Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Balanced act of a leading strand DNA polymerase specific domain and its exonuclease domain promotes genome-wide replication fork symmetry

ABSTRACT: During genome replication, the leading strand DNA polymerase conducts continuous synthesis over long stretches of DNA within each replicon. Processive DNA synthesis supports symmetric progression of sister replication forks and efficient genome duplication. To address the mechanisms underlying leading strand polymerase-mediated synthesis, we examine one of its conserved domains, referred to as POPS, in the budding yeast Pol2 enzyme. We provide evidence that POPS supports replication fork symmetry and efficient genome replication via balancing the function of the Pol2 exonuclease domain. We found that the defective growth, slow S phase progression, and impaired genome synthesis associated with a POPS mutation were rescued by abolishing the Pol2 exonuclease activity. The suppressive effects further extended to the increased DNA re-arrangements in the POPS mutant and its negative genetic interactions with mutants of other genome maintenance factors. Significantly, our single molecule replicon-seq data demonstrate that the POPS mutant exhibited genome-wide replication fork asymmetry, and this defect was improved by eliminating the Pol2 exonuclease activity, thus providing a basis for its rescuing of a range of POPS mutant phenotypes. Collectively, these data suggest a model in which balanced activity between a unique Pol2 catalytic domain, and its exonuclease domain facilitates replication fork symmetry and genome maintenance.

ORGANISM(S): Saccharomyces cerevisiae

PROVIDER: GSE212101 | GEO | 2022/09/02

REPOSITORIES: GEO

ACCESS DATA

Json Xml

Similar Datasets

Checkpoint-mediated DNA polymerase e exonuclease activity curbing counteracts resection-driven fork collapse

Project description:DNA polymerase epsilon (Pole) carries out leading strand synthesis with high fidelity owing to its exonuclease activity. Pole polymerase and exonuclease activities are in balance, due to partitioning of nascent strands between catalytic sites, so that net end resection occurs when synthesis is impaired. Stalling of chromosomal DNA synthesis activates replication checkpoint kinases, required to preserve the functional integrity of replication forks. We found that Pole is phosphorylated in a Rad53CHK1-dependent manner upon fork stalling, likely to limit Pole-driven nascent strand resection that causes replication fork collapse. In stress conditions Pole phosphorylation occurs on serine 430 of the Pol2 catalytic subunit. A S430 phosphomimic limits strand partitioning and exonucleolytic processivity, while non-phosphorylatable Pol2-S430A bypasses checkpoint regulation causing stalled fork resection and collapse. We propose that checkpoint kinases switch Pole to an exonuclease-safe mode by curbing active site partitioning thus preventing nascent strand resection and stabilizing stalled replication forks.

2021-04-30 | GSE156480 | GEO

SAMHD1 degrades nascent DNA at stalled Forks to promote replication restart

Project description:SAMHD1 is a triphosphohydrolase and a 3'-5' exonuclease that restricts HIV-1 infection in non-cycling cells. It is also mutated in the Aicardi-Goutières Syndrome (AGS) and in different cancers, including chronic lymphotic leukemia. We report herre that SAMHD1 localizes to DNA replication foci during S phase suggesting that it is involved in DNA synthesis. Using microarray analysis, we examine the replication timing program an we show that SAMHD1 regulates the fork progression but also the replication timing program.

2017-07-29 | GSE77392 | GEO

Rif1 inhibits replication fork progression and controls copy number in Drosophila.

Project description:Control of DNA copy number is essential to maintain genome stability and ensure proper cell and tissue function. In Drosophila, the SNF2-domain-containing SUUR protein inhibits replication fork progression within specific regions of the genome to promote DNA underreplication. While dissecting the function of SUUR’s SNF2 domain, we identified a physical interaction between SUUR and Rif1. Rif1 has many roles in DNA metabolism and regulates the replication timing program. We demonstrate that repression of DNA replication is dependent on Rif1. Rif1 localizes to active replication forks in an SUUR-dependent manner and directly regulates replication fork progression. Importantly, SUUR associates with replication forks in the absence of Rif1, indicating that Rif1 acts downstream of SUUR to inhibit fork progression. Our findings uncover an unrecognized function of the Rif1 protein as a direct regulator of replication fork progression suggesting developmental regulation of Rif1 activity.

2018-10-15 | GSE114370 | GEO

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

Rad53 checkpoint kinase couples leading and lagging strand DNA synthesis

Project description:In response to DNA replication stress, DNA replication checkpoint is activated to maintain fork stability, a process critical for maintenance of genome stability. However, how DNA replication checkpoint regulates replication forks remain elusive. Here we show that Rad53, a highly conserved replication checkpoint kinase, functions to couple leading and lagging strand DNA synthesis. In wild type cells under HU induced replication stress, synthesis of lagging strand, which contains ssDNA gaps, is comparable to leading strand DNA. In contrast, synthesis of lagging strand is much more than leading strand, and consequently, leading template ssDNA coated with ssDNA binding protein RPA was detected in rad53-1 mutant cells, suggesting that synthesis of leading strand and lagging strand DNA is uncoupled. Mechanistically, we show that replicative helicase MCM and leading strand DNA polymerase Pole move beyond actual DNA synthesis and that an increase in dNTP pools largely suppresses the uncoupled leading and lagging strand DNA synthesis. Our studies reveal an unexpected mechanism whereby Rad53 regulates replication fork stability.

2018-02-11 | GSE89721 | GEO

Replication Timing in Yeast: CGH microarrays: S vs G1 cells in various stress conditions

Project description:Replication timing in Saccharomyces cerevisiae in various replication stress conditions using CGH microarrays of S vs G1 cells. Replication of the eukaryotic genomes follows a characteristic temporal program, with different regions replicating at different times in S phase. We describe a stochastic model of DNA replication that naturally explains the replication profiles of a dozen of S. cerevisiae mutants. Each profile is described by a single parameter that differ between the mutants: the ratio between the fork velocity and the typical initiation rate (the “replicon length”), while all other origin-specific parameters remain unchanged. We devise an analytical method to infer the replicon lengths from the available data, and predicted its value in different mutant backgrounds. Further experiments verified model’s predictions. Our study supports a stochastic model of DNA replication in S. cerevisiae and explains the apparent robustness of this profile to perturbations that extend S phase.

2011-09-09 | GSE32002 | GEO

Replication Timing in Yeast: CGH microarrays: S vs G1 cells in various stress conditions

2011-09-08 | E-GEOD-32002 | biostudies-arrayexpress

Regulation of Rad52-dependent replication fork recovery through serine ADP-ribosylation of PolD3

Project description:Although Poly(ADP-ribose)-polymerases (PARPs) are key regulators of genome stability, how site-specific ADP-ribosylation regulates DNA repair is unclear. Here, we describe a novel role for PARP1 and PARP2 in regulating Rad52-dependent replication fork repair to maintain cell viability when HR is dysfunctional, suppress replication-associated DNA damage, and maintain genome stability. Mechanistically, Mre11 is required for induction of PARP activity in response to replication stress that in turn promotes break-induced replication (BIR) through assembly of Rad52 at stalled/damaged replication forks. Further, by mapping ADP-ribosylation sites induced upon replication stress, we identify that PolD3 is a target for PARP1/PARP2 and importantly, that its site-specific ADP-ribosylation is required for BIR activity, replication fork recovery and genome stability. Overall, these data identify a critical role for Mre11-dependent PARP activation and site-specific ADP-ribosylation in regulating BIR to maintain genome integrity during DNA synthesis.

2023-10-24 | PXD035661 | Pride

Adenovirus E1A oncogene induces re-replication of cellular DNA and alters DNA replication dynamics

Project description:Human adenovirus type 5 oncogene E1A was shown to induce massive re-replication of cellular DNA in late S phase. Using DNA combing analysis, it was shown that DNA replication dynamics including replicon length, fork velocity and inter-origin distance were dramatically altered in E1A expressing cells as compared to that of normal serum stimulated cells. It was also shown that c-Myc that is induced in E1A expressing cells is essential for the efficient entry of E1A expressing cells into S phase.

2013-05-22 | GSE47140 | GEO

Metalloprotease SPRTN/DVC1 Orchestrates Replication-Coupled DNA-Protein Crosslink Removal

Project description:Cytotoxicity of DNA-protein crosslinks (DPCs) is ascribed largely to their ability to block the progression of DNA replication fork. DPCs are frequently occurring in cells, either as a consequence of metabolism or exogenous agents. The mechanism of DPCs removal is not completely understood. Here, we characterize SPRTN (DVC1) as specialised DNA-dependent metalloprotease for DPC removal in humans. SPRTN has an N-terminal metalloprotease domain that cleaves various DNA binding substrate during S-phase progression. SPRTN is a part of replisome and removes DPCs during DNA replication fork progression, thus protecting proliferative cells from DPCs toxicity. Ruijs-Aalfs Syndrome (RJALS) patient cells with monogenic mutations in SPRTN are hypersensitive to DPC-inducing agents due to DPC removal defect and DNA replication fork stalling. We propose a model where SPRTN protease forms specialised DNA-replication coupled DPC removal pathway essential for DNA replication fork progression and genome stability. We conclude RJALS is the first human syndrome linked to this pathway

2017-03-29 | MSV000080743 | MassIVE

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data