Differential Requirements for the RAD51 Paralogs in Genome Repair and Maintenance in Human Cells

Deficiency in several of the classical human RAD51 paralogs [RAD51B, RAD51C, RAD51D, XRCC2 and XRCC3] is associated with cancer predisposition and Fanconi anemia. To investigate their functions, isogenic disruption mutants for each were generated in non-transformed MCF10A mammary epithelial cells and in transformed U2OS and HEK293 cells. In U2OS and HEK293 cells, viable ablated clones were readily isolated for each RAD51 paralog; in contrast, with the exception of RAD51B, RAD51 paralogs are cell-essential in MCF10A cells. Underlining their importance for genomic stability, mutant cell lines display variable growth defects, impaired sister chromatid recombination, reduced levels of stable RAD51 nuclear foci, and hyper-sensitivity to mitomycin C and olaparib, with the weakest phenotypes observed in RAD51B-deficient cells. Altogether these observations underscore the contributions of RAD51 paralogs in diverse DNA repair processes, and demonstrate essential differences in different cell types. Finally, this study will provide useful reagents to analyze patient-derived mutations and to investigate mechanisms of chemotherapeutic resistance deployed by cancers.
Author summary The five classical RAD51 paralogs [RAD51B, RAD51C, RAD51D, XRCC2 and XRCC3] are important components of the homologous recombination pathway that preserves genomic integrity and protects against cancer. Recently, two RAD51 paralogs have been clearly identified as tumor suppressors and the other three paralogs have been found to be mutated in some tumors. As well, two RAD51 paralogs have been reported to be Fanconi anemia proteins. Thus, there is renewed interest in the RAD51 paralogs from a human health concern and the need for isogenic human cell lines to evaluate tumor-derived mutations and support biochemical approaches aimed at understanding their molecular functions. Here we provide three coherent sets of isogenic mutants, both in transformed and non-transformed human cells. Importantly, using these mutant lines, we report the unanticipated result that RAD51B has a less crucial role in homologous recombination than the other four paralogs, and find that all RAD51 paralogs are critically important for early functions during homologous recombination.