Replication fork reversal triggers fork degradation in BRCA2-defective cells

Sofija Mijic,Nagaraja Chappidi,Massimo Lopes,Andre Nussenzweig,Kurt Jacobs,Karun Mutreja,Pavel Janscak,Ralph Zellweger,Sebastian Ursich,Matteo Berti,Arnab Ray Chaudhuri

doi:10.1038/s41467-017-01164-5

Sofija Mijic, Nagaraja Chappidi + Show 9 more

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https://doi.org/10.1038/s41467-017-01164-5

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Abstract

Besides its role in homologous recombination, the tumor suppressor BRCA2 protects stalled replication forks from nucleolytic degradation. Defective fork stability contributes to chemotherapeutic sensitivity of BRCA2-defective tumors by yet-elusive mechanisms. Using DNA fiber spreading and direct visualization of replication intermediates, we report that reversed replication forks are entry points for fork degradation in BRCA2-defective cells. Besides MRE11 and PTIP, we show that RAD52 promotes stalled fork degradation and chromosomal breakage in BRCA2-defective cells. Inactivation of these factors restores reversed fork frequency and chromosome integrity in BRCA2-defective cells. Conversely, impairing fork reversal prevents fork degradation, but increases chromosomal breakage, uncoupling fork protection, and chromosome stability. We propose that BRCA2 is dispensable for RAD51-mediated fork reversal, but assembles stable RAD51 nucleofilaments on regressed arms, to protect them from degradation. Our data uncover the physiopathological relevance of fork reversal and illuminate a complex interplay of homologous recombination factors in fork remodeling and stability.

Highlights

Besides its role in homologous recombination, the tumor suppressor BRCA2 protects stalled replication forks from nucleolytic degradation
Replication forks can be effectively reversed upon HU treatment in the absence of BRCA2, but they are targeted by MRE11-dependent degradation
We did not detect a specific accumulation of ssDNA on regressed arms in HU-treated BRCA2-defective cells (Supplementary Fig. 1a), extended ssDNA stretches were observed upon BRCA2 downregulation at standard three-way fork junctions and were suppressed by mirin treatment in HU-treated cells (Fig. 1c, d)

Summary

Introduction

Besides its role in homologous recombination, the tumor suppressor BRCA2 protects stalled replication forks from nucleolytic degradation. Recent work has uncovered a second, genetically separable function for BRCA2 in protecting stalled replication forks from extensive nucleolytic degradation[4] This concept was later extended to several additional HR factors, as well as factors mutated in the cancer predisposition syndrome Fanconi anemia (FA)[5]. While controlled nucleolytic degradation of stalled replication forks likely plays a physiological role to tolerate replication stress, uncontrolled fork degradation upon HR/FA defects is detrimental for genome stability and affects cellular resistance to replication inhibitors[4, 6,7,8] Most recently, this uncontrolled fork degradation—as opposed to the classical DSB repair defect—was linked both to the lethality of BRCA2defective embryonic stem cells and to the exquisite sensitivity of BRCA-defective cells to certain chemotherapeutic treatments, elucidating a novel crucial mechanism of therapy resistance of BRCA-defective tumors[9]. We provide evidence that, albeit priming fork degradation, reversal of stalled forks is essential to prevent excessive chromosomal breakage in BRCA2-defective tumor cells

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