Homologous recombination (HR) is a major mechanism utilized to repair blockage of DNA replication forks. Here, we report that a sister chromatid exchange (SCE) generated by crossover-associated HR efficiently occurs in response to replication fork stalling before any measurable DNA double-strand breaks (DSBs). Interestingly, SCE produced by replication fork collapse following DNA DSBs creation is specifically suppressed by ATR, a central regulator of the replication checkpoint. BRCA1 depletion leads to decreased RPA2 phosphorylation (RPA2-P) following replication fork stalling but has no obvious effect on RPA2-P following replication fork collapse. Importantly, we found that BRCA1 promotes RAD51 recruitment and SCE induced by replication fork stalling independent of ATR. In contrast, BRCA1 depletion leads to a more profound defect in RAD51 recruitment and SCE induced by replication fork collapse when ATR is depleted. We concluded that BRCA1 plays a dual role in two distinct HR-mediated repair upon replication fork stalling and collapse. Our data established a molecular basis for the observation that defective BRCA1 leads to a high sensitivity to agents that cause replication blocks without being associated with DSBs, and also implicate a novel mechanism by which loss of cell cycle checkpoints promotes BRCA1-associated tumorigenesis via enhancing HR defect resulting from BRCA1 deficiency.

Alternative non-homologous end joining (alt-NHEJ) was originally identified as a backup repair mechanism in the absence of classical NHEJ (c-NHEJ) factors but recent studies have demonstrated that alt-NHEJ is active even when c-NHEJ as well as homologous recombination is available. The functions of 53BP1 in NHEJ processes are not well understood. Here, we report that 53BP1 promotes DNA double-strand break (DSB) repair and genomic stability not only in c-NHEJ-proficient but also -deficient human G1-phase cells. Using an array of repair substrates we show that these effects of 53BP1 are correlated with a promotion of microhomology-mediated end-joining (MMEJ), a subtype of alt-NHEJ, in G1-phase. Consistent with a specific role in MMEJ we confirm that 53BP1 status does not affect c-NHEJ. 53BP1 supports sequence deletion during MMEJ consistent with a putative role in facilitating end-resection. Interestingly, promotion of MMEJ by 53BP1 in G1-phase cells is only observed in the presence of functional BRCA1. Depletion of both 53BP1 and BRCA1 increases repair needing microhomology usage and augments loss of DNA sequence, suggesting that MMEJ is a highly regulated DSB repair process. Together, these findings significantly expand our understanding of the cell-cycle-dependent roles of 53BP1 in DSB repair.