The Rad51 paralogs are required for homologous recombination (HR) and the maintenance of genomic stability. The molecular mechanisms by which the five vertebrate Rad51 paralogs regulate HR and genomic integrity remain unclear. The Rad51 paralogs associate with one another in two distinct complexes: Rad51B-Rad51C-Rad51D-XRCC2 (BCDX2) and Rad51C-XRCC3 (CX3). We find that the BCDX2 and CX3 complexes act at different stages of the HR pathway. In response to DNA damage, the BCDX2 complex acts downstream of BRCA2 recruitment but upstream of Rad51 recruitment. In contrast, the CX3 complex acts downstream of Rad51 recruitment but still has a marked impact on the measured frequency of homologous recombination. Both complexes are epistatic with BRCA2 and synthetically lethal with Rad52. We conclude that human Rad51 paralogs facilitate BRCA2-Rad51-dependent homologous recombination at different stages in the pathway and function independently of Rad52. Homologous recombination (HR) is an error-free mechanism of repairing double-strand breaks (DSBs) that can occur during the mitotic cell cycle that arise from endogenous DNA damage or exogenously by exposure to radiation and other DNA-damaging agents (1). Abnormalities of HR are associated with a number of genetic diseases, including ataxia-telangiectasia, Nijmegen break syndrome, Fanconi anemia, and Bloom's syndrome (2). HR abrogation in these diseases often leads to cancer susceptibility, which highlights the critical role of HR in maintaining genomic integrity (2). A key step in HR is the polymerization of Rad51 at 3= single-stranded DNA (ssDNA) that is exposed after DSB processing by the Mre11-Rad50-Nbs1 (MRN) complex, CtIP, Exo1, and DNA2 nucleases (3). Rad51 nucleoprotein filament formation is required for subsequent homology search and strand invasion (4). In eukaryotic cells, BRCA2 is the primary mediator that facilitates Rad51 filament formation (5). Recent results from our laboratory now demonstrate that in the absence of BRCA2, Rad52 provides a secondary pathway to promote Rad51-mediated HR (6). Depletion of both BRCA2 and Rad52 is synthetically lethal, mirroring the phenotype of Rad51 inactivation. The recombinase activities of Rad51 in both pathways are well defined; however, the molecular mechanism by which the five Rad51 paralogs (Rad51B, Rad51C, Rad51D, XRCC2, and XRCC3) regulate HR and genomic integrity remains unclear.Multiple approaches have demonstrated that Rad51 paralogs are required for HR repair. Hamster cells or chicken DT40 B-lymphocyte cells defective in any of the Rad51 paralogs are sensitive to DNA cross-linking agents and to ionizing radiation (IR) (7-11). These mutant cells also display chromosomal aberrations, reduced frequencies of HR-mediated gene targeting and DSB repair, and reduced frequencies of sister chromatid exchanges (7-11). Rad51 paralogs may play an early role in the HR pathway, as they have been shown to bind single-stranded DNA (ssDNA) and single-stranded regions on duplex DNA (12). Importantly, Rad51 paralog deficiency signific...

DNA double-stranded breaks (DSBs) are deleterious lesions, and their incorrect repair can drive cancer development1. HELQ is a superfamily 2 helicase with 3′ to 5′ polarity, and its disruption in mice confers germ cells loss, infertility and increased predisposition to ovarian and pituitary tumours2–4. At the cellular level, defects in HELQ result in hypersensitivity to cisplatin and mitomycin C, and persistence of RAD51 foci after DNA damage3,5. Notably, HELQ binds to RPA and the RAD51-paralogue BCDX2 complex, but the relevance of these interactions and how HELQ functions in DSB repair remains unclear3,5,6. Here we show that HELQ helicase activity and a previously unappreciated DNA strand annealing function are differentially regulated by RPA and RAD51. Using biochemistry analyses and single-molecule imaging, we establish that RAD51 forms a complex with and strongly stimulates HELQ as it translocates during DNA unwinding. By contrast, RPA inhibits DNA unwinding by HELQ but strongly stimulates DNA strand annealing. Mechanistically, we show that HELQ possesses an intrinsic ability to capture RPA-bound DNA strands and then displace RPA to facilitate annealing of complementary sequences. Finally, we show that HELQ deficiency in cells compromises single-strand annealing and microhomology-mediated end-joining pathways and leads to bias towards long-tract gene conversion tracts during homologous recombination. Thus, our results implicate HELQ in multiple arms of DSB repair through co-factor-dependent modulation of intrinsic translocase and DNA strand annealing activities.

Double strand break (DSB) repair primarily occurs through 3 pathways: non-homologous end-joining (NHEJ), alternative end-joining (Alt-EJ), and homologous recombination (HR). Typical methods to measure pathway usage include integrated cassette reporter assays or visualization of DNA damage induced nuclear foci. It is now well understood that repair of Cas9-induced breaks also involves NHEJ, Alt-EJ, and HR pathways, providing a new format to measure pathway usage. Here, we have developed a simple Cas9-based system with validated repair outcomes that accurately represent each pathway and then converted it to a droplet digital PCR (ddPCR) readout, thus obviating the need for Next Generation Sequencing and bioinformatic analysis with the goal to make Cas9-based system accessible to more laboratories. The assay system has reproduced several important insights. First, absence of the key Alt-EJ factor Pol θ only abrogates ∼50% of total Alt-EJ. Second, single-strand templated repair (SSTR) requires BRCA1 and MRE11 activity, but not BRCA2, establishing that SSTR commonly used in genome editing is not conventional HR. Third, BRCA1 promotes Alt-EJ usage at two-ended DSBs in contrast to BRCA2. This assay can be used in any system, which permits Cas9 delivery and, importantly, allows rapid genotype-to-phenotype correlation in isogenic cell line pairs.