Brca2 deficiency causes Mre11-dependent degradation of nascent DNA at stalled forks, leading to cell lethality. To understand the molecular mechanisms underlying this process, we isolated Xenopus laevis Brca2. We demonstrated that Brca2 protein prevents single-stranded DNA gap accumulation at replication fork junctions and behind them by promoting Rad51 binding to replicating DNA. Without Brca2, forks with persistent gaps are converted by Smarcal1 into reversed forks, triggering extensive Mre11-dependent nascent DNA degradation. Stable Rad51 nucleofilaments, but not RPA or Rad51T131P mutant proteins, directly prevent Mre11-dependent DNA degradation. Mre11 inhibition instead promotes reversed fork accumulation in the absence of Brca2. Rad51 directly interacts with the Pol α N-terminal domain, promoting Pol α and δ binding to stalled replication forks. This interaction likely promotes replication fork restart and gap avoidance. These results indicate that Brca2 and Rad51 prevent formation of abnormal DNA replication intermediates, whose processing by Smarcal1 and Mre11 predisposes to genome instability.

Breaks at common fragile sites (CFS) are a recognized source of genome instability in pre-neoplastic lesions, but how such checkpoint-proficient cells escape surveillance and continue cycling is unknown. Here we show, in lymphocytes and fibroblasts, that moderate replication stresses like those inducing breaks at CFSs trigger chromatin loading of sensors and mediators of the ATR pathway but fail to activate Chk1 or p53. Consistently, we found that cells depleted of ATR, but not of Chk1, accumulate single-stranded DNA upon Mre11-dependent resection of collapsed forks. Partial activation of the pathway under moderate stress thus takes steps against fork disassembly but tolerates S-phase progression and mitotic onset. We show that fork protection by ATR is crucial to CFS integrity, specifically in the cell type where a given site displays paucity in backup replication origins. Tolerance to mitotic entry with under-replicated CFSs therefore results in chromosome breaks, providing a pool of cells committed to further instability. Accurate genome duplication is crucial at each cell generation to maintain genetic information. However, replication forks routinely face lesions on the DNA template and/or travel through sequences intrinsically difficult to replicate, such as common fragile sites (CFS). To help the fork to proceed, the cells have evolved the DNA damage checkpoint that senses different types of damage and triggers well-adapted cellular responses. We have studied the DNA damage response of human lymphoblastoid cells and normal fibroblasts to various levels of fork slowing. We showed that a two- to ten-fold reduction of fork speed leads to global chromatin recruitment of sensors and mediators of the ATR pathway without substantial activation of Chk1, ATM or p53. Analysis of the phenotype of cells depleted of ATR or Chk1 and submitted to moderate levels of stress shows that ATR, but not Chk1, is crucial to CFS integrity. We propose a model explaining how fork speed thresholds direct fine-tuned checkpoint responses that protect genome integrity without blocking cell cycle progression upon moderate replication fork impediment. Tolerance to mitotic entry with under-replicated CFSs therefore results in chromosome breaks, providing a pool of cells committed to further instability.