Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “stalled replication fork”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 217 records · Page 12Linked to original sources
PubMed2004

DNA repair nucleases.

Stability of DNA largely depends on accuracy of repair mechanisms, which remove structural anomalies induced by exogenous and endogenous agents or introduced by DNA metabolism, such as replication. Most repair mechanisms include nucleolytic processing of DNA, where nucleases cleave a phosphodiester bond between a deoxyribose and a phosphate residue, thereby producing 5'-terminal phosphate and 3'-terminal hydroxyl groups. Exonucleases hydrolyse nucleotides from either the 5' or 3' end of DNA, while endonucleases incise internal sites of DNA. Flap endonucleases cleave DNA flap structures at or near the junction between single-stranded and double-stranded regions. DNA nucleases play a crucial role in mismatch repair, nucleotide excision repair, base excision repair and double-strand break repair. In addition, nucleolytic repair functions are required during replication to remove misincorporated nucleotides, Okazaki fragments and 3' tails that may be formed after repair of stalled replication forks.

Bacterial Proteins↗
PubMed2004

The DNA damage response: sensing and signaling.

The protein kinases ATM and ATR are central components of the checkpoint mechanisms that signal the presence of damaged DNA and stalled replication forks. Recent studies have provided important new insights into how these kinases work together with their regulatory subunits, DNA repair proteins and adaptor proteins to sense abnormal DNA structures and implement the appropriate DNA damage response. These advances have provided a more detailed understanding of the interface between damaged DNA and the checkpoint sensor proteins.

Animals↗
PubMed2004

Choreography of the DNA damage response: spatiotemporal relationships among checkpoint and repair proteins.

DNA repair is an essential process for preserving genome integrity in all organisms. In eukaryotes, recombinational repair is choreographed by multiprotein complexes that are organized into centers (foci). Here, we analyze the cellular response to DNA double-strand breaks (DSBs) and replication stress in Saccharomyces cerevisiae. The Mre11 nuclease and the ATM-related Tel1 kinase are the first proteins detected at DSBs. Next, the Rfa1 single-strand DNA binding protein relocalizes to the break and recruits other key checkpoint proteins. Later and only in S and G2 phase, the homologous recombination machinery assembles at the site. Unlike the response to DSBs, Mre11 and recombination proteins are not recruited to hydroxyurea-stalled replication forks unless the forks collapse. The cellular response to DSBs and DNA replication stress is likely directed by the Mre11 complex detecting and processing DNA ends in conjunction with Sae2 and by RP-A recognizing single-stranded DNA and recruiting additional checkpoint and repair proteins.

Cell Cycle Proteins↗
PubMed2006

Polo-like kinase-1 controls proteasome-dependent degradation of Claspin during checkpoint recovery.

DNA-damage checkpoints maintain genomic integrity by mediating a cell-cycle delay in response to genotoxic stress or stalled replication forks. In response to damage, the checkpoint kinase ATR phosphorylates and activates its effector kinase Chk1 in a process that critically depends on Claspin . However, it is not known how exactly this kinase cascade is silenced. Here we demonstrate that the abundance of Claspin is regulated through proteasomal degradation. In response to DNA damage, Claspin is transiently stabilized, and its expression depends on Chk1 kinase activity. In addition, we show that Claspin is degraded upon mitotic entry, a process that depends on the beta-TrCP-SCF ubiquitin ligase and Polo-like kinase-1 (Plk1). We demonstrate that Claspin interacts with both beta-TrCP and Plk1 and that inactivation of these components or the beta-TrCP recognition motif in Claspin prevents its mitotic degradation. Interestingly, expression of a nondegradable Claspin mutant inhibits recovery from a DNA-damage-induced checkpoint arrest. Thus, we conclude that Claspin levels are tightly regulated, both during unperturbed cell cycles and after DNA damage. Moreover, our data demonstrate that the degradation of Claspin at the onset of mitosis is an essential step for the recovery of a cell from a DNA-damage-induced cell-cycle arrest.

Adaptor Proteins, Signal Transducing↗
PubMed2005

Conservation of RecG activity from pathogens to hyperthermophiles.

Maintaining the integrity of the genome is essential for the survival of all organisms. RecG helicase plays an important part in this process in Escherichia coli, promoting recombination and DNA repair, and providing ways to rescue stalled replication forks by way of a Holliday junction intermediate. We purified RecG proteins from three other species: two Gram-positive mesophiles, Bacillus subtilis and Streptococcus pneumoniae, and one extreme thermophile, Aquifex aeolicus. All three proteins bind and unwind replication fork and Holliday junction DNA molecules with efficiencies similar to the E. coli protein. Proteins from the Gram-positive species promote DNA repair in E. coli, indicating either that RecG acts alone or that any necessary protein-protein interactions are conserved. The S. pneumoniae RecG reduces plasmid copy number when expressed in E. coli, indicating that like the E. coli protein it unwinds plasmid R loop structures used to prime replication. This effect is not seen with B. subtilis RecG; the protein either lacks R loop unwinding activity or is compromised by having insufficient ATP. The A. aeolicus protein unwinds DNA well at 60 degrees C but is less efficient at 37 degrees C, explaining its inability to function in E. coli at this temperature. The N-terminal extension present in this protein was investigated and found to be dispensable for activity and thermo-stability. The results presented suggest that the role of RecG in DNA replication and repair is likely to be conserved throughout all bacteria, which underlines the importance of this protein in genome duplication and cell survival.

Bacteria↗
PubMed2006

Phosphorylation of replication protein A by S-phase checkpoint kinases.

The major eukaryotic single-stranded DNA (ssDNA) binding protein, replication protein A (RPA), is a heterotrimer with subunits of 70, 32 and 14 kDa (RPA70, RPA32 and RPA14). RPA-coated ssDNA has been implicated as one of the triggers for intra-S-phase checkpoint activation. Phosphorylation of RPA occurs in cells with damaged DNA or stalled replication forks. Here we show that human RPA70 and RPA32 can be phosphorylated by purified S-phase checkpoint kinases, ATR and Chk1. While ATR phosphorylates the N-terminus of RPA70, Chk1 preferentially phosphorylates RPA's major ssDNA binding domain. Chk1 phosphorylated RPA70 shows reduced ssDNA binding activity, and binding of RPA to ssDNA blocks Chk1 phosphorylation, suggesting that Chk1 and ssDNA compete for RPA's major ssDNA binding domain. ssDNA stimulates RPA32 phosphorylation by ATR in a length dependent manner. Furthermore, 3'-, but not 5'-, recessed single strand/double strand DNA junctions produce an even stronger stimulatory effect on RPA32 phosphorylation by ATR. This stimulation occurs for both RNA and DNA recessed ends. RPA's DNA binding polarity and its interaction to 3'-primer-template junctions contribute to efficient RPA32 phosphorylation. Progression of DNA polymerase is able to block the accessibility of the 3'-recessed ends and prevent the stimulatory effects of primer-template junctions on RPA phosphorylation by ATR. We propose models for the role of RPA phosphorylation by Chk1 in S-phase checkpoint pathways, and the possible regulation of ATR activity by different nucleic acid structures.

Ataxia Telangiectasia Mutated Proteins↗
PubMed2006

Multiple solutions to inefficient lesion bypass by T7 DNA polymerase.

We hypothesize that enzymatic switching during translesion synthesis (TLS) to relieve stalled replication forks occurs during transitions from preferential to disfavored use of damaged primer-templates, and that the polymerase or 3'-exonuclease used for each successive nucleotide incorporated is the one whose properties result in the highest efficiency and the highest fidelity of bypass. Testing this hypothesis requires quantitative determination of the relative lesion bypass ability of both TLS polymerases and major replicative polymerases. As a model of the latter, here we measure the efficiency and fidelity of cis-syn TT dimer and abasic site bypass using the structurally well-characterized T7 DNA polymerase. No bypass of either lesion occurred during a single round of synthesis, and the exonuclease activity of wild-type T7 DNA polymerase was critical in preventing TLS. When repetitive cycling of the exonuclease-deficient enzyme was allowed, limited bypass did occur but hundreds to thousands of cycles were required to achieve even a single bypass event. Analysis of TLS fidelity indicated that these rare bypass events involved rearrangements of the template and primer strands, insertions opposite the lesion, and combinations of these events, with the choice among these strongly depending on the sequence context of the lesion. Moreover, the presence of a lesion affected the fidelity of copying adjacent undamaged template bases, even when lesion bypass itself was correct. The results also indicate that a TT dimer presents a different type of block to the polymerase than an abasic site, even though both lesions are extremely potent blocks to processive synthesis. The approaches used here to quantify the efficiency and fidelity of TLS can be applied to other polymerase-lesion combinations, to provide guidance as to which of many possible polymerases is most likely to bypass various lesions in biological contexts.

DNA Damage↗
PubMed2004

Switching from high-fidelity replicases to low-fidelity lesion-bypass polymerases.

Replication of damaged DNA often requires a DNA polymerase in addition to the cell's normal replicase. Recent research has begun to shed light on the switch from a high-fidelity replicative polymerase to a low-fidelity translesion polymerase that occurs at a stalled replication fork. A picture is emerging in which eukaryotic replicative clamps are posttranslationally modified by ubiquitination, SUMOylation or phosphorylation. It is believed that such modifications help to regulate the access of translesion polymerases to the nascent primer terminus.

DNA Damage↗
PubMed2004

Redundancy, insult-specific sensors and thresholds: unlocking the S-phase checkpoint response.

DNA damage that is not properly repaired during genomic replication is a major source of gross chromosomal rearrangements and sequence loss during cell proliferation. In higher eukaryotes such mutations increase the risk of cancer. Eukaryotic cells have multiple checkpoint responses activated by DNA damage and stalled replication forks. We focus here on fork-associated events that activate and respond to S-phase checkpoint kinases.

Animals↗
PubMed2006

A new molecular model of cellular aging based on Werner syndrome.

In the Hayflick model, a decrease in the number of cells capable of undergoing proliferation constitutes the main criterion of cellular aging and is closely linked to organismal aging. Evidence suggests that a reduction of DNA replication capacity or a failure in the regulation systems of DNA replication occurs in aging cells, which leads to cellular replicative senescence. DNA replication depends on two parameters: the number of active replicons and the rate of chain elongation. Epigenetic parameters, in particular methylation, would be able to, either directly or indirectly, regulate replication origin activity of normal mammalian cells, as well as subsequent DNA replication. Werner syndrome (WS) is an autosomal recessive disorder that results in premature aging and is considered to be a model system for the study of cellular senescence and aging. WRN could involve DNA replication initiation, replication foci establishment, and the resolution of stalled replication forks during replication. In this paper, a molecular model of in vitro cellular aging is presented in which changes in DNA methylation, in particular, global hypomethylation related to methyltransferase Dnmt1 downregulation, and specific hypermethylation related to methyltransferase Dnmt3b upregulation as seen during cellular aging, could be responsible for the inactivation of replication origins or foci and the subsequent documented reduction in DNA replication capacity and increased mutations that are observed in senescent cells. Thus, Werner syndrome cells could be mimicking what is observed in normal aging in an accelerated form.

Aging↗
PubMed2003

Precocious G1/S transitions and genomic instability: the origin connection.

Both in yeast and in higher eukaryotes, genomic instability often ensues when the G1/S transition machinery is deregulated and cells are forced to enter S phase prematurely. This case of acquired mutability is particularly important, since a majority of genes mutated in human cancers encode factors that influence the G1/S transition. The precocious G1/S transition often results in a sub-optimal S phase. Moreover, the problems generated in such an S phase can escape detection by the cellular surveillance systems, allowing undeterred mitosis. This review focuses primarily on budding yeast data, where progress has been made in the past couple of years towards a mechanistic understanding of the underlying processes. A dual surveillance system is discussed, which relies on the presence of licensed but unfired origins and stalled replication forks to deter mitosis until replication is complete. Normally, this dual surveillance system allows S phase to be flexible in duration in a variety of growth conditions, when the fork density and/or fork progression rates can vary widely. However, precocious exit from G1 can have a disabling effect on this surveillance system. Premature exit from G1 can cut short the licensing of origins and the accumulation of resources for the upcoming replication, while giving a cell a false indication that it is metabolically ready to conduct S phase.

Animals↗
PubMed2006

DNA structures, repeat expansions and human hereditary disorders.

Expansions of simple DNA repeats are responsible for more than two dozen hereditary disorders in humans, including fragile X syndrome, myotonic dystrophy, Huntington's disease, various spinocerebellar ataxias, Friedreich's ataxia and others. During the past decade, it became clear that unusual structural features of expandable repeats greatly contribute to their instability and could lead to their expansion. Furthermore, DNA replication, repair and recombination are implicated in the formation of repeat expansions, as shown in various experimental systems. The replication model of repeat expansion stipulates that unusual structures of expandable repeats stall replication fork progression, whereas extra repeats are added during replication fork restart. It also explains the bias toward repeat expansion or contraction that was observed in different organisms.

DNA↗
PubMed2004

Error-prone replication for better or worse.

Precise genome duplication requires accurate copying by DNA polymerases and the elimination of occasional mistakes by proofreading exonucleases and mismatch repair enzymes. The commonly held belief that 'if something is worth doing, then it's worth doing well' normally applies to DNA replication and repair, however, there are exceptions. This review describes elements that are crucial to cell fitness, evolution and survival in the recently discovered error-prone DNA polymerases. Large numbers of errant DNA polymerases, spanning microorganisms to humans, are used to rescue stalled replication forks by copying damaged DNA and even undamaged DNA to generate 'purposeful' mutations that generate genetic diversity in times of stress. Here we focus on low-fidelity polymerases from bacteria, comparing Escherichia coli, archeabacteria and those most recently discovered in Gram-positive Bacilli, Streptococcus, pathogenic Mycobacterium and intein-containing cyanobacteria.

Adaptation, Biological↗
PubMed2004

Replication protein A and gamma-H2AX foci assembly is triggered by cellular response to DNA double-strand breaks.

Human replication protein A (RPA p34), a crucial component of diverse DNA excision repair pathways, is implicated in DNA double-strand break (DSB) repair. To evaluate its role in DSB repair, the intranuclear dynamics of RPA was investigated after DNA damage and replication blockage in human cells. Using two different agents [ionizing radiation (IR) and hydroxyurea (HU)] to generate DSBs, we found that RPA relocated into distinct nuclear foci and colocalized with a well-known DSB binding factor, gamma-H2AX, at the sites of DNA damage in a time-dependent manner. Colocalization of RPA and gamma-H2AX foci peaked at 2 h after IR treatment and subsequently declined with increasing postrecovery times. The time course of RPA and gamma-H2AX foci association correlated well with the DSB repair activity detected by a neutral comet assay. A phosphatidylinositol-3 (PI-3) kinase inhibitor, wortmannin, completely abolished both RPA and gamma-H2AX foci formation triggered by IR. Additionally, radiosensitive ataxia telangiectasia (AT) cells harboring mutations in ATM gene product were found to be deficient in RPA and gamma-H2AX colocalization after IR. Transfection of AT cells with ATM cDNA fully restored the association of RPA foci with gamma-H2AX illustrating the requirement of ATM gene product for this process. The exact coincidence of RPA and gamma-H2AX in response to HU specifically in S-phase cells supports their role in DNA replication checkpoint control. Depletion of RPA by small interfering RNA (SiRNA) substantially elevated the frequencies of IR-induced micronuclei (MN) and apoptosis in human cells suggestive of a role for RPA in DSB repair. We propose that RPA in association with gamma-H2AX contributes to both DNA damage checkpoint control and repair in response to strand breaks and stalled replication forks in human cells.

Apoptosis↗
PubMed2003

RAD51 is involved in repair of damage associated with DNA replication in mammalian cells.

The RAD51 protein, a eukaryotic homologue of the Escherichia coli RecA protein, plays an important role in the repair of DNA double-strand breaks (DSBs) by homologous recombination (HR) in mammalian cells. Recent findings suggest that HR may be important in repair following replication arrest in mammalian cells. Here, we have investigated the role of RAD51 in the repair of different types of damage induced during DNA replication with etoposide, hydroxyurea or thymidine. We show that etoposide induces DSBs at newly replicated DNA more frequently than gamma-rays, and that these DSBs are different from those induced by hydroxyurea. No DSB was found following treatment with thymidine. Although these compounds appear to induce different DNA lesions during DNA replication, we show that a cell line overexpressing RAD51 is resistant to all of them, indicating that RAD51 is involved in repair of a wide range of DNA lesions during DNA replication. We observe fewer etoposide-induced DSBs in RAD51-overexpressing cells and that HR repair of etoposide-induced DSBs is faster. Finally, we show that induced long-tract HR in the hprt gene is suppressed in RAD51-overexpressing cells, although global HR appears not to be suppressed. This suggests that overexpression of RAD51 prevents long-tract HR occurring during DNA replication. We discuss our results in light of recent models suggested for HR at stalled replication forks.

Animals↗
PubMed2002

Replication of damaged DNA in mammalian cells: new solutions to an old problem.

All cells need not only to remove damage from their DNA, but also to be able to replicate DNA containing unrepaired damage. In mammalian cells, the major process by which cells are able to replicate damaged templates is translesion synthesis, the direct synthesis of DNA past altered bases. Crucial to this process is a series of recently discovered DNA polymerases. Most of them belong to a new family of polymerases designated the Y-family, which have conserved sequences in the catalytic N-terminal half of the proteins. These polymerases have different efficiencies and specificities in vitro depending on the type of damage in the template.One of them, DNA polymerase eta, is defective in xeroderma pigmentosum variants, and overwhelming evidence suggests that this is the polymerase that carries out translesion synthesis past UV-induced cyclobutane pyrimidine dimers in vivo. DNA polymerase eta is localised in replication factories during DNA replication and accumulates at sites of stalled replication forks. Many studies have been carried out on the properties of the other polymerases in vitro, but there is as yet very little evidence for their specific roles in vivo.

Cells, Cultured↗
PubMed2001

Mus81-Eme1 are essential components of a Holliday junction resolvase.

Mus81, a fission yeast protein related to the XPF subunit of ERCC1-XPF nucleotide excision repair endonuclease, is essential for meiosis and important for coping with stalled replication forks. These processes require resolution of X-shaped DNA structures known as Holliday junctions. We report that Mus81 and an associated protein Eme1 are components of an endonuclease that resolves Holliday junctions into linear duplex products. Mus81 and Eme1 are required during meiosis at a late step of meiotic recombination. The mus81 meiotic defect is rescued by expression of a bacterial Holliday junction resolvase. These findings constitute strong evidence that Mus81 and Eme1 are subunits of a nuclear Holliday junction resolvase.

Amino Acid Sequence↗
PubMedDate not supplied

Inhibition of DNA synthesis is not sufficient to cause mutagenesis in Chinese hamster cells.

Experiments were designed to test whether the inhibition of DNA synthesis in Chinese hamster V79 cells would result in increased mutagenesis by a mechanism similar to "SOS repair" in E. coli. Treatment of cells for 16 hours with excess of the deoxynucleosides TdR, UdR, AdR and GdR was mutagenic, whereas treatment with hydroxyurea demonstrated no mutagenic effect. The mutagenicity of TdR could be reversed by the addition of CdR. In E. coli, inhibition of DNA synthesis by a short exposure to hydroxyurea resulted in the induction of lambda prophage and increased mutagenesis. These results show that whereas the presence of a stalled replication fork in E. coli can result in mutagenesis via induction of the "SOS system", the same phenomenon does not seem to occur in Chinese hamster V79 cells. The mutagenic mechanism of high concentrations of deoxynucleosides in Chinese hamster V79 cells is likely to be due to replication errors which result from alterations in deoxynucleotide pools.

Animals↗