general, is required for effective fork reversal and limits DSB formation. These data identify fork reversal as a means to prevent chromosome breakage upon exogenous replication stress and implicate proteins involved in fork reversal or restart as factors modulating the cytotoxicity of replication stress-inducing chemotherapeutics. PMID:22388737

  7. Analysis of protein dynamics at active, stalled, and collapsed replication forks.

    PubMed

    Sirbu, Bianca M; Couch, Frank B; Feigerle, Jordan T; Bhaskara, Srividya; Hiebert, Scott W; Cortez, David

    2011-06-15

    Successful DNA replication and packaging of newly synthesized DNA into chromatin are essential to maintain genome integrity. Defects in the DNA template challenge genetic and epigenetic inheritance. Unfortunately, tracking DNA damage responses (DDRs), histone deposition, and chromatin maturation at replication forks is difficult in mammalian cells. Here we describe a technology called iPOND (isolation of proteins on nascent DNA) to analyze proteins at active and damaged replication forks at high resolution. Using this methodology, we define the timing of histone deposition and chromatin maturation. Class 1 histone deacetylases are enriched at replisomes and remove predeposition marks on histone H4. Chromatin maturation continues even when decoupled from replisome movement. Furthermore, fork stalling causes changes in the recruitment and phosphorylation of proteins at the damaged fork. Checkpoint kinases catalyze H2AX phosphorylation, which spreads from the stalled fork to include a large chromatin domain even prior to fork collapse and double-strand break formation. Finally, we demonstrate a switch in the DDR at persistently stalled forks that includes MRE11-dependent RAD51 assembly. These data reveal a dynamic recruitment of proteins and post-translational modifications at damaged forks and surrounding chromatin. Furthermore, our studies establish iPOND as a useful methodology to study DNA replication and chromatin maturation.

  8. 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

  9. The DNA helicase Pfh1 promotes fork merging at replication termination sites to ensure genome stability.

    PubMed

    Steinacher, Roland; Osman, Fekret; Dalgaard, Jacob Z; Lorenz, Alexander; Whitby, Matthew C

    2012-03-15

    Bidirectionally moving DNA replication forks merge at termination sites composed of accidental or programmed DNA-protein barriers. If merging fails, then regions of unreplicated DNA can result in the breakage of DNA during mitosis, which in turn can give rise to genome instability. Despite its importance, little is known about the mechanisms that promote the final stages of fork merging in eukaryotes. Here we show that the Pif1 family DNA helicase Pfh1 plays a dual role in promoting replication fork termination. First, it facilitates replication past DNA-protein barriers, and second, it promotes the merging of replication forks. A failure of these processes in Pfh1-deficient cells results in aberrant chromosome segregation and heightened genome instability.

  10. Replication fork bypass of a pyrimidine dimer blocking leading strand DNA synthesis.

    PubMed

    Cordeiro-Stone, M; Zaritskaya, L S; Price, L K; Kaufmann, W K

    1997-05-23

    We constructed a double-stranded plasmid containing a single cis, syn-cyclobutane thymine dimer (T[c,s]T) 385 base pairs from the center of the SV40 origin of replication. This circular DNA was replicated in vitro by extracts from several types of human cells. The dimer was placed on the leading strand template of the first replication fork to encounter the lesion. Two-dimensional gel electrophoresis of replication intermediates documented the transient arrest of the replication fork by the dimer. Movement of the replication fork beyond the dimer was recognized by the appearance of a single fork arc in DNA sequences located between the T[c,s]T and the half-way point around the circular template (180 degrees from the origin). Upon completion of plasmid replication, the T[c,s]T was detected by T4 endonuclease V in about one-half (46 +/- 9%) of the closed circular daughter molecules. Our results demonstrate that extracts prepared from HeLa cells and SV40-transformed human fibroblasts (SV80, IDH4), including a cell line defective in nucleotide-excision repair (XPA), were competent for leading strand DNA synthesis opposite the pyrimidine dimer and replication fork bypass. In contrast, dimer bypass was severely impaired in otherwise replication-competent extracts from two different xeroderma pigmentosum variant cell lines.

  11. Substrate-selective repair and restart of replication forks by DNA translocases.

    PubMed

    Bétous, Rémy; Couch, Frank B; Mason, Aaron C; Eichman, Brandt F; Manosas, Maria; Cortez, David

    2013-06-27

    Stalled replication forks are sources of genetic instability. Multiple fork-remodeling enzymes are recruited to stalled forks, but how they work to promote fork restart is poorly understood. By combining ensemble biochemical assays and single-molecule studies with magnetic tweezers, we show that SMARCAL1 branch migration and DNA-annealing activities are directed by the single-stranded DNA-binding protein RPA to selectively regress stalled replication forks caused by blockage to the leading-strand polymerase and to restore normal replication forks with a lagging-strand gap. We unveil the molecular mechanisms by which RPA enforces SMARCAL1 substrate preference. E. coli RecG acts similarly to SMARCAL1 in the presence of E. coli SSB, whereas the highly related human protein ZRANB3 has different substrate preferences. Our findings identify the important substrates of SMARCAL1 in fork repair, suggest that RecG and SMARCAL1 are functional orthologs, and provide a comprehensive model of fork repair by these DNA translocases.

  12. Strand-specific analysis shows protein binding at replication forks and PCNA unloading from lagging strands when forks stall.

    PubMed

    Yu, Chuanhe; Gan, Haiyun; Han, Junhong; Zhou, Zhi-Xiong; Jia, Shaodong; Chabes, Andrei; Farrugia, Gianrico; Ordog, Tamas; Zhang, Zhiguo

    2014-11-20

    In eukaryotic cells, DNA replication proceeds with continuous synthesis of leading-strand DNA and discontinuous synthesis of lagging-strand DNA. Here we describe a method, eSPAN (enrichment and sequencing of protein-associated nascent DNA), which reveals the genome-wide association of proteins with leading and lagging strands of DNA replication forks. Using this approach in budding yeast, we confirm the strand specificities of DNA polymerases delta and epsilon and show that the PCNA clamp is enriched at lagging strands compared with leading-strand replication. Surprisingly, at stalled forks, PCNA is unloaded specifically from lagging strands. PCNA unloading depends on the Elg1-containing alternative RFC complex, ubiquitination of PCNA, and the checkpoint kinases Mec1 and Rad53. Cells deficient in PCNA unloading exhibit increased chromosome breaks. Our studies provide a tool for studying replication-related processes and reveal a mechanism whereby checkpoint kinases regulate strand-specific unloading of PCNA from stalled replication forks to maintain genome stability.

  13. The ColE1 unidirectional origin acts as a polar replication fork pausing site.

    PubMed

    Viguera, E; Hernández, P; Krimer, D B; Boistov, A S; Lurz, R; Alonso, J C; Schvartzman, J B

    1996-09-13

    Co-orientation of replication origins is the most common organization found in nature for multimeric plasmids. Streptococcus pyogenes broad-host-range plasmid pSM19035 and Escherichia coli pPI21 are among the exceptions. pPI21, which is a derivative of pSM19035 and pBR322, has two long inverted repeats, each one containing a potentially active ColE1 unidirectional origin. Analysis of pPI21 replication intermediates (RIs) by two-dimensional agarose gel electrophoresis and electron microscopy revealed the accumulation of a specific RI containing a single internal bubble. The data obtained demonstrated that initiation of DNA replication occurred at a single origin in pPI21. Progression of the replicating fork initiated at either of the two potential origins was transiently stalled at the other inversely oriented silent ColE1 origin of the plasmid. The accumulated RIs, containing an internal bubble, occurred as a series of stereoisomers with different numbers of knots in their replicated portion. These observations provide one of the first functional explanations for the disadvantage of head-to-head plasmid multimers with respect to head-to-tail ones.

  14. 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

  15. 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

  16. Human single-stranded DNA binding protein 1 (hSSB1/NABP2) is required for the stability and repair of stalled replication forks.

    PubMed

    Bolderson, Emma; Petermann, Eva; Croft, Laura; Suraweera, Amila; Pandita, Raj K; Pandita, Tej K; Helleday, Thomas; Khanna, Kum Kum; Richard, Derek J

    2014-06-01

    Aberrant DNA replication is a primary cause of mutations that are associated with pathological disorders including cancer. During DNA metabolism, the primary causes of replication fork stalling include secondary DNA structures, highly transcribed regions and damaged DNA. The restart of stalled replication forks is critical for the timely progression of the cell cycle and ultimately for the maintenance of genomic stability. Our previous work has implicated the single-stranded DNA binding protein, hSSB1/NABP2, in the repair of DNA double-strand breaks via homologous recombination. Here, we demonstrate that hSSB1 relocates to hydroxyurea (HU)-damaged replication forks where it is required for ATR and Chk1 activation and recruitment of Mre11 and Rad51. Consequently, hSSB1-depleted cells fail to repair and restart stalled replication forks. hSSB1 deficiency causes accumulation of DNA strand breaks and results in chromosome aberrations observed in mitosis, ultimately resulting in hSSB1 being required for survival to HU and camptothecin. Overall, our findings demonstrate the importance of hSSB1 in maintaining and repairing DNA replication forks and for overall genomic stability.

  17. Characterization of the ATPase activity of RecG and RuvAB proteins on model fork structures reveals insight into stalled DNA replication fork repair.

    PubMed

    Abd Wahab, Syafiq; Choi, Meerim; Bianco, Piero R

    2013-09-13

    RecG and RuvAB are proposed to act at stalled DNA replication forks to facilitate replication restart. To clarify the roles of these proteins in fork regression, we used a coupled spectrophotometric ATPase assay to determine how these helicases act on two groups of model fork substrates: the first group mimics nascent stalled forks, whereas the second mimics regressed fork structures. The results show that RecG is active on the substrates in group 1, whereas these are poor substrates for RuvAB. In addition, in the presence of group 1 forks, the single-stranded DNA-binding protein (SSB) enhances the activity of RecG and enables it to compete with excess RuvA. In contrast, SSB inhibits the activity of RuvAB on these substrates. Results also show that the preferred regressed fork substrate for RuvAB is a Holliday junction, not a forked DNA. The active form of the enzyme on the Holliday junction contains a single RuvA tetramer. In contrast, although the enzyme is active on a regressed fork structure, RuvB loading by a single RuvA tetramer is impaired, and full activity requires the cooperative binding of two forked DNA substrate molecules. Collectively, the data support a model where RecG is responsible for stalled DNA replication fork regression. SSB ensures that if the nascent fork has single-stranded DNA character RuvAB is inhibited, whereas the activity of RecG is preferentially enhanced. Only once the fork has been regressed and the DNA is relaxed can RuvAB bind to a RecG-extruded Holliday junction.

  18. 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

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

    NASA Astrophysics Data System (ADS)

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

    2016-08-01

    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.

  20. 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

  1. Replication fork blockage by RTS1 at an ectopic site promotes recombination in fission yeast.

    PubMed

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

    2005-06-01

    Homologous recombination is believed to play important roles in processing stalled/blocked replication forks in eukaryotes. In accordance with this, recombination is induced by replication fork barriers (RFBs) within the rDNA locus. However, the rDNA locus is a specialised region of the genome, and therefore the action of recombinases at its RFBs may be atypical. We show here for the first time that direct repeat recombination, dependent on Rad22 and Rhp51, is induced by replication fork blockage at a site-specific RFB (RTS1) within a 'typical' genomic locus in fission yeast. Importantly, when the RFB is positioned between the direct repeat, conservative gene conversion events predominate over deletion events. This is consistent with recombination occurring without breakage of the blocked fork. In the absence of the RecQ family DNA helicase Rqh1, deletion events increase dramatically, which correlates with the detection of one-sided DNA double-strand breaks at or near RTS1. These data indicate that Rqh1 acts to prevent blocked replication forks from collapsing and thereby inducing deletion events.

  2. 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

  3. BLM SUMOylation regulates ssDNA accumulation at stalled replication forks.

    PubMed

    Ouyang, Karen J; Yagle, Mary K; Matunis, Michael J; Ellis, Nathan A

    2013-01-01

    Polymerase stalling results in uncoupling of DNA polymerase and the replicative helicase, which generates single-stranded DNA (ssDNA). After stalling, RAD51 accumulates at stalled replication forks to stabilize the fork and to repair by homologous recombination (HR) double-strand breaks (DSBs) that accumulate there. We showed recently that SUMO modification of the BLM helicase is required in order for RAD51 to accumulate at stalled forks. In order to investigate how BLM SUMOylation controls RAD51 accumulation, we characterized the function of HR proteins and ssDNA-binding protein RPA in cells that stably expressed either normal BLM (BLM+) or SUMO-mutant BLM (SM-BLM). In HU-treated SM-BLM cells, mediators BRCA2 and RAD52, which normally substitute RAD51 for RPA on ssDNA, failed to accumulate normally at stalled forks; instead, excess RPA accumulated. SM-BLM cells also exhibited higher levels of HU-induced chromatin-bound RPA than BLM+ cells did. The excess RPA did not result from excessive intrinsic BLM helicase activity, because in vitro SUMOylated BLM unwound similar amounts of replication-fork substrate as unSUMOylated BLM. Nor did BLM SUMOylation inhibit binding of RPA to BLM in vitro; however, in immunoprecipitation experiments, more BLM-RPA complex formed in HU-treated SM-BLM cells, indicating that BLM SUMOylation controls the amount of BLM-RPA complex normally formed at stalled forks. Together, these results showed that BLM SUMOylation regulates the amount of ssDNA that accumulates during polymerase stalling. We conclude that BLM SUMOylation functions as a licensing mechanism that permits and regulates HR at damaged replication forks.

  4. RecG interacts directly with SSB: implications for stalled replication fork regression.

    PubMed

    Buss, Jackson A; Kimura, Yuji; Bianco, Piero R

    2008-12-01

    RecG and RuvAB are proposed to act at stalled DNA replication forks to facilitate replication restart. To define the roles of these proteins in fork regression, we used a combination of assays to determine whether RecG, RuvAB or both are capable of acting at a stalled fork. The results show that RecG binds to the C-terminus of single-stranded DNA binding protein (SSB) forming a stoichiometric complex of 2 RecG monomers per SSB tetramer. This binding occurs in solution and to SSB protein bound to single stranded DNA (ssDNA). The result of this binding is stabilization of the interaction of RecG with ssDNA. In contrast, RuvAB does not bind to SSB. Side-by-side analysis of the catalytic efficiency of the ATPase activity of each enzyme revealed that (-)scDNA and ssDNA are potent stimulators of the ATPase activity of RecG but not for RuvAB, whereas relaxed circular DNA is a poor cofactor for RecG but an excellent one for RuvAB. Collectively, these data suggest that the timing of repair protein access to the DNA at stalled forks is determined by the nature of the DNA available at the fork. We propose that RecG acts first, with RuvAB acting either after RecG or in a separate pathway following protein-independent fork regression.

  5. 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.

  6. 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

  7. 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

  8. Strand specificity in the interactions of Escherichia coli primary replicative helicase DnaB protein with a replication fork.

    PubMed

    Jezewska, M J; Rajendran, S; Bujalowski, W

    1997-08-19

    The interactions of the Escherichia coli primary replicative helicase DnaB protein, with synthetic DNA replication fork substrates, having either a single arm or both arms, have been studied using the thermodynamically rigorous fluorescence titration techniques. This approach allows us to obtain absolute stoichiometries of the formed complexes and interaction parameters without any assumptions about the relationship between the observed signal (fluorescence) and the degree of binding. Subsequently, the formation of the complexes, with different replication fork substrates, has also been characterized using the sedimentation velocity technique. To our knowledge, this is the first quantitative characterization of interactions of a hexameric helicase with replication fork substrates. In the presence of the ATP nonhydrolyzable analog, AMP-PNP, the E. coli DnaB helicase preferentially binds to the 5' arm of the single-arm fork substrate with an intrinsic affinity 6-fold higher than its affinity for the 3' arm. ATP hydrolysis is not necessary for formation of the helicase-fork complex. The asymmetric interactions are consistent with the 5' --> 3' directionality of the helicase activity of the DnaB protein and most probably reflects a preferential 5' --> 3' polarity in the helicase binding to ssDNA, with respect to the ssDNA backbone. The double-stranded part of the fork contributes little to the free energy of binding. The data indicate a rather passive role of the duplex part of the fork in the binding of the helicase. This role seems to be limited to impose steric hindrance in the formation of nonproductive complexes of the enzyme with the fork. Quantitative analysis of binding of the helicase to the two-arm fork substrate shows that two DnaB hexamers can bind to the fork, with each single hexamer associated with a single arm of the fork. In this complex, the intrinsic affinity of the DnaB hexamer for the 5' arm in a two-arm fork is not affected by the presence of the

  9. Growth rate dependent numbers of SeqA structures organize the multiple replication forks in rapidly growing Escherichia coli.

    PubMed

    Morigen; Odsbu, Ingvild; Skarstad, Kirsten

    2009-05-01

    When the bacterium Escherichia coli is grown in rich medium, the replication and segregation periods may span two, three or four generations and cells may contain up to 24 replication forks. The newly synthesized, hemimethylated DNA at each fork is bound by SeqA protein. The SeqA-DNA structures form distinct foci that can be observed by immunofluorescence microscopy. The numbers of foci were lower than the numbers of replication forks indicating fork co-localization. The extent of co-localization correlated with the extent of replication cycle overlap in wild-type cells. No abrupt increase in the numbers of foci occurred at the time of initiation of replication, suggesting that new replication forks bind to existing SeqA structures. Manipulations with replication control mechanisms that led to extension or reduction of the replication period and number of forks, did not lead to changes in the numbers of SeqA foci per cell. The results indicate that the number of SeqA foci is not directly governed by the number of replication forks, and supports the idea that new DNA may be 'captured' by existing SeqA structures.

  10. Cleavage of stalled forks by fission yeast Mus81/Eme1 in absence of DNA replication checkpoint.

    PubMed

    Froget, Benoît; Blaisonneau, Joël; Lambert, Sarah; Baldacci, Giuseppe

    2008-02-01

    During replication arrest, the DNA replication checkpoint plays a crucial role in the stabilization of the replisome at stalled forks, thus preventing the collapse of active forks and the formation of aberrant DNA structures. How this checkpoint acts to preserve the integrity of replication structures at stalled fork is poorly understood. In Schizosaccharomyces pombe, the DNA replication checkpoint kinase Cds1 negatively regulates the structure-specific endonuclease Mus81/Eme1 to preserve genomic integrity when replication is perturbed. Here, we report that, in response to hydroxyurea (HU) treatment, the replication checkpoint prevents S-phase-specific DNA breakage resulting from Mus81 nuclease activity. However, loss of Mus81 regulation by Cds1 is not sufficient to produce HU-induced DNA breaks. Our results suggest that unscheduled cleavage of stalled forks by Mus81 is permitted when the replisome is not stabilized by the replication checkpoint. We also show that HU-induced DNA breaks are partially dependent on the Rqh1 helicase, the fission yeast homologue of BLM, but are independent of its helicase activity. This suggests that efficient cleavage of stalled forks by Mus81 requires Rqh1. Finally, we identified an interplay between Mus81 activity at stalled forks and the Chk1-dependent DNA damage checkpoint during S-phase when replication forks have collapsed.

  11. Cleavage of a model DNA replication fork by a methyl-specific endonuclease.

    PubMed

    Ishikawa, Ken; Handa, Naofumi; Sears, Lauren; Raleigh, Elisabeth A; Kobayashi, Ichizo

    2011-07-01

    Epigenetic DNA methylation is involved in many biological processes. An epigenetic status can be altered by gain or loss of a DNA methyltransferase gene or its activity. Repair of DNA damage can also remove DNA methylation. In response to such alterations, DNA endonucleases that sense DNA methylation can act and may cause cell death. Here, we explored the possibility that McrBC, a methylation-dependent DNase of Escherichia coli, cleaves DNA at a replication fork. First, we found that in vivo restriction by McrBC of bacteriophage carrying a foreign DNA methyltransferase gene is increased in the absence of homologous recombination. This suggests that some cleavage events are repaired by recombination and must take place during or after replication. Next, we demonstrated that the enzyme can cleave a model DNA replication fork in vitro. Cleavage of a fork required methylation on both arms and removed one, the other or both of the arms. Most cleavage events removed the methylated sites from the fork. This result suggests that acquisition of even rarely occurring modification patterns will be recognized and rejected efficiently by modification-dependent restriction systems that recognize two sites. This process might serve to maintain an epigenetic status along the genome through programmed cell death.

  12. Replication forks of Escherichia coli are not the preferred sites for lysogenic integration of bacteriophage Mu.

    PubMed Central

    Sivan, S; Zaritsky, A; Kagan-Zur, V

    1988-01-01

    The question of whether bacteriophage Mu prefers replication forks for lysogenic integration into Escherichia coli chromosomes was tested by using two different systems. In the first, inactivation of genes was scored in synchronized cultures infected by Mu at various times. No increase in the mutation frequency of a gene was found after infection at the time of its replication. In the second, the composition of colonies formed by bacteria lysogenized by Mu was determined; the newly formed lysogens should give rise to mixed colonies (containing lysogenized as well as nonlysogenized bacteria), uniform colonies, or both, depending on the mode of integration. Both types of colonies were found, and the fraction of uniform colonies was proportional to the relative length of the unreplicated segment of an average chromosome in the culture. The results in both systems clearly preclude the possibility that a lysogenizing Mu integrates with high preference at the chromosome replication forks. PMID:2968339

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

    PubMed

    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

    2015-12-01

    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.

  14. 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

  15. SUMO modification regulates BLM and RAD51 interaction at damaged replication forks.

    PubMed

    Ouyang, Karen J; Woo, Leslie L; Zhu, Jianmei; Huo, Dezheng; Matunis, Michael J; Ellis, Nathan A

    2009-12-01

    The gene mutated in Bloom's syndrome, BLM, is important in the repair of damaged replication forks, and it has both pro- and anti-recombinogenic roles in homologous recombination (HR). At damaged forks, BLM interacts with RAD51 recombinase, the essential enzyme in HR that catalyzes homology-dependent strand invasion. We have previously shown that defects in BLM modification by the small ubiquitin-related modifier (SUMO) cause increased gamma-H2AX foci. Because the increased gamma-H2AX could result from defective repair of spontaneous DNA damage, we hypothesized that SUMO modification regulates BLM's function in HR repair at damaged forks. To test this hypothesis, we treated cells that stably expressed a normal BLM (BLM+) or a SUMO-mutant BLM (SM-BLM) with hydroxyurea (HU) and examined the effects of stalled replication forks on RAD51 and its DNA repair functions. HU treatment generated excess gamma-H2AX in SM-BLM compared to BLM+ cells, consistent with a defect in replication-fork repair. SM-BLM cells accumulated increased numbers of DNA breaks and were hypersensitive to DNA damage. Importantly, HU treatment failed to induce sister-chromatid exchanges in SM-BLM cells compared to BLM+ cells, indicating a specific defect in HR repair and suggesting that RAD51 function could be compromised. Consistent with this hypothesis, RAD51 localization to HU-induced repair foci was impaired in SM-BLM cells. These data suggested that RAD51 might interact noncovalently with SUMO. We found that in vitro RAD51 interacts noncovalently with SUMO and that it interacts more efficiently with SUMO-modified BLM compared to unmodified BLM. These data suggest that SUMOylation controls the switch between BLM's pro- and anti-recombinogenic roles in HR. In the absence of BLM SUMOylation, BLM perturbs RAD51 localization at damaged replication forks and inhibits fork repair by HR. Conversely, BLM SUMOylation relieves its inhibitory effects on HR, and it promotes RAD51 function.

  16. Tracking of controlled Escherichia coli replication fork stalling and restart at repressor-bound DNA in vivo.

    PubMed

    Possoz, Christophe; Filipe, Sergio R; Grainge, Ian; Sherratt, David J

    2006-06-01

    We report an efficient, controllable, site-specific replication roadblock that blocks cell proliferation, but which can be rapidly and efficiently reversed, leading to recovery of viability. Escherichia coli replication forks of both polarities stalled in vivo within the first 500 bp of a 10 kb repressor-bound array of operator DNA-binding sites. Controlled release of repressor binding led to rapid restart of the blocked replication fork without the participation of homologous recombination. Cytological tracking of fork stalling and restart showed that the replisome-associated SSB protein remains associated with the blocked fork for extended periods and that duplication of the fluorescent foci associated with the blocked operator array occurs immediately after restart, thereby demonstrating a lack of sister cohesion in the region of the array. Roadblocks positioned near oriC or the dif site did not prevent replication and segregation of the rest of the chromosome.

  17. Reorganization of terminator DNA upon binding replication terminator protein: implications for the functional replication fork arrest complex.

    PubMed

    Kralicek, A V; Wilson, P K; Ralston, G B; Wake, R G; King, G F

    1997-02-01

    Termination of DNA replication in Bacillus subtilis involves the polar arrest of replication forks by a specific complex formed between the replication terminator protein (RTP) and DNA terminator sites. While determination of the crystal structure of RTP has facilitated our understanding of how a single RTP dimer interacts with terminator DNA, additional information is required in order to understand the assembly of a functional fork arrest complex, which requires an interaction between two RTP dimers and the terminator site. In this study, we show that the conformation of the major B.subtilis DNA terminator,TerI, becomes considerably distorted upon binding RTP. Binding of the first dimer of RTP to the B site of TerI causes the DNA to become slightly unwound and bent by approximately 40 degrees. Binding of a second dimer of RTP to the A site causes the bend angle to increase to approximately 60 degrees . We have used this new data to construct two plausible models that might explain how the ternary terminator complex can block DNA replication in a polar manner. In the first model, polarity of action is a consequence of the two RTP-DNA half-sites having different conformations. These different conformations result from different RTP-DNA contacts at each half-site (due to the intrinsic asymmetry of the terminator DNA), as well as interactions (direct or indirect) between the RTP dimers on the DNA. In the second model, polar fork arrest activity is a consequence of the different affinities of RTP for the A and B sites of the terminator DNA, modulated significantly by direct or indirect interactions between the RTP dimers.

  18. Minding the gap: The underground functions of BRCA1 and BRCA2 at stalled replication forks

    PubMed Central

    Nagaraju, Ganesh; Scully, Ralph

    2010-01-01

    The hereditary breast and ovarian cancer predisposition genes, BRCA1 and BRCA2, participate in the repair of DNA double strand breaks by homologous recombination. Circumstantial evidence implicates these genes in recombinational responses to DNA polymerase stalling during the S phase of the cell cycle. These responses play a key role in preventing genomic instability and cancer. Here, we review the current literature implicating the BRCA pathway in HR at stalled replication forks and explore the hypothesis that BRCA1 and BRCA2 participate in the recombinational resolution of single stranded DNA lesions termed “daughter strand gaps”, generated during replication across a damaged DNA template. PMID:17379580

  19. A DNA replication origin and a replication fork barrier used in vivo in the circular plasmid pKD1.

    PubMed

    Fabiani, L; Irene, C; Aragona, M; Newlon, C S

    2001-10-01

    As in other yeasts, ARS-containing plasmids can be maintained extrachromosomally in Kluyveromyces lactis. Although some fragments of K. lactis DNA have ARS activity in both K. lactis and Saccharomyces cerevisiae, it appears that the sequences required for ARS activity in the two yeasts are different. As an approach to a better understanding of ARS structure and function in K. lactis, we analyzed the replication of the circular plasmid pKD1. We identified a 159-bp sequence able to promote autonomous replication of pKD1 in both yeasts; this fragments contains both a sequence related to the S. cerevisiae ARS consensus sequence and a region of 53% identity to the 40-bp sequence essential for K. lactis KARS101 function. By the analysis of in vivo replication intermediates we provide the first direct evidence that DNA replication initiates at or near the K. lactis ARS element. Replication terminates at the cisacting stability locus of pKD1, which functions as a replication fork barrier (RFB) and is necessary for proper plasmid segregation. RFB activity requires the pKDI gene products that are important for plasmid segregation, suggesting a link between DNA replication termination and plasmid segregation in a eukaryotic organism.

  20. 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

  1. A Reduction in Ribonucleotide Reductase Activity Slows Down the Chromosome Replication Fork but Does Not Change Its Localization

    PubMed Central

    Odsbu, Ingvild; Morigen; Skarstad, Kirsten

    2009-01-01

    Background It has been proposed that the enzymes of nucleotide biosynthesis may be compartmentalized or concentrated in a structure affecting the organization of newly replicated DNA. Here we have investigated the effect of changes in ribonucleotide reductase (RNR) activity on chromosome replication and organization of replication forks in Escherichia coli. Methodology/Principal Findings Reduced concentrations of deoxyribonucleotides (dNTPs) obtained by reducing the activity of wild type RNR by treatment with hydroxyurea or by mutation, resulted in a lengthening of the replication period. The replication fork speed was found to be gradually reduced proportionately to moderate reductions in nucleotide availability. Cells with highly extended C periods showed a “delay” in cell division i.e. had a higher cell mass. Visualization of SeqA structures by immunofluorescence indicated no change in organization of the new DNA upon moderate limitation of RNR activity. Severe nucleotide limitation led to replication fork stalling and reversal. Well defined SeqA structures were not found in situations of extensive replication fork repair. In cells with stalled forks obtained by UV irradiation, considerable DNA compaction was observed, possibly indicating a reorganization of the DNA into a “repair structure” during the initial phase of the SOS response. Conclusion/Significance The results indicate that the replication fork is slowed down in a controlled manner during moderate nucleotide depletion and that a change in the activity of RNR does not lead to a change in the organization of newly replicated DNA. Control of cell division but not control of initiation was affected by the changes in replication elongation. PMID:19898675

  2. 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

  3. 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

  4. Establishment of a replication fork barrier following induction of DNA binding in mammalian cells

    PubMed Central

    Beuzer, Paolo; Quivy, Jean-Pierre; Almouzni, Geneviève

    2014-01-01

    Understanding the mechanisms that lead to replication fork blocks (RFB) and the means to bypass them is important given the threat that they represent for genome stability if inappropriately handled. Here, to study this issue in mammals, we use integrated arrays of the LacO and/or TetO as a tractable system to follow in time a process in an individual cell and at a single locus. Importantly, we show that induction of the binding by LacI and TetR proteins, and not the presence of the repeats, is key to form the RFB. We find that the binding of the proteins to the arrays during replication causes a prolonged persistence of replication foci at the site. This, in turn, induces a local DNA damage repair (DDR) response, with the recruitment of proteins involved in double-strand break (DSB) repair such as TOPBP1 and 53BP1, and the phosphorylation of H2AX. Furthermore, the appearance of micronuclei and DNA bridges after mitosis is consistent with an incomplete replication. We discuss how the many DNA binding proteins encountered during replication can be dealt with and the consequences of incomplete replication. Future studies exploiting this type of system should help analyze how an RFB, along with bypass mechanisms, are controlled in order to maintain genome integrity. PMID:24675882

  5. Differential regulation of the anti-crossover and replication fork regression activities of Mph1 by Mte1

    PubMed Central

    Xue, Xiaoyu; Papusha, Alma; Choi, Koyi; Bonner, Jaclyn N.; Kumar, Sandeep; Niu, Hengyao; Kaur, Hardeep; Zheng, Xiao-Feng; Donnianni, Roberto A.; Lu, Lucy; Lichten, Michael; Zhao, Xiaolan; Ira, Grzegorz; Sung, Patrick

    2016-01-01

    We identified Mte1 (Mph1-associated telomere maintenance protein 1) as a multifunctional regulator of Saccharomyces cerevisiae Mph1, a member of the FANCM family of DNA motor proteins important for DNA replication fork repair and crossover suppression during homologous recombination. We show that Mte1 interacts with Mph1 and DNA species that resemble a DNA replication fork and the D loop formed during recombination. Biochemically, Mte1 stimulates Mph1-mediated DNA replication fork regression and branch migration in a model substrate. Consistent with this activity, genetic analysis reveals that Mte1 functions with Mph1 and the associated MHF complex in replication fork repair. Surprisingly, Mte1 antagonizes the D-loop-dissociative activity of Mph1–MHF and exerts a procrossover role in mitotic recombination. We further show that the influence of Mte1 on Mph1 activities requires its binding to Mph1 and DNA. Thus, Mte1 differentially regulates Mph1 activities to achieve distinct outcomes in recombination and replication fork repair. PMID:26966246

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

    PubMed Central

    Kuong, Kawai J; Kuzminov, Andrei

    2014-01-01

    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

  7. 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

  8. Breathing fluctuations in position-specific DNA base pairs are involved in regulating helicase movement into the replication fork.

    PubMed

    Jose, Davis; Weitzel, Steven E; von Hippel, Peter H

    2012-09-01

    We previously used changes in the near-UV circular dichroism and fluorescence spectra of DNA base analogue probes placed site specifically to show that the first three base pairs at the fork junction in model replication fork constructs are significantly opened by "breathing" fluctuations under physiological conditions. Here, we use these probes to provide mechanistic snapshots of the initial interactions of the DNA fork with a tight-binding replication helicase in solution. The primosome helicase of bacteriophage T4 was assembled from six (gp41) helicase subunits, one (gp61) primase subunit, and nonhydrolyzable GTPγS. When bound to a DNA replication fork construct this complex advances one base pair into the duplex portion of the fork and forms a stably bound helicase "initiation complex." Replacement of GTPγS with GTP permits the completion of the helicase-driven unwinding process. Our spectroscopic probes show that the primosome in this stable helicase initiation complex binds the DNA of the fork primarily via backbone contacts and holds the first complementary base pair of the fork in an open conformation, whereas the second, third, and fourth base pairs of the duplex show essentially the breathing behavior that previously characterized the first three base pairs of the free fork. These spectral changes, together with dynamic fluorescence quenching results, are consistent with a primosome-binding model in which the lagging DNA strand passes through the central hole of the hexagonal helicase, the leading strand binds to the "outside" surfaces of subunits of the helicase hexamer, and the single primase subunit interacts with both strands.

  9. 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

  10. An antitumor drug-induced topoisomerase cleavage complex blocks a bacteriophage T4 replication fork in vivo.

    PubMed

    Hong, G; Kreuzer, K N

    2000-01-01

    Many antitumor and antibacterial drugs inhibit DNA topoisomerases by trapping covalent enzyme-DNA cleavage complexes. Formation of cleavage complexes is important for cytotoxicity, but evidence suggests that cleavage complexes themselves are not sufficient to cause cell death. Rather, active cellular processes such as transcription and/or replication are probably necessary to transform cleavage complexes into cytotoxic lesions. Using defined plasmid substrates and two-dimensional agarose gel analysis, we examined the collision of an active replication fork with an antitumor drug-trapped cleavage complex. Discrete DNA molecules accumulated on the simple Y arc, with branch points very close to the topoisomerase cleavage site. Accumulation of the Y-form DNA required the presence of a topoisomerase cleavage site, the antitumor drug, the type II topoisomerase, and a T4 replication origin on the plasmid. Furthermore, all three arms of the Y-form DNA were replicated, arguing strongly that these are trapped replication intermediates. The Y-form DNA appeared even in the absence of two important phage recombination proteins, implying that Y-form DNA is the result of replication rather than recombination. This is the first direct evidence that a drug-induced topoisomerase cleavage complex blocks the replication fork in vivo. Surprisingly, these blocked replication forks do not contain DNA breaks at the topoisomerase cleavage site, implying that the replication complex was inactivated (at least temporarily) and that topoisomerase resealed the drug-induced DNA breaks. The replication fork may behave similarly at other types of DNA lesions, and thus cleavage complexes could represent a useful (site-specific) model for chemical- and radiation-induced DNA damage.

  11. 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.

  12. 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

  13. DNA polymerase κ-dependent DNA synthesis at stalled replication forks is important for CHK1 activation

    PubMed Central

    Bétous, Rémy; Pillaire, Marie-Jeanne; Pierini, Laura; van der Laan, Siem; Recolin, Bénédicte; Ohl-Séguy, Emma; Guo, Caixia; Niimi, Naoko; Grúz, Petr; Nohmi, Takehiko; Friedberg, Errol; Cazaux, Christophe; Maiorano, Domenico; Hoffmann, Jean-Sébastien

    2013-01-01

    Formation of primed single-stranded DNA at stalled replication forks triggers activation of the replication checkpoint signalling cascade resulting in the ATR-mediated phosphorylation of the Chk1 protein kinase, thus preventing genomic instability. By using siRNA-mediated depletion in human cells and immunodepletion and reconstitution experiments in Xenopus egg extracts, we report that the Y-family translesion (TLS) DNA polymerase kappa (Pol κ) contributes to the replication checkpoint response and is required for recovery after replication stress. We found that Pol κ is implicated in the synthesis of short DNA intermediates at stalled forks, facilitating the recruitment of the 9-1-1 checkpoint clamp. Furthermore, we show that Pol κ interacts with the Rad9 subunit of the 9-1-1 complex. Finally, we show that this novel checkpoint function of Pol κ is required for the maintenance of genomic stability and cell proliferation in unstressed human cells. PMID:23799366

  14. Hypermutagenesis in mutA cells is mediated by mistranslational corruption of polymerase, and is accompanied by replication fork collapse.

    PubMed

    Al Mamun, Abu Amar M; Gautam, Satyendra; Humayun, M Zafri

    2006-12-01

    Elevated mistranslation induces a mutator response termed translational stress-induced mutagenesis (TSM) that is mediated by an unidentified modification of DNA polymerase III. Here we address two questions: (i) does TSM result from direct polymerase corruption, or from an indirect pathway triggered by increased protein turnover? (ii) Why are homologous recombination functions required for the expression of TSM under certain conditions, but not others? We show that replication of bacteriophage T4 in cells expressing the mutA allele of the glyVtRNA gene (Asp-Gly mistranslation), leads to both increased mutagenesis, and to an altered mutational specificity, results that strongly support mistranslational corruption of DNA polymerase. We also show that expression of mutA, which confers a recA-dependent mutator phenotype, leads to increased lambdoid prophage induction (selectable in vivo expression technology assay), suggesting that replication fork collapse occurs more frequently in mutA cells relative to control cells. No such increase in prophage induction is seen in cells expressing alaVGlu tRNA (Glu-->Ala mistranslation), in which the mutator phenotype is recA-independent. We propose that replication fork collapse accompanies episodic hypermutagenic replication cycles in mutA cells, requiring homologous recombination functions for fork recovery, and therefore, for mutation recovery. These findings highlight hitherto under-appreciated links among translation, replication and recombination, and suggest that translational fidelity, which is affected by genetic and environmental signals, is a key modulator of replication fidelity.

  15. Mycobacterium tuberculosis RecG protein but not RuvAB or RecA protein is efficient at remodeling the stalled replication forks: implications for multiple mechanisms of replication restart in mycobacteria.

    PubMed

    Thakur, Roshan Singh; Basavaraju, Shivakumar; Khanduja, Jasbeer Singh; Muniyappa, K; Nagaraju, Ganesh

    2015-10-01

    Aberrant DNA replication, defects in the protection, and restart of stalled replication forks are major causes of genome instability in all organisms. Replication fork reversal is emerging as an evolutionarily conserved physiological response for restart of stalled forks. Escherichia coli RecG, RuvAB, and RecA proteins have been shown to reverse the model replication fork structures in vitro. However, the pathways and the mechanisms by which Mycobacterium tuberculosis, a slow growing human pathogen, responds to different types of replication stress and DNA damage are unclear. Here, we show that M. tuberculosis RecG rescues E. coli ΔrecG cells from replicative stress. The purified M. tuberculosis RecG (MtRecG) and RuvAB (MtRuvAB) proteins catalyze fork reversal of model replication fork structures with and without a leading strand single-stranded DNA gap. Interestingly, single-stranded DNA-binding protein suppresses the MtRecG- and MtRuvAB-mediated fork reversal with substrates that contain lagging strand gap. Notably, our comparative studies with fork structures containing template damage and template switching mechanism of lesion bypass reveal that MtRecG but not MtRuvAB or MtRecA is proficient in driving the fork reversal. Finally, unlike MtRuvAB, we find that MtRecG drives efficient reversal of forks when fork structures are tightly bound by protein. These results provide direct evidence and valuable insights into the underlying mechanism of MtRecG-catalyzed replication fork remodeling and restart pathways in vivo.

  16. FANCD2-controlled chromatin access of the Fanconi-associated nuclease FAN1 is crucial for the recovery of stalled replication forks.

    PubMed

    Chaudhury, Indrajit; Stroik, Daniel R; Sobeck, Alexandra

    2014-11-01

    Fanconi anemia (FA) is a cancer predisposition syndrome characterized by cellular hypersensitivity to DNA interstrand cross-links (ICLs). Within the FA pathway, an upstream core complex monoubiquitinates and recruits the FANCD2 protein to ICLs on chromatin. Ensuing DNA repair involves the Fanconi-associated nuclease 1 (FAN1), which interacts selectively with monoubiquitinated FANCD2 (FANCD2(Ub)) at ICLs. Importantly, FANCD2 has additional independent functions: it binds chromatin and coordinates the restart of aphidicolin (APH)-stalled replication forks in concert with the BLM helicase, while protecting forks from nucleolytic degradation by MRE11. We identified FAN1 as a new crucial replication fork recovery factor. FAN1 joins the BLM-FANCD2 complex following APH-mediated fork stalling in a manner dependent on MRE11 and FANCD2, followed by FAN1 nuclease-mediated fork restart. Surprisingly, APH-induced activation and chromatin recruitment of FAN1 occur independently of the FA core complex or the FAN1 UBZ domain, indicating that the FANCD2(Ub) isoform is dispensable for functional FANCD2-FAN1 cross talk during stalled fork recovery. In the absence of FANCD2, MRE11 exonuclease-promoted access of FAN1 to stalled forks results in severe FAN1-mediated nucleolytic degradation of nascent DNA strands. Thus, FAN1 nuclease activity at stalled replication forks requires tight regulation: too little inhibits fork restart, whereas too much causes fork degradation.

  17. FANCD2-Controlled Chromatin Access of the Fanconi-Associated Nuclease FAN1 Is Crucial for the Recovery of Stalled Replication Forks

    PubMed Central

    Chaudhury, Indrajit; Stroik, Daniel R.

    2014-01-01

    Fanconi anemia (FA) is a cancer predisposition syndrome characterized by cellular hypersensitivity to DNA interstrand cross-links (ICLs). Within the FA pathway, an upstream core complex monoubiquitinates and recruits the FANCD2 protein to ICLs on chromatin. Ensuing DNA repair involves the Fanconi-associated nuclease 1 (FAN1), which interacts selectively with monoubiquitinated FANCD2 (FANCD2Ub) at ICLs. Importantly, FANCD2 has additional independent functions: it binds chromatin and coordinates the restart of aphidicolin (APH)-stalled replication forks in concert with the BLM helicase, while protecting forks from nucleolytic degradation by MRE11. We identified FAN1 as a new crucial replication fork recovery factor. FAN1 joins the BLM-FANCD2 complex following APH-mediated fork stalling in a manner dependent on MRE11 and FANCD2, followed by FAN1 nuclease-mediated fork restart. Surprisingly, APH-induced activation and chromatin recruitment of FAN1 occur independently of the FA core complex or the FAN1 UBZ domain, indicating that the FANCD2Ub isoform is dispensable for functional FANCD2-FAN1 cross talk during stalled fork recovery. In the absence of FANCD2, MRE11 exonuclease-promoted access of FAN1 to stalled forks results in severe FAN1-mediated nucleolytic degradation of nascent DNA strands. Thus, FAN1 nuclease activity at stalled replication forks requires tight regulation: too little inhibits fork restart, whereas too much causes fork degradation. PMID:25135477

  18. DNA gyrase-mediated wrapping of the DNA strand is required for the replication fork arrest by the DNA gyrase-quinolone-DNA ternary complex.

    PubMed

    Hiasa, H; Shea, M E

    2000-11-01

    The ability of DNA gyrase (Gyr) to wrap the DNA strand around itself allows Gyr to introduce negative supercoils into DNA molecules. It has been demonstrated that the deletion of the C-terminal DNA-binding domain of the GyrA subunit abolishes the ability of Gyr to wrap the DNA strand and catalyze the supercoiling reaction (Kampranis, S. C., and Maxwell, A. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 14416-14421). By using this mutant Gyr, Gyr (A59), we have studied effects of Gyr-mediated wrapping of the DNA strand on its replicative function and its interaction with the quinolone antibacterial drugs. We find that Gyr (A59) can support oriC DNA replication in vitro. However, Gyr (A59)-catalyzed decatenation activity is not efficient enough to complete the decatenation of replicating daughter DNA molecules. As is the case with topoisomerase IV, the active cleavage and reunion activity of Gyr is required for the formation of the ternary complex that can arrest replication fork progression in vitro. Although the quinolone drugs stimulate the covalent Gyr (A59)-DNA complex formation, the Gyr (A59)-quinolone-DNA ternary complexes do not arrest the progression of replication forks. Thus, the quinolone-induced covalent topoisomerase-DNA complex formation is necessary but not sufficient to cause the inhibition of DNA replication. We also assess the stability of ternary complexes formed with Gyr (A59), the wild type Gyr, or topoisomerase IV. The ternary complexes formed with Gyr (A59) are more sensitive to salt than those formed with either the wild type Gyr or topoisomerase IV. Furthermore, a competition experiment demonstrates that the ternary complexes formed with Gyr (A59) readily disassociate from the DNA, whereas the ternary complexes formed with either the wild type Gyr or topoisomerase IV remain stably bound. Thus, Gyr-mediated wrapping of the DNA strand is required for the formation of the stable Gyr-quinolone-DNA ternary complex that can arrest replication fork

  19. Mre11-dependent degradation of stalled DNA replication forks is prevented by BRCA2 and PARP1.

    PubMed

    Ying, Songmin; Hamdy, Freddie C; Helleday, Thomas

    2012-06-01

    PARP inhibitors are currently being used in clinical trials to treat BRCA1- or BRCA2-defective tumors, based on the synthetic lethal interaction between PARP1 and BRCA1/2-mediated homologous recombination (HR). However, the molecular mechanisms that drive this synthetic lethality remain unclear. Here, we show increased levels of Mre11, a key component of MRN (Mre11-Rad50-Nbs1) complex that plays a role in the restart of stalled replication forks and enhanced resection at stalled replication forks in BRCA2-deficient cells. BRCA2-deficient cells also showed hypersensitivity to the Mre11 inhibitor mirin. Interestingly, PARP1 activity was required to protect stalled forks from Mre11-dependent degradation. Resistance to PARP inhibition in BRCA2-mutant cells led to reduced levels of Mre11 foci and also rescued their sensitivity to mirin. Taken together, our findings not only show that Mre11 activity is required for the survival of BRCA2 mutant cells but also elucidate roles for both the BRCA2 and PARP1 proteins in protecting stalled replication forks, which offers insight into the molecular mechanisms of the synthetic lethality between BRCA2 and PARP1.

  20. Binding of the replication terminator protein Fob1p to the Ter sites of yeast causes polar fork arrest.

    PubMed

    Mohanty, Bidyut K; Bastia, Deepak

    2004-01-16

    Fob1p protein has been implicated in the termination of replication forks at the two tandem termini present in the non-transcribed spacer region located between the sequences encoding the 35 S and the 5 S RNAs of Saccharomyces cerevisiae. However, the biochemistry and mode of action of this protein were previously unknown. We have purified the Fob1p protein to near-homogeneity, and we developed a novel technique to show that it binds specifically to the Ter1 and Ter2 sequences. Interestingly, the two sequences share no detectable homology. We present two lines of evidence showing that the interaction of the Fob1p with the Ter sites causes replication termination. First, a mutant of FOB1, L104S, that significantly reduced the binding of the mutant form of the protein to the tandem Ter sites, also failed to promote replication termination in vivo. The mutant did not diminish nucleolar transport, and interaction of the mutant form of Fob1p with itself and with another protein encoded in the locus YDR026C suggested that the mutation did not cause global misfolding of the protein. Second, DNA site mutations in the Ter sequences that separately and specifically abolished replication fork arrest at Ter1 or Ter2 also eliminated sequence-specific binding of the Fob1p to the two sites. The work presented here definitively established Ter DNA-Fob1p interaction as an important step in fork arrest.

  1. Replication of the Escherichia coli chromosome in RNase HI-deficient cells: multiple initiation regions and fork dynamics.

    PubMed

    Maduike, Nkabuije Z; Tehranchi, Ashley K; Wang, Jue D; Kreuzer, Kenneth N

    2014-01-01

    DNA replication in Escherichia coli is normally initiated at a single origin, oriC, dependent on initiation protein DnaA. However, replication can be initiated elsewhere on the chromosome at multiple ectopic oriK sites. Genetic evidence indicates that initiation from oriK depends on RNA-DNA hybrids (R-loops), which are normally removed by enzymes such as RNase HI to prevent oriK from misfiring during normal growth. Initiation from oriK sites occurs in RNase HI-deficient mutants, and possibly in wild-type cells under certain unusual conditions. Despite previous work, the locations of oriK and their impact on genome stability remain unclear. We combined 2D gel electrophoresis and whole genome approaches to map genome-wide oriK locations. The DNA copy number profiles of various RNase HI-deficient strains contained multiple peaks, often in consistent locations, identifying candidate oriK sites. Removal of RNase HI protein also leads to global alterations of replication fork migration patterns, often opposite to normal replication directions, and presumably eukaryote-like replication fork merging. Our results have implications for genome stability, offering a new understanding of how RNase HI deficiency results in R-loop-mediated transcription-replication conflict, as well as inappropriate replication stalling or blockage at Ter sites outside of the terminus trap region and at ribosomal operons.

  2. RecQ and RecJ process blocked replication forks prior to the resumption of replication in UV-irradiated Escherichia coli.

    PubMed

    Courcelle, J; Hanawalt, P C

    1999-10-01

    The accurate recovery of replication following DNA damage and repair is critical for the maintenance of genomic integrity. In Escherichia coli, the recovery of replication following UV-induced DNA damage is dependent upon several proteins in the recF pathway, including RecF, RecO, and RecR. Two other recF pathway proteins, the RecQ helicase and the RecJ exonuclease, have been shown to affect the sites and frequencies at which illegitimate rearrangements occur following UV-induced DNA damage, suggesting that they also may function during the recovery of replication. We show here that RecQ and RecJ process the nascent DNA at blocked replication forks prior to the resumption of DNA synthesis. The processing involves selective degradation of the nascent lagging DNA strand and it requires both RecQ and RecJ. We suggest that this processing may serve to lengthen the substrate that can be recognized and stabilized by the RecA protein at the replication fork, thereby helping to ensure the accurate recovery of replication after the obstructing lesion has been repaired. PMID:10589843

  3. Implication of RPA32 phosphorylation in S-phase checkpoint signalling at replication forks stalled with aphidicolin in Xenopus egg extracts.

    PubMed

    Recolin, Bénédicte; Maiorano, Domenico

    2012-11-01

    Activation of the replication checkpoint relies upon uncoupling of DNA polymerases and helicase activities at replication forks, which in multicellular organism results in production of long stretches of single-stranded DNA bound by the trimeric, single stranded DNA binding protein, the RPA complex. Binding of RPA to this substrate promotes synthesis of replication intermediates that contributes to checkpoint activation by allowing binding of the 9-1-1 checkpoint clamp. The RPA32kDa subunit is also phosphorylated during this process but its role in checkpoint signalling is unclear. Here we have investigated the requirement for RPA32 phosphorylation in checkpoint activation in Xenopus egg extracts. We show that phospho-deficient mutants of RPA32 stimulate checkpoint signalling at replication forks arrested with aphidicolin at both the initiation and the elongation step of DNA replication, without affecting DNA synthesis. In contrast, we show that phospho-mimetic RPA32 mutants do not stimulate checkpoint activation at unwound forks. These results indicate that the hypophosphorylated, replication fork-associated form of RPA32 functions in S-phase-dependent checkpoint signalling at unwound forks in Xenopus egg extracts while RPA32 phosphorylation may be implicated in other pathways such as repair or restart of arrested replication forks.

  4. Thermostable flap endonuclease from the archaeon, Pyrococcus horikoshii, cleaves the replication fork-like structure endo/exonucleolytically.

    PubMed

    Matsui, E; Kawasaki, S; Ishida, H; Ishikawa, K; Kosugi, Y; Kikuchi, H; Kawarabayashi, Y; Matsui, I

    1999-06-25

    The flap endonuclease gene homologue from the hyperthermophilic archaeon, Pyrococcus horikoshii, was overexpressed in Escherichia coli and purified. The results of gel filtration indicated that this protein was a 41-kDa monomer. P. horikoshii flap endonuclease (phFEN) cleaves replication fork-like substrates (RF) and 5' double-strand flap structures (DF) using both flap endonuclease and 5'-3'-exonuclease activities. The mammalian flap endonuclease (mFEN) is a single-strand flap-specific endonuclease (Harrington, J. J., and Lieber, M. R. (1994) EMBO J. 13, 1235-1246), but the action patterns of phFEN appear to be quite different from those of mFEN at this point. The DF-specific flap endonuclease and 5'-exonuclease activities have not yet been reported. Therefore, this is the first report of the specific endo/exonuclease activities of phFEN. The DF-specific 5'-exonuclease activity degraded the downstream primer of 3' single-flap structure and was 15 times higher than the activities against nicked substrates without 3' flap strand. DF-specific flap endonuclease cleaved the 5' double-flap strand in DF and the lagging strand in RF at the junction portion. Because the RF appears to be the intermediate structure, due to the arrest of the replication fork, the double strand breaks after the arrests of the replication forks are probably caused by phFEN.

  5. Structural basis of the 3'-end recognition of a leading strand in stalled replication forks by PriA.

    PubMed

    Sasaki, Kaori; Ose, Toyoyuki; Okamoto, Naoaki; Maenaka, Katsumi; Tanaka, Taku; Masai, Hisao; Saito, Mihoko; Shirai, Tsuyoshi; Kohda, Daisuke

    2007-05-16

    In eubacteria, PriA helicase detects the stalled DNA replication forks. This critical role of PriA is ascribed to its ability to bind to the 3' end of a nascent leading DNA strand in the stalled replication forks. The crystal structures in complexes with oligonucleotides and the combination of fluorescence correlation spectroscopy and mutagenesis reveal that the N-terminal domain of PriA possesses a binding pocket for the 3'-terminal nucleotide residue of DNA. The interaction with the deoxyribose 3'-OH is essential for the 3'-terminal recognition. In contrast, the direct interaction with 3'-end nucleobase is unexpected, considering the same affinity for oligonucleotides carrying the four bases at the 3' end. Thus, the N-terminal domain of PriA recognizes the 3'-end base in a base-non-selective manner, in addition to the deoxyribose and 5'-side phosphodiester group, of the 3'-terminal nucleotide to acquire both sufficient affinity and non-selectivity to find all of the stalled replication forks generated during DNA duplication. This unique feature is prerequisite for the proper positioning of the helicase domain of PriA on the unreplicated double-stranded DNA. PMID:17464287

  6. Replication fork arrest and rDNA silencing are two independent and separable functions of the replication terminator protein Fob1 of Saccharomyces cerevisiae.

    PubMed

    Bairwa, Narendra K; Zzaman, Shamsu; Mohanty, Bidyut K; Bastia, Deepak

    2010-04-23

    The replication terminator protein Fob1 of Saccharomyces cerevisiae is multifunctional, and it not only promotes polar replication fork arrest at the tandem Ter sites located in the intergenic spacer region of rDNA but also loads the NAD-dependent histone deacetylase Sir2 at Ter sites via a protein complex called RENT (regulator of nucleolar silencing and telophase exit). Sir2 is a component of the RENT complex, and its loading not only silences intrachromatid recombination in rDNA but also RNA polymerase II-catalyzed transcription. Here, we present three lines of evidence showing that the two aforementioned activities of Fob1 are independent of each other as well as functionally separable. First, a Fob1 ortholog of Saccharomyces bayanus expressed in a fob1Delta strain of S. cerevisiae restored polar fork arrest at Ter but not rDNA silencing. Second, a mutant form (I407T) of S. cerevisiae Fob1 retained normal fork arresting activity but was partially defective in rDNA silencing. We further show that the silencing defect of S. bayanus Fob1 and the Iota407Tau mutant of S. cerevisiae Fob1 were caused by the failure of the proteins to interact with two members of the S. cerevisiae RENT complex, namely S. cerevisiae Sir2 and S. cerevisiae Net1. Third, deletions of the intra-S phase checkpoint proteins Tof1 and Csm3 abolished fork arrest by Fob1 at Ter without causing loss of silencing. Taken together, the data support the conclusion that unlike some other functions of Fob1, rDNA silencing at Ter is independent of fork arrest.

  7. Inhibition of histone deacetylase in cancer cells slows down replication forks, activates dormant origins and induces DNA damage

    PubMed Central

    Conti, Chiara; Leo, Elisabetta; Eichler, Gabriel S.; Sordet, Olivier; Martin, Melvenia M.; Fan, Angela; Aladjem, Mirit I.; Pommier, Yves

    2010-01-01

    Protein acetylation is a reversible process regulated by histone deacetylases (HDACs) that is often altered in human cancers. SAHA (suberoylanilide hydroxamic acid) is the first histone deacetylase inhibitor (HDACi) to be approved for clinical use as an anticancer agent. Given that histone acetylation is a key determinant of chromatin structure, we investigated how SAHA may affect DNA replication and integrity to gain deeper insights into the basis for its anticancer activity. Nuclear replication factories were visualized with confocal immunofluorescence microscopy and with single-replicon analyses conducted by genome-wide molecular combing after pulse labeling with two thymidine-analogues. Additionally, nascent strand real-time polymerase chain reaction (RT-PCR) in the human β-globin locus was used to assess the effects of SAHA on replication fork origin firing. We found that pharmacological concentrations of SAHA induce replication-mediated DNA damage, on the basis of single-cell and single-DNA molecule analyses. Molecular combing indicated slowdown in replication speed along with activation of dormant replication origins in response to SAHA. Similar results were obtained using siRNA-mediated depletion of HDAC3 expression, implicating this HDAC member as a likely target in the SAHA response. Activation of dormant origins was confirmed by molecular analyses of the β-globin locus control region. Our findings indicate that SAHA produces profound alterations in DNA replication that cause DNA damage, establishing a critical link between robust chromatin acetylation and DNA replication in human cancer cells. PMID:20460513

  8. RecO acts with RecF and RecR to protect and maintain replication forks blocked by UV-induced DNA damage in Escherichia coli.

    PubMed

    Chow, Kin-Hoe; Courcelle, Justin

    2004-01-30

    In Escherichia coli, recF and recR are required to stabilize and maintain replication forks arrested by UV-induced DNA damage. In the absence of RecF, replication fails to recover, and the nascent lagging strand of the arrested replication fork is extensively degraded by the RecQ helicase and RecJ nuclease. recO mutants are epistatic with recF and recR with respect to recombination and survival assays after DNA damage. In this study, we show that RecO functions with RecF and RecR to protect the nascent lagging strand of arrested replication forks after UV-irradiation. In the absence of RecO, the nascent DNA at arrested replication forks is extensively degraded and replication fails to recover. The extent of nascent DNA degradation is equivalent in single, double, or triple mutants of recF, recO, or recR, and the degradation is dependent upon RecJ and RecQ functions. Because RecF has been shown to protect the nascent lagging strand from degradation, these observations indicate that RecR and RecO function with RecF to protect the same nascent strand of the arrested replication fork and are likely to act at a common point during the recovery process. We discuss these results in relation to the biochemical and cellular properties of RecF, RecO, and RecR and their potential role in loading RecA filaments to maintain the replication fork structure after the arrest of replication by UV-induced DNA damage. PMID:14625283

  9. 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.

  10. DNA polymerase IV mediates efficient and quick recovery of replication forks stalled at N2-dG adducts

    PubMed Central

    Ikeda, Mio; Furukohri, Asako; Philippin, Gaelle; Loechler, Edward; Akiyama, Masahiro Tatsumi; Katayama, Tsutomu; Fuchs, Robert P.; Maki, Hisaji

    2014-01-01

    Escherichia coli DNA polymerase IV (Pol IV, also known as DinB) is a Y-family DNA polymerase capable of catalyzing translesion DNA synthesis (TLS) on certain DNA lesions, and accumulating data suggest that Pol IV may play an important role in copying various kinds of spontaneous DNA damage including N2-dG adducts and alkylated bases. Pol IV has a unique ability to coexist with Pol III on the same β clamp and to positively dissociate Pol III from β clamp in a concentration-dependent manner. Reconstituting the entire process of TLS in vitro using E. coli replication machinery and Pol IV, we observed that a replication fork stalled at (−)-trans-anti-benzo[a]pyrene-N2-dG lesion on the leading strand was efficiently and quickly recovered via two sequential switches from Pol III to Pol IV and back to Pol III. Our results suggest that TLS by Pol IV smoothes the way for the replication fork with minimal interruption. PMID:24957605

  11. Tandem repeat recombination induced by replication fork defects in Escherichia coli requires a novel factor, RadC.

    PubMed

    Saveson, C J; Lovett, S T

    1999-05-01

    DnaB is the helicase associated with the DNA polymerase III replication fork in Escherichia coli. Previously we observed that the dnaB107(ts) mutation, at its permissive temperature, greatly stimulated deletion events at chromosomal tandem repeats. This stimulation required recA, which suggests a recombinational mechanism. In this article we examine the genetic dependence of recombination stimulated by the dnaB107 mutation. Gap repair genes recF, recO, and recR were not required. Mutations in recB, required for double-strand break repair, and in ruvC, the Holliday junction resolvase gene, were synthetically lethal with dnaB107, causing enhanced temperature sensitivity. The hyperdeletion phenotype of dnaB107 was semidominant, and in dnaB107/dnaB+ heterozygotes recB was partially required for enhanced deletion, whereas ruvC was not. We believe that dnaB107 causes the stalling of replication forks, which may become broken and require repair. Misalignment of repeated sequences during RecBCD-mediated repair may account for most, but not all, of deletion stimulated by dnaB107. To our surprise, the radC gene, like recA, was required for virtually all recombination stimulated by dnaB107. The biochemical function of RadC is unknown, but is reported to be required for growth-medium-dependent repair of DNA strand breaks. Our results suggest that RadC functions specifically in recombinational repair that is associated with the replication fork. PMID:10224240

  12. Transcription Termination Factor reb1p Causes Two Replication Fork Barriers at Its Cognate Sites in Fission Yeast Ribosomal DNA In Vivo

    PubMed Central

    Sánchez-Gorostiaga, Alicia; López-Estraño, Carlos; Krimer, Dora B.; Schvartzman, Jorge B.; Hernández, Pablo

    2004-01-01

    Polar replication fork barriers (RFBs) near the 3′ end of the rRNA transcriptional unit are a conserved feature of ribosomal DNA (rDNA) replication in eukaryotes. In the mouse, in vivo studies indicate that the cis-acting Sal boxes required for rRNA transcription termination are also involved in replication fork blockage. On the contrary, in the budding yeast Saccharomyces cerevisiae, the rRNA transcription termination factors are not required for RFBs. Here we characterized the rDNA RFBs in the fission yeast Schizosaccharomyces pombe. S. pombe rDNA contains three closely spaced polar replication barriers named RFB1, RFB2, and RFB3 in the 3′ to 5′ order. The transcription termination protein reb1 and its two binding sites, present at the 3′ end of the coding region, were required for fork arrest at RFB2 and RFB3 in vivo. On the other hand, fork arrest at the strongest RFB1 barrier was independent of the above transcription termination factors. Therefore, RFB2 and RFB3 resemble the barriers present in the mouse rDNA, whereas RFB1 is similar to the budding yeast RFBs. These results suggest that during evolution, cis- and trans-acting factors required for rRNA transcription termination became involved in replication fork blockage also. S. pombe is suggested to be a transitional species in which both mechanisms coexist. PMID:14673172

  13. Deletion of the Tetrahymena thermophila rDNA replication fork barrier region disrupts macronuclear rDNA excision and creates a fragile site in the micronuclear genome.

    PubMed

    Yakisich, J S; Kapler, G M

    2006-01-01

    During macronuclear development the Tetrahymena thermophila ribosomal RNA gene is excised from micronuclear chromosome 1 by site-specific cleavage at chromosome breakage sequence (Cbs) elements, rearranged into a 'palindromic' 21 kb minichromosome and extensively amplified. Gene amplification initiates from origins in the 5' non-transcribed spacer, and forks moving toward the center of the palindrome arrest at a developmentally regulated replication fork barrier (RFB). The RFB is inactive during vegetative cell divisions, suggesting a role in the formation or amplification of macronuclear rDNA. Using micronuclear (germline) transformation, we show that the RFB region facilitates Cbs-mediated excision. Deletion of the RFB inhibits chromosome breakage in a sub-population of developing macronuclei and promotes alternative processing by a Cbs-independent mechanism. Remarkably, the RFB region prevents spontaneous breakage of chromosome 1 in the diploid micronucleus. Strains heterozygous for DeltaRFB and wild-type rDNA lose the DeltaRFB allele and distal left arm of chromosome 1 during vegetative propagation. The wild-type chromosome is subsequently fragmented near the rDNA locus, and both homologs are progressively eroded, suggesting that broken micronuclear chromosomes are not 'healed' by telomerase. Deletion of this 363 bp segment effectively creates a fragile site in the micronuclear genome, providing the first evidence for a non-telomere cis-acting determinant that functions to maintain the structural integrity of a mitotic eukaryotic chromosome.

  14. 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.

  15. 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

  16. The major roles of DNA polymerases epsilon and delta at the eukaryotic replication fork are evolutionarily conserved.

    PubMed

    Miyabe, Izumi; Kunkel, Thomas A; Carr, Antony M

    2011-12-01

    Coordinated replication of eukaryotic genomes is intrinsically asymmetric, with continuous leading strand synthesis preceding discontinuous lagging strand synthesis. Here we provide two types of evidence indicating that, in fission yeast, these two biosynthetic tasks are performed by two different replicases. First, in Schizosaccharomyces pombe strains encoding a polδ-L591M mutator allele, base substitutions in reporter genes placed in opposite orientations relative to a well-characterized replication origin are strand-specific and distributed in patterns implying that Polδ is primarily involved in lagging strand replication. Second, in strains encoding a polε-M630F allele and lacking the ability to repair rNMPs in DNA due to a defect in RNase H2, rNMPs are selectively observed in nascent leading strand DNA. The latter observation demonstrates that abundant rNMP incorporation during replication can be tolerated and that they are normally removed in an RNase H2-dependent manner. This provides strong physical evidence that Polε is the primary leading strand replicase. Collectively, these data and earlier results in budding yeast indicate that the major roles of Polδ and Polε at the eukaryotic replication fork are evolutionarily conserved.

  17. ATR checkpoint kinase and CRL1βTRCP collaborate to degrade ASF1a and thus repress genes overlapping with clusters of stalled replication forks.

    PubMed

    Im, Jun-Sub; Keaton, Mignon; Lee, Kyung Yong; Kumar, Pankaj; Park, Jonghoon; Dutta, Anindya

    2014-04-15

    Many agents used for chemotherapy, such as doxorubicin, interfere with DNA replication, but the effect of this interference on transcription is largely unknown. Here we show that doxorubicin induces the firing of dense clusters of neoreplication origins that lead to clusters of stalled replication forks in gene-rich parts of the genome, particularly on expressed genes. Genes that overlap with these clusters of stalled forks are actively dechromatinized, unwound, and repressed by an ATR-dependent checkpoint pathway. The ATR checkpoint pathway causes a histone chaperone normally associated with the replication fork, ASF1a, to degrade through a CRL1(βTRCP)-dependent ubiquitination/proteasome pathway, leading to the localized dechromatinization and gene repression. Therefore, a globally active checkpoint pathway interacts with local clusters of stalled forks to specifically repress genes in the vicinity of the stalled forks, providing a new mechanism of action of chemotherapy drugs like doxorubicin. Finally, ASF1a-depleted cancer cells are more sensitive to doxorubicin, suggesting that the 7%-10% of prostate adenocarcinomas and adenoid cystic carcinomas reported to have homozygous deletion or significant underexpression of ASF1a should be tested for high sensitivity to doxorubicin.

  18. Nuclear DNA synthesis in vitro is mediated via stable replication forks assembled in a temporally specific fashion in vivo.

    PubMed Central

    Heintz, N H; Stillman, B W

    1988-01-01

    A cell-free nuclear replication system that is S-phase specific, that requires the activity of DNA polymerase alpha, and that is stimulated three- to eightfold by cytoplasmic factors from S-phase cells was used to examine the temporal specificity of chromosomal DNA synthesis in vitro. Temporal specificity of DNA synthesis in isolated nuclei was assessed directly by examining the replication of restriction fragments derived from the amplified 200-kilobase dihydrofolate reductase domain of methotrexate-resistant CHOC 400 cells as a function of the cell cycle. In nuclei prepared from cells collected at the G1/S boundary of the cell cycle, synthesis of amplified sequences commenced within the immediate dihydrofolate reductase origin region and elongation continued for 60 to 80 min. The order of synthesis of amplified restriction fragments in nuclei from early S-phase cells in vitro appeared to be indistinguishable from that in vivo. Nuclei prepared from CHOC 400 cells poised at later times in the S phase synthesized characteristic subsets of other amplified fragments. The specificity of fragment labeling patterns was stable to short-term storage at 4 degrees C. The occurrence of stimulatory factors in cytosol extracts was cell cycle dependent in that minimal stimulation was observed with early G1-phase extracts, whereas maximal stimulation was observed with cytosol extracts from S-phase cells. Chromosomal synthesis was not observed in nuclei from G1 cells, nor did cytosol extracts from S-phase cells induce chromosomal replication in G1 nuclei. In contrast to chromosomal DNA synthesis, mitochondrial DNA replication in vitro was not stimulated by cytoplasmic factors and occurred at equivalent rates throughout the G1 and S phases. These studies show that chromosomal DNA replication in isolated nuclei is mediated by stable replication forks that are assembled in a temporally specific fashion in vivo and indicate that the synthetic mechanisms observed in vitro accurately

  19. ATM and the Mre11-Rad50-Nbs1 complex respond to nucleoside analogue-induced stalled replications forks and contribute to drug resistance

    PubMed Central

    Ewald, Brett; Sampath, Deepa; Plunkett, William

    2008-01-01

    The Mre11-Rad50-Nbs1 complex and autophosphorylated Ser1981-ATM are involved in recognizing and repairing DNA damage, such as double-strand breaks (DSBs). However, the role of these factors in response to stalled replication forks is not clear. Nucleoside analogues are agents that are incorporated into DNA during replication, which cause stalling of replication forks. The molecular mechanisms that sense these events may signal for DNA repair and contribute to survival, but are poorly understood. Cellular responses to both DSBs and stalled replication forks are marked by H2AX phosphorylation on Ser139 (γ-H2AX), which forms nuclear foci at sites of DNA damage. Here, concentrations of the nucleoside analogues, ara-C, gemcitabine, and troxacitabine, that inhibited DNA synthesis by 90% within 2 h were determined for each agent. Using γ-H2AX as a marker for changes in chromatin structure, we demonstrate that Mre11, Rad50, Nbs1 and phosphorylated ATM respond to nucleoside analogue-induced stalled replication forks by forming nuclear foci that co-localize with γ-H2AX within 2 h. Since neither DSBs nor single-strand breaks were detectable after nucleoside analogue exposure, we conclude that this molecular response is not due to the presence of DNA breaks. Deficiencies in ATM, Mre11, or Rad50 led to a two- to five-fold increase in clonogenic sensitization to gemcitabine, while Nbs1 and H2AX deficiency did not effect reproductive growth. Taken together, these results suggest that ATM, Mre11, and Rad50 are required for survival after replication fork stalling, whereas Nbs1 and H2AX are in-consequential. PMID:18829552

  20. RecG and UvsW catalyse robust DNA rewinding critical for stalled DNA replication fork rescue

    PubMed Central

    Manosas, Maria; Perumal, Senthil K.; Bianco, Piero; Ritort, Felix; Benkovic, Stephen J.; Croquette, Vincent

    2013-01-01

    Helicases that both unwind and rewind DNA have central roles in DNA repair and genetic recombination. In contrast to unwinding, DNA rewinding by helicases has proved difficult to characterize biochemically because of its thermodynamically downhill nature. Here we use single-molecule assays to mechanically destabilize a DNA molecule and follow, in real time, unwinding and rewinding by two DNA repair helicases, bacteriophage T4 UvsW and Escherichia coli RecG. We find that both enzymes are robust rewinding enzymes, which can work against opposing forces as large as 35 pN, revealing their active character. The generation of work during the rewinding reaction allows them to couple rewinding to DNA unwinding and/or protein displacement reactions central to the rescue of stalled DNA replication forks. The overall results support a general mechanism for monomeric rewinding enzymes. PMID:24013402

  1. Breast cancer proteins PALB2 and BRCA2 stimulate polymerase η in recombination-associated DNA synthesis at blocked replication forks.

    PubMed

    Buisson, Rémi; Niraj, Joshi; Pauty, Joris; Maity, Ranjan; Zhao, Weixing; Coulombe, Yan; Sung, Patrick; Masson, Jean-Yves

    2014-02-13

    One envisioned function of homologous recombination (HR) is to find a template for DNA synthesis from the resected 3'-OH molecules that occur during double-strand break (DSB) repair at collapsed replication forks. However, the interplay between DNA synthesis and HR remains poorly understood in higher eukaryotic cells. Here, we reveal functions for the breast cancer proteins BRCA2 and PALB2 at blocked replication forks and show a role for these proteins in stimulating polymerase η (Polη) to initiate DNA synthesis. PALB2, BRCA2, and Polη colocalize at stalled or collapsed replication forks after hydroxyurea treatment. Moreover, PALB2 and BRCA2 interact with Polη and are required to sustain the recruitment of Polη at blocked replication forks. PALB2 and BRCA2 stimulate Polη-dependent DNA synthesis on D loop substrates. We conclude that PALB2 and BRCA2, in addition to their functions in D loop formation, play crucial roles in the initiation of recombination-associated DNA synthesis by Polη-mediated DNA repair.

  2. Inactivation of the DnaB helicase leads to the collapse and degradation of the replication fork: a comparison to UV-induced arrest.

    PubMed

    Belle, Jerilyn J; Casey, Andrew; Courcelle, Charmain T; Courcelle, Justin

    2007-08-01

    Replication forks face a variety of structurally diverse impediments that can prevent them from completing their task. The mechanism by which cells overcome these hurdles is likely to vary depending on the nature of the obstacle and the strand in which the impediment is encountered. Both UV-induced DNA damage and thermosensitive replication proteins have been used in model systems to inhibit DNA replication and characterize the mechanism by which it recovers. In this study, we examined the molecular events that occur at replication forks following inactivation of a thermosensitive DnaB helicase and found that they are distinct from those that occur following arrest at UV-induced DNA damage. Following UV-induced DNA damage, the integrity of replication forks is maintained and protected from extensive degradation by RecA, RecF, RecO, and RecR until replication can resume. By contrast, inactivation of DnaB results in extensive degradation of the nascent and leading-strand template DNA and a loss of replication fork integrity as monitored by two-dimensional agarose gel analysis. The degradation that occurs following DnaB inactivation partially depends on several genes, including recF, recO, recR, recJ, recG, and xonA. Furthermore, the thermosensitive DnaB allele prevents UV-induced DNA degradation from occurring following arrest even at the permissive temperature, suggesting a role for DnaB prior to loading of the RecFOR proteins. We discuss these observations in relation to potential models for both UV-induced and DnaB(Ts)-mediated replication inhibition.

  3. Inactivation of the DnaB helicase leads to the collapse and degradation of the replication fork: a comparison to UV-induced arrest.

    PubMed

    Belle, Jerilyn J; Casey, Andrew; Courcelle, Charmain T; Courcelle, Justin

    2007-08-01

    Replication forks face a variety of structurally diverse impediments that can prevent them from completing their task. The mechanism by which cells overcome these hurdles is likely to vary depending on the nature of the obstacle and the strand in which the impediment is encountered. Both UV-induced DNA damage and thermosensitive replication proteins have been used in model systems to inhibit DNA replication and characterize the mechanism by which it recovers. In this study, we examined the molecular events that occur at replication forks following inactivation of a thermosensitive DnaB helicase and found that they are distinct from those that occur following arrest at UV-induced DNA damage. Following UV-induced DNA damage, the integrity of replication forks is maintained and protected from extensive degradation by RecA, RecF, RecO, and RecR until replication can resume. By contrast, inactivation of DnaB results in extensive degradation of the nascent and leading-strand template DNA and a loss of replication fork integrity as monitored by two-dimensional agarose gel analysis. The degradation that occurs following DnaB inactivation partially depends on several genes, including recF, recO, recR, recJ, recG, and xonA. Furthermore, the thermosensitive DnaB allele prevents UV-induced DNA degradation from occurring following arrest even at the permissive temperature, suggesting a role for DnaB prior to loading of the RecFOR proteins. We discuss these observations in relation to potential models for both UV-induced and DnaB(Ts)-mediated replication inhibition. PMID:17526695

  4. Spi-1/PU.1 oncogene accelerates DNA replication fork elongation and promotes genetic instability in the absence of DNA breakage.

    PubMed

    Rimmelé, Pauline; Komatsu, Jun; Hupé, Philippe; Roulin, Christophe; Barillot, Emmanuel; Dutreix, Marie; Conseiller, Emmanuel; Bensimon, Aaron; Moreau-Gachelin, Françoise; Guillouf, Christel

    2010-09-01

    The multistage process of cancer formation is driven by the progressive acquisition of somatic mutations. Replication stress creates genomic instability in mammals. Using a well-defined multistep leukemia model driven by Spi-1/PU.1 overexpression in the mouse and Spi-1/PU.1-overexpressing human leukemic cells, we investigated the relationship between DNA replication and cancer progression. Here, using DNA molecular combing and flow cytometry methods, we show that Spi-1 increases the speed of replication by acting specifically on elongation rather than enhancing origin firing. This shortens the S-phase duration. Combining data from Spi-1 knockdown in murine cells with Spi-1 overexpression in human cells, we provide evidence that inappropriate Spi-1 expression is directly responsible for the replication alteration observed. Importantly, the acceleration of replication progression coincides with an increase in the frequency of genomic mutations without inducing DNA breakage. Thus, we propose that the hitherto unsuspected role for spi-1 oncogene in promoting replication elongation and genomic mutation promotes blastic progression during leukemic development.

  5. Degradation of p12 subunit by CRL4Cdt2 E3 ligase inhibits fork progression after DNA damage.

    PubMed

    Terai, Kenta; Shibata, Etsuko; Abbas, Tarek; Dutta, Anindya

    2013-10-18

    After acute DNA damage, the cell arrests S-phase progression by inhibiting origin initiation and fork progression to repair damaged DNA. The intra-S-phase checkpoint kinase Chk1 phosphorylates Cdc25A to target the latter for degradation by CRL1(β-TrCP) and so inhibit origin firing. The mechanism for inhibiting fork progression, however, has not been identified. Here, we show that degradation of p12, the fourth subunit of DNA polymerase δ, is critical for inhibiting fork progression. CRL4(Cdt2) is an E3 ligase that ubiquitinates and degrades p12 after UV treatment. Cells expressing a stable form of p12 exhibit UV-resistant DNA synthesis. DNA fiber assay and alkaline-sucrose gradient assay demonstrate that the impairment of fork progression after DNA damage requires p12 degradation. These results suggest that ubiquitination of p12 through CRL4(Cdt2) and subsequent degradation form one mechanism by which a cell responds to DNA damage to inhibit fork progression.

  6. The Slx4-Dpb11 scaffold complex: coordinating the response to replication fork stalling in S-phase and the subsequent mitosis.

    PubMed

    Princz, Lissa N; Gritenaite, Dalia; Pfander, Boris

    2015-01-01

    Replication fork stalling at DNA lesions is a common problem during the process of DNA replication. One way to allow the bypass of these lesions is via specific recombination-based mechanisms that involve switching of the replication template to the sister chromatid. Inherent to these mechanisms is the formation of DNA joint molecules (JMs) between sister chromatids. Such JMs need to be disentangled before chromatid separation in mitosis and the activity of JM resolution enzymes, which is under stringent cell cycle control, is therefore up-regulated in mitosis. An additional layer of control is facilitated by scaffold proteins. In budding yeast, specifically during mitosis, Slx4 and Dpb11 form a cell cycle kinase-dependent complex with the Mus81-Mms4 structure-selective endonuclease, which allows efficient JM resolution by Mus81. Furthermore, Slx4 and Dpb11 interact even prior to joining Mus81 and respond to replication fork stalling in S-phase. This S-phase complex is involved in the regulation of the DNA damage checkpoint as well as in early steps of template switch recombination. Similar interactions and regulatory principles are found in human cells suggesting that Slx4 and Dpb11 may have an evolutionary conserved role organizing the cellular response to replication fork stalling.

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

    PubMed

    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.

  8. 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

  9. During replication stress, non-SMC element 5 (NSE5) is required for Smc5/6 protein complex functionality at stalled forks.

    PubMed

    Bustard, Denise E; Menolfi, Demis; Jeppsson, Kristian; Ball, Lindsay G; Dewey, Sidney Carter; Shirahige, Katsuhiko; Sjögren, Camilla; Branzei, Dana; Cobb, Jennifer A

    2012-03-30

    The Smc5/6 complex belongs to the SMC (structural maintenance of chromosomes) family, which also includes cohesin and condensin. In Saccharomyces cerevisiae, the Smc5/6 complex contains six essential non-Smc elements, Nse1-6. Very little is known about how these additional elements contribute to complex function except for Nse2/Mms21, which is an E3 small ubiquitin-like modifier (SUMO) ligase important for Smc5 sumoylation. Characterization of two temperature-sensitive mutants, nse5-ts1 and nse5-ts2, demonstrated the importance of Nse5 within the Smc5/6 complex for its stability and functionality at forks during hydroxyurea-induced replication stress. Both NSE5 alleles showed a marked reduction in Smc5 sumoylation to levels lower than those observed with mms21-11, a mutant of Mms21 that is deficient in SUMO ligase activity. However, a phenotypic comparison of nse5-ts1 and nse5-ts2 revealed a separation of importance between Smc5 sumoylation and the function of the Smc5/6 complex during replication. Only cells carrying the nse5-ts1 allele exhibited defects such as dissociation of the replisome from stalled forks, formation of fork-associated homologous recombination intermediates, and hydroxyurea sensitivity that is additive with mms21-11. These defects are attributed to a failure in Smc5/6 localization to forks in nse5-ts1 cells. Overall, these data support the premise that Nse5 is important for vital interactions between components within the Smc5/6 complex, and for its functionality during replication stress. PMID:22303010

  10. RecO and RecR are necessary for RecA loading in response to DNA damage and replication fork stress.

    PubMed

    Lenhart, Justin S; Brandes, Eileen R; Schroeder, Jeremy W; Sorenson, Roderick J; Showalter, Hollis D; Simmons, Lyle A

    2014-08-01

    RecA is central to maintaining genome integrity in bacterial cells. Despite the near-ubiquitous conservation of RecA in eubacteria, the pathways that facilitate RecA loading and repair center assembly have remained poorly understood in Bacillus subtilis. Here, we show that RecA rapidly colocalizes with the DNA polymerase complex (replisome) immediately following DNA damage or damage-independent replication fork arrest. In Escherichia coli, the RecFOR and RecBCD pathways serve to load RecA and the choice between these two pathways depends on the type of damage under repair. We found in B. subtilis that the rapid localization of RecA to repair centers is strictly dependent on RecO and RecR in response to all types of damage examined, including a site-specific double-stranded break and damage-independent replication fork arrest. Furthermore, we provide evidence that, although RecF is not required for RecA repair center formation in vivo, RecF does increase the efficiency of repair center assembly, suggesting that RecF may influence the initial stages of RecA nucleation or filament extension. We further identify single-stranded DNA binding protein (SSB) as an additional component important for RecA repair center assembly. Truncation of the SSB C terminus impairs the ability of B. subtilis to form repair centers in response to damage and damage-independent fork arrest. With these results, we conclude that the SSB-dependent recruitment of RecOR to the replisome is necessary for loading and organizing RecA into repair centers in response to DNA damage and replication fork arrest. PMID:24891441

  11. During replication stress, non-SMC element 5 (NSE5) is required for Smc5/6 protein complex functionality at stalled forks.

    PubMed

    Bustard, Denise E; Menolfi, Demis; Jeppsson, Kristian; Ball, Lindsay G; Dewey, Sidney Carter; Shirahige, Katsuhiko; Sjögren, Camilla; Branzei, Dana; Cobb, Jennifer A

    2012-03-30

    The Smc5/6 complex belongs to the SMC (structural maintenance of chromosomes) family, which also includes cohesin and condensin. In Saccharomyces cerevisiae, the Smc5/6 complex contains six essential non-Smc elements, Nse1-6. Very little is known about how these additional elements contribute to complex function except for Nse2/Mms21, which is an E3 small ubiquitin-like modifier (SUMO) ligase important for Smc5 sumoylation. Characterization of two temperature-sensitive mutants, nse5-ts1 and nse5-ts2, demonstrated the importance of Nse5 within the Smc5/6 complex for its stability and functionality at forks during hydroxyurea-induced replication stress. Both NSE5 alleles showed a marked reduction in Smc5 sumoylation to levels lower than those observed with mms21-11, a mutant of Mms21 that is deficient in SUMO ligase activity. However, a phenotypic comparison of nse5-ts1 and nse5-ts2 revealed a separation of importance between Smc5 sumoylation and the function of the Smc5/6 complex during replication. Only cells carrying the nse5-ts1 allele exhibited defects such as dissociation of the replisome from stalled forks, formation of fork-associated homologous recombination intermediates, and hydroxyurea sensitivity that is additive with mms21-11. These defects are attributed to a failure in Smc5/6 localization to forks in nse5-ts1 cells. Overall, these data support the premise that Nse5 is important for vital interactions between components within the Smc5/6 complex, and for its functionality during replication stress.

  12. RecO and RecR Are Necessary for RecA Loading in Response to DNA Damage and Replication Fork Stress

    PubMed Central

    Lenhart, Justin S.; Brandes, Eileen R.; Schroeder, Jeremy W.; Sorenson, Roderick J.; Showalter, Hollis D.

    2014-01-01

    RecA is central to maintaining genome integrity in bacterial cells. Despite the near-ubiquitous conservation of RecA in eubacteria, the pathways that facilitate RecA loading and repair center assembly have remained poorly understood in Bacillus subtilis. Here, we show that RecA rapidly colocalizes with the DNA polymerase complex (replisome) immediately following DNA damage or damage-independent replication fork arrest. In Escherichia coli, the RecFOR and RecBCD pathways serve to load RecA and the choice between these two pathways depends on the type of damage under repair. We found in B. subtilis that the rapid localization of RecA to repair centers is strictly dependent on RecO and RecR in response to all types of damage examined, including a site-specific double-stranded break and damage-independent replication fork arrest. Furthermore, we provide evidence that, although RecF is not required for RecA repair center formation in vivo, RecF does increase the efficiency of repair center assembly, suggesting that RecF may influence the initial stages of RecA nucleation or filament extension. We further identify single-stranded DNA binding protein (SSB) as an additional component important for RecA repair center assembly. Truncation of the SSB C terminus impairs the ability of B. subtilis to form repair centers in response to damage and damage-independent fork arrest. With these results, we conclude that the SSB-dependent recruitment of RecOR to the replisome is necessary for loading and organizing RecA into repair centers in response to DNA damage and replication fork arrest. PMID:24891441

  13. Reversible Top1 cleavage complexes are stabilized strand-specifically at the ribosomal replication fork barrier and contribute to ribosomal DNA stability.

    PubMed

    Krawczyk, Claudia; Dion, Vincent; Schär, Primo; Fritsch, Olivier

    2014-04-01

    Various topological constraints at the ribosomal DNA (rDNA) locus impose an extra challenge for transcription and DNA replication, generating constant torsional DNA stress. The topoisomerase Top1 is known to release such torsion by single-strand nicking and re-ligation in a process involving transient covalent Top1 cleavage complexes (Top1cc) with the nicked DNA. Here we show that Top1ccs, despite their usually transient nature, are specifically targeted to and stabilized at the ribosomal replication fork barrier (rRFB) of budding yeast, establishing a link with previously reported Top1 controlled nicks. Using ectopically engineered rRFBs, we establish that the rRFB sequence itself is sufficient for induction of DNA strand-specific and replication-independent Top1ccs. These Top1ccs accumulate only in the presence of Fob1 and Tof2, they are reversible as they are not subject to repair by Tdp1- or Mus81-dependent processes, and their presence correlates with Top1 provided rDNA stability. Notably, the targeted formation of these Top1ccs accounts for the previously reported broken replication forks at the rRFB. These findings implicate a novel and physiologically regulated mode of Top1 action, suggesting a mechanism by which Top1 is recruited to the rRFB and stabilized in a reversible Top1cc configuration to preserve the integrity of the rDNA.

  14. 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

  15. FANC pathway promotes UV-induced stalled replication forks recovery by acting both upstream and downstream Polη and Rev1.

    PubMed

    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.

  16. 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

  17. 3D replicon distributions arise from stochastic initiation and domino-like DNA replication progression.

    PubMed

    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.

  18. The C-terminal domain of the bacterial SSB protein acts as a DNA maintenance hub at active chromosome replication forks.

    PubMed

    Costes, Audrey; Lecointe, François; McGovern, Stephen; Quevillon-Cheruel, Sophie; Polard, Patrice

    2010-01-01

    We have investigated in vivo the role of the carboxy-terminal domain of the Bacillus subtilis Single-Stranded DNA Binding protein (SSB(Cter)) as a recruitment platform at active chromosomal forks for many proteins of the genome maintenance machineries. We probed this SSB(Cter) interactome using GFP fusions and by Tap-tag and biochemical analysis. It includes at least 12 proteins. The interactome was previously shown to include PriA, RecG, and RecQ and extended in this study by addition of DnaE, SbcC, RarA, RecJ, RecO, XseA, Ung, YpbB, and YrrC. Targeting of YpbB to active forks appears to depend on RecS, a RecQ paralogue, with which it forms a stable complex. Most of these SSB partners are conserved in bacteria, while others, such as the essential DNA polymerase DnaE, YrrC, and the YpbB/RecS complex, appear to be specific to B. subtilis. SSB(Cter) deletion has a moderate impact on B. subtilis cell growth. However, it markedly affects the efficiency of repair of damaged genomic DNA and arrested replication forks. ssbΔCter mutant cells appear deficient in RecA loading on ssDNA, explaining their inefficiency in triggering the SOS response upon exposure to genotoxic agents. Together, our findings show that the bacterial SSB(Cter) acts as a DNA maintenance hub at active chromosomal forks that secures their propagation along the genome. PMID:21170359

  19. 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.

  20. Dynamics of Escherichia coli chromosome segregation during multifork replication.

    PubMed

    Nielsen, Henrik J; Youngren, Brenda; Hansen, Flemming G; Austin, Stuart

    2007-12-01

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

  1. 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

  2. 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

  3. 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).

  4. Hyperactive Cdc2 kinase interferes with the response to broken replication forks by trapping S.pombe Crb2 in its mitotic T215 phosphorylated state.

    PubMed

    Mahyous Saeyd, Salah Adam; Ewert-Krzemieniewska, Katarzyna; Liu, Boyin; Caspari, Thomas

    2014-07-01

    Although it is well established that Cdc2 kinase phosphorylates the DNA damage checkpoint protein Crb2(53BP1) in mitosis, the full impact of this modification is still unclear. The Tudor-BRCT domain protein Crb2 binds to modified histones at DNA lesions to mediate the activation of Chk1 by Rad3ATR kinase. We demonstrate here that fission yeast cells harbouring a hyperactive Cdc2CDK1 mutation (cdc2.1w) are specifically sensitive to the topoisomerase 1 inhibitor camptothecin (CPT) which breaks DNA replication forks. Unlike wild-type cells, which delay only briefly in CPT medium by activating Chk1 kinase, cdc2.1w cells bypass Chk1 to enter an extended cell-cycle arrest which depends on Cds1 kinase. Intriguingly, the ability to bypass Chk1 requires the mitotic Cdc2 phosphorylation site Crb2-T215. This implies that the presence of the mitotic phosphorylation at Crb2-T215 channels Rad3 activity towards Cds1 instead of Chk1 when forks break in S phase. We also provide evidence that hyperactive Cdc2.1w locks cells in a G1-like DNA repair mode which favours non-homologous end joining over interchromosomal recombination. Taken together, our data support a model such that elevated Cdc2 activity delays the transition of Crb2 from its G1 to its G2 mode by blocking Srs2 DNA helicase and Casein Kinase 1 (Hhp1).

  5. Dpb11 protein helps control assembly of the Cdc45·Mcm2-7·GINS replication fork helicase.

    PubMed

    Dhingra, Nalini; Bruck, Irina; Smith, Skye; Ning, Boting; Kaplan, Daniel L

    2015-03-20

    Dpb11 is required for the initiation of DNA replication in budding yeast. Dpb11 binds to S-phase cyclin-dependent kinase-phosphorylated Sld2 and Sld3 to form a ternary complex during S phase. The replication fork helicase in eukaryotes is composed of Cdc45, Mcm2-7, and GINS. We show here, using purified proteins from budding yeast, that Dpb11 alone binds to Mcm2-7 and that Dpb11 also competes with GINS for binding to Mcm2-7. Furthermore, Dpb11 binds directly to single-stranded DNA (ssDNA), and ssDNA inhibits the Dpb11 interaction with Mcm2-7. We also found that Dpb11 can recruit Cdc45 to Mcm2-7. We identified a mutant of the BRCT4 motif of Dpb11 that remains bound to Mcm2-7 in the presence of ssDNA (dpb11-m1,m2,m3,m5), and this mutant exhibits a DNA replication defect when expressed in budding yeast cells. Expression of this mutant results in increased interaction between Dpb11 and Mcm2-7 during S phase, impaired GINS interaction with Mcm2-7 during S phase, and decreased replication protein A (RPA) interaction with origin DNA during S phase. We propose a model in which Dpb11 first recruits Cdc45 to Mcm2-7. Dpb11, although bound to Cdc45·Mcm2-7, can block the interaction between GINS and Mcm2-7. Upon extrusion of ssDNA from the central channel of Mcm2-7, Dpb11 dissociates from Mcm2-7, and Dpb11 binds to ssDNA, thereby allowing GINS to bind to Cdc45·Mcm2-7. Finally, we propose that Dpb11 functions with Sld2 and Sld3 to help control the assembly of the replication fork helicase.

  6. 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

  7. Dimerization of the ATRIP protein through the coiled-coil motif and its implication to the maintenance of stalled replication forks.

    PubMed

    Itakura, Eisuke; Sawada, Isao; Matsuura, Akira

    2005-12-01

    ATR (ATM and Rad3-related), a PI kinase-related kinase (PIKK), has been implicated in the DNA structure checkpoint in mammalian cells. ATR associates with its partner protein ATRIP to form a functional complex in the nucleus. In this study, we investigated the role of the ATRIP coiled-coil domain in ATR-mediated processes. The coiled-coil domain of human ATRIP contributes to self-dimerization in vivo, which is important for the stable translocation of the ATR-ATRIP complex to nuclear foci that are formed after exposure to genotoxic stress. The expression of dimerization-defective ATRIP diminishes the maintenance of replication forks during treatment with replication inhibitors. By contrast, it does not compromise the G2/M checkpoint after IR-induced DNA damage. These results show that there are two critical functions of ATR-ATRIP after the exposure to genotoxic stress: maintenance of the integrity of replication machinery and execution of cell cycle arrest, which are separable and are achieved via distinct mechanisms. The former function may involve the concentrated localization of ATR to damaged sites for which the ATRIP coiled-coil motif is critical.

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

    PubMed

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

    2015-11-15

    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.

  9. Recent Progress in Understanding Coxsackievirus Replication, Dissemination, and Pathogenesis

    PubMed Central

    Sin, Jon; Mangale, Vrushali; Thienphrapa, Wdee; Gottlieb, Roberta A.; Feuer, Ralph

    2015-01-01

    Coxsackieviruses (CVs) are relatively common viruses associated with a number of serious human diseases, including myocarditis and meningo-encephalitis. These viruses are considered cytolytic yet can persist for extended periods of time within certain host tissues requiring evasion from the host immune response and a greatly reduced rate of replication. A member of Picornaviridae family, CVs have been historically considered non-enveloped viruses – although recent evidence suggest that CV and other picornaviruses hijack host membranes and acquire an envelope. Acquisition of an envelope might provide distinct benefits to CV virions, such as resistance to neutralizing antibodies and efficient nonlytic viral spread. CV exhibits a unique tropism for progenitor cells in the host which may help to explain the susceptibility of the young host to infection and the establishment of chronic disease in adults. CVs have also been shown to exploit autophagy to maximize viral replication and assist in unconventional release from target cells. In this article, we review recent progress in clarifying virus replication and dissemination within the host cell, identifying determinants of tropism, and defining strategies utilized by the virus to evade the host immune response. Also, we will highlight unanswered questions and provide future perspectives regarding the potential mechanisms of CV pathogenesis. PMID:26142496

  10. 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

  11. 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

  12. A unique binding mode enables MCM2 to chaperone histones H3–H4 at replication forks

    PubMed Central

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

    2015-01-01

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

  13. 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.

  14. ATP-dependent chromatin remodeling shapes the DNA replication landscape

    PubMed Central

    Vincent, Jack A.; Kwong, Tracey J.; Tsukiyama, Toshio

    2009-01-01

    Summary The eukaryotic DNA replication machinery must traverse every nucleosome in the genome during S phase. As nucleosomes are generally inhibitory to DNA-dependent processes, chromatin structure must undergo extensive reorganization to facilitate DNA synthesis. However, the identity of chromatin-remodeling factors involved in replication and how they affect DNA synthesis is largely unknown. Here we show that two highly conserved ATP-dependent chromatin-remodeling complexes in Saccharomyces cerevisiae, Isw2 and Ino80, function in parallel to promote replication fork progression. As a result, Isw2 and Ino80 play especially important roles for replication of late-replicating regions during periods of replication stress. Both Isw2 and Ino80 complexes are enriched at sites of replication, suggesting that these complexes act directly to promote fork progression. These findings identify ATP-dependent chromatin-remodeling complexes promoting DNA replication, and define a specific stage of replication that requires remodeling for normal function. PMID:18408730

  15. 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

  16. 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

  17. Water quality of Fremont Lake and New Fork Lakes, western Wyoming; a progress report

    USGS Publications Warehouse

    Peterson, D.A.; Averett, R.C.; Mora, K.L.

    1987-01-01

    Fremont Lake and New Fork Lakes in the New Fork River drainage of western Wyoming were selected for a comprehensive study of hydrologic processes affecting mountain lakes in the Rocky Mountains. Information is needed about lakes in this area to assess their response to existing and planned development. The concerns include regional issues such as acid precipitation from gas-sweetening plants, coal-fired powerplants, and smelters, as well as local issues, such as shoreline development and raising outlet control structures. Onsite measurements indicated strong thermal stratification in the lakes during the summer. Isothermal conditions occurred during December 1983 and May 1984. Mean phytoplankton concentrations were less than 5,000 cells/ml, and chlorophyll a concentrations were weakly correlated with phytoplankton concentrations. Zooplankton concentrations were small, less than 6 organisms/L. The numbers of benthic invertebrates/unit area in Fremont Lake were extremely small. The lake waters and inflow and outflow streams were chemically dilute solutions. Mean dissolved-solids concentrations were 13 mg/L in Fremont Lake and 24 mg/L in New Fork Lakes. Calcium and bicarbonate were the predominant ions. Concentrations of phosphorus and nitrogen usually were less than detection limits. Trace-metals concentrations in the lakes were similar to those in precipitation and generally were small. Dissolved organic-carbon concentrations were about 1 mg/L. Concentrations of fulvic and humic acids were relatively large in the inlet of Fremont Lake during the spring. Pine Creek has deposited 800 metric tons of sediment, on an annual average, to the delta of Fremont Lake. Most sediment is deposited during spring runoff. (USGS)

  18. Nuclear DNA synthesis in vitro is mediated via stable replication forks assembled in a temporally specific fashion in vivo

    SciTech Connect

    Heintz, N.H.; Stillman, B.W.

    1988-05-01

    A cell-free nuclear replication system that is S-phase specific, that requires the activity of DNA polymerase alpha, and that is stimulated three- to eightfold by cytoplasmic factors from S-phase cells was used to examine the temporal specificity of chromosomal DNA synthesis in vitro. Temporal specificity of DNA synthesis in isolated nuclei was assessed directly by examining the replication of restriction fragments derived from the amplified 200-kilobase dihydrofolate reductase domain of methotrexate-resistant CHOC 400 cells as a function of the cell cycle. In nuclei prepared from cells collected at the G1/S boundary of the cell cycle, synthesis of amplified sequences commenced within the immediate dihydrofolate reductase origin region and elongation continued for 60 to 80 min. The order of synthesis of amplified restriction fragments in nuclei from early S-phase cells in vitro appeared to be indistinguishable from that in vivo. Nuclei prepared from CHOC 400 cells poised at later times in the S phase synthesized characteristic subsets of other amplified fragments. The specificity of fragment labeling patterns was stable to short-term storage at 4/sup 0/C. The occurrence of stimulatory factors in cytosol extracts was cell cycle dependent in that minimal stimulation was observed with early G1-phase extracts, whereas maximal stimulation was observed with cytosol extracts from S-phase cells. Chromosomal synthesis was not observed in nuclei from G1 cells, nor did cytosol extracts from S-phase cells induce chromosomal replication in G1 nuclei. In contrast to chromosomal DNA synthesis, mitochondrial DNA replication in vitro was not stimulated by cytoplasmic factors and occurred at equivalent rates throughout the G1 and S phases.

  19. Replication fork-stimulated eIF-4A from Plasmodium cynomolgi unwinds DNA in the 3' to 5' direction and is inhibited by DNA-interacting compounds.

    PubMed

    Tuteja, Renu; Tuteja, Narendra; Malhotra, Pawan; Singh Chauhan, Virander

    2003-06-01

    Plasmodium cynomolgi DEAD-box DNA helicase 45 (PcDDH45) is an ATP-dependent DNA-unwinding enzyme with intrinsic DNA-dependent ATPase activity and is highly homologous to eIF-4A. In this study, we have further characterized and tested the effect of various DNA-interacting compounds on the DNA-unwinding activity of PcDDH45. The results show that PcDDH45 translocates in the 3' to 5' direction along the bound strand, a replication fork-like structure of the substrate stimulates its DNA-unwinding activity, and it failed to unwind blunt-ended duplex DNA. Of various compounds tested, only cisplatin, 4',6'-diamidino-2-phenylindole, daunorubicin, and nogalamycin were inhibitory to the unwinding activity of PcDDH45 with apparent IC(50) values of 1.0, 4.0, 7.5, and 1.7 microM, respectively. These results suggest that the interaction of these compounds with duplex DNA generate a complex that probably impedes the translocation of PcDDH45, resulting in inhibition of unwinding activity. This study is one of the first to demonstrate the effect of various DNA-binding compounds on a malaria parasite DNA helicase and should make an important contribution to our better understanding of the nucleic acid transactions in the parasite.

  20. Evidence for sequential and increasing activation of replication origins along replication timing gradients in the human genome.

    PubMed

    Guilbaud, Guillaume; Rappailles, Aurélien; Baker, Antoine; Chen, Chun-Long; Arneodo, Alain; Goldar, Arach; d'Aubenton-Carafa, Yves; Thermes, Claude; Audit, Benjamin; Hyrien, Olivier

    2011-12-01

    Genome-wide replication timing studies have suggested that mammalian chromosomes consist of megabase-scale domains of coordinated origin firing separated by large originless transition regions. Here, we report a quantitative genome-wide analysis of DNA replication kinetics in several human cell types that contradicts this view. DNA combing in HeLa cells sorted into four temporal compartments of S phase shows that replication origins are spaced at 40 kb intervals and fire as small clusters whose synchrony increases during S phase and that replication fork velocity (mean 0.7 kb/min, maximum 2.0 kb/min) remains constant and narrowly distributed through S phase. However, multi-scale analysis of a genome-wide replication timing profile shows a broad distribution of replication timing gradients with practically no regions larger than 100 kb replicating at less than 2 kb/min. Therefore, HeLa cells lack large regions of unidirectional fork progression. Temporal transition regions are replicated by sequential activation of origins at a rate that increases during S phase and replication timing gradients are set by the delay and the spacing between successive origin firings rather than by the velocity of single forks. Activation of internal origins in a specific temporal transition region is directly demonstrated by DNA combing of the IGH locus in HeLa cells. Analysis of published origin maps in HeLa cells and published replication timing and DNA combing data in several other cell types corroborate these findings, with the interesting exception of embryonic stem cells where regions of unidirectional fork progression seem more abundant. These results can be explained if origins fire independently of each other but under the control of long-range chromatin structure, or if replication forks progressing from early origins stimulate initiation in nearby unreplicated DNA. These findings shed a new light on the replication timing program of mammalian genomes and provide a general

  1. 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

  2. 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

  3. 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

  4. Replisome speed determines the efficiency of the Tus-Ter replication termination barrier.

    PubMed

    Elshenawy, Mohamed M; Jergic, Slobodan; Xu, Zhi-Qiang; Sobhy, Mohamed A; Takahashi, Masateru; Oakley, Aaron J; Dixon, Nicholas E; Hamdan, Samir M

    2015-09-17

    In all domains of life, DNA synthesis occurs bidirectionally from replication origins. Despite variable rates of replication fork progression, fork convergence often occurs at specific sites. Escherichia coli sets a 'replication fork trap' that allows the first arriving fork to enter but not to leave the terminus region. The trap is set by oppositely oriented Tus-bound Ter sites that block forks on approach from only one direction. However, the efficiency of fork blockage by Tus-Ter does not exceed 50% in vivo despite its apparent ability to almost permanently arrest replication forks in vitro. Here we use data from single-molecule DNA replication assays and structural studies to show that both polarity and fork-arrest efficiency are determined by a competition between rates of Tus displacement and rearrangement of Tus-Ter interactions that leads to blockage of slower moving replisomes by two distinct mechanisms. To our knowledge this is the first example where intrinsic differences in rates of individual replisomes have different biological outcomes.

  5. 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.

  6. 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

  7. Replicating nucleosomes

    PubMed Central

    Ramachandran, Srinivas; Henikoff, Steven

    2015-01-01

    Eukaryotic replication disrupts each nucleosome as the fork passes, followed by reassembly of disrupted nucleosomes and incorporation of newly synthesized histones into nucleosomes in the daughter genomes. In this review, we examine this process of replication-coupled nucleosome assembly to understand how characteristic steady-state nucleosome landscapes are attained. Recent studies have begun to elucidate mechanisms involved in histone transfer during replication and maturation of the nucleosome landscape after disruption by replication. A fuller understanding of replication-coupled nucleosome assembly will be needed to explain how epigenetic information is replicated at every cell division. PMID:26269799

  8. The chromatin assembly factor 1 promotes Rad51-dependent template switches at replication forks by counteracting D-loop disassembly by the RecQ-type helicase Rqh1.

    PubMed

    Pietrobon, Violena; Fréon, Karine; Hardy, Julien; Costes, Audrey; Iraqui, Ismail; Ochsenbein, Françoise; Lambert, Sarah A E

    2014-10-01

    At blocked replication forks, homologous recombination mediates the nascent strands to switch template in order to ensure replication restart, but faulty template switches underlie genome rearrangements in cancer cells and genomic disorders. Recombination occurs within DNA packaged into chromatin that must first be relaxed and then restored when recombination is completed. The chromatin assembly factor 1, CAF-1, is a histone H3-H4 chaperone involved in DNA synthesis-coupled chromatin assembly during DNA replication and DNA repair. We reveal a novel chromatin factor-dependent step during replication-coupled DNA repair: Fission yeast CAF-1 promotes Rad51-dependent template switches at replication forks, independently of the postreplication repair pathway. We used a physical assay that allows the analysis of the individual steps of template switch, from the recruitment of recombination factors to the formation of joint molecules, combined with a quantitative measure of the resulting rearrangements. We reveal functional and physical interplays between CAF-1 and the RecQ-helicase Rqh1, the BLM homologue, mutations in which cause Bloom's syndrome, a human disease associating genome instability with cancer predisposition. We establish that CAF-1 promotes template switch by counteracting D-loop disassembly by Rqh1. Consequently, the likelihood of faulty template switches is controlled by antagonistic activities of CAF-1 and Rqh1 in the stability of the D-loop. D-loop stabilization requires the ability of CAF-1 to interact with PCNA and is thus linked to the DNA synthesis step. We propose that CAF-1 plays a regulatory role during template switch by assembling chromatin on the D-loop and thereby impacting the resolution of the D-loop. PMID:25313826

  9. [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.

  10. Progress in mask replication using jet and flash imprint lithography

    NASA Astrophysics Data System (ADS)

    Selinidis, Kosta S.; Brooks, Cynthia B.; Doyle, Gary F.; Brown, Laura; Jones, Chris; Imhof, Joseph; LaBrake, Dwayne L.; Resnick, Douglas J.; Sreenivasan, S. V.

    2011-04-01

    The Jet and Flash Imprint Lithography (J-FILTM) process uses drop dispensing of UV curable resists to assist high resolution patterning for subsequent dry etch pattern transfer. The technology is actively being used to develop solutions for memory markets including Flash memory and patterned media for hard disk drives. It is anticipated that the lifetime of a single template (for patterned media) or mask (for semiconductor) will be on the order of 104 - 105imprints. This suggests that tens of thousands of templates/masks will be required to satisfy the needs of a manufacturing environment. Electron-beam patterning is too slow to feasibly deliver these volumes, but instead can provide a high quality "master" mask which can be replicated many times with an imprint lithography tool. This strategy has the capability to produce the required supply of "working" templates/masks. In this paper, we review the development of the mask form factor, imprint replication tools and processes specifically for semiconductor applications. The requirements needed for semiconductors dictate the need for a well defined form factor for both master and replica masks which is also compatible with the existing mask infrastructure established for the 6025 semi standard, 6" x 6" x 0.25" photomasks. Complying with this standard provides the necessary tooling needed for mask fabrication processes, cleaning, metrology, and inspection. The replica form factor has additional features specific to imprinting such as a pre-patterned mesa. A PerfectaTM MR5000 mask replication tool has been developed specifically to pattern replica masks from an e-beam written master. The system specifications include a throughput of four replicas per hour with an added image placement component of 5nm, 3sigma and a critical dimension uniformity error of less than 1nm, 3sigma. A new process has been developed to fabricate replicas with high contrast alignment marks so that designs for imprint can fit within current

  11. DNA recombination: the replication connection.

    PubMed

    Haber, J E

    1999-07-01

    Chromosomal double-strand breaks (DSBs) arise after exposure to ionizing radiation or enzymatic cleavage, but especially during the process of DNA replication itself. Homologous recombination plays a critical role in repair of such DSBs. There has been significant progress in our understanding of two processes that occur in DSB repair: gene conversion and recombination-dependent DNA replication. Recent evidence suggests that gene conversion and break-induced replication are related processes that both begin with the establishment of a replication fork in which both leading- and lagging-strand synthesis occur. There has also been much progress in characterization of the biochemical roles of recombination proteins that are highly conserved from yeast to humans.

  12. 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

  13. Along Middle Fork Road toward North Fork of the Crazy ...

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

    Along Middle Fork Road toward North Fork of the Crazy Woman Creek Bridge, view to west - North Fork of Crazy Woman Creek Bridge, Spanning North Fork of Crazy Woman Creek at Middle Fork Road, Buffalo, Johnson County, WY

  14. 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

  15. Checkpoints are blind to replication restart and recombination intermediates that result in gross chromosomal rearrangements

    PubMed Central

    Mohebi, Saed; Mizuno, Ken’Ichi; Watson, Adam; Carr, Antony M.; Murray, Johanne M.

    2015-01-01

    Replication fork inactivation can be overcome by homologous recombination, but this can cause gross chromosomal rearrangements that subsequently missegregate at mitosis, driving further chromosome instability. It is unclear when the chromosome rearrangements are generated and whether individual replication problems or the resulting recombination intermediates delay the cell cycle. Here we have investigated checkpoint activation during HR-dependent replication restart using a site-specific replication fork-arrest system. Analysis during a single cell cycle shows that HR-dependent replication intermediates arise in S phase, shortly after replication arrest, and are resolved into acentric and dicentric chromosomes in G2. Despite this, cells progress into mitosis without delay. Neither the DNA damage nor the intra-S phase checkpoints are activated in the first cell cycle, demonstrating that these checkpoints are blind to replication and recombination intermediates as well as to rearranged chromosomes. The dicentrics form anaphase bridges that subsequently break, inducing checkpoint activation in the second cell cycle. PMID:25721418

  16. Replication of simian virus 40 DNA after UV irradiation: evidence of growing fork blockage and single-stranded gaps in daughter strands

    SciTech Connect

    Mezzina, M.; Menck, C.F.M.; Courtin, P.; Sarasin, A.

    1988-11-01

    The molecular mechanisms of in vivo inhibition of mammalian DNA replication by exposure to UV light (at 254 nm) was studied in monkey and human cells infected with simian virus 40. Analysis of viral DNA by electron microscopy and sucrose gradients confirmed that the presence of UV-induced lesions severely blocks DNA synthesis, and thus the conversion of replicative intermediates (RIs) into fully replicated form I DNA is inhibited by UV irradiation. These blocked RI molecules present several special features when visualized by electron microscopy. In excision repair-proficient monkey and human cells they are composed of a double-stranded circular DNA with a double-stranded tail whose size corresponds to the average interpyrimidine dimer distance, as determined by the dimer-specific T4 endonuclease V. In excision repair-deficient human cells from patients with xeroderma pigmentosum, UV-irradiated RIs present a Carins-like structure similar to that observed for replicating molecules obtained from unirradiated infected cells. Single-stranded gaps are visualized in the replicated portions of UV-irradiated RI molecules; such regions are detected and clearly distinguishable from double-stranded DNA when probed by a specific single-stranded DNA-binding protein such as the bacteriophage T4 gene 32 product. Consistent with the presence of gaps in UV-irradiated RI molecules, single-strand-specific S1 nuclease digestion causes a shift in their sedimentation properties when analyzed in neutral sucrose gradients compared with undamaged molecules.

  17. 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

  18. 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

  19. 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

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

    PubMed

    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

  1. 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

  2. Replication landscape of the human genome.

    PubMed

    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

  3. The DNA replication program is altered at the FMR1 locus in fragile X embryonic stem cells.

    PubMed

    Gerhardt, Jeannine; Tomishima, Mark J; Zaninovic, Nikica; Colak, Dilek; Yan, Zi; Zhan, Qiansheng; Rosenwaks, Zev; Jaffrey, Samie R; Schildkraut, Carl L

    2014-01-01

    Fragile X syndrome (FXS) is caused by a CGG repeat expansion in the FMR1 gene that appears to occur during oogenesis and during early embryogenesis. One model proposes that repeat instability depends on the replication fork direction through the repeats such that (CNG)n hairpin-like structures form, causing DNA polymerase to stall and slip. Examining DNA replication fork progression on single DNA molecules at the endogenous FMR1 locus revealed that replication forks stall at CGG repeats in human cells. Furthermore, replication profiles of FXS human embryonic stem cells (hESCs) compared to nonaffected hESCs showed that fork direction through the repeats is altered at the FMR1 locus in FXS hESCs, such that predominantly the CCG strand serves as the lagging-strand template. This is due to the absence of replication initiation that would typically occur upstream of FMR1, suggesting that altered replication origin usage combined with fork stalling promotes repeat instability during early embryonic development.

  4. 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

  5. The Western Progression of Lyme Disease: Infectious and Nonclonal Borrelia burgdorferi Sensu Lato Populations in Grand Forks County, North Dakota

    PubMed Central

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

    2014-01-01

    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

  6. 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

  7. 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

  8. The MMS22L-TONSL complex mediates recovery from replication stress and homologous recombination

    PubMed Central

    O’Donnell, Lara; Panier, Stephanie; Wildenhain, Jan; Tkach, Johnny M.; Al-Hakim, Abdallah; Landry, Marie-Claude; Escribano-Diaz, Cristina; Szilard, Rachel K.; Young, Jordan T. F.; Munro, Meagan; Canny, Marella D.; Kolas, Nadine K.; Zhang, Wei; Harding, Shane M.; Ylanko, Jarkko; Mendez, Megan; Mullin, Michael; Sun, Thomas; Habermann, Bianca; Datti, Alessandro; Bristow, Robert G.; Gingras, Anne-Claude; Tyers, Michael D.; Brown, Grant W.; Durocher, Daniel

    2010-01-01

    Summary Genome integrity is jeopardized each time DNA replication forks stall or collapse. Here, we report the identification of a complex composed of MMS22L (C6ORF167) and TONSL (NFKBIL2) that participates in the recovery from replication stress. MMS22L and TONSL are homologous to yeast Mms22 and plant Tonsoku/Brushy1, respectively. MMS22L-TONSL accumulates at regions of ssDNA associated with distressed replication forks or at processed DNA breaks, and its depletion results in high levels of endogenous DNA double-strand breaks caused by an inability to complete DNA synthesis after replication fork collapse. Moreover, cells depleted of MMS22L are highly sensitive to camptothecin, a topoisomerase I poison that impairs DNA replication progression. Finally, MMS22L and TONSL are necessary for the efficient formation of RAD51 foci after DNA damage and their depletion impairs homologous recombination. These results indicate that MMS22L and TONSL are genome caretakers that stimulate the recombination-dependent repair of stalled or collapsed replication forks. PMID:21055983

  9. Ku Protein Levels, Localization and Association to Replication Origins in Different Stages of Breast Tumor Progression

    PubMed Central

    Abdelbaqi, Khalil; Di Paola, Domenic; Rampakakis, Emmanouil; Zannis-Hadjopoulos, Maria

    2013-01-01

    Human origins of DNA replication are specific sequences within the genome whereby DNA replication is initiated. A select group of proteins, known as the pre-replication (pre-RC) complex, in whose formation the Ku protein (Ku70/Ku86) was shown to play a role, bind to replication origins to initiate DNA replication. In this study, we have examined the involvement of Ku in breast tumorigenesis and tumor progression and found that the Ku protein expression levels in human breast metastatic (MCF10AC1a) cells were higher in the chromatin fraction compared to hyperplastic (MCF10AT) and normal (MCF10A) human breast cells, but remained constant in both the nuclear and cytoplasmic fractions. In contrast, in human intestinal cells, the Ku expression level was relatively constant for all cell fractions. Nascent DNA abundance and chromatin association of Ku70/86 revealed that the c-myc origin activity in MCF10AC1a is 2.5 to 5-fold higher than in MCF10AT and MCF10A, respectively, and Ku was bound to the c-myc origin more abundantly in MCF10AC1a, by approximately 1.5 to 4.2-fold higher than in MCF10AT and MCF10A, respectively. In contrast, similar nascent DNA abundance and chromatin association was found for all cell lines for the lamin B2 origin, associated with the constitutively active housekeeping lamin B2 gene. Electrophoretic mobility shift assays (EMSAs) performed on the nuclear extracts (NEs) of the three cell types revealed the presence of protein-DNA replication complexes on both the c-myc and lamin B2 origins, but an increase in binding activity was observed from normal, to transformed, to cancer cells for the c-myc origin, whereas no such difference was seen for the lamin B2 origin. Overall, the results suggest that increased Ku chromatin association, beyond wild type levels, alters cellular processes, which have been implicated in tumorigenesis. PMID:23781282

  10. Ku protein levels, localization and association to replication origins in different stages of breast tumor progression.

    PubMed

    Abdelbaqi, Khalil; Di Paola, Domenic; Rampakakis, Emmanouil; Zannis-Hadjopoulos, Maria

    2013-01-01

    Human origins of DNA replication are specific sequences within the genome whereby DNA replication is initiated. A select group of proteins, known as the pre-replication (pre-RC) complex, in whose formation the Ku protein (Ku70/Ku86) was shown to play a role, bind to replication origins to initiate DNA replication. In this study, we have examined the involvement of Ku in breast tumorigenesis and tumor progression and found that the Ku protein expression levels in human breast metastatic (MCF10AC1a) cells were higher in the chromatin fraction compared to hyperplastic (MCF10AT) and normal (MCF10A) human breast cells, but remained constant in both the nuclear and cytoplasmic fractions. In contrast, in human intestinal cells, the Ku expression level was relatively constant for all cell fractions. Nascent DNA abundance and chromatin association of Ku70/86 revealed that the c-myc origin activity in MCF10AC1a is 2.5 to 5-fold higher than in MCF10AT and MCF10A, respectively, and Ku was bound to the c-myc origin more abundantly in MCF10AC1a, by approximately 1.5 to 4.2-fold higher than in MCF10AT and MCF10A, respectively. In contrast, similar nascent DNA abundance and chromatin association was found for all cell lines for the lamin B2 origin, associated with the constitutively active housekeeping lamin B2 gene. Electrophoretic mobility shift assays (EMSAs) performed on the nuclear extracts (NEs) of the three cell types revealed the presence of protein-DNA replication complexes on both the c-myc and lamin B2 origins, but an increase in binding activity was observed from normal, to transformed, to cancer cells for the c-myc origin, whereas no such difference was seen for the lamin B2 origin. Overall, the results suggest that increased Ku chromatin association, beyond wild type levels, alters cellular processes, which have been implicated in tumorigenesis.

  11. Tuning fork decay.

    PubMed

    Miller, G W

    1979-03-01

    Tuning fork tests are used routinely by many otologists. A different group of otologists find the tests inconsistent and unreliable. This controversy has probably developed because the audiometer has replaced the tuning fork in hearing measurement. As a result, the art of use of the tuning fork is poorly learned. This study examines decay, one of the physical parameters of tuning forks. Measurements of acoustic (sound wave) and vibration (stem movement) decay were made. Alteration in decay due to pressure changes on the fork stem were studied. Acoustic signals were generated in an anechoic chamber. Vibration measurements were obtained using an artificial mastoid. Analysis of the signals was accomplished by a system of amplifiers, filters, tape recorders, and a graphic recorder. Tuning fork sound decay is a property of the instrument which occurs every time the fork is struck. The decay is a constant in decibels per second. The acoustic mode and the vibration mode decay at similar rates for the same fork. The strike frequency (a higher frequency than the fundamental produced when the fork is struck) also has a constant decay rate in decibels per second, and it is reported here for the first time. Force of 800 gm. and less applied to the bottom of the stem in vibration measurement caused minimal decay constant changes. When the physical parameters of the tuning fork (including this information on damping) are fully studied, tuning fork testing should become more of a science and less of an art.

  12. Kinase-independent function of checkpoint kinase 1 (Chk1) in the replication of damaged DNA.

    PubMed

    Speroni, Juliana; Federico, María Belén; Mansilla, Sabrina F; Soria, Gastón; Gottifredi, Vanesa

    2012-05-01

    The checkpoint kinases Chk1 and ATR are broadly known for their role in the response to the accumulation of damaged DNA. Because Chk1 activation requires its phosphorylation by ATR, it is expected that ATR or Chk1 down-regulation should cause similar alterations in the signals triggered by DNA lesions. Intriguingly, we found that Chk1, but not ATR, promotes the progression of replication forks after UV irradiation. Strikingly, this role of Chk1 is independent of its kinase-domain and of its partnership with Claspin. Instead, we demonstrate that the ability of Chk1 to promote replication fork progression on damaged DNA templates relies on its recently identified proliferating cell nuclear antigen-interacting motif, which is required for its release from chromatin after DNA damage. Also supporting the importance of Chk1 release, a histone H2B-Chk1 chimera, which is permanently immobilized in chromatin, is unable to promote the replication of damaged DNA. Moreover, inefficient chromatin dissociation of Chk1 impairs the efficient recruitment of the specialized DNA polymerase η (pol η) to replication-associated foci after UV. Given the critical role of pol η during translesion DNA synthesis (TLS), these findings unveil an unforeseen facet of the regulation by Chk1 of DNA replication. This kinase-independent role of Chk1 is exclusively associated to the maintenance of active replication forks after UV irradiation in a manner in which Chk1 release prompts TLS to avoid replication stalling.

  13. Kinase-independent function of checkpoint kinase 1 (Chk1) in the replication of damaged DNA

    PubMed Central

    Speroni, Juliana; Federico, María Belén; Mansilla, Sabrina F.; Soria, Gastón; Gottifredi, Vanesa

    2012-01-01

    The checkpoint kinases Chk1 and ATR are broadly known for their role in the response to the accumulation of damaged DNA. Because Chk1 activation requires its phosphorylation by ATR, it is expected that ATR or Chk1 down-regulation should cause similar alterations in the signals triggered by DNA lesions. Intriguingly, we found that Chk1, but not ATR, promotes the progression of replication forks after UV irradiation. Strikingly, this role of Chk1 is independent of its kinase-domain and of its partnership with Claspin. Instead, we demonstrate that the ability of Chk1 to promote replication fork progression on damaged DNA templates relies on its recently identified proliferating cell nuclear antigen-interacting motif, which is required for its release from chromatin after DNA damage. Also supporting the importance of Chk1 release, a histone H2B-Chk1 chimera, which is permanently immobilized in chromatin, is unable to promote the replication of damaged DNA. Moreover, inefficient chromatin dissociation of Chk1 impairs the efficient recruitment of the specialized DNA polymerase η (pol η) to replication-associated foci after UV. Given the critical role of pol η during translesion DNA synthesis (TLS), these findings unveil an unforeseen facet of the regulation by Chk1 of DNA replication. This kinase-independent role of Chk1 is exclusively associated to the maintenance of active replication forks after UV irradiation in a manner in which Chk1 release prompts TLS to avoid replication stalling. PMID:22529391

  14. Kinase-independent function of checkpoint kinase 1 (Chk1) in the replication of damaged DNA.

    PubMed

    Speroni, Juliana; Federico, María Belén; Mansilla, Sabrina F; Soria, Gastón; Gottifredi, Vanesa

    2012-05-01

    The checkpoint kinases Chk1 and ATR are broadly known for their role in the response to the accumulation of damaged DNA. Because Chk1 activation requires its phosphorylation by ATR, it is expected that ATR or Chk1 down-regulation should cause similar alterations in the signals triggered by DNA lesions. Intriguingly, we found that Chk1, but not ATR, promotes the progression of replication forks after UV irradiation. Strikingly, this role of Chk1 is independent of its kinase-domain and of its partnership with Claspin. Instead, we demonstrate that the ability of Chk1 to promote replication fork progression on damaged DNA templates relies on its recently identified proliferating cell nuclear antigen-interacting motif, which is required for its release from chromatin after DNA damage. Also supporting the importance of Chk1 release, a histone H2B-Chk1 chimera, which is permanently immobilized in chromatin, is unable to promote the replication of damaged DNA. Moreover, inefficient chromatin dissociation of Chk1 impairs the efficient recruitment of the specialized DNA polymerase η (pol η) to replication-associated foci after UV. Given the critical role of pol η during translesion DNA synthesis (TLS), these findings unveil an unforeseen facet of the regulation by Chk1 of DNA replication. This kinase-independent role of Chk1 is exclusively associated to the maintenance of active replication forks after UV irradiation in a manner in which Chk1 release prompts TLS to avoid replication stalling. PMID:22529391

  15. Human immunodeficiency virus type 1 variants with increased replicative capacity develop during the asymptomatic stage before disease progression.

    PubMed

    Connor, R I; Ho, D D

    1994-07-01

    We examined the replicative properties of a series of sequential isolates and biological clones of human immunodeficiency virus type 1 (HIV-1) obtained from an individual who progressed from seroconversion to AIDS in approximately 5 years. HIV-1 isolated soon after seroconversion replicated slowly and to low levels in cultures of peripheral blood mononuclear cells; however, subsequent isolates obtained during asymptomatic infection showed a marked increase in replication kinetics. This was examined in more detail by using a panel of 35 biological clones of HIV-1 generated from sequential patient peripheral blood mononuclear cell samples. Each clone was evaluated for replication in primary macrophages and CD4+ T lymphocytes and for the ability to induce syncytium formation in MT-2 cell cultures. Consistent with earlier observations, we found that all of the clones isolated just after seroconversion were slowly replicating and non-syncytium inducing (NSI). However, NSI variants with increased replication kinetics in macrophages were identified soon thereafter. These variants preceded the appearance of NSI and syncytium-inducing variants, with rapid replication in both macrophages and CD4+ T lymphocytes. To determine whether changes in the rate of replication could be traced to the early stages of the virus life cycle, PCR assays were used to evaluate entry and reverse transcription of selected biological clones in macrophages and CD4+ T lymphocytes. We found there was no inherent block to entry or reverse transcription for the slowly replicating variants; however, this does not preclude the possibility that small differences in the rate of entry may account for larger differences in the replication kinetics over many cycles. Overall, our results demonstrate that rapidly replicating variants of HIV-1 emerge during the asymptomatic period in a patient who subsequently progressed clinically, suggesting that these variants may play an important role in HIV-1 pathogenesis

  16. Human immunodeficiency virus type 1 variants with increased replicative capacity develop during the asymptomatic stage before disease progression.

    PubMed Central

    Connor, R I; Ho, D D

    1994-01-01

    We examined the replicative properties of a series of sequential isolates and biological clones of human immunodeficiency virus type 1 (HIV-1) obtained from an individual who progressed from seroconversion to AIDS in approximately 5 years. HIV-1 isolated soon after seroconversion replicated slowly and to low levels in cultures of peripheral blood mononuclear cells; however, subsequent isolates obtained during asymptomatic infection showed a marked increase in replication kinetics. This was examined in more detail by using a panel of 35 biological clones of HIV-1 generated from sequential patient peripheral blood mononuclear cell samples. Each clone was evaluated for replication in primary macrophages and CD4+ T lymphocytes and for the ability to induce syncytium formation in MT-2 cell cultures. Consistent with earlier observations, we found that all of the clones isolated just after seroconversion were slowly replicating and non-syncytium inducing (NSI). However, NSI variants with increased replication kinetics in macrophages were identified soon thereafter. These variants preceded the appearance of NSI and syncytium-inducing variants, with rapid replication in both macrophages and CD4+ T lymphocytes. To determine whether changes in the rate of replication could be traced to the early stages of the virus life cycle, PCR assays were used to evaluate entry and reverse transcription of selected biological clones in macrophages and CD4+ T lymphocytes. We found there was no inherent block to entry or reverse transcription for the slowly replicating variants; however, this does not preclude the possibility that small differences in the rate of entry may account for larger differences in the replication kinetics over many cycles. Overall, our results demonstrate that rapidly replicating variants of HIV-1 emerge during the asymptomatic period in a patient who subsequently progressed clinically, suggesting that these variants may play an important role in HIV-1 pathogenesis

  17. RecFOR proteins target RecA protein to a DNA gap with either DNA or RNA at the 5' terminus: implication for repair of stalled replication forks.

    PubMed

    Morimatsu, Katsumi; Wu, Yun; Kowalczykowski, Stephen C

    2012-10-12

    The repair of single-stranded gaps in duplex DNA by homologous recombination requires the proteins of the RecF pathway. The assembly of RecA protein onto gapped DNA (gDNA) that is complexed with the single-stranded DNA-binding protein is accelerated by the RecF, RecO, and RecR (RecFOR) proteins. Here, we show the RecFOR proteins specifically target RecA protein to gDNA even in the presence of a thousand-fold excess of single-stranded DNA (ssDNA). The binding constant of RecF protein, in the presence of the RecOR proteins, to the junction of ssDNA and dsDNA within a gap is 1-2 nm, suggesting that a few RecF molecules in the cell are sufficient to recognize gDNA. We also found that the nucleation of a RecA filament on gDNA in the presence of the RecFOR proteins occurs at a faster rate than filament elongation, resulting in a RecA nucleoprotein filament on ssDNA for 1000-2000 nucleotides downstream (5' → 3') of the junction with duplex DNA. Thus, RecA loading by RecFOR is localized to a region close to a junction. RecFOR proteins also recognize RNA at the 5'-end of an RNA-DNA junction within an ssDNA gap, which is compatible with their role in the repair of lagging strand gaps at stalled replication forks. PMID:22902627

  18. Pol eta is required for DNA replication during nucleotide deprivation by hydroxyurea.

    PubMed

    de Feraudy, S; Limoli, C L; Giedzinski, E; Karentz, D; Marti, T M; Feeney, L; Cleaver, J E

    2007-08-23

    Hydroxyurea reduces DNA replication by nucleotide deprivation, whereas UV damage generates DNA photoproducts that directly block replication fork progression. We show that the low fidelity class Y polymerase Pol eta is recruited to proliferating cell nuclear antigen at replication forks both by hydroxyurea and UV light. Under nucleotide deprivation, Pol eta allows cells to accumulate at the G1/S boundary by facilitating slow S-phase progression and promotes apoptosis. Normal cells consequently enter apoptosis at a faster rate than Pol eta-deficient cells. Coincident with hydroxyurea-induced S-phase delay, Pol eta-deficient cells undergo more replication fork breakage and accumulate more foci of the Mre11/Rad50/Nbs1 complex and phosphorylated histone H2AX. We conclude that under conditions of nucleotide deprivation, Pol eta is required for S-phase progression but is proapoptotic. However, as Pol eta is reported to require higher nucleotide concentrations than class B replicative polymerases, its recruitment by hydroxyurea requires it to function under suboptimal conditions. Our results suggest that hydroxyurea-induced apoptosis occurs at the G1/S boundary and that initiation of the S-phase requires greater nucleotide concentrations than does S-phase progression. PMID:17369853

  19. 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

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

    PubMed

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

    2003-02-01

    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

  1. PrimPol bypasses UV photoproducts during eukaryotic chromosomal DNA replication.

    PubMed

    Bianchi, Julie; Rudd, Sean G; Jozwiakowski, Stanislaw K; Bailey, Laura J; Soura, Violetta; Taylor, Elaine; Stevanovic, Irena; Green, Andrew J; Stracker, Travis H; Lindsay, Howard D; Doherty, Aidan J

    2013-11-21

    DNA damage can stall the DNA replication machinery, leading to genomic instability. Thus, numerous mechanisms exist to complete genome duplication in the absence of a pristine DNA template, but identification of the enzymes involved remains incomplete. Here, we establish that Primase-Polymerase (PrimPol; CCDC111), an archaeal-eukaryotic primase (AEP) in eukaryotic cells, is involved in chromosomal DNA replication. PrimPol is required for replication fork progression on ultraviolet (UV) light-damaged DNA templates, possibly mediated by its ability to catalyze translesion synthesis (TLS) of these lesions. This PrimPol UV lesion bypass pathway is not epistatic with the Pol η-dependent pathway and, as a consequence, protects xeroderma pigmentosum variant (XP-V) patient cells from UV-induced cytotoxicity. In addition, we establish that PrimPol is also required for efficient replication fork progression during an unperturbed S phase. These and other findings indicate that PrimPol is an important player in replication fork progression in eukaryotic cells. PMID:24267451

  2. DNA damage-induced replication arrest in Xenopus egg extracts

    PubMed Central

    Stokes, Matthew P.; Michael, W. Matthew

    2003-01-01

    Chromosomal replication is sensitive to the presence of DNA-damaging alkylating agents, such as methyl methanesulfonate (MMS). MMS is known to inhibit replication though activation of the DNA damage checkpoint and through checkpoint-independent slowing of replication fork progression. Using Xenopus egg extracts, we now report an additional pathway that is stimulated by MMS-induced damage. We show that, upon incubation in egg extracts, MMS-treated DNA activates a diffusible inhibitor that blocks, in trans, chromosomal replication. The downstream effect of the inhibitor is a failure to recruit proliferating cell nuclear antigen, but not DNA polymerase α, to the nascent replication fork. Thus, alkylation damage activates an inhibitor that intercepts the replication pathway at a point between the polymerase α and proliferating cell nuclear antigen execution steps. We also show that activation of the inhibitor does not require the DNA damage checkpoint; rather, stimulation of the pathway described here results in checkpoint activation. These data describe a novel replication arrest pathway, and they also provide an example of how subpathways within the DNA damage response network are integrated to promote efficient cell cycle arrest in response to damaged DNA. PMID:14581453

  3. Analysis of Replicating Yeast Chromosomes by DNA Combing.

    PubMed

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

    2016-02-01

    Molecular combing of DNA fibers is a powerful technique to monitor origin usage and DNA replication fork progression in the budding yeast Saccharomyces cerevisiae. In contrast to traditional flow cytometry, microarray, or sequencing techniques, which provide population-level data, DNA combing provides DNA replication profiles of individual molecules. DNA combing uses yeast strains that express human thymidine kinase, which facilitates the incorporation of thymidine analogs into nascent DNA. First, DNA is isolated and stretched uniformly onto silanized glass coverslips. Following immunodetection with antibodies that recognize the thymidine analog and the DNA, the DNA fibers are imaged using a fluorescence microscope. Finally, the lengths of newly replicated DNA tracks are measured and converted to base pairs, allowing calculations of the speed of the replication fork and of interorigin distances. DNA combing can be applied to monitor replication defects caused by gene mutations or by chemical agents that induce replication stress. Here, we present a methodology for studying replicating yeast chromosomes by molecular DNA combing. We begin with procedures for the preparation of silanized coverslips and for assembly of a DNA combing machine (DCM) and conclude by presenting a detailed protocol for molecular DNA combing in yeast.

  4. 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

  5. 4. VIEW UPSTREAM ALONG NORTH FORK OF MIDDLE FORK OF ...

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

    4. VIEW UPSTREAM ALONG NORTH FORK OF MIDDLE FORK OF TULE RIVER, LOOKING TOWARD DOWNSTREAM SIDE OF BRIDGE AND POWERHOUSE; FLUME AT LEFT. LOOKING EAST. 90mm lens - Tule River Hydroelectric Complex, Tule River Bridge, Spanning North Fork of Middle Fork of Tule River, Springville, Tulare County, CA

  6. CDK2-dependent phosphorylation of Suv39H1 is involved in control of heterochromatin replication during cell cycle progression

    PubMed Central

    Park, Su Hyung; Yu, Seung Eun; Chai, Young Gyu; Jang, Yeun Kyu

    2014-01-01

    Although several studies have suggested that the functions of heterochromatin regulators may be regulated by post-translational modifications during cell cycle progression, regulation of the histone methyltransferase Suv39H1 is not fully understood. Here, we demonstrate a direct link between Suv39H1 phosphorylation and cell cycle progression. We show that CDK2 phosphorylates Suv39H1 at Ser391 and these phosphorylation levels oscillate during the cell cycle, peaking at S phase and maintained during S-G2-M phase. The CDK2-mediated phosphorylation of Suv39H1 at Ser391 results in preferential dissociation from chromatin. Furthermore, phosphorylation-mediated dissociation of Suv39H1 from chromatin causes an enhanced occupancy of JMJD2A histone demethylase on heterochromatin and alterations in inactive histone marks. Overexpression of phospho-mimic Suv39H1 induces early replication of heterochromatin, suggesting the importance of Suv39H1 phosphorylation in the replication of heterochromatin. Moreover, overexpression of phospho-defective Suv39H1 caused altered replication timing of heterochromatin and increases sensitivity to replication stress. Collectively, our data suggest that phosphorylation-mediated modulation of Suv39H1-chromatin association may be an initial step in heterochromatin replication. PMID:24728993

  7. 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

  8. 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

  9. Direct Visualization of DNA Replication Dynamics in Zebrafish Cells.

    PubMed

    Kuriya, Kenji; Higashiyama, Eriko; Avşar-Ban, Eriko; Tamaru, Yutaka; Ogata, Shin; Takebayashi, Shin-ichiro; Ogata, Masato; Okumura, Katsuzumi

    2015-12-01

    Spatiotemporal regulation of DNA replication in the S-phase nucleus has been extensively studied in mammalian cells because it is tightly coupled with the regulation of other nuclear processes such as transcription. However, little is known about the replication dynamics in nonmammalian cells. Here, we analyzed the DNA replication processes of zebrafish (Danio rerio) cells through the direct visualization of replicating DNA in the nucleus and on DNA fiber molecules isolated from the nucleus. We found that zebrafish chromosomal DNA at the nuclear interior was replicated first, followed by replication of DNA at the nuclear periphery, which is reminiscent of the spatiotemporal regulation of mammalian DNA replication. However, the relative duration of interior DNA replication in zebrafish cells was longer compared to mammalian cells, possibly reflecting zebrafish-specific genomic organization. The rate of replication fork progression and ori-to-ori distance measured by the DNA combing technique were ∼ 1.4 kb/min and 100 kb, respectively, which are comparable to those in mammalian cells. To our knowledge, this is a first report that measures replication dynamics in zebrafish cells.

  10. 1. NORTH FORK OF THE TULE RIVER MIDDLE FORK BRANCH ...

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

    1. NORTH FORK OF THE TULE RIVER MIDDLE FORK BRANCH FLUME AND CONCRETE DIVERSION DAM SPILLING WATER. CONCRETE ABUTMENTS OF THE ORIGINAL HIGHWAY 190 BRIDGE OVER THE NORTH FORK ARE VISIBLE ON EITHER SIDE OF THE DAM. NEW HIGHWAY 190 BRIDGE IS VISIBLE ACROSS TOP OF PHOTO. VIEW TO NORTH. - Tule River Hydroelectric Project, Water Conveyance System, Middle Fork Tule River, Springville, Tulare County, CA

  11. 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

  12. Force regulated dynamics of RPA on a DNA fork.

    PubMed

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

    2016-07-01

    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 Mg(2+) concentrations, such that human RPA can melt DNA in absence of force.

  13. Myc and Ras oncogenes engage different energy metabolism programs and evoke distinct patterns of oxidative and DNA replication stress.

    PubMed

    Maya-Mendoza, Apolinar; Ostrakova, Jitka; Kosar, Martin; Hall, Arnaldur; Duskova, Pavlina; Mistrik, Martin; Merchut-Maya, Joanna Maria; Hodny, Zdenek; Bartkova, Jirina; Christensen, Claus; Bartek, Jiri

    2015-03-01

    Both Myc and Ras oncogenes impact cellular metabolism, deregulate redox homeostasis and trigger DNA replication stress (RS) that compromises genomic integrity. However, how are such oncogene-induced effects evoked and temporally related, to what extent are these kinetic parameters shared by Myc and Ras, and how are these cellular changes linked with oncogene-induced cellular senescence in different cell context(s) remain poorly understood. Here, we addressed the above-mentioned open questions by multifaceted comparative analyses of human cellular models with inducible expression of c-Myc and H-RasV12 (Ras), two commonly deregulated oncoproteins operating in a functionally connected signaling network. Our study of DNA replication parameters using the DNA fiber approach and time-course assessment of perturbations in glycolytic flux, oxygen consumption and production of reactive oxygen species (ROS) revealed the following results. First, overabundance of nuclear Myc triggered RS promptly, already after one day of Myc induction, causing slow replication fork progression and fork asymmetry, even before any metabolic changes occurred. In contrast, Ras overexpression initially induced a burst of cell proliferation and increased the speed of replication fork progression. However, after several days of induction Ras caused bioenergetic metabolic changes that correlated with slower DNA replication fork progression and the ensuing cell cycle arrest, gradually leading to senescence. Second, the observed oncogene-induced RS and metabolic alterations were cell-type/context dependent, as shown by comparative analyses of normal human BJ fibroblasts versus U2-OS sarcoma cells. Third, the energy metabolic reprogramming triggered by Ras was more robust compared to impact of Myc. Fourth, the detected oncogene-induced oxidative stress was due to ROS (superoxide) of non-mitochondrial origin and mitochondrial OXPHOS was reduced (Crabtree effect). Overall, our study provides novel

  14. 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

  15. Recombination and Replication

    PubMed Central

    Syeda, Aisha H.; Hawkins, Michelle; McGlynn, Peter

    2014-01-01

    The links between recombination and replication have been appreciated for decades and it is now generally accepted that these two fundamental aspects of DNA metabolism are inseparable: Homologous recombination is essential for completion of DNA replication and vice versa. This review focuses on the roles that recombination enzymes play in underpinning genome duplication, aiding replication fork movement in the face of the many replisome barriers that challenge genome stability. These links have many conserved features across all domains of life, reflecting the conserved nature of the substrate for these reactions, DNA. PMID:25341919

  16. Replication restart: a pathway for (CTG).(CAG) repeat deletion in Escherichia coli.

    PubMed

    Kim, Seung-Hwan; Pytlos, Małgorzata J; Sinden, Richard R

    2006-03-20

    (CTG)n.(CAG)n repeats undergo deletion at a high rate in plasmids in Escherichia coli in a process that involves RecA and RecB. In addition, DNA replication fork progression can be blocked during synthesis of (CTG)n.(CAG)n repeats. Replication forks stalled at (CTG)n.(CAG)n repeats may be rescued by replication restart that involves recombination as well as enzymes involved in replication and DNA repair, and this process may be responsible for the high rate of repeat deletion in E. coli. To test this hypothesis (CAG)n.(CTG)n deletion rates were measured in several E. coli strains carrying mutations involved in replication restart. (CAG)n.(CTG)n deletion rates were decreased, relative to the rates in wild type cells, in strains containing mutations in priA, recG, ruvAB, and recO. Mutations in priB and priC resulted in small reductions in deletion rates. In a recF strain, rates were decreased when (CAG)n comprised the leading template strand, but rates were increased when (CTG)n comprised the leading template. Deletion rates were increased slightly in a recJ strain. The mutational spectra for most mutant strains were altered relative to those in parental strains. In addition, purified PriA and RecG proteins showed unexpected binding to single-stranded, duplex, and forked DNAs containing (CAG)n and/or (CTG)n loop-outs in various positions. The results presented are consistent with an interpretation that the high rates of trinucleotide repeat instability observed in E. coli result from the attempted restart of replication forks stalled at (CAG)n.(CTG)n repeats. PMID:16472829

  17. Activation of mammalian Chk1 during DNA replication arrest

    PubMed Central

    Feijoo, Carmen; Hall-Jackson, Clare; Wu, Rong; Jenkins, David; Leitch, Jane; Gilbert, David M.; Smythe, Carl

    2001-01-01

    Checkpoints maintain order and fidelity in the cell cycle by blocking late-occurring events when earlier events are improperly executed. Here we describe evidence for the participation of Chk1 in an intra-S phase checkpoint in mammalian cells. We show that both Chk1 and Chk2 are phosphorylated and activated in a caffeine-sensitive signaling pathway during S phase, but only in response to replication blocks, not during normal S phase progression. Replication block–induced activation of Chk1 and Chk2 occurs normally in ataxia telangiectasia (AT) cells, which are deficient in the S phase response to ionizing radiation (IR). Resumption of synthesis after removal of replication blocks correlates with the inactivation of Chk1 but not Chk2. Using a selective small molecule inhibitor, cells lacking Chk1 function show a progressive change in the global pattern of replication origin firing in the absence of any DNA replication. Thus, Chk1 is apparently necessary for an intra-S phase checkpoint, ensuring that activation of late replication origins is blocked and arrested replication fork integrity is maintained when DNA synthesis is inhibited. PMID:11535615

  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. 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

  20. 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

  1. 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.

  2. 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

  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. Accessory replicative helicases and the replication of protein-bound DNA.

    PubMed

    Brüning, Jan-Gert; Howard, Jamieson L; McGlynn, Peter

    2014-12-12

    Complete, accurate duplication of the genetic material is a prerequisite for successful cell division. Achieving this accuracy is challenging since there are many barriers to replication forks that may cause failure to complete genome duplication or result in possibly catastrophic corruption of the genetic code. One of the most important types of replicative barriers are proteins bound to the template DNA, especially transcription complexes. Removal of these barriers demands energy input not only to separate the DNA strands but also to disrupt multiple bonds between the protein and DNA. Replicative helicases that unwind the template DNA for polymerases at the fork can displace proteins bound to the template. However, even occasional failures in protein displacement by the replicative helicase could spell disaster. In such circumstances, failure to restart replication could result in incomplete genome duplication. Avoiding incomplete genome duplication via the repair and restart of blocked replication forks also challenges viability since the involvement of recombination enzymes is associated with the risk of genome rearrangements. Organisms have therefore evolved accessory replicative helicases that aid replication fork movement along protein-bound DNA. These helicases reduce the dangers associated with replication blockage by protein-DNA complexes, aiding clearance of blocks and resumption of replication by the same replisome thus circumventing the need for replication repair and restart. This review summarises recent work in bacteria and eukaryotes that has begun to delineate features of accessory replicative helicases and their importance in genome stability.

  5. Replication, recombination, and repair: going for the gold.

    PubMed

    Klein, Hannah L; Kreuzer, Kenneth N

    2002-03-01

    DNA recombination is now appreciated to be integral to DNA replication and cell survival. Recombination allows replication to successfully maneuver through the roadblocks of damaged or collapsed replication forks. The signals and controls that permit cells to transition between replication and recombination modes are now being identified.

  6. Recombination-restarted replication makes inverted chromosome fusions at inverted repeats.

    PubMed

    Mizuno, Ken'Ichi; Miyabe, Izumi; Schalbetter, Stephanie A; Carr, Antony M; Murray, Johanne M

    2013-01-10

    Impediments to DNA replication are known to induce gross chromosomal rearrangements (GCRs) and copy-number variations (CNVs). GCRs and CNVs underlie human genomic disorders and are a feature of cancer. During cancer development, environmental factors and oncogene-driven proliferation promote replication stress. Resulting GCRs and CNVs are proposed to contribute to cancer development and therapy resistance. When stress arrests replication, the replisome remains associated with the fork DNA (stalled fork) and is protected by the inter-S-phase checkpoint. Stalled forks efficiently resume when the stress is relieved. However, if the polymerases dissociate from the fork (fork collapse) or the fork structure breaks (broken fork), replication restart can proceed either by homologous recombination or microhomology-primed re-initiation. Here we ascertain the consequences of replication with a fork restarted by homologous recombination in fission yeast. We identify a new mechanism of chromosomal rearrangement through the observation that recombination-restarted forks have a considerably high propensity to execute a U-turn at small inverted repeats (up to 1 in 40 replication events). We propose that the error-prone nature of restarted forks contributes to the generation of GCRs and gene amplification in cancer, and to non-recurrent CNVs in genomic disorders.

  7. 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.

  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. 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

  13. 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.

  14. How dormant origins promote complete genome replication.

    PubMed

    Blow, J Julian; Ge, Xin Quan; Jackson, Dean A

    2011-08-01

    Many replication origins that are licensed by loading MCM2-7 complexes in G1 are not normally used. Activation of these dormant origins during S phase provides a first line of defence for the genome if replication is inhibited. When replication forks fail, dormant origins are activated within regions of the genome currently engaged in replication. At the same time, DNA damage-response kinases activated by the stalled forks preferentially suppress the assembly of new replication factories, thereby ensuring that chromosomal regions experiencing replicative stress complete synthesis before new regions of the genome are replicated. Mice expressing reduced levels of MCM2-7 have fewer dormant origins, are cancer-prone and are genetically unstable, demonstrating the importance of dormant origins for preserving genome integrity. We review the function of dormant origins, the molecular mechanism of their regulation and their physiological implications.

  15. 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

  16. 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

  17. 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

  18. Tuning fork tests: forgotten art.

    PubMed

    Girgis, T F; Shambaugh, G E

    1988-01-01

    Four examples are cited in which tuning fork tests helped in proper selection of patients for surgery, after audiometric air and bone tests were equivocal or gave the wrong diagnostic and prognostic indication.

  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. Transcriptional Regulators Compete with Nucleosomes Post-replication.

    PubMed

    Ramachandran, Srinivas; Henikoff, Steven

    2016-04-21

    Every nucleosome across the genome must be disrupted and reformed when the replication fork passes, but how chromatin organization is re-established following replication is unknown. To address this problem, we have developed Mapping In vivo Nascent Chromatin with EdU and sequencing (MINCE-seq) to characterize the genome-wide location of nucleosomes and other chromatin proteins behind replication forks at high temporal and spatial resolution. We find that the characteristic chromatin landscape at Drosophila promoters and enhancers is lost upon replication. The most conspicuous changes are at promoters that have high levels of RNA polymerase II (RNAPII) stalling and DNA accessibility and show specific enrichment for the BRM remodeler. Enhancer chromatin is also disrupted during replication, suggesting a role for transcription factor (TF) competition in nucleosome re-establishment. Thus, the characteristic nucleosome landscape emerges from a uniformly packaged genome by the action of TFs, RNAPII, and remodelers minutes after replication fork passage. PMID:27062929

  1. 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

  2. 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.

  3. 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

  4. Dormant origins licensed by excess Mcm2-7 are required for human cells to survive replicative stress.

    PubMed

    Ge, Xin Quan; Jackson, Dean A; Blow, J Julian

    2007-12-15

    In late mitosis and early G1, Mcm2-7 complexes are loaded onto DNA to license replication origins for use in the upcoming S phase. However, the amount of Mcm2-7 loaded is in significant excess over the number of origins normally used. We show here that in human cells, excess chromatin-bound Mcm2-7 license dormant replication origins that do not fire during normal DNA replication, in part due to checkpoint activity. Dormant origins were activated within active replicon clusters if replication fork progression was inhibited, despite the activation of S-phase checkpoints. After lowering levels of chromatin-bound Mcm2-7 in human cells by RNA interference (RNAi), the use of dormant origins was suppressed in response to replicative stress. Although cells with lowered chromatin-bound Mcm2-7 replicated at normal rates, when challenged with replication inhibitors they had dramatically reduced rates of DNA synthesis and reduced viability. These results suggest that the use of dormant origins licensed by excess Mcm2-7 is a new and physiologically important mechanism that cells utilize to maintain DNA replication rates under conditions of replicative stress. We propose that checkpoint kinase activity can preferentially suppress initiation within inactive replicon clusters, thereby directing new initiation events toward active clusters that are experiencing replication problems.

  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. 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

  8. 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

  9. 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

  10. 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

  11. 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

  12. 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

  13. Identification of an origin of bidirectional DNA replication in mammalian chromosomes.

    PubMed

    Burhans, W C; Vassilev, L T; Caddle, M S; Heintz, N H; DePamphilis, M L

    1990-09-01

    Mechanistically, an origin of bidirectional DNA replication (OBR) can be defined by the transition from discontinuous to continuous DNA synthesis that must occur on each template strand at the site where replication forks originate. This results from synthesis of Okazaki fragments predominantly on the retrograde arms of forks. We have identified these transitions at a specific site within a 0.45 kb sequence approximately 17 kb downstream from the 3' end of the dihydrofolate reductase gene in Chinese hamster ovary chromosomes. At least 80% of the replication forks in a 27 kb region emanated from this OBR. Thus, initiation of DNA replication in mammalian chromosomes uses the same replication fork mechanism previously described in a variety of prokaryotic and eukaryotic genomes, suggesting that mammalian chromosomes also utilize specific cis-acting sequences as origins of DNA replication.

  14. Modeling DNA Replication Intermediates

    SciTech Connect

    Broyde, S.; Roy, D.; Shapiro, R.

    1997-06-01

    While there is now available a great deal of information on double stranded DNA from X-ray crystallography, high resolution NMR and computer modeling, very little is known about structures that are representative of the DNA core of replication intermediates. DNA replication occurs at a single strand/double strand junction and bulged out intermediates near the junction can lead to frameshift mutations. The single stranded domains are particularly challenging. Our interest is focused on strategies for modeling the DNA of these types of replication intermediates. Modeling such structures presents special problems in addressing the multiple minimum problem and in treating the electrostatic component of the force field. We are testing a number of search strategies for locating low energy structures of these types and we are also investigating two different distance dependent dielectric functions in the coulombic term of the force field. We are studying both unmodified DNA and DNA damaged by aromatic amines, carcinogens present in the environment in tobacco smoke, barbecued meats and automobile exhaust. The nature of the structure adopted by the carcinogen modified DNA at the replication fork plays a key role in determining whether the carcinogen will cause a mutation during replication that can initiate the carcinogenic process. In the present work results are presented for unmodified DNA.

  15. Mechanism and physiological significance of programmed replication termination.

    PubMed

    Bastia, Deepak; Zaman, Shamsu

    2014-06-01

    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.

  16. 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.

  17. 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

  18. 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

    2011-05-06

    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.

  19. An ADAM33 Polymorphism Associates with Progression of Preschool Wheeze into Childhood Asthma: A Prospective Case-Control Study with Replication in a Birth Cohort Study

    PubMed Central

    Klaassen, Ester M. M.; Penders, John; Jöbsis, Quirijn; van de Kant, Kim D. G.; Thijs, Carel; Mommers, Monique; van Schayck, Constant P.; van Eys, Guillaume; Koppelman, Gerard H.; Dompeling, Edward

    2015-01-01

    Background The influence of asthma candidate genes on the development from wheeze to asthma in young children still needs to be defined. Objective To link genetic variants in asthma candidate genes to progression of wheeze to persistent wheeze into childhood asthma. Materials and Methods In a prospective study, children with recurrent wheeze from the ADEM (Asthma DEtection and Monitoring) study were followed until the age of six. At that age a classification (transient wheeze or asthma) was based on symptoms, lung function and medication use. In 198 children the relationship between this classification and 30 polymorphisms in 16 asthma candidate genes was assessed by logistic regression. In case of an association based on a p<0.10, replication analysis was performed in an independent birth cohort study (KOALA study, n = 248 included for the present analysis). Results In the ADEM study, the minor alleles of ADAM33 rs511898 and rs528557 and the ORMDL3/GSDMB rs7216389 polymorphisms were negatively associated, whereas the minor alleles of IL4 rs2243250 and rs2070874 polymorphisms were positively associated with childhood asthma. When replicated in the KOALA study, ADAM33 rs528557 showed a negative association of the CG/GG-genotype with progression of recurrent wheeze into childhood asthma (0.50 (0.26-0.97) p = 0.04) and no association with preschool wheeze. Conclusion Polymorphisms in ADAM33, ORMDL3/GSDMB and IL4 were associated with childhood asthma in a group of children with recurrent wheeze. The replication of the negative association of the CG/GG-genotype of rs528557 ADAM33 with childhood asthma in an independent birth cohort study confirms that a compromised ADAM33 gene may be implicated in the progression of wheeze into childhood asthma. PMID:25768087

  20. 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

  1. Transcriptional coactivator HCF-1 couples the histone chaperone Asf1b to HSV-1 DNA replication components.

    PubMed

    Peng, Hua; Nogueira, Mauricio L; Vogel, Jodi L; Kristie, Thomas M

    2010-02-01

    The cellular transcriptional coactivator HCF-1 interacts with numerous transcription factors as well as other coactivators and is a component of multiple chromatin modulation complexes. The protein is essential for the expression of the immediate early genes of both herpes simplex virus (HSV) and varicella zoster virus and functions, in part, by coupling chromatin modification components including the Set1 or MLL1 histone methyltransferases and the histone demethylase LSD1 to promote the installation of positive chromatin marks and the activation of viral immediately early gene transcription. Although studies have investigated the role of HCF-1 in both cellular and viral transcription, little is known about other processes that the protein may be involved in. Here we demonstrate that HCF-1 localizes to sites of HSV replication late in infection. HCF-1 interacts directly and simultaneously with both HSV DNA replication proteins and the cellular histone chaperone Asf1b, a protein that regulates the progression of cellular DNA replication forks via chromatin reorganization. Asf1b localizes with HCF-1 in viral replication foci and depletion of Asf1b results in significantly reduced viral DNA accumulation. The results support a model in which the transcriptional coactivator HCF-1 is a component of the HSV DNA replication assembly and promotes viral DNA replication by coupling Asf1b to DNA replication components. This coupling provides a novel function for HCF-1 and insights into the mechanisms of modulating chromatin during DNA replication.

  2. An AT-rich sequence in human common fragile site FRA16D causes fork stalling and chromosome breakage in S. cerevisiae.

    PubMed

    Zhang, Haihua; Freudenreich, Catherine H

    2007-08-01

    Common fragile sites are regions of human chromosomes prone to breakage. Fragile site FRA16D spans the WWOX/FOR tumor suppressor gene and has been linked to cancer-causing deletions and translocations. Using a genetic assay in yeast, we found that a short AT-rich region (Flex1) within FRA16D increases chromosome fragility, whereas three other sequences within FRA16D do not. To our knowledge, this is the first identification of a sequence element within a common fragile site that increases chromosome fragility. The fragility of Flex1 was exacerbated by the absence of Rad52 or the presence of hydroxyurea. Flex1 contains a polymorphic AT repeat predicted to form a DNA structure, and two-dimensional gel analysis showed accumulation of stalled replication forks at the Flex1 sequence that was dependent on AT length. Our data suggest that the FRA16D Flex1 sequence causes increased chromosome breakage by forming secondary structures that stall replication fork progression.

  3. DNA replication and spindle checkpoints cooperate during S phase to delay mitosis and preserve genome integrity.

    PubMed

    Magiera, Maria M; Gueydon, Elisabeth; Schwob, Etienne

    2014-01-20

    Deoxyribonucleic acid (DNA) replication and chromosome segregation must occur in ordered sequence to maintain genome integrity during cell proliferation. Checkpoint mechanisms delay mitosis when DNA is damaged or upon replication stress, but little is known on the coupling of S and M phases in unperturbed conditions. To address this issue, we postponed replication onset in budding yeast so that DNA synthesis is still underway when cells should enter mitosis. This delayed mitotic entry and progression by transient activation of the S phase, G2/M, and spindle assembly checkpoints. Disabling both Mec1/ATR- and Mad2-dependent controls caused lethality in cells with deferred S phase, accompanied by Rad52 foci and chromosome missegregation. Thus, in contrast to acute replication stress that triggers a sustained Mec1/ATR response, multiple pathways cooperate to restrain mitosis transiently when replication forks progress unhindered. We suggest that these surveillance mechanisms arose when both S and M phases were coincidently set into motion by a unique ancestral cyclin-Cdk1 complex.

  4. Enhanced SIV replication and accelerated progression to AIDS in macaques primed to mount a CD4 T cell response to the SIV envelope protein

    PubMed Central

    Staprans, Silvija I.; Barry, Ashley P.; Silvestri, Guido; Safrit, Jeffrey T.; Kozyr, Natalia; Sumpter, Beth; Nguyen, Hanh; McClure, Harold; Montefiori, David; Cohen, Jeffrey I.; Feinberg, Mark B.

    2004-01-01

    Given the dual role of CD4 T cells as both immune effectors and targets for HIV infection, the balance of CD4 versus CD8 T cell-mediated responses induced by candidate AIDS vaccines may be critical in determining postvaccination infection outcomes. An attenuated recombinant varicella-zoster virus vaccine expressing the simian immunodeficiency virus (SIV) envelope (Env) elicited nonneutralizing Env-binding antibodies and little if any cytotoxic T lymphocyte responses in rhesus macaques (Macaca mulatta). After challenge with SIV, Env vaccinees manifested increased levels of SIV replication, more rapid CD4 depletion, and accelerated progression to AIDS compared with controls. Enhanced SIV replication correlated with increased CD4 T cell proliferation soon after SIV challenge, apparently the result of an anamnestic response to SIV antigens. Thus activation of virus-specific CD4 T cells at the time of exposure to a CD4 T cell-tropic lentivirus, in the absence of an effective CD8 response, may enhance virus replication and disease. These data suggest suggest that candidate AIDS vaccines may not simply be either efficacious or neutral; they may also have the potential to be harmful. PMID:15326293

  5. 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

  6. 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.

  7. 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

  8. Dicer in action at replication-transcription collisions.

    PubMed

    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

  9. Approach view of the North Fork Butter Creek Bridge, view ...

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

    Approach view of the North Fork Butter Creek Bridge, view looking south - North Fork Butter Creek Bridge, Spanning North Fork Butter Creek Bridge at Milepost 76.63 on Heppner Highway (Oregon Route 74), Pilot Rock, Umatilla County, OR

  10. Elevation view of the North Fork Butter Creek Bridge, view ...

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

    Elevation view of the North Fork Butter Creek Bridge, view looking west - North Fork Butter Creek Bridge, Spanning North Fork Butter Creek Bridge at Milepost 76.63 on Heppner Highway (Oregon Route 74), Pilot Rock, Umatilla County, OR

  11. Detail perspective view of the North Fork Butter Creek Bridge, ...

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

    Detail perspective view of the North Fork Butter Creek Bridge, view looking southwest - North Fork Butter Creek Bridge, Spanning North Fork Butter Creek Bridge at Milepost 76.63 on Heppner Highway (Oregon Route 74), Pilot Rock, Umatilla County, OR

  12. General perspective view of the North Fork Butter Creek Bridge, ...

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

    General perspective view of the North Fork Butter Creek Bridge, view looking north - North Fork Butter Creek Bridge, Spanning North Fork Butter Creek Bridge at Milepost 76.63 on Heppner Highway (Oregon Route 74), Pilot Rock, Umatilla County, OR

  13. General perspective view of the North Fork Butter Creek Bridge, ...

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

    General perspective view of the North Fork Butter Creek Bridge, view looking south - North Fork Butter Creek Bridge, Spanning North Fork Butter Creek Bridge at Milepost 76.63 on Heppner Highway (Oregon Route 74), Pilot Rock, Umatilla County, OR

  14. General perspective view of the North Fork Butter Creek Bridge, ...

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

    General perspective view of the North Fork Butter Creek Bridge, view looking southwest - North Fork Butter Creek Bridge, Spanning North Fork Butter Creek Bridge at Milepost 76.63 on Heppner Highway (Oregon Route 74), Pilot Rock, Umatilla County, OR

  15. Approach view of the North Fork Butter Creek Bridge, view ...

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

    Approach view of the North Fork Butter Creek Bridge, view looking north - North Fork Butter Creek Bridge, Spanning North Fork Butter Creek Bridge at Milepost 76.63 on Heppner Highway (Oregon Route 74), Pilot Rock, Umatilla County, OR

  16. The Replication System of Bacteriophage T7.

    PubMed

    Kulczyk, A W; Richardson, C C

    2016-01-01

    The replication system of bacteriophage T7 is remarkable in that the 40,000 nucleotide genome is replicated over 100-fold in a matter of minutes. In order to accomplish this feat T7 has evolved an efficient and economical process for the replication of its DNA. The T7 replisome provides a model system to study DNA replication. Four proteins are sufficient for reconstitution of the functional replication complex, yet the assembled replisome recapitulates all the key features of more complex prokaryotic and eukaryotic systems. In this review, we describe chemical mechanisms employed by individual proteins at the replication fork. Integration of structural, biochemical, and single-molecule data reveals a compelling view on how a nearly 1-MDa molecular machine acts as a unit to synthetize the two antiparallel DNA strands in a coordinated fashion.

  17. 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...

  18. 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 NEUROLOGICAL DEVICES Neurological Diagnostic Devices § 882.1525 Tuning fork. (a) Identification. A tuning...

  19. 21 CFR 882.1525 - Tuning fork.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 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...

  20. 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 NEUROLOGICAL DEVICES Neurological Diagnostic Devices § 882.1525 Tuning fork. (a) Identification. A tuning...

  1. 21 CFR 882.1525 - Tuning fork.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 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...

  2. 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…

  3. 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.

  4. Replicative fitness of transmitted HIV-1 drives acute immune activation, proviral load in memory CD4+ T cells, and disease progression.

    PubMed

    Claiborne, Daniel T; Prince, Jessica L; Scully, Eileen; Macharia, Gladys; Micci, Luca; Lawson, Benton; Kopycinski, Jakub; Deymier, Martin J; Vanderford, Thomas H; Nganou-Makamdop, Krystelle; Ende, Zachary; Brooks, Kelsie; Tang, Jianming; Yu, Tianwei; Lakhi, Shabir; Kilembe, William; Silvestri, Guido; Douek, Daniel; Goepfert, Paul A; Price, Matthew A; Allen, Susan A; Paiardini, Mirko; Altfeld, Marcus; Gilmour, Jill; Hunter, Eric

    2015-03-24

    HIV-1 infection is characterized by varying degrees of chronic immune activation and disruption of T-cell homeostasis, which impact the rate of disease progression. A deeper understanding of the factors that influence HIV-1-induced immunopathology and subsequent CD4(+) T-cell decline is critical to strategies aimed at controlling or eliminating the virus. In an analysis of 127 acutely infected Zambians, we demonstrate a dramatic and early impact of viral replicative capacity (vRC) on HIV-1 immunopathogenesis that is independent of viral load (VL). Individuals infected with high-RC viruses exhibit a distinct inflammatory cytokine profile as well as significantly elevated T-cell activation, proliferation, and CD8(+) T-cell exhaustion, during the earliest months of infection. Moreover, the vRC of the transmitted virus is positively correlated with the magnitude of viral burden in naive and central memory CD4(+) T-cell populations, raising the possibility that transmitted viral phenotypes may influence the size of the initial latent viral reservoir. Taken together, these findings support an unprecedented role for the replicative fitness of the founder virus, independent of host protective genes and VL, in influencing multiple facets of HIV-1-related immunopathology, and that a greater focus on this parameter could provide novel approaches to clinical interventions.

  5. The structure of supercoiled intermediates in DNA replication.

    PubMed

    Peter, B J; Ullsperger, C; Hiasa, H; Marians, K J; Cozzarelli, N R

    1998-09-18

    We studied the structure of replication intermediates accumulated by Tus-induced arrest of plasmid DNA replication at termination sites. For intermediates generated both in vitro with purified components and in vivo, superhelical stress is distributed throughout the entire partially replicated molecule; daughter DNA segments are wound around each other, and the unreplicated region is supercoiled. Thus, unlinking of parental DNA strands by topoisomerases can be carried out both behind and in front of the replication fork. We explain why previous studies with prokaryotic and eukaryotic replication intermediates discerned only supercoiling in the unreplicated portion.

  6. NCOA4 transcriptional coactivator inhibits activation of DNA replication origins.

    PubMed

    Bellelli, Roberto; Castellone, Maria Domenica; Guida, Teresa; Limongello, Roberto; Dathan, Nina Alayne; Merolla, Francesco; Cirafici, Anna Maria; Affuso, Andrea; Masai, Hisao; Costanzo, Vincenzo; Grieco, Domenico; Fusco, Alfredo; Santoro, Massimo; Carlomagno, Francesca

    2014-07-01

    NCOA4 is a transcriptional coactivator of nuclear hormone receptors that undergoes gene rearrangement in human cancer. By combining studies in Xenopus laevis egg extracts and mouse embryonic fibroblasts (MEFs), we show here that NCOA4 is a minichromosome maintenance 7 (MCM7)-interacting protein that is able to control DNA replication. Depletion-reconstitution experiments in Xenopus laevis egg extracts indicate that NCOA4 acts as an inhibitor of DNA replication origin activation by regulating CMG (CDC45/MCM2-7/GINS) helicase. NCOA4(-/-) MEFs display unscheduled origin activation and reduced interorigin distance; this results in replication stress, as shown by the presence of fork stalling, reduction of fork speed, and premature senescence. Together, our findings indicate that NCOA4 acts as a regulator of DNA replication origins that helps prevent inappropriate DNA synthesis and replication stress.

  7. 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.

  8. 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

  9. Mechanistic studies on the impact of transcription on sequence-specific termination of DNA replication and vice versa.

    PubMed

    Mohanty, B K; Sahoo, T; Bastia, D

    1998-01-30

    Since DNA replication and transcription often temporally and spatially overlap each other, the impact of one process on the other is of considerable interest. We have reported previously that transcription is impeded at the replication termini of Escherichia coli and Bacillus subtilis in a polar mode and that, when transcription is allowed to invade a replication terminus from the permissive direction, arrest of replication fork at the terminus is abrogated. In the present report, we have addressed four significant questions pertaining to the mechanism of transcription impedance by the replication terminator proteins. Is transcription arrested at the replication terminus or does RNA polymerase dissociate from the DNA causing authentic transcription termination? How does transcription cause abrogation of replication fork arrest at the terminus? Are the points of arrest of the replication fork and transcription the same or are these different? Are eukaryotic RNA polymerases also arrested at prokaryotic replication termini? Our results show that replication terminator proteins of E. coli and B. subtilis arrest but do not terminate transcription. Passage of an RNA transcript through the replication terminus causes the dissociation of the terminator protein from the terminus DNA, thus causing abrogation of replication fork arrest. DNA and RNA chain elongation are arrested at different locations on the terminator sites. Finally, although bacterial replication terminator proteins blocked yeast RNA polymerases in a polar fashion, a yeast transcription terminator protein (Reb1p) was unable to block T7 RNA polymerase and E. coli DnaB helicase.

  10. 16 CFR 1512.13 - Requirements for front fork.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 16 Commercial Practices 2 2012-01-01 2012-01-01 false Requirements for front fork. 1512.13 Section... 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. 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

  12. 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

  13. 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

  14. 16 CFR 1512.13 - Requirements for front fork.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 16 Commercial Practices 2 2014-01-01 2014-01-01 false Requirements for front fork. 1512.13 Section... 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...

  15. 16 CFR 1512.13 - Requirements for front fork.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 16 Commercial Practices 2 2013-01-01 2013-01-01 false Requirements for front fork. 1512.13 Section... 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...

  16. 16 CFR 1512.13 - Requirements for front fork.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 16 Commercial Practices 2 2010-01-01 2010-01-01 false Requirements for front fork. 1512.13 Section... 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...

  17. 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

  18. 16 CFR 1512.13 - Requirements for front fork.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 16 Commercial Practices 2 2011-01-01 2011-01-01 false Requirements for front fork. 1512.13 Section... 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...

  19. 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.

  20. 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.

  1. SMARCAL1 maintains telomere integrity during DNA replication

    PubMed Central

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

    2015-01-01

    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

  2. Enhanced Viral Replication by Cellular Replicative Senescence

    PubMed Central

    Kim, Ji-Ae; Seong, Rak-Kyun

    2016-01-01

    Cellular replicative senescence is a major contributing factor to aging and to the development and progression of aging-associated diseases. In this study, we sought to determine viral replication efficiency of influenza virus (IFV) and Varicella Zoster Virus (VZV) infection in senescent cells. Primary human bronchial epithelial cells (HBE) or human dermal fibroblasts (HDF) were allowed to undergo numbers of passages to induce replicative senescence. Induction of replicative senescence in cells was validated by positive senescence-associated β-galactosidase staining. Increased susceptibility to both IFV and VZV infection was observed in senescent HBE and HDF cells, respectively, resulting in higher numbers of plaque formation, along with the upregulation of major viral antigen expression than that in the non-senescent cells. Interestingly, mRNA fold induction level of virus-induced type I interferon (IFN) was attenuated by senescence, whereas IFN-mediated antiviral effect remained robust and potent in virus-infected senescent cells. Additionally, we show that a longevity-promoting gene, sirtuin 1 (SIRT1), has antiviral role against influenza virus infection. In conclusion, our data indicate that enhanced viral replication by cellular senescence could be due to senescence-mediated reduction of virus-induced type I IFN expression. PMID:27799874

  3. Mycobacterium tuberculosis modulates the gene interactions to activate the HIV replication and faster disease progression in a co-infected host.

    PubMed

    Toor, Jaideep S; Singh, Sukhvinder; Sharma, Aman; Arora, Sunil K

    2014-01-01

    Understanding of the chronic immune activation, breakdown of immune defense and synergistic effect between HIV and Mycobacterium tuberculosis (Mtb) may provide essential information regarding key factors involved in the pathogenesis of HIV disease. In this study, we aimed to highlight a few of the immunological events that may influence and accelerate the progression of HIV disease in the presence of co-infecting Mtb. A cross-sectional study was performed on cohorts, including anti-tubercular therapy (ATT) naïve active pulmonary tuberculosis (PTB) patients, antiretroviral therapy (ART) naïve HIV-1 infected individuals at different stages of disease, ATT and ART naïve HIV-PTB co-infected individuals and healthy controls. A significantly higher T-regulatory cell (Treg) frequency coupled with the high FoxP3 expression in the CD4 T-cells indicated an immunosuppressive environment in the advance stage of HIV-1 infection. This is further substantiated by high HO-1 expression favoring TB co-infection. Functionally, this change in Treg frequency in HIV-1 infected individuals correlated well with suppression of T-cell proliferation. Mtb infection seems to facilitate the expansion of the Treg pool along with increased expression of FoxP3, specifically the variant-1, as evident from the data in HIV-1 co-infected as well as in patients with only PTB. A significantly lower expression of HO-1 in co-infected individuals compared to patients with only HIV-infection having comparable CD4 count correlated well with increased expression of CCR5 and CxCR4 as well as NF-κB and inflammatory cytokines IL-6 and TNF-α, which collectively may contribute to enhanced viral replication and increased cell death, hence faster disease progression in co-infected individuals.

  4. 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

  5. Role of replication protein A as sensor in activation of the S-phase checkpoint in Xenopus egg extracts.

    PubMed

    Recolin, Bénédicte; Van der Laan, Siem; Maiorano, Domenico

    2012-04-01

    Uncoupling between DNA polymerases and helicase activities at replication forks, induced by diverse DNA lesions or replication inhibitors, generate long stretches of primed single-stranded DNA that is implicated in activation of the S-phase checkpoint. It is currently unclear whether nucleation of the essential replication factor RPA onto this substrate stimulates the ATR-dependent checkpoint response independently of its role in DNA synthesis. Using Xenopus egg extracts to investigate the role of RPA recruitment at uncoupled forks in checkpoint activation we have surprisingly found that in conditions in which DNA synthesis occurs, RPA accumulation at forks stalled by either replication stress or UV irradiation is dispensable for Chk1 phosphorylation. In contrast, when both replication fork uncoupling and RPA hyperloading are suppressed, Chk1 phosphorylation is inhibited. Moreover, we show that extracts containing reduced levels of RPA accumulate ssDNA and induce spontaneous, caffeine-sensitive, Chk1 phosphorylation in S-phase. These results strongly suggest that disturbance of enzymatic activities of replication forks, rather than RPA hyperloading at stalled forks, is a critical determinant of ATR activation. PMID:22187152

  6. 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

  7. Roles of histone chaperone CIA/Asf1 in nascent DNA elongation during nucleosome replication.

    PubMed

    Ishikawa, Katsuyuki; Ohsumi, Tatsuya; Tada, Shusuke; Natsume, Ryo; Kundu, Lena Rani; Nozaki, Naohito; Senda, Toshiya; Enomoto, Takemi; Horikoshi, Masami; Seki, Masayuki

    2011-10-01

    The nucleosome, which is composed of DNA wrapped around a histone octamer, is a fundamental unit of chromatin and is duplicated during the eukaryotic DNA replication process. The evolutionarily conserved histone chaperone cell cycle gene 1 (CCG1) interacting factor A/anti-silencing function 1 (CIA/Asf1) is involved in histone transfer and nucleosome reassembly during DNA replication. CIA/Asf1 has been reported to split the histone (H3-H4)(2) tetramer into histone H3-H4 dimer(s) in vitro, raising a possibility that, in DNA replication, CIA/Asf1 is involved in nucleosome disassembly and the promotion of semi-conservative histone H3-H4 dimer deposition onto each daughter strand in vivo. Despite numerous studies on the functional roles of CIA/Asf1, its mechanistic role(s) remains elusive because of lack of biochemical analyses. The biochemical studies described here show that a V94R CIA/Asf1 mutant, which lacks histone (H3-H4)(2) tetramer splitting activity, does not form efficiently a quaternary complex with histones H3-H4 and the minichromosome maintenance 2 (Mcm2) subunit of the Mcm2-7 replicative DNA helicase. Interestingly, the mutant enhances nascent DNA strand synthesis in a cell-free chromosomal DNA replication system using Xenopus egg extracts. These results suggest that CIA/Asf1 in the CIA/Asf1-H3-H4-Mcm2 complex, which is considered to be an intermediate in histone transfer during DNA replication, negatively regulates the progression of the replication fork.

  8. 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

  9. 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

  10. Topological analysis of plasmid DNA replication intermediates using two-dimensional agarose gels.

    PubMed

    Hyrien, Olivier

    2009-01-01

    A fundamental process in DNA replication is the disentangling of the two parental strands by DNA topoisomerases. In this chapter, I detail the topological analysis of plasmid replication intermediates using two-dimensional (2D) agarose gels. The method can resolve replication intermediates according to mass and topology, and can resolve unlinked monomeric circles from catenated dimers of varying topology. The method has been used, alone or in combination with a procedure for purifying covalent protein-DNA complexes, to analyse the effect oftopoisomerase inhibitors on the topology of replication intermediates, to map the location of drug-stabilized topoisomerase cleavage complexes with respect to replication forks and to detect the breakage and repair of replication forks following collision with cleavage complexes. Other applications include the detection of knots that form independently of, or concomitantly with, DNA replication.

  11. 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.

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

    PubMed

    Sabatinos, Sarah A; Forsburg, Susan L

    2015-01-01

    Replication fork stalling generates a variety of responses, most of which cause an increase in single-stranded DNA. ssDNA is a primary signal of replication distress that activates cellular checkpoints. It is also a potential source of genome instability and a substrate for mutation and recombination. Therefore, managing ssDNA levels is crucial to chromosome integrity. Limited ssDNA accumulation occurs in wild-type cells under stress. In contrast, cells lacking the replication checkpoint cannot arrest forks properly and accumulate large amounts of ssDNA. This likely occurs when the replication fork polymerase and helicase units are uncoupled. Some cells with mutations in the replication helicase (mcm-ts) mimic checkpoint-deficient cells, and accumulate extensive areas of ssDNA to trigger the G2-checkpoint. Another category of helicase mutant (mcm4-degron) causes fork stalling in early S-phase due to immediate loss of helicase function. Intriguingly, cells realize that ssDNA is present, but fail to detect that they accumulate ssDNA, and continue to divide. Thus, the cellular response to replication stalling depends on checkpoint activity and the time that replication stress occurs in S-phase. In this review we describe the signs, signals, and symptoms of replication arrest from an ssDNA perspective. We explore the possible mechanisms for these effects. We also advise the need for caution when detecting and interpreting data related to the accumulation of ssDNA. PMID:26393661

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

    PubMed Central

    Sabatinos, Sarah A.; Forsburg, Susan L.

    2015-01-01

    Replication fork stalling generates a variety of responses, most of which cause an increase in single-stranded DNA. ssDNA is a primary signal of replication distress that activates cellular checkpoints. It is also a potential source of genome instability and a substrate for mutation and recombination. Therefore, managing ssDNA levels is crucial to chromosome integrity. Limited ssDNA accumulation occurs in wild-type cells under stress. In contrast, cells lacking the replication checkpoint cannot arrest forks properly and accumulate large amounts of ssDNA. This likely occurs when the replication fork polymerase and helicase units are uncoupled. Some cells with mutations in the replication helicase (mcm-ts) mimic checkpoint-deficient cells, and accumulate extensive areas of ssDNA to trigger the G2-checkpoint. Another category of helicase mutant (mcm4-degron) causes fork stalling in early S-phase due to immediate loss of helicase function. Intriguingly, cells realize that ssDNA is present, but fail to detect that they accumulate ssDNA, and continue to divide. Thus, the cellular response to replication stalling depends on checkpoint activity and the time that replication stress occurs in S-phase. In this review we describe the signs, signals, and symptoms of replication arrest from an ssDNA perspective. We explore the possible mechanisms for these effects. We also advise the need for caution when detecting and interpreting data related to the accumulation of ssDNA. PMID:26393661

  14. 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

  15. Tuning fork shear-force feedback.

    PubMed

    Ruiter, A G; van der Werf, K O; Veerman, J A; Garcia-Parajo, M F; Rensen, W H; van Hulst, N F

    1998-03-01

    Investigations have been performed on the dynamics of a distance regulation system based on an oscillating probe at resonance. This was examined at a tuning fork shear-force feedback system, which is used as a distance control mechanism in near-field scanning optical microscopy. In this form of microscopy, a tapered optical fiber is attached to the tuning fork and scanned over the sample surface to be imaged. Experiments were performed measuring both amplitude and phase of the oscillation of the tuning fork as a function of driving frequency and tip-sample distance. These experiments reveal that the resonance frequency of the tuning fork changes upon approaching the sample. Both the amplitude and the phase of the tuning fork can be used as distance control parameter in the feedback system. Using the amplitude a second-order behavior is observed, while with phase only a first-order behavior is observed. Numerical calculations confirm these observations. This first-order behavior results in an improved stability of the feedback system. As an example, a sample consisting of DNA strands on mica was imaged which showed the height of the DNA as 1.4 +/- 0.2 nm.

  16. 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

  17. 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

  18. 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

  19. 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

  20. 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

  1. 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

  2. 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

  3. 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

  4. 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

  5. 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

  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. 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

  8. 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

  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. 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

  11. 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

  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. Resonant tuning fork detector for electromagnetic radiation.

    PubMed

    Pohlkötter, Andreas; Willer, Ulrike; Bauer, Christoph; Schade, Wolfgang

    2009-02-01

    A mechanical quartz microresonator (tuning fork) is used to detect electromagnetic radiation. The detection scheme is based on forces created due to the incident electromagnetic radiation on the piezoelectric tuning fork. A force can be created due to the transfer of the photon momentum of the incident electromagnetic radiation. If the surfaces of the tuning fork are nonuniformly heated, a second force acts on it, the so-called photophoretic force. These processes occur for all wavelengths of the incident radiation, making the detector suitable for sensing of ultraviolet, visible, and mid-infrared light, even THz-radiation. Here the detector is characterized in the visible range; noise analysis is performed for 650 nm and 5.26 microm. A linear power characteristic and the dependence on pulse lengths of the incoming light are shown. Examples for applications for the visible and mid-infrared spectral region are given by 2f and absorption spectroscopy of oxygen and nitric oxide, respectively.

  14. 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

  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. 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... North Fork of Roanoke. (a) Name. The name of the viticultural area described in this section is “North Fork of Roanoke.” (b) Approved maps. The appropriate maps for determining the boundaries of the...

  17. 27 CFR 9.65 - North Fork of Roanoke.

    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 Roanoke. 9... North Fork of Roanoke. (a) Name. The name of the viticultural area described in this section is “North Fork of Roanoke.” (b) Approved maps. The appropriate maps for determining the boundaries of the...

  18. 27 CFR 9.65 - North Fork of Roanoke.

    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 Roanoke. 9... North Fork of Roanoke. (a) Name. The name of the viticultural area described in this section is “North Fork of Roanoke.” (b) Approved maps. The appropriate maps for determining the boundaries of the...

  19. 27 CFR 9.65 - North Fork of Roanoke.

    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 Roanoke. 9... North Fork of Roanoke. (a) Name. The name of the viticultural area described in this section is “North Fork of Roanoke.” (b) Approved maps. The appropriate maps for determining the boundaries of the...

  20. 27 CFR 9.65 - North Fork of Roanoke.

    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 Roanoke. 9... North Fork of Roanoke. (a) Name. The name of the viticultural area described in this section is “North Fork of Roanoke.” (b) Approved maps. The appropriate maps for determining the boundaries of the...

  1. CLEAR FORK OF THE BRAZOS SUSPENSION BRIDGE, CIRCA 1896, SHOWING ...

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

    CLEAR FORK OF THE BRAZOS SUSPENSION BRIDGE, CIRCA 1896, SHOWING INCLINED STAY CABLES EXTENDING FROM TOP OF TOWER TO DECK. 3/4 VIEW FROM BELOW. - Clear Fork of Brazos River Suspension Bridge, Spanning Clear Fork of Brazos River at County Route 179, Albany, Shackelford County, TX

  2. 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...

  3. 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...

  4. 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...

  5. 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...

  6. 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...

  7. Topographic view of the North Fork Butter Creek Bridge (located ...

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

    Topographic view of the North Fork Butter Creek Bridge (located center of frame), view looking west - North Fork Butter Creek Bridge, Spanning North Fork Butter Creek Bridge at Milepost 76.63 on Heppner Highway (Oregon Route 74), Pilot Rock, Umatilla County, OR

  8. 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.

  9. 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

  10. 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.

  11. 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

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

    PubMed

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

    2015-06-01

    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 are

  13. PARG is dispensable for recovery from transient replicative stress but required to prevent detrimental accumulation of poly(ADP-ribose) upon prolonged replicative stress

    PubMed Central

    Illuzzi, Giuditta; Fouquerel, Elise; Amé, Jean-Christophe; Noll, Aurélia; Rehmet, Kristina; Nasheuer, Heinz-Peter; Dantzer, Françoise; Schreiber, Valérie

    2014-01-01

    Poly(ADP-ribosyl)ation is involved in numerous bio-logical processes including DNA repair, transcription and cell death. Cellular levels of poly(ADP-ribose) (PAR) are regulated by PAR polymerases (PARPs) and the degrading enzyme PAR glycohydrolase (PARG), controlling the cell fate decision between life and death in response to DNA damage. Replication stress is a source of DNA damage, leading to transient stalling of replication forks or to their collapse followed by the generation of double-strand breaks (DSB). The involvement of PARP-1 in replicative stress response has been described, whereas the consequences of a deregulated PAR catabolism are not yet well established. Here, we show that PARG-deprived cells showed an enhanced sensitivity to the replication inhibitor hydroxyurea. PARG is dispensable to recover from transient replicative stress but is necessary to avoid massive PAR production upon prolonged replicative stress, conditions leading to fork collapse and DSB. Extensive PAR accumulation impairs replication protein A association with collapsed forks resulting in compromised DSB repair via homologous recombination. Our results highlight the critical role of PARG in tightly controlling PAR levels produced upon genotoxic stress to prevent the detrimental effects of PAR over-accumulation. PMID:24906880

  14. 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

  15. Correlation between ribonucleoside-diphosphate reductase and three replication proteins in Escherichia coli

    PubMed Central

    2010-01-01

    Background There has long been evidence supporting the idea that RNR and the dNTP-synthesizing complex must be closely linked to the replication complex or replisome. We contributed to this body of evidence in proposing the hypothesis of the replication hyperstructure. A recently published work called this postulate into question, reporting that NrdB is evenly distributed throughout the cytoplasm. Consequently we were interested in the localization of RNR protein and its relationship with other replication proteins. Results We tagged NrdB protein with 3×FLAG epitope and detected its subcellular location by immunofluorescence microscopy. We found that this protein is located in nucleoid-associated clusters, that the number of foci correlates with the number of replication forks at any cell age, and that after the replication process ends the number of cells containing NrdB foci decreases. We show that the number of NrdB foci is very similar to the number of SeqA, DnaB, and DnaX foci, both in the whole culture and in different cell cycle periods. In addition, interfoci distances between NrdB and three replication proteins are similar to the distances between two replication protein foci. Conclusions NrdB is present in nucleoid-associated clusters during the replication period. These clusters disappear after replication ends. The number of these clusters is closely related to the number of replication forks and the number of three replication protein clusters in any cell cycle period. Therefore we conclude that NrdB protein, and most likely RNR protein, is closely linked to the replication proteins or replisome at the replication fork. These results clearly support the replication hyperstructure model. PMID:20102606

  16. 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.

  17. Brc1-dependent recovery from replication stress

    PubMed Central

    Bass, Kirstin L.; Murray, Johanne M.; O'Connell, Matthew J.

    2012-01-01

    BRCT-containing protein 1 (Brc1) is a multi-BRCT (BRCA1 carboxyl terminus) domain protein in Schizosaccharomyces pombe that is required for resistance to chronic replicative stress, but whether this reflects a repair or replication defect is unknown and the subject of this study. We show that brc1Δ cells are significantly delayed in recovery from replication pausing, though this does not activate a DNA damage checkpoint. DNA repair and recombination protein Rad52 is a homologous recombination protein that loads the Rad51 recombinase at resected double-stranded DNA (dsDNA) breaks and is also recruited to stalled replication forks, where it may stabilize structures through its strand annealing activity. Rad52 is required for the viability of brc1Δ cells, and brc1Δ cells accumulate Rad52 foci late in S phase that are potentiated by replication stress. However, these foci contain the single-stranded DNA (ssDNA) binding protein RPA, but not Rad51 or γH2A. Further, these foci are not associated with increased recombination between repeated sequences, or increased post-replication repair. Thus, these Rad52 foci do not represent sites of recombination. Following the initiation of DNA replication, the induction of these foci by replication stress is suppressed by defects in origin recognition complex (ORC) function, which is accompanied by loss of viability and severe mitotic defects. This suggests that cells lacking Brc1 undergo an ORC-dependent rescue of replication stress, presumably through the firing of dormant origins, and this generates RPA-coated ssDNA and recruits Rad52. However, as Rad51 is not recruited, and the checkpoint effector kinase Chk1 is not activated, these structures must not contain the unprotected primer ends found at sites of DNA damage that are required for recombination and checkpoint activation. PMID:22366461

  18. USP7 is a SUMO deubiquitinase essential for DNA replication.

    PubMed

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

    2016-04-01

    Post-translational modification of proteins by ubiquitin (Ub) and Ub-like modifiers regulates DNA replication. We have previously shown that chromatin around replisomes is rich in SUMO and poor in Ub, whereas mature chromatin exhibits an opposite pattern. How this SUMO-rich, Ub-poor environment is maintained at sites of DNA replication in mammalian cells remains unexplored. Here we identify USP7 as a replisome-enriched SUMO deubiquitinase that is essential for DNA replication. By acting on SUMO and SUMOylated proteins, USP7 counteracts their ubiquitination. Inhibition or genetic deletion of USP7 leads to the accumulation of Ub on SUMOylated proteins, which are displaced away from replisomes. Our findings provide a model explaining the differential accumulation of SUMO and Ub at replication forks and identify an essential role of USP7 in DNA replication that should be considered in the development of USP7 inhibitors as anticancer agents. PMID:26950370

  19. Break-seq reveals hydroxyurea-induced chromosome fragility as a result of unscheduled conflict between DNA replication and transcription

    PubMed Central

    Hoffman, Elizabeth A.; McCulley, Andrew; Haarer, Brian; Arnak, Remigiusz

    2015-01-01

    We have previously demonstrated that in Saccharomyces cerevisiae replication, checkpoint inactivation via a mec1 mutation leads to chromosome breakage at replication forks initiated from virtually all origins after transient exposure to hydroxyurea (HU), an inhibitor of ribonucleotide reductase. Here we sought to determine whether all replication forks containing single-stranded DNA gaps have equal probability of producing double-strand breaks (DSBs) when cells attempt to recover from HU exposure. We devised a new methodology, Break-seq, that combines our previously described DSB labeling with next generation sequencing to map chromosome breaks with improved sensitivity and resolution. We show that DSBs preferentially occur at genes transcriptionally induced by HU. Notably, different subsets of the HU-induced genes produced DSBs in MEC1 and mec1 cells as replication forks traversed a greater distance in MEC1 cells than in mec1 cells during recovery from HU. Specifically, while MEC1 cells exhibited chromosome breakage at stress-response transcription factors, mec1 cells predominantly suffered chromosome breakage at transporter genes, many of which are the substrates of those transcription factors. We propose that HU-induced chromosome fragility arises at higher frequency near HU-induced genes as a result of destabilized replication forks encountering transcription factor binding and/or the act of transcription. We further propose that replication inhibitors can induce unscheduled encounters between replication and transcription and give rise to distinct patterns of chromosome fragile sites. PMID:25609572

  20. 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

  1. 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

  2. Induction of the Bovine Papillomavirus Origin “Onion Skin”-Type DNA Replication at High E1 Protein Concentrations In Vivo

    PubMed Central

    Männik, Andres; Rünkorg, Kertu; Jaanson, Nele; Ustav, Mart; Ustav, Ene

    2002-01-01

    We have studied the replication of plasmids composed of bovine papillomavirus type 1 (BPV1) origin of replication and expression cartridges for viral proteins E1 and E2 in hamster and mouse cells. We found that the replication mode changed dramatically at different expression levels of the E1 protein. At high levels of the E1 protein, overreplication of the origin region of the plasmid was observed. Analysis of the replication products by one-dimensional and two-dimensional gel electrophoresis suggested that initially “onion skin”-type replication intermediates were generated, presumably resulting from initiation of the new replication forks before the leading fork completed the synthesis of the DNA on the episomal plasmid. These replication intermediates served as templates for generation of a heterogeneous set of origin region-containing linear fragments by displacement synthesis at the partially replicated plasmid. Additionally, the linear fragments may have been generated by DNA break-up of the onion skin-type intermediates. Analysis of replication products indicated that generated linear fragments recombined and formed concatemers or circular molecules, which presumably were able to replicate in an E1- and E2-dependent fashion. At moderate and low levels of E1, generated by transcription of the E1 open reading frame using weaker promoters, DNA replication was initiated at much lower levels, which allowed elongation of the replication fork starting from the origin to be more balanced and resulted in the generation of full-sized replication products. PMID:11992014

  3. 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

  4. 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.

  5. The effect of the intra-S-phase checkpoint on origins of replication in human cells.

    PubMed

    Karnani, Neerja; Dutta, Anindya

    2011-03-15

    Although many chemotherapy drugs activate the intra-S-phase checkpoint pathway to block S-phase progression, not much is known about how and where the intra-S-phase checkpoint regulates origins of replication in human chromosomes. A genomic analysis of replication in human cells in the presence of hydroxyurea (HU) revealed that only the earliest origins fire, but the forks stall within 2 kb and neighboring clusters of dormant origins are activated. The initiation events are located near expressed genes with a preference for transcription start and end sites, and when they are located in intergenic regions they are located near regulatory factor-binding regions (RFBR). The activation of clustered neo-origins by HU suggests that there are many potential replication initiation sites in permissive parts of the genome, most of which are not used in a normal S phase. Consistent with this redundancy, we see multiple sites bound to MCM3 (representative of the helicase) in the region flanking three out of three origins studied in detail. Bypass of the intra-S-phase checkpoint by caffeine activates many new origins in mid- and late-replicating parts of the genome. The intra-S-phase checkpoint suppresses origin firing after the loading of Mcm10, but before the recruitment of Cdc45 and AND-1/CTF4; i.e., after helicase loading but before helicase activation and polymerase loading. Interestingly, Cdc45 recruitment upon checkpoint bypass was accompanied by the restoration of global Cdk2 kinase activity and decrease in both global and origin-bound histone H3 Lys 4 trimethylation (H3K4me3), consistent with the suggestion that both of these factors are important for Cdc45 recruitment.

  6. 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

  7. PriA mediates DNA replication pathway choice at recombination intermediates.

    PubMed

    Xu, Liewei; Marians, Kenneth J

    2003-03-01

    We report the reconstitution of the initial steps of the double-strand break-repair pathway where joint molecule formation between a duplex DNA fragment and a circular template by the combined action of RecA, RecBCD, and the single-stranded DNA binding protein provides the substrate for replication fork formation by the restart primosome and the DNA polymerase III holoenzyme. We show that PriA dictates the pathway of replication from the recombination intermediate by inhibiting a nonspecific, strand displacement DNA synthesis reaction and favoring the formation of a bona fide replication fork. Furthermore, we find that RecO and RecR significantly stimulate this recombination-directed DNA replication reaction, and that this stimulation is modulated by the presence of RecF, suggesting that the latter protein may also act as a regulator of the pathway of resolution of the recombination intermediate. PMID:12667462

  8. 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.

  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. BRCA1 is Required for Post-replication Repair After UV-induced DNA Damage

    PubMed Central

    Pathania, Shailja; Nguyen, Jenna; Hill, Sarah J.; Scully, Ralph; Feunteun, Jean; Livingston, David M.

    2011-01-01

    BRCA1 contributes to the response to UV irradiation. Utilizing its BRCT motifs, it is recruited during S/G2 to UV-damaged sites in a DNA replication-dependent, but nucleotide excision repair- independent manner. More specifically, at UV- stalled replication forks, it promotes photoproduct excision, suppression of translesion synthesis, and the localization and activation of replication factor C complex (RFC) subunits. The last function, in turn, triggers post-UV checkpoint activation and post- replicative repair. These BRCA1 functions differ from those required for DSBR. PMID:21963239

  11. 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

  12. Chromatin structural changes precede replication in initiated replicons during inhibition of DNA elongation

    SciTech Connect

    D'Anna, J.A.; Grady, D.L.; Tobey, R.A.

    1988-01-01

    Partial inhibition of replicative DNA synthesis by hydroxyurea or other agents produces changes in the composition and structure of bulk chromatin. We have begun to investigate the structural changes in specific regions of the genome using synchronized cells and cloned genomic probes. Current results indicate changes in chromatin structure occur preferentially in initiated replicons and can precede the replication fork during inhibition of DNA elongation. 4 refs., 2 figs.

  13. Bedload measurements, East Fork River, Wyoming

    PubMed Central

    Leopold, Luna B.; Emmett, William W.

    1976-01-01

    A bedload trap in the riverbed provided direct quantitative measurement of debris-transport rate in the East Fork River, Wyoming, a basin of 466 km2 drainage area. Traction load moves only during the spring snow melt season. Data collected in three spring runoff seasons during which a peak flow of 45 m3/s occurred showed that transport rate is correlated with power expenditure of the flowing water and at high flows becomes directly proportional to power as suggested by Bagnold. PMID:16592302

  14. 33 CFR 117.307 - Miami River, North Fork.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 33 Navigation and Navigable Waters 1 2014-07-01 2014-07-01 false Miami River, North Fork. 117.307 Section 117.307 Navigation and Navigable Waters COAST GUARD, DEPARTMENT OF HOMELAND SECURITY BRIDGES DRAWBRIDGE OPERATION REGULATIONS Specific Requirements Florida § 117.307 Miami River, North Fork. The draw...

  15. 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. 207.370 Section 207.370 Navigation and Navigable Waters CORPS OF ENGINEERS, DEPARTMENT OF THE ARMY, DEPARTMENT OF DEFENSE NAVIGATION REGULATIONS § 207.370 Big Fork River, Minn.; logging. (a) During the...

  16. 33 CFR 117.307 - Miami River, North Fork.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 33 Navigation and Navigable Waters 1 2010-07-01 2010-07-01 false Miami River, North Fork. 117.307 Section 117.307 Navigation and Navigable Waters COAST GUARD, DEPARTMENT OF HOMELAND SECURITY BRIDGES DRAWBRIDGE OPERATION REGULATIONS Specific Requirements Florida § 117.307 Miami River, North Fork. The draw...

  17. 33 CFR 117.307 - Miami River, North Fork.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 33 Navigation and Navigable Waters 1 2012-07-01 2012-07-01 false Miami River, North Fork. 117.307 Section 117.307 Navigation and Navigable Waters COAST GUARD, DEPARTMENT OF HOMELAND SECURITY BRIDGES DRAWBRIDGE OPERATION REGULATIONS Specific Requirements Florida § 117.307 Miami River, North Fork. The draw...

  18. 33 CFR 117.315 - New River, South Fork.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 33 Navigation and Navigable Waters 1 2012-07-01 2012-07-01 false New River, South Fork. 117.315 Section 117.315 Navigation and Navigable Waters COAST GUARD, DEPARTMENT OF HOMELAND SECURITY BRIDGES DRAWBRIDGE OPERATION REGULATIONS Specific Requirements Florida § 117.315 New River, South Fork. (a) The...

  19. 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. 207.370 Section 207.370 Navigation and Navigable Waters CORPS OF ENGINEERS, DEPARTMENT OF THE ARMY, DEPARTMENT OF DEFENSE NAVIGATION REGULATIONS § 207.370 Big Fork River, Minn.; logging. (a) During the...

  20. 33 CFR 117.307 - Miami River, North Fork.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 33 Navigation and Navigable Waters 1 2013-07-01 2013-07-01 false Miami River, North Fork. 117.307 Section 117.307 Navigation and Navigable Waters COAST GUARD, DEPARTMENT OF HOMELAND SECURITY BRIDGES DRAWBRIDGE OPERATION REGULATIONS Specific Requirements Florida § 117.307 Miami River, North Fork. The draw...

  1. 33 CFR 117.307 - Miami River, North Fork.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 33 Navigation and Navigable Waters 1 2011-07-01 2011-07-01 false Miami River, North Fork. 117.307 Section 117.307 Navigation and Navigable Waters COAST GUARD, DEPARTMENT OF HOMELAND SECURITY BRIDGES DRAWBRIDGE OPERATION REGULATIONS Specific Requirements Florida § 117.307 Miami River, North Fork. The draw...

  2. 33 CFR 117.315 - New River, South Fork.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 33 Navigation and Navigable Waters 1 2014-07-01 2014-07-01 false New River, South Fork. 117.315 Section 117.315 Navigation and Navigable Waters COAST GUARD, DEPARTMENT OF HOMELAND SECURITY BRIDGES DRAWBRIDGE OPERATION REGULATIONS Specific Requirements Florida § 117.315 New River, South Fork. (a) The...

  3. Musical-grade tuning forks for emergent audiometric screening.

    PubMed

    McCormick, Rodger J; Poling, Mikaela I

    2015-06-11

    We discuss our experience using high-quality, musical-grade tuning forks for emergent audiometric screening in a 22-year-old woman with sudden-onset unilateral hearing sensation loss. We present a framework for using this method when proper audiometric equipment is unavailable but where musical-grade tuning forks can be rapidly supplied.

  4. 33 CFR 117.1063 - Willapa River South Fork.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 33 Navigation and Navigable Waters 1 2012-07-01 2012-07-01 false Willapa River South Fork. 117.1063 Section 117.1063 Navigation and Navigable Waters COAST GUARD, DEPARTMENT OF HOMELAND SECURITY... Fork. (a) The draw of the Washington State Parks and Recreation Commission bridge across the South...

  5. 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. 207.370 Section 207.370 Navigation and Navigable Waters CORPS OF ENGINEERS, DEPARTMENT OF THE ARMY, DEPARTMENT OF DEFENSE NAVIGATION REGULATIONS § 207.370 Big Fork River, Minn.; logging. (a) During the...

  6. 33 CFR 117.1063 - Willapa River South Fork.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 33 Navigation and Navigable Waters 1 2013-07-01 2013-07-01 false Willapa River South Fork. 117.1063 Section 117.1063 Navigation and Navigable Waters COAST GUARD, DEPARTMENT OF HOMELAND SECURITY... Fork. (a) The draw of the Washington State Parks and Recreation Commission bridge across the South...

  7. 33 CFR 117.315 - New River, South Fork.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 33 Navigation and Navigable Waters 1 2010-07-01 2010-07-01 false New River, South Fork. 117.315 Section 117.315 Navigation and Navigable Waters COAST GUARD, DEPARTMENT OF HOMELAND SECURITY BRIDGES DRAWBRIDGE OPERATION REGULATIONS Specific Requirements Florida § 117.315 New River, South Fork. (a) The...

  8. 33 CFR 117.1063 - Willapa River South Fork.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 33 Navigation and Navigable Waters 1 2011-07-01 2011-07-01 false Willapa River South Fork. 117.1063 Section 117.1063 Navigation and Navigable Waters COAST GUARD, DEPARTMENT OF HOMELAND SECURITY... Fork. (a) The draw of the Washington State Parks and Recreation Commission bridge across the South...

  9. 33 CFR 117.315 - New River, South Fork.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 33 Navigation and Navigable Waters 1 2011-07-01 2011-07-01 false New River, South Fork. 117.315 Section 117.315 Navigation and Navigable Waters COAST GUARD, DEPARTMENT OF HOMELAND SECURITY BRIDGES DRAWBRIDGE OPERATION REGULATIONS Specific Requirements Florida § 117.315 New River, South Fork. (a) The...

  10. 33 CFR 117.315 - New River, South Fork.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 33 Navigation and Navigable Waters 1 2013-07-01 2013-07-01 false New River, South Fork. 117.315 Section 117.315 Navigation and Navigable Waters COAST GUARD, DEPARTMENT OF HOMELAND SECURITY BRIDGES DRAWBRIDGE OPERATION REGULATIONS Specific Requirements Florida § 117.315 New River, South Fork. (a) The...

  11. 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. 207.370 Section 207.370 Navigation and Navigable Waters CORPS OF ENGINEERS, DEPARTMENT OF THE ARMY, DEPARTMENT OF DEFENSE NAVIGATION REGULATIONS § 207.370 Big Fork River, Minn.; logging. (a) During the...

  12. 33 CFR 117.1063 - Willapa River South Fork.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 33 Navigation and Navigable Waters 1 2010-07-01 2010-07-01 false Willapa River South Fork. 117.1063 Section 117.1063 Navigation and Navigable Waters COAST GUARD, DEPARTMENT OF HOMELAND SECURITY... Fork. (a) The draw of the Washington State Parks and Recreation Commission bridge across the South...

  13. 33 CFR 117.1063 - Willapa River South Fork.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 33 Navigation and Navigable Waters 1 2014-07-01 2014-07-01 false Willapa River South Fork. 117.1063 Section 117.1063 Navigation and Navigable Waters COAST GUARD, DEPARTMENT OF HOMELAND SECURITY... Fork. (a) The draw of the Washington State Parks and Recreation Commission bridge across the South...

  14. 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

  15. 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

  16. 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

  17. 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

  18. 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

  19. 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

  20. 9. 'CRIB DAM IN LAKE FORK RIVER AT HEADING OF ...

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

    9. 'CRIB DAM IN LAKE FORK RIVER AT HEADING OF LAKE FORK CANAL, UINTAH PROJECT. TWO SLUICEWAYS TWENTY FEET WIDE HAVE BEEN LEFT IN THE DAM TO PASS BOULDERS DURING HIGH WATER. THESE SLUICEWAYS ARE CLOSED BY LOGS AND HAY DURING LOW WATER.' Date unknown - Irrigation Canals in the Uinta Basin, Duchesne, Duchesne County, UT

  1. On the nature of origins of DNA replication in eukaryotes.

    PubMed

    Benbow, R M; Zhao, J; Larson, D D

    1992-10-01

    Chromosomal origins of DNA replication in higher eukaryotes differ significantly from those of E. coli (oriC) and the tumor virus, SV40 (ori sequence). Initiation events appear to occur throughout broad zones rather than at specific origin sequences. Analysis of four chromosomal origin regions reveals that they share common modular sequence elements. These include DNA unwinding elements, pyrimidine tracts that may serve as strong DNA polymerase-primase start sites, scaffold associated regions, transcriptional regulatory sequences, and, possibly, initiator protein binding sites and inherently destabilized regions. Based on the novel organization of chromosomal origin regions, we propose a model for initiation of DNA replication in higher eukaryotes. Unwinding of duplex DNA during initiation may be uncoupled, both temporally and spatially, from DNA synthesis, resulting in transient single-stranded intermediates that function in lieu of conventional replication forks during chromosomal DNA replication. DNA synthesis begins subsequently at multiple sites within the unwound regions rather than at specific origin sequences.

  2. 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

  3. Initiation of DNA Replication within oriP Is Dispensable for Stable Replication of the Latent Epstein-Barr Virus Chromosome after Infection of Established Cell Lines

    PubMed Central

    Norio, Paolo; Schildkraut, Carl L.; Yates, John L.

    2000-01-01

    The 165-kb circularized chromosome of Epstein-Barr virus (EBV) is replicated in latently infected cells once per cell cycle by host proteins during S phase. Replication initiates at multiple sites on latent EBV chromosomes, including within a 1.8-kb region called oriP, which can provide both replication and stabilization for recombinant plasmids in the presence of the EBV-encoded protein, EBNA-1. Replication initiates at or near the dyad symmetry component (DS) of oriP, which depends on multiple EBNA-1 binding sites for activity. To test the importance of the replication function of oriP, the DS was deleted from the viral genome. EBV mutants lacking the DS and carrying a selectable gene could establish latent infections in BL30 cells, in which circular, mutant viral chromosomes were stably maintained. Analysis of replication fork movement using two-dimensional gel electrophoresis showed that the deletion of the DS reduced the initiation events to an undetectable level within the oriP region so that this segment was replicated exclusively by forks entering the region from either direction. A significant slowing or stalling of replication forks that occurs normally at the approximate position of the DS was also eliminated by deletion of the DS. The results confirm the DS as both a replication origin and a place where replication forks pause. Since the replication function of oriP is dispensable at least in certain cell lines, the essential role of EBNA-1 for infection of these cell lines is likely to be that of stabilizing the EBV chromosome by associating with the 30-bp repeats of oriP. The results also imply that in established cell lines, the EBV chromosome can be efficiently replicated entirely from origins that are activated by cellular factors. Presumably, initiation of replication at the DS, mediated by EBNA-1, is important for the natural life cycle of EBV, perhaps in establishing latent infections of normal B cells. PMID:10954558

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

    PubMed

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

    2015-07-01

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

  5. 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.

  6. Electron microscopic analysis of partially replicated bacteriophage T7 DNA.

    PubMed Central

    Burck, K B; Scraba, D G; Miller, R C

    1979-01-01

    Partially replicated bacteriophage T7 DNA was isolated from Escherichia coli infected with UV-irradiated T7 bacteriophage and was analyzed by electron microscopy. The analysis determined the distribution of eye forms and forks in the partially replicated molecules. Eye forms and forks in unit length molecules were aligned with respect to the left end of the T7 genome, and segments were scored for replication in each molecule. The resulting histogram showed that only the left 25 to 30% of the molecules was replicated. Several different origins of DNA replication were used to initiate replication in the UV-irradiated experiments in which 32P-labeled progeny DNA from UV-irradiated phage was annealed with ordered restriction fragments of T7 DNA (K. B. Burck and R. C. Miller, Jr., Proc. Natl. Acad. Sci. U.S.A. 75:6144--6148, 1978). Both analyses support partial-replica hypotheses (N. A. Barricelli and A. H. Doermann, Virology 13:460--476, 1961; Doermann et al., J. Cell. comp. Physiol. 45[Suppl.]:51--74, 1955) as an explanation for the distribution of marker rescue frequencies during cross-reactivation; i.e., replication proceeds in a bidirectional manner from an origin to a site of UV damage, and those regions of the genome which replicate most efficiently are rescued most efficiently by a coinfecting phage. In addition, photoreactivation studies support the hypothesis that thymine dimers are the major UV damage blocking cross-reactivation in the right end of the T7 genome. Images PMID:291738

  7. fork head domain genes in zebrafish.

    PubMed

    Odenthal, J; Nüsslein-Volhard, C

    1998-07-01

    Nine members of the fork head domain gene family (fkd1-fkd9) were isolated from early cDNA libraries in the zebrafish. They show unique expression patterns in whole-mount RNA in situ hybridization during the first 24 h of embryonic development. These fkd genes fall into three of ten classes, based on sequence similarities within the DNA-binding domain, whereas members for the other seven classes described in other vertebrates were not found. In addition to conserved residues at certain positions in the fork head domain, characteristic transcription activation domains as well as similarities in expression patterns were found for members of the different classes. Members of class I (fkd1/axial, fkd2/Zffkh1, fkd4 and fkd7) are differentially transcribed in unsegmented dorsal axial structures such as the floor plate, the notochord, the hypochord and, in addition, the endoderm. Transcripts of fkd3 and fkd5 (class II) are mainly detected in the cells of the ectoderm which form neural tissues, as is the case for genes of this class in other species. RNAs of the three members of class V (fkd6, fkd8 and fkd9) are expressed in the paraxial mesoderm and transiently in the neuroectoderm. fkd6 is strongly expressed in neural crest cells from early stages on, whereas fkd2 and fkd7 are transcribed in individual neural crest cells in the pharyngula period.

  8. Double-strand end repair via the RecBC pathway in Escherichia coli primes DNA replication

    PubMed Central

    Kuzminov, Andrei; Stahl, Franklin W.

    1999-01-01

    To study the relationship between homologous recombination and DNA replication in Escherichia coli, we monitored the behavior of phage λ chromosomes, repressed or not for λ gene activities. Recombination in our system is stimulated both by DNA replication and by experimentally introduced double-strand ends, supporting the idea that DNA replication generates occasional double-strand ends. We report that the RecBC recombinational pathway of E. coli uses double-strand ends to prime DNA synthesis, implying a circular relationship between DNA replication and recombination and suggesting that the primary role of recombination is in the repair of disintegrated replication forks arising during vegetative reproduction. PMID:9990858

  9. Measuring DNA Replication in Hypoxic Conditions.

    PubMed

    Foskolou, Iosifina P; Biasoli, Deborah; Olcina, Monica M; Hammond, Ester M

    2016-01-01

    It is imperative that dividing cells maintain replication fork integrity in order to prevent DNA damage and cell death. The investigation of DNA replication is of high importance as alterations in this process can lead to genomic instability, a known causative factor of tumor development. A simple, sensitive, and informative technique which enables the study of DNA replication, is the DNA fiber assay, an adaptation of which is described in this chapter. The DNA fiber method is a powerful tool, which allows the quantitative and qualitative analysis of DNA replication at the single molecule level. The sequential pulse labeling of live cells with two thymidine analogues and the subsequent detection with specific antibodies and fluorescence imaging allows direct examination of sites of DNA synthesis. In this chapter, we describe how this assay can be performed in conditions of low oxygen levels (hypoxia)-a physiologically relevant stress that occurs in most solid tumors. Moreover, we suggest ways on how to overcome the technical problems that arise while using the hypoxic chambers.

  10. 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.

  11. 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.

    PubMed

    Chakravarthy, M V; Abraha, T W; Schwartz, R J; Fiorotto, M L; Booth, F W

    2000-11-17

    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.

  12. 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

  13. 78 FR 29202 - Environmental Impact Statement: Grand Forks County, North Dakota and Polk County, Minnesota

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-05-17

    ... Federal Highway Administration Environmental Impact Statement: Grand Forks County, North Dakota and Polk... prepared for a proposed highway project in Grand Forks County, North Dakota and Polk County, Minnesota. FOR... Sorlie Bridge over the Red River between Grand Forks, North Dakota and East Grand Forks,...

  14. 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... Fork American River Hydroelectric Project (FERC No. 2079), located on the Middle Fork of the...

  15. New interpretation of Clarks Fork field, northern Bighorn basin, Montana

    SciTech Connect

    Johnson, J.S.; Lindsley-Griffin, N.

    1986-08-01

    Clarks Fork field is located at the northern edge of the Bighorn basin (T9S, R22E) in Carbon County, Montana. Production was first established in 1944 by General Petroleum Corporation in the Cretaceous Peay Sandstone (basal Frontier) and was later extended to the Cretaceous Greybull (1949) and Lakota (1956) sandstones by British American. Total cumulative hydrocarbons from this field are 1,1789,193 bbl of oil and 3,061,522 mcf of gas, with Lakota sandstones being most productive. Lakota production occurs from a structural-stratigraphic trap in an east-west-trending channel on the axis of Clarks Fork anticline, geographically near the center of the township. Our structural reinterpretation of Clarks Fork field suggests that Elk Basin anticline is a northwest extension of the Elk Basin field anticline. The Elk Basin thrust truncates the north limb of the fold and does not strike to the northwest, as shown by earlier interpretations. They interpret a northwest-striking thrust in the center of the township as a splay off the Elk Basin thrust, and have named it the Clarks Fork thrust. The Clarks Fork anticline is located on the hanging wall of Clarks Fork thrust. Subsurface maps indicate the Clarks Fork area has not been fully developed. Stratigraphic traps in the Lakota and Greybull sandstones are present in several areas of the township. Structural traps in the center and northwest portions of the township may also exist.

  16. The Fork+ burnup measurement system: Design and first measurement campaign

    SciTech Connect

    Olson, C.E.; Bronowski, D.R.; McMurtry, W.; Ewing, R.; Jordan, R.; Rivard, D.

    1998-12-31

    Previous work with the original Fork detector showed that burnup as determined by reactor records could be accurately allocated to spent nuclear fuel assemblies. The original Fork detector, designed by Los Alamos National Laboratory, used an ion chamber to measure gross gamma count and a fission chamber to measure neutrons from an activation source, {sup 244}Cm. In its review of the draft Topical Report on Burnup Credit, the US Nuclear Regulatory Commission indicated it felt uncomfortable with a measurement system that depended on reactor records for calibration. The Fork+ system was developed at Sandia National Laboratories under the sponsorship of the Electric Power Research Institute with the aim of providing this independent measurement capability. The initial Fork+ prototype was used in a measurement campaign at the Maine Yankee reactor. The campaign confirmed the applicability of the sensor approach in the Fork+ system and the efficiency of the hand-portable Fork+ prototype in making fuel assembly measurements. It also indicated potential design modifications that will be necessary before the Fork+ can be used effectively on high-burnup spent fuel.

  17. Development of Tuning Fork Based Probes for Atomic Force Microscopy

    NASA Astrophysics Data System (ADS)

    Jalilian, Romaneh; Yazdanpanah, Mehdi M.; Torrez, Neil; Alizadeh, Amirali; Askari, Davood

    2014-03-01

    This article reports on the development of tuning fork-based AFM/STM probes in NaugaNeedles LLC for use in atomic force microscopy. These probes can be mounted on different carriers per customers' request. (e.g., RHK carrier, Omicron carrier, and tuning fork on a Sapphire disk). We are able to design and engineer tuning forks on any type of carrier used in the market. We can attach three types of tips on the edge of a tuning fork prong (i.e., growing Ag2Ga nanoneedles at any arbitrary angle, cantilever of AFM tip, and tungsten wire) with lengths from 100-500 μm. The nanoneedle is located vertical to the fork. Using a suitable insulation and metallic coating, we can make QPlus sensors that can detect tunneling current during the AFM scan. To make Qplus sensors, the entire quartz fork will be coated with an insulating material, before attaching the nanoneedle. Then, the top edge of one prong is coated with a thin layer of conductive metal and the nanoneedle is attached to the fork end of the metal coated prong. The metal coating provides electrical connection to the tip for tunneling current readout and to the electrodes and used to read the QPlus current. Since the amount of mass added to the fork is minimal, the resonance frequency spectrum does not change and still remains around 32.6 KHz and the Q factor is around 1,200 in ambient condition. These probes can enhance the performance of tuning fork based atomic microscopy.

  18. Antiviral CD8+ T cells in the genital tract control viral replication and delay progression to AIDS after vaginal SIV challenge in rhesus macaques immunized with virulence attenuated SHIV 89.6

    PubMed Central

    Genescà, M.; McChesney, M.B.; Miller, C.J.

    2009-01-01

    Genescà M, McChesney MB, Miller CJ (Center for Comparative Medicine and California National Primate Research Center, University of California, Davis, CA, USA). Antiviral CD8+ T cells in the genital tract control viral replication and delay progression to AIDS after vaginal SIV challenge in rhesus macaques immunized with virulence attenuated SHIV 89.6 (Review). The recently failed clinical efficacy trial of an acquired immunodeficiency syndrome (AIDS) vaccine that elicits antiviral CD8+ T-cell responses has emphasized the challenge of producing an effective vaccine against human immunodeficiency virus (HIV). In the simian immunodeficiency virus (SIV)/rhesus monkey model of AIDS, live-attenuated lentivirus ‘vaccines’ provide the best protection from uncontrolled viral replication and clinical disease after pathogenic SIV challenge. This review summarizes a recent series of studies in which we show that after vaginal SIV challenge of rhesus macaques immunized with an attenuated lentivirus protection from uncontrolled viral replication is primarily mediated by CD8+ T cells in the vaginal mucosa. Immunization with a chimeric simian/human immunodeficiency virus (SHIV) results in a systemic infection that induces a moderate population of SIV-specific CD8+ and CD4+ T cells with cytolytic potential in the vaginal mucosa. Depletion of CD8+ T cells at the time of SIV challenge completely abrogates the protection mediated by prior infection with attenuated SHIV. Further after vaginal SIV challenge, the only significant expansion of SIV-specific T cells occurs in the vagina in these animals. No significant expansion of T-cell responses was observed in systemic lymphoid tissues. Thus, the presence of SIV-specific CD8+ T cells in the vagina on the day of vaginal SIV challenge and a modest expansion of local effector T cells is sufficient to stop uncontrolled SIV replication. It seems that T-cell based vaccine strategies that can elicit mucosal effector CD8+ T

  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. 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

  1. Chemical and biological sensing using tuning forks

    SciTech Connect

    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.

  2. 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

  3. 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

  4. 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

  5. Cytoplasmic Viral Replication Complexes

    PubMed Central

    den Boon, Johan A.; Diaz, Arturo; Ahlquist, Paul

    2010-01-01

    Many viruses that replicate in the cytoplasm compartmentalize their genome replication and transcription in organelle-like structures that enhance replication efficiency and protection from host defenses. In particular, recent studies with diverse positive-strand RNA viruses have further elucidated the ultrastructure of membrane-bounded RNA replication complexes and their close coordination with virion assembly and budding. The structure, function and assembly of some positive-strand RNA virus replication complexes have parallels and potential evolutionary links with the replicative cores of double-strand RNA virus and retrovirus virions, and more general similarities with the replication factories of cytoplasmic DNA viruses. PMID:20638644

  6. 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.

  7. 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

  8. Cyclin-Dependent Kinase Suppression by WEE1 Kinase Protects the Genome through Control of Replication Initiation and Nucleotide Consumption

    PubMed Central

    Beck, Halfdan; Nähse-Kumpf, Viola; Larsen, Marie Sofie Yoo; O'Hanlon, Karen A.; Patzke, Sebastian; Holmberg, Christian; Mejlvang, Jakob; Groth, Anja; Nielsen, Olaf

    2012-01-01

    Activation of oncogenes or inhibition of WEE1 kinase deregulates cyclin-dependent kinase (CDK) activity and leads to replication stress; however, the underlying mechanism is not understood. We now show that elevation of CDK activity by inhibition of WEE1 kinase rapidly increases initiation of replication. This leads to nucleotide shortage and reduces replication fork speed, which is followed by SLX4/MUS81-mediated DNA double-strand breakage. Fork speed is normalized and DNA double-strand break (DSB) formation is suppressed when CDT1, a key factor for replication initiation, is depleted. Furthermore, addition of nucleosides counteracts the effects of unscheduled CDK activity on fork speed and DNA DSB formation. Finally, we show that WEE1 regulates the ionizing radiation (IR)-induced S-phase checkpoint, consistent with its role in control of replication initiation. In conclusion, these results suggest that deregulated CDK activity, such as that occurring following inhibition of WEE1 kinase or activation of oncogenes, induces replication stress and loss of genomic integrity through increased firing of replication origins and subsequent nucleotide shortage. PMID:22907750

  9. Liver microRNA hsa-miR-125a-5p in HBV chronic infection: correlation with HBV replication and disease progression.

    PubMed

    Coppola, Nicola; Potenza, Nicoletta; Pisaturo, Mariantonietta; Mosca, Nicola; Tonziello, Gilda; Signoriello, Giuseppe; Messina, Vincenzo; Sagnelli, Caterina; Russo, Aniello; Sagnelli, Evangelista

    2013-01-01

    To study in HBsAg chronic carriers the expression of liver hsa-miR-125a-5p and its correlation with liver HBV-DNA values and clinical presentation, 27 consecutive Caucasian, HBsAg/anti-HBe/HBV-DNA-positive patients who were naive to nucleos(t)ide analogues and interferon therapy and had no marker of HCV, HDV or HIV infection and no history of alcohol intake were enrolled. For each patient, liver HBV DNA and liver hsa-miR-125a-5p were quantified by real-time PCR in relation to β-globin DNA or RNU6B, respectively. Liver fibrosis and necroinflammation were graded by applying Ishak's scoring system. Liver hsa-miR-125a-5p was detected in all patients enrolled and a correlation between its concentration and liver HBV DNA was demonstrated (p<0.0001). Higher liver hsa-miR-125a-5p concentrations were observed in patients with HBV-DNA plasma level >10(3) IU/ml (p<0.02), in those with HAI >6 (p = 0.02) and those with fibrosis score >2 (p<0.02) than in patients with lower scores. Higher HBV-DNA liver concentrations were found in patients with abnormal AST (p = 0.005) and ALT serum levels (p = 0.05), in those with serum HBV DNA higher than 10E3 IU/mL (p = 0.001) and those with fibrosis score >2 (p = 0.02) than in patients with a lower load. By multivariate logistic regression analysis, liver hsa-miR-125a-5p was identified as an independent predictor of disease progression: O.R. = 4.21, C.I. 95% = 1.08-16.43, p<0.05, for HAI >6; O.R. = 3.12, C.I. 95% = 1.17-8.27, p<0.05, for fibrosis score >2. In conclusion, in HBsAg/anti-HBe-positive patients, the liver hsa-miR-125a-5p level correlated with liver and plasma HBV-DNA values and was associated to a more severe disease progression. PMID:23843939

  10. 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

  11. 26. MOORSE DRILL CABINET AND FORK ART FABRICATED AT SHOP ...

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

    26. MOORSE DRILL CABINET AND FORK ART FABRICATED AT SHOP (L TO R)- LOOKING SOUTHEAST. - W. A. Young & Sons Foundry & Machine Shop, On Water Street along Monongahela River, Rices Landing, Greene County, PA

  12. 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

  13. 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

  14. 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)

  15. 8. VIEW OF WHEEL RACK FOR BORING MILL. Fork loading ...

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

    8. VIEW OF WHEEL RACK FOR BORING MILL. Fork loading crane, manufactured by Cleveland Tramrail, 2-1/2 ton capacity. - Juniata Shops, Erecting Shop & Machine Shop, East of Fourth Avenue, between Fourth & Fifth Streets, Altoona, Blair County, PA

  16. 9. VIEW OF WHEEL RACK FOR BORING MILL. Fork loading ...

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

    9. VIEW OF WHEEL RACK FOR BORING MILL. Fork loading crane, manufactured by Cleveland Tramrail, 2-1/2 ton capacity. - Juniata Shops, Erecting Shop & Machine Shop, East of Fourth Avenue, between Fourth & Fifth Streets, Altoona, Blair County, PA

  17. 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

  18. Initiation preference at a yeast origin of replication.

    PubMed Central

    Brewer, B J; Fangman, W L

    1994-01-01

    Replication origins in the yeast Saccharomyces cerevisiae are identified as autonomous replication sequence (ARS) elements. To examine the effect of origin density on replication initiation, we have analyzed the replication of a plasmid that contains two copies of the same origin, ARS1. The activation of origins and the direction that replication forks move through flanking sequences can be physically determined by analyzing replication intermediates on two-dimensional agarose gels. We find that only one of the two identical ARSs on the plasmid initiates replication on any given plasmid molecule; that is, this close spacing of ARSs results in an apparent interference between the potential origins. Moreover, in the particular plasmid that we constructed, one of the two identical copies of ARS1 is used four times more frequently than the other one. These results show that the plasmid context is critical for determining the preferred origin. This origin preference is also exhibited when the tandem copies of ARS1 are introduced into a yeast chromosome. The sequences responsible for establishing the origin preference have been identified by deletion analysis and are found to reside in a portion of the yeast URA3 gene. Images PMID:8159762

  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. INTEGRATED OUTCROP AND SUBSURFACE STUDIES OF THE INTERWELL ENVIRONMENT OF CARBONATE RESERVOIRS: CLEAR FORK (LEONARDIAN AGE) RESERVOIRS, WEST TEXAS AND NEW MEXICO

    SciTech Connect

    F. Jerry Lucia; Stephen E. Laubach

    2000-04-01

    We have completed 18 months of this project and are reporting progress we have made in the areas of fracture and reservoir modeling. Task 1a: Construct Model of Outcrop Stratigraphic Framework has been completed. Task 1b: Construct Model of Fine-Scale Petrophysical Heterogeneity has been completed. Task 1c: Construct Model of Fracture Porosity and Permeability the object of this task is to characterize fracture porosity and distribution in the Wasson Clear Fork reservoir and to determine the effects of fractures on well performance. The approach is to measure fracture attributes in an analog outcrop, to develop models of fracture spacing and aperture, and to apply this information to the South Wasson Clear Fork reservoir. A data set has been collected from outcrops of the Clear Fork Formation, Apache Canyon, Sierra Diablo Mountains, West Texas, and a summary of the results is presented.