Interplay Between the Two DNA Binding Domains of BRCA2

Overview & Synopsis

Germline mutations in the BRCA2 gene predispose to increased risk of breast and ovarian cancer. BRCA2 is one of the key mediators of Homologous Recombination (HR), the pathway that most accurately repairs DNA double-strand breaks (DSBs). BRCA2 is also involved in protecting stalled replication forks from excessive nucleolytic degradation by MRE11. BRCA2 contains a DNA binding domain at the C-terminus (CTD). Interestingly, our laboratory recently reported a novel DNA binding site in the N-terminus (NTD). The NTD binds both ssDNA and dsDNA however what are the functions of both DNA binding domains in the context of the full-length protein and in the cell remains unclear. We hypothesized that these two DNA binding domains of BRCA2 may have evolved to repair specific types of DNA damage. To test this hypothesis, we generated human stable cell lines expressing BRCA2 variants identified in breast cancer patients altering the DNA binding activity of the canonical CTD or the NTD. We then used functional assays to characterize their phenotype in the context of DSB repair and replication stress. We demonstrated that, in contrast to the CTD variant bearing cells, cells expressing the NTD variants are HR proficient and, accordingly, they are not sensitive to PARP inhibitor treatment. Interestingly, we observed an increased sensitivity to replication stress in cells expressing NTD variants, which was not observed in cells expressing the CTD variant. Moreover, using in situ proximity ligation assay we showed that BRCA2 is recruited to active and HU-stalled replication forks and cells expressing BRCA2 NTD variants display a defect in localization of both BRCA2 and RAD51 to the forks. In contrast, cells expressing CTD variant exhibits no defect in BRCA2 recruitment and mild defect in RAD51 localization. Interestingly, only the cells expressing NTD variants accumulate ssDNA gaps after HU induced Replication stress. Our data strongly suggest that BRCA2 CTD domain is required for the HR (Double-strand break repair) and that the NTD is involved in the replication stress response and prevention of ssDNA gap accumulation that could ultimately lead to genomic instability.