Replication fork reversal triggers fork degradation in BRCA2-defective cells

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.

Replication Fork Reversal: Players and Guardians

“…Interestingly, Mijic et al (2017) also provide new insight into the mechanism of MRE11 recruitment to stalled forks by showing that RAD52 is another factor, together with PTIP and MLL4 (Ray Chaudhuri et al, 2016), required to load MRE11 and prime MRE11-dependent fork resection in BRCA2-deficient cells. At the same time, Lemacon et al (2017) further define the exact sequence of events leading to reversed fork resection in BRCA2-deficient cells by showing that the CtIP protein initiates MRE11-dependent degradation of the regressed arms, which is then extended by the EXO1 nuclease.…”

“…However, the mechanisms leading to this extensive fork degradation phenotype, as well as the structure of the replication intermediates targeted by nucleases in the absence of BRCA1 or BRCA2, remained unclear. The studies reviewed here provide important clues on the molecular basis for this extended fork degradation phenotype (Kolinjivadi et al, 2017; Lemacon et al, 2017; Mijic et al, 2017; Taglialatela et al, 2017). One of their major breakthroughs is the finding that unprotected reversed forks are the structures targeted by MRE11 in a BRCA-deficient background, suggesting that BRCA1 and BRCA2 play a crucial role in reversed fork protection.…”

“…However, the molecular determinants required to protect the integrity of regressed arms until forks are restarted are unknown. Here, we review recent articles that provide a fresh perspective on these important issues by defining a key function of two translocases of the SWI/SNF protein family, i.e., ZRANB3 and SMARCAL1, in reversed fork formation (Kolinjivadi et al, 2017; Taglialatela et al, 2017; Vujanovic et al, 2017) and a key function of the breast cancer susceptibility proteins BRCA1 and BRCA2 in reversed fork protection (Kolinjivadi et al, 2017; Lemacon et al, 2017; Mijic et al, 2017; Taglialatela et al, 2017). …”

“…Altogether, these studies suggest that RAD51 has two distinct functions during replication stress: a BRCA-independent function in promoting the initial step of reversed fork formation, as discussed above, and a BRCA-dependent function whereby BRCA proteins protect the already formed reversed forks from nucleolytic degradation by stabilizing the RAD51 filament on the regressed arm. In BRCA2-deficient cells, this second function is lost, leading to the nascent strand degradation phenotype observed with BRCA2 mutants unable to stabilize RAD51 on ssDNA (Schlacher et al, 2011), with RAD51 mutants that destabilize the RAD51 nucleofilament (Kolinjivadi et al, 2017; Mijic et al, 2017; Zadorozhny et al, 2017), or with small molecules that inhibit RAD51 DNA binding activity (Taglialatela et al, 2017) . …”

“…However, Mijic et al (2017) showed that impairing the initial formation of reversed forks leads to increased chromosomal breakage, suggesting that fork reversal is an important transaction to preserve replication fork integrity, whereas Taglialatela et al (2017) suggest that depletion of factors required fork reversal is accompanied by a marked reduction of genomic instability. This apparent discrepancy likely reflects the use of cancer cells in one study (Mijic et al, 2017) and mammalian epithelial cells in the other (Taglialatela et al, 2017), or possibly other differences in the experimental set-up, and should be the subject of further investigation in the future. In this regard, another recent study suggests that the role of BRCA2 in homologous recombination, but not in stalled replication fork protection, is primarily associated with supporting human mammary epithelial cell viability and preventing replication stress, a hallmark of precancerous cell lesions (Feng and Jasin, 2017).…”