RAD51-MEDIATED ABASIC SITES PROTECTION PREVENTS REPLICATION FORK COLLAPSE.

Genomic DNA is continuously subjected to damage caused by endogenous and environmental agents. This damage, produces as a result DNA lesions, among which abasic (AP) sites are the most prevalent in genomic DNA, occurring at an estimated rate of 10,000 to 20,000 per cell per day. Under normal conditions, AP sites on double-stranded DNA are primarily repaired by the base-excision repair (BER) pathway. However, AP sites that escape BER can be cleaved by endonucleases, causing as result DNA single-strand or double-strand breaks. These DNA breaks can further increase genomic instability and contribute to cell death if not properly repaired. While different molecular pathways have been described which seamless repair abasic sites over double-stranded DNA, the mechanisms by which AP sites are repaired within ssDNA to avoid catastrophic chromosomal breakage remain unclear. Genes encoding for some BER proteins are synthetically lethal with BRCA1/2, which are frequently mutated in several type of cancers, including ovarian and breast cancer. In this study, we utilized the Xenopus laevis egg extract-based in vitro DNA replication system and transmission electron microscopy (TEM) to reveal a new role for the homologous recombination (HR) protein RAD51 in AP site protection and repair during genomic DNA replication. Our findings provide direct evidence that unrepaired AP sites generate discontinuous ssDNA stretches at replication forks onto which RAD51 binds preventing MRE11-RAD50 nuclease-mediated AP site cleavage. Additionally, our results demonstrate that Polymerase Theta (θ) is involved in filling in AP sites over genomic DNA regions containing ssDNA gaps, thereby suppressing MRE11-RAD50 nuclease-dependent fork cleavage. Overall, this study uncovers a novel role for RAD51 recombinase in protecting against unrepaired abasic sites on ssDNA templates, thereby preventing replication fork collapse and preserving genomic stability. Furthermore, this work provides molecular insights into the synthetic lethality observed in the absence of BER proteins over a BRCA-mutated background, suggesting new molecular pathways to be potential targets in BRCA-mutated cancers.