Genome-wide fork-collapse sites in MEFs from ATR inhibition [mouse]
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA475584 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR7290883.fastq.gz | Fastqsanger.gz | |||
| SRR7290884.fastq.gz | Fastqsanger.gz | |||
| SRR7290885.fastq.gz | Fastqsanger.gz | |||
| SRR7290886.fastq.gz | Fastqsanger.gz | |||
| SRR7290887.fastq.gz | Fastqsanger.gz |
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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 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:Genome-wide fork-collapse sites in MDA-MB-231 cells from ATR inhibition [human]
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Project description:Genome-wide fork-collapse sites in MEF and MDA-MB-231 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 that become processed into persistent double-strand breaks across the human genome from ATR inhibition.
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Project description:Genome-wide fork-collapse sites in MEFs from ATR inhibition
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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:The ATR kinase is a master regulator of cellular responses to DNA damage and replication stress that is activated by a complex mechanism involving its binding partner ATRIP, RPA-coated ssDNA, the 9-1-1 complex and TopBP1. Here, we discovered a new ATR activation mechanism in human cells, mediated by the uncharacterized protein ETAA1 (Ewing’s tumor-associated antigen 1). From a comprehensive proteomic survey of protein recruitment to DNA double-strand break-modified chromatin, we identified ETAA1 as a factor that accumulates at DNA damage sites via an RPA-binding motif, and which has an important role in promoting cell survival and preventing replication fork breakage after genotoxic insults. Mechanistically, we show that ETAA1 harbors a conserved domain that potently and directly stimulates ATR kinase activity independent of TopBP1 and 9-1-1, and which is essential for the function of ETAA1 in the DNA damage response. Consistently, ablation of ETAA1 shows profound synthetic lethality with TopBP1 knockdown, resulting from massive replication fork collapse and abrogation of ATR-dependent signaling. Finally, we show that ETAA1 levels in cancer cell lines inversely correlate with their dependency on TopBP1 for ATR activation, and that overexpression of ETAA1 partially restores ATR-dependent signaling after loss of TopBP1. Together, these findings establish a new mechanism of ATR activation in human cells that operates in parallel with, but independent of, the canonical TopBP1-mediated pathway.
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Project description:Transcription hinders replication fork progression and stability, and the Mec1/ATR checkpoint protects fork integrity. Examining checkpoint-dependent mechanisms controlling fork stability, we find that fork reversal or dormant origin firing owing to checkpoint defects are rescued in checkpoint mutants lacking THO, TREX-2 or inner basket nucleoporins. Gene gating tethers transcribed genes to the nuclear periphery and is counteracted by checkpoint kinases through phosphorylation of nucleoporins such as Mlp1. Checkpoint mutants fail to detach transcribed genes from nuclear pores, thus generating topological impediments for incoming forks. Releasing this topological complexity by introducing a double-strand break between a fork and a transcribed unit prevents fork collapse. Mlp1 mutants mimicking constitutive checkpoint-dependent phosphorylation also alleviate checkpoint defects. We propose that the checkpoint assists fork progression and stability at transcribed genes by phosphorylating key nucleoporins and counteracting gene gating, thus neutralizing the topological tension generated at nuclear pore gated genes.
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