Abstract
The breast cancer susceptibility proteins BRCA1 and BRCA2 have emerged as key stabilizing factors for the maintenance of replication fork integrity following replication stress. In their absence, stalled replication forks are extensively degraded by the MRE11 nuclease, leading to chemotherapeutic sensitivity. Here we report that BRCA proteins prevent nucleolytic degradation by protecting replication forks that have undergone fork reversal upon drug treatment. The unprotected regressed arms of reversed forks are the entry point for MRE11 in BRCA-deficient cells. The CtIP protein initiates MRE11-dependent degradation, which is extended by the EXO1 nuclease. Next, we show that the initial limited resection of the regressed arms establishes the substrate for MUS81 in BRCA2-deficient cells. In turn, MUS81 cleavage of regressed forks with a ssDNA tail promotes POLD3-dependent fork rescue. We propose that targeting this pathway may represent a new strategy to modulate BRCA2-deficient cancer cell response to chemotherapeutics that cause fork degradation.
Highlights
The breast cancer susceptibility proteins BRCA1 and BRCA2 have emerged as key stabilizing factors for the maintenance of replication fork integrity following replication stress
We show that the main function of BRCA proteins in this context is to protect the regressed arms of replication forks that have reversed upon drug treatment from nucleolytic degradation
This work defines the mechanism that leads to the extensive fork degradation phenotype observed in BRCA2-deficient cells and provides novel insights into the molecular steps that rescue the resected forks upon drug removal
Summary
The breast cancer susceptibility proteins BRCA1 and BRCA2 have emerged as key stabilizing factors for the maintenance of replication fork integrity following replication stress In their absence, stalled replication forks are extensively degraded by the MRE11 nuclease, leading to chemotherapeutic sensitivity. We show that the main function of BRCA proteins in this context is to protect the regressed arms of replication forks that have reversed upon drug treatment from nucleolytic degradation In their absence, CtIP initiates the MRE11-dependent degradation of the unprotected regressed arms and EXO1 contributes to extend fork degradation. The same results were obtained by treating BRCA2-deficient cells with DNA damaging agents such as cisplatin or UV-C, supporting the notion that different genotoxic agents trigger a similar fork resection mechanism whereby MRE11 and EXO1 extensively degrade replication forks in the absence of key HR factors (Supplementary Fig. 3a, b). These results suggest that human EXO1, but not DNA2, contributes to replication fork degradation in BRCA1and BRCA2-deficient cells
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