Structure and repair of replication-coupled DNA breaks
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE269694 | GEO | 2025/06/11
REPOSITORIES: GEO
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Project description:Mutations in the ATM tumor suppressor gene confer cellular hypersensitivity to various DNA-damaging chemotherapeutic agents. To explore genetic resistance mechanisms towards such drugs, we performed genome-wide CRISPR-Cas9 loss-of-function screens in cells treated with the DNA topoisomerase I poison topotecan. Our ensuing characterizations of hits established that loss of terminal components of the non-homologous end joining (NHEJ) machinery or components of the BRCA1-A complex specifically confer topotecan resistance to ATM-deficient cells. Our findings indicate that hypersensitivity of ATM-mutant cells to topotecan or the poly-(ADP-ribose) polymerase (PARP) inhibitor olaparib is due to delayed engagement of homologous recombination repair (HRR) at a subset of DNA-replication-fork associated single ended double-strand breaks (seDSBs), which allows non-homologous end joining (NHEJ) mediated repair, resulting in toxic chromosome fusions. Thus, restoration of legitimate repair in ATM-deficient cells – either by preventing the DNA ligation step of NHEJ or by enhancing HRR engagement by deregulating the BRCA1-A complex – markedly suppresses this toxicity. We conclude that the crucial role for ATM at seDSBs is to prevent toxic LIG4-mediated NHEJ at damaged replication forks. Furthermore, our observation that suppressor mutations in ATM-mutant backgrounds are fundamentally different to those that operate in BRCA1-mutant scenarios suggests new opportunities for patient stratification in the clinic, as well as additional therapeutic vulnerabilities that might be exploited in drug-resistant cancers.
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Project description:Heterozygous germline mutations in BRCA1 or BRCA2 predispose to breast, ovarian, as well as other cancers. The main physiological function of BRCA1 and BRCA2 is to facilitate DNA replication, by stabilising and re-starting stalled replication forks. Consequently, inactivation of either BRCA1 or BRCA2 genes leads to replication pathologies and accumulation of DNA double strand breaks. Similar to BRCA1/2 inactivation, oncogene overexpression interferes with replication and inflicts DNA damage. It remains unclear how the replication defects in the two settings differ from each other and what is their combined effect on cell viability. Here, we report that accumulation of β-catenin, an oncogene of the WNT signalling pathway, is synthetically lethal with BRCA1/2 inactivation. We identify two transcription-dependent mechanisms that mediate oncogene toxicity in the context of BRCA1/2 deficiency. Firstly, accumulation of -catenin activates E2F-dependent transcription, which accelerates G1/S transition, thus counteracting the delayed S-phase entry caused by BRCA1/2 abrogation. This, in turn, triggers illegitimate origin firing leading to fork collapse, DNA damage and deregulation of gene expression. Secondly, we find that β-catenin accumulation suppresses transcription of CDKN1A gene encoding p21, a modulator of fork progression. Decreased replication rates represent a hallmark of BRCA2 deficiency. Through p21 downregulation, β-catenin accelerates replication fork progression in BRCA2-deficient cells, thereby inflicting DNA damage and triggering cell death. Thus, our results demonstrate that oncogene-driven premature S-phase entry and increased replication rate are lethal in the context of BRCA1/2 abrogation, which explains the mutual exclusivity between oncogene activation and BRCA1/2 gene mutations in human tumours.
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Project description:Heterozygous germline mutations in BRCA1 or BRCA2 predispose to breast, ovarian, as well as other cancers. The main physiological function of BRCA1 and BRCA2 is to facilitate DNA replication, by stabilising and re-starting stalled replication forks. Consequently, inactivation of either BRCA1 or BRCA2 genes leads to replication pathologies and accumulation of DNA double strand breaks. Similar to BRCA1/2 inactivation, oncogene overexpression interferes with replication and inflicts DNA damage. It remains unclear how the replication defects in the two settings differ from each other and what is their combined effect on cell viability. Here, we report that accumulation of β-catenin, an oncogene of the WNT signalling pathway, is synthetically lethal with BRCA1/2 inactivation. We identify two transcription-dependent mechanisms that mediate oncogene toxicity in the context of BRCA1/2 deficiency. Firstly, accumulation of β-catenin activates E2F-dependent transcription, which accelerates G1/S transition, thus counteracting the delayed S-phase entry caused by BRCA1/2 abrogation. This, in turn, triggers illegitimate origin firing leading to fork collapse, DNA damage and deregulation of gene expression. Secondly, we find that β-catenin accumulation suppresses transcription of CDKN1A gene encoding p21, a modulator of fork progression. Decreased replication rates represent a hallmark of BRCA2 deficiency. Through p21 downregulation, β-catenin accelerates replication fork progression in BRCA2-deficient cells, thereby inflicting DNA damage and triggering cell death. Thus, our results demonstrate that oncogene-driven premature S-phase entry and increased replication rate are lethal in the context of BRCA1/2 abrogation, which explains the mutual exclusivity between oncogene activation and BRCA1/2 gene mutations in human tumours.
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Project description:PARP1 mediates poly-ADP-ribosylation of proteins on chromatin in response to different types of DNA lesions. PARP inhibitors are used for the treatment of BRCA1/2-deficient breast, ovarian, and prostate cancer. Loss of DNA replication fork protection is proposed as one mechanism that contributes to the vulnerability of BRCA1/2-deficient cells to PARP inhibitors. However, the mechanisms that regulate PARP1 activity at stressed replication forks remain poorly understood. Here, we performed proximity proteomics of PARP1 and isolation of proteins on stressed replication forks to map putative PARP1 regulators. We identified TPX2 as a direct PARP1-binding protein that regulates the auto-ADP-ribosylation activity of PARP1. TPX2 interacts with DNA damage response proteins and promotes homology-directed repair of DNA double-strand breaks. Moreover, TPX2 mRNA levels are increased in BRCA1/2-mutated breast and prostate cancers, and high TPX2 expression levels correlate with the sensitivity of cancer cells to PARP-trapping inhibitors. We propose that TPX2 confers a mitosis-independent function in the cellular response to replication stress by interacting with PARP1.
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