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Mrc1 and DNA polymerase epsilon function together in linking DNA replication and the S phase checkpoint.

ABSTRACT: Yeast Mrc1, ortholog of metazoan Claspin, is both a central component of normal DNA replication forks and a mediator of the S phase checkpoint. We report that Mrc1 interacts with Pol2, the catalytic subunit of DNA polymerase epsilon, essential for leading-strand DNA replication and for the checkpoint. In unperturbed cells, Mrc1 interacts independently with both the N-terminal and C-terminal halves of Pol2 (Pol2N and Pol2C). Strikingly, phosphorylation of Mrc1 during the S phase checkpoint abolishes Pol2N binding, but not Pol2C interaction. Mrc1 is required to stabilize Pol2 at replication forks stalled in HU. The bimodal Mrc1/Pol2 interaction may be an additional step in regulating the S phase checkpoint response to DNA damage on the leading strand. We propose that Mrc1, which also interacts with the MCMs, may modulate coupling of polymerization and unwinding at the replication fork.

SUBMITTER: Lou H

PROVIDER: S-EPMC2699584 | biostudies-literature | 2008 Oct

REPOSITORIES: biostudies-literature

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Mrc1 and DNA polymerase epsilon function together in linking DNA replication and the S phase checkpoint.

Lou Huiqiang H Komata Makiko M Katou Yuki Y Guan Zhiyun Z Reis Clara C CC Budd Martin M Shirahige Katsuhiko K Campbell Judith L JL

Molecular cell 20081001 1

Yeast Mrc1, ortholog of metazoan Claspin, is both a central component of normal DNA replication forks and a mediator of the S phase checkpoint. We report that Mrc1 interacts with Pol2, the catalytic subunit of DNA polymerase epsilon, essential for leading-strand DNA replication and for the checkpoint. In unperturbed cells, Mrc1 interacts independently with both the N-terminal and C-terminal halves of Pol2 (Pol2N and Pol2C). Strikingly, phosphorylation of Mrc1 during the S phase checkpoint abolis ...[more]

PMID: 18851837

Similar Datasets

Mrc1 and DNA polymerase ε function together in linking DNA replication and the S phase checkpoint

Project description:Yeast Mrc1, ortholog of metazoan Claspin, is both a central component of normal DNA replication forks and a mediator of the S phase checkpoint. We report that Mrc1 interacts with Pol2, the catalytic subunit of DNA polymerase ε, essential for leading strand DNA replication and for the checkpoint. In unperturbed cells, Mrc1 interacts independently with both the N-terminal and C-terminal halves of Pol2 (Pol2N and Pol2C). Strikingly, phosphorylation of Mrc1 during the S phase checkpoint abolishes Pol2N binding but not Pol2C interaction. Mrc1 is required to stabilize Pol2 at replication forks stalled in HU. The bimodal Mrc1/Pol2 interaction may identify a novel step in regulating the S phase checkpoint response to DNA damage on the leading strand. We propose that Mrc1, which also interacts with the MCMs, may modulate coupling of polymerization and unwinding at the replication fork.

2008-10-10 | GSE12138 | GEO

Schizosaccharomyces pombe cdc20+ encodes DNA polymerase epsilon and is required for chromosomal replication but not for the S phase checkpoint.

Project description:In fission yeast both DNA polymerase alpha (pol alpha) and delta (pol delta) are required for DNA chromosomal replication. Here we demonstrate that Schizosaccharomyces pombe cdc20+ encodes the catalytic subunit of DNA polymerase epsilon (pol epsilon) and that this enzyme is also required for DNA replication. Following a shift to the restrictive temperature, cdc20 temperature-sensitive mutant cells block at the onset of DNA replication, suggesting that cdc20+ is required early in S phase very near to the initiation step. In the budding yeast Saccharomyces cerevisiae, it has been reported that in addition to its proposed role in chromosomal replication, DNA pol epsilon (encoded by POL2) also functions directly as an S phase checkpoint sensor [Navas, T. A., Zhou, Z. & Elledge, S. J. (1995) Cell 80, 29-39]. We have investigated whether cdc20+ is required for the checkpoint control operating in fission yeast, and our data indicate that pol epsilon does not have a role as a checkpoint sensor coordinating S phase with mitosis. In contrast, germinating spores disrupted for the gene encoding pol alpha rapidly enter mitosis in the absence of DNA synthesis, suggesting that in the absence of pol alpha, normal coordination between S phase and mitosis is lost. We propose that the checkpoint signal operating in S phase depends on assembly of the replication initiation complex, and that this signal is generated prior to the elongation stage of DNA synthesis.

| S-EPMC25014 | biostudies-literature

Rad53 checkpoint kinase regulation of DNA replication fork rate via Mrc1 phosphorylation.

Project description:The Rad53 DNA checkpoint protein kinase plays multiple roles in the budding yeast cell response to DNA replication stress. Key amongst these is its enigmatic role in safeguarding DNA replication forks. Using DNA replication reactions reconstituted with purified proteins, we show Rad53 phosphorylation of Sld3/7 or Dbf4-dependent kinase blocks replication initiation whilst phosphorylation of Mrc1 or Mcm10 slows elongation. Mrc1 phosphorylation is necessary and sufficient to slow replication forks in complete reactions; Mcm10 phosphorylation can also slow replication forks, but only in the absence of unphosphorylated Mrc1. Mrc1 stimulates the unwinding rate of the replicative helicase, CMG, and Rad53 phosphorylation of Mrc1 prevents this. We show that a phosphorylation-mimicking Mrc1 mutant cannot stimulate replication in vitro and partially rescues the sensitivity of a rad53 null mutant to genotoxic stress in vivo. Our results show that Rad53 protects replication forks in part by antagonising Mrc1 stimulation of CMG unwinding.

| S-EPMC8387023 | biostudies-literature

Differential regulation of homologous recombination at DNA breaks and replication forks by the Mrc1 branch of the S-phase checkpoint.

Project description:The Rad52 pathway has a central function in the recombinational repair of chromosome breaks and in the recovery from replication stress. Tolerance to replication stress also depends on the Mec1 kinase, which activates the DNA replication checkpoint in an Mrc1-dependent manner in response to fork arrest. Although the Mec1 and Rad52 pathways are initiated by the same single-strand DNA (ssDNA) intermediate, their interplay at stalled forks remains largely unexplored. Here, we show that the replication checkpoint suppresses the formation of Rad52 foci in an Mrc1-dependent manner and prevents homologous recombination (HR) at chromosome breaks induced by the HO endonuclease. This repression operates at least in part by impeding resection of DNA ends, which is essential to generate 3' ssDNA tails, the primary substrate of HR. Interestingly, we also observed that the Mec1 pathway does not prevent recombination at stalled forks, presumably because they already contain ssDNA. Taken together, these data indicate that the DNA replication checkpoint suppresses genomic instability in S phase by blocking recombination at chromosome breaks and permitting helpful recombination at stalled forks.

| S-EPMC2683710 | biostudies-literature

Mutations in the Non-Catalytic Subunit Dpb2 of DNA Polymerase Epsilon Affect the Nrm1 Branch of the DNA Replication Checkpoint.

Project description:To preserve genome integrity, the S-phase checkpoint senses damaged DNA or nucleotide depletion and when necessary, arrests replication progression and delays cell division. Previous studies, based on two pol2 mutants have suggested the involvement of DNA polymerase epsilon (Pol ?) in sensing DNA replication accuracy in Saccharomyces cerevisiae. Here we have studied the involvement of Pol ? in sensing proper progression of DNA replication, using a mutant in DPB2, the gene coding for a non-catalytic subunit of Pol ?. Under genotoxic conditions, the dpb2-103 cells progress through S phase faster than wild-type cells. Moreover, the Nrm1-dependent branch of the checkpoint, which regulates the expression of many replication checkpoint genes, is impaired in dpb2-103 cells. Finally, deletion of DDC1 in the dpb2-103 mutant is lethal supporting a model of strand-specific activation of the replication checkpoint. This lethality is suppressed by NRM1 deletion. We postulate that improper activation of the Nrm1-branch may explain inefficient replication checkpoint activation in Pol ? mutants.

| S-EPMC5291541 | biostudies-literature

Synthetic Lethality between DNA Polymerase Epsilon and RTEL1 in Metazoan DNA Replication.

Project description:Genome stability requires coordination of DNA replication origin activation and replication fork progression. RTEL1 is a regulator of homologous recombination (HR) implicated in meiotic cross-over control and DNA repair in C. elegans. Through a genome-wide synthetic lethal screen, we uncovered an essential genetic interaction between RTEL1 and DNA polymerase (Pol) epsilon. Loss of POLE4, an accessory subunit of Pol epsilon, has no overt phenotype in worms. In contrast, the combined loss of POLE-4 and RTEL-1 results in embryonic lethality, accumulation of HR intermediates, genome instability, and cessation of DNA replication. Similarly, loss of Rtel1 in Pole4-/- mouse cells inhibits cellular proliferation, which is associated with persistent HR intermediates and incomplete DNA replication. We propose that RTEL1 facilitates genome-wide fork progression through its ability to metabolize DNA secondary structures that form during DNA replication. Loss of this function becomes incompatible with cell survival under conditions of reduced origin activation, such as Pol epsilon hypomorphy.

| S-EPMC7262601 | biostudies-literature

Mrc1 and Tof1 regulate DNA replication forks in different ways during normal S phase.

Project description:The Mrc1 and Tof1 proteins are conserved throughout evolution, and in budding yeast they are known to associate with the MCM helicase and regulate the progression of DNA replication forks. Previous work has shown that Mrc1 is important for the activation of checkpoint kinases in responses to defects in S phase, but both Mrc1 and Tof1 also regulate the normal process of chromosome replication. Here, we show that these two important factors control the normal progression of DNA replication forks in distinct ways. The rate of progression of DNA replication forks is greatly reduced in the absence of Mrc1 but much less affected by loss of Tof1. In contrast, Tof1 is critical for DNA replication forks to pause at diverse chromosomal sites where nonnucleosomal proteins bind very tightly to DNA, and this role is not shared with Mrc1.

| S-EPMC1995724 | biostudies-literature

Studies on human DNA polymerase epsilon and GINS complex and their role in DNA replication.

Project description:In eukaryotic cells, DNA replication is carried out by the coordinated action of three DNA polymerases (Pols), Pol ?, ?, and ?. In this report, we describe the reconstitution of the human four-subunit Pol ? and characterization of its catalytic properties in comparison with Pol ? and Pol ?. Human Pol ? holoenzyme is a monomeric complex containing stoichiometric subunit levels of p261/Pol 2, p59, p17, and p12. We show that the Pol ? p261 N-terminal catalytic domain is solely responsible for its ability to catalyze DNA synthesis. Importantly, human Pol (hPol) ? was found more processive than hPol ? in supporting proliferating cell nuclear antigen-dependent elongation of DNA chains, which is in keeping with proposed roles for hPol ? and hPol ? in the replication of leading and lagging strands, respectively. Furthermore, GINS, a component of the replicative helicase complex that is composed of Sld5, Psf1, Psf2, and Psf3, was shown to interact weakly with all three replicative DNA Pols (?, ?, and ?) and to markedly stimulate the activities of Pol ? and Pol ?. In vivo studies indicated that siRNA-targeted depletion of hPol ? and/or hPol ? reduced cell cycle progression and the rate of fork progression. Under the conditions used, we noted that depletion of Pol ? had a more pronounced inhibitory effect on cellular DNA replication than depletion of Pol ?. We suggest that reduction in the level of Pol ? may be less deleterious because of its collision-and-release role in lagging strand synthesis.

| S-EPMC3190704 | biostudies-literature

Exonuclease mutations in DNA polymerase epsilon reveal replication strand specific mutation patterns and human origins of replication.

Project description:Tumors with somatic mutations in the proofreading exonuclease domain of DNA polymerase epsilon (POLE-exo*) exhibit a novel mutator phenotype, with markedly elevated TCT?TAT and TCG?TTG mutations and overall mutation frequencies often exceeding 100 mutations/Mb. Here, we identify POLE-exo* tumors in numerous cancers and classify them into two groups, A and B, according to their mutational properties. Group A mutants are found only in POLE, whereas Group B mutants are found in POLE and POLD1 and appear to be nonfunctional. In Group A, cell-free polymerase assays confirm that mutations in the exonuclease domain result in high mutation frequencies with a preference for C?A mutation. We describe the patterns of amino acid substitutions caused by POLE-exo* and compare them to other tumor types. The nucleotide preference of POLE-exo* leads to increased frequencies of recurrent nonsense mutations in key tumor suppressors such as TP53, ATM, and PIK3R1. We further demonstrate that strand-specific mutation patterns arise from some of these POLE-exo* mutants during genome duplication. This is the first direct proof of leading strand-specific replication by human POLE, which has only been demonstrated in yeast so far. Taken together, the extremely high mutation frequency and strand specificity of mutations provide a unique identifier of eukaryotic origins of replication.

| S-EPMC4216916 | biostudies-literature

DNA polymerase epsilon is required for coordinated and efficient chromosomal DNA replication in Xenopus egg extracts.

Project description:DNA polymerase epsilon (Pol epsilon) is thought to be involved in DNA replication, repair, and cell-cycle checkpoint control in eukaryotic cells. Although the requirement of other replicative DNA polymerases, DNA polymerases alpha and delta (Pol alpha and delta), for chromosomal DNA replication has been well documented by genetic and biochemical studies, the precise role, if any, of Pol epsilon in chromosomal DNA replication is still obscure. Here we show, with the use of a cell-free replication system with Xenopus egg extracts, that Xenopus Pol epsilon is indeed required for chromosomal DNA replication. In Pol epsilon-depleted extracts, the elongation step of chromosomal DNA replication is markedly impaired, resulting in significant reduction of the overall DNA synthesis as well as accumulation of small replication intermediates. Moreover, despite the decreased DNA synthesis, excess amounts of Pol alpha are loaded onto the chromatin template in Pol epsilon-depleted extracts, indicative of the failure of proper assembly of DNA synthesis machinery at the fork. These findings strongly suggest that Pol epsilon, along with Pol alpha and Pol delta, is necessary for coordinated chromosomal DNA replication in eukaryotic cells.

| S-EPMC33149 | biostudies-literature

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