Checkpoint-mediated DNA polymerase e exonuclease activity curbing counteracts resection-driven fork collapse
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ABSTRACT:
PROVIDER: PRJNA658081 | ENA |
REPOSITORIES: ENA
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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.
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Project description:DNA polymerase ε (Polε) carries out high-fidelity leading strand synthesis owing to its exonuclease activity. Polε polymerase and exonuclease activities are balanced, because of partitioning of nascent DNA strands between catalytic sites, so that net resection occurs when synthesis is impaired. In vivo, DNA synthesis stalling activates replication checkpoint kinases, which act to preserve the functional integrity of replication forks. We show that stalled Polε drives nascent strand resection causing fork functional collapse, averted via checkpoint-dependent phosphorylation. Polε catalytic subunit Pol2 is phosphorylated on serine 430, influencing partitioning between polymerase and exonuclease active sites. A phosphormimetic S430D change reduces exonucleolysis in vitro and counteracts fork collapse. Conversely, non-phosphorylatable pol2-S430A expression causes resection-driven stressed fork defects. Our findings reveal that checkpoint kinases switch Polε to an exonuclease-safe mode preventing nascent strand resection and stabilizing stalled replication forks. Elective partitioning suppression has implications for the diverse Polε roles in genome integrity maintenance.
| S-EPMC7612761 | biostudies-literature
Project description:Transient obstruction of DNA polymerase progression activates the ATR checkpoint kinase, which suppresses fork breakage, strand resection, and RPA accumulation. Herein, we use RPA ChIP-Seq to identify replication-problematic loci (RPLs) across the mammalian genome from ATR inhibition.
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Project description:Transient obstruction of DNA polymerase progression activates the ATR checkpoint kinase, which suppresses fork breakage, strand resection, and RPA accumulation. Herein, we use a developed DNA break-detection assay, BrITL, to identify replication-problematic loci (RPLs) that become processed into persistent double-strand breaks across the mammalian genome from ATR inhibition.
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Project description: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.
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Project description:Transient obstruction of DNA polymerase progression activates the ATR checkpoint kinase, which suppresses fork breakage, strand resection, and RPA accumulation. Herein, we use a developed DNA break-detection assay, BrITL, to identify replication-problematic loci that become processed into persistent double-strand breaks across the human genome from ATR inhibition.
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Project description:Safeguarding replication fork stability in transcriptionally active regions, which require high DNA replication accuracy, is crucial for precise DNA replication and prevention of mutations. However, how cells ensure the stability of replication forks in these regions remains a critical challenge. Here, we discovered the pervasive existence of replication forks-associated RNA-DNA hybrids (RF-RDs) within transcriptionally active regions, where they act as a protective barrier against DNA2-mediated nascent DNA degradation and prevent replication fork collapse upon replication stress. Subsequently, the RNA helicase DDX39A dismantles RF-RDs, facilitating proper DNA2-mediated DNA resection and replication fork restart. Excessive dissolution of RF-RDs causes replication fork collapse and genomic instability, while insufficient dissolution of RF-RDs under replication stress increases fork stability, resulting in chemoresistance that can be reversed by eliminating RF-RDs. In summary, we elucidated the prevalence of RF-RDs at replication forks within transcriptionally active regions, revealed their pivotal role in safeguarding replication fork stability, and proposed that targeting RF-RDs holds promise for augmenting chemotherapeutic efficacy.
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Project description:Safeguarding replication fork stability in transcriptionally active regions, which require high DNA replication accuracy, is crucial for precise DNA replication and prevention of mutations. However, how cells ensure the stability of replication forks in these regions remains a critical challenge. Here, we discovered the pervasive existence of replication forks-associated RNA-DNA hybrids (RF-RDs) within transcriptionally active regions, where they act as a protective barrier against DNA2-mediated nascent DNA degradation and prevent replication fork collapse upon replication stress. Subsequently, the RNA helicase DDX39A dismantles RF-RDs, facilitating proper DNA2-mediated DNA resection and replication fork restart. Excessive dissolution of RF-RDs causes replication fork collapse and genomic instability, while insufficient dissolution of RF-RDs under replication stress increases fork stability, resulting in chemoresistance that can be reversed by eliminating RF-RDs. In summary, we elucidated the prevalence of RF-RDs at replication forks within transcriptionally active regions, revealed their pivotal role in safeguarding replication fork stability, and proposed that targeting RF-RDs holds promise for augmenting chemotherapeutic efficacy.
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Project description:Safeguarding replication fork stability in transcriptionally active regions, which require high DNA replication accuracy, is crucial for precise DNA replication and prevention of mutations. However, how cells ensure the stability of replication forks in these regions remains a critical challenge. Here, we discovered the pervasive existence of replication forks-associated RNA-DNA hybrids (RF-RDs) within transcriptionally active regions, where they act as a protective barrier against DNA2-mediated nascent DNA degradation and prevent replication fork collapse upon replication stress. Subsequently, the RNA helicase DDX39A dismantles RF-RDs, facilitating proper DNA2-mediated DNA resection and replication fork restart. Excessive dissolution of RF-RDs causes replication fork collapse and genomic instability, while insufficient dissolution of RF-RDs under replication stress increases fork stability, resulting in chemoresistance that can be reversed by eliminating RF-RDs. In summary, we elucidated the prevalence of RF-RDs at replication forks within transcriptionally active regions, revealed their pivotal role in safeguarding replication fork stability, and proposed that targeting RF-RDs holds promise for augmenting chemotherapeutic efficacy.
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