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

Abstract

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 hypersensitivity to mitomycin C and olaparib. 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.

Drug-gene interaction screens coupled to tumour data analyses identify the most clinically-relevant cancer vulnerabilities driving sensitivity to PARP inhibition

“…Importantly, and in agreement with previous reports (32), RAD51B inactivation resulted in a more modest reduction in RAD51 foci formation both in SKOV3 and DU145 cells despite efficient DNA damage induction ( Fig 2C-D ), consistent with the reduced sensitivity of RAD51B KO cells to olaparib treatment. Also as previously reported, RAD54L deficiency led to an increase in RAD51 foci formation ( Fig 2D ), which has been linked to the impaired removal of RAD51 molecules upon resolution of HRR and/or the increased number of RAD51 pre-synaptic complexes required to find homology on themdonor DNA strand (35,36).…”

“…Interestingly, from the list of 110 confidence genes, XRCC3 was the most frequently altered gene in our analyses specifically in PARPi-approved settings ( Fig 3A, C ). XRCC3 is one of the five RAD51 paralogs encoded in the human genome (the others being RAD51B, RAD51C, RAD51D and XRCC2) and its function in HRR is well described (32). Accordingly, statistical analyses of the most altered genes in our dataset showed that LoF of XRCC3 is mutually exclusive with either BRCA1 (Fisher’s exact p value 0.002) or BRCA2 (Fisher’s exact p value 0.01) LoF when considering the pan-cancer dataset ( Supp Fig S4A-B ), an effect that becomes even more significant when limiting the analysis to ovarian, breast, pancreatic and prostate tumours ( BRCA1 p value 4.1E-07; BRCA2 p value 6.4E-04; Fig 4A ).…”

“…XRCC3 forms a functional complex (the CX3 complex) with another RAD51 paralog, RAD51C, whose LoF events are also mainly caused by gene silencing, in this case in breast cancer samples ( Supp Table S3 ). RAD51C forms another protein complex (the BCDX2 complex) with other RAD51 paralogs (RAD51B, RAD51D and XRCC2) that does not include XRCC3 (32). Given that XRCC3 deficiency causes a high level of sensitivity to olaparib in the DU145 prostate cancer cell line, and that RAD51C loss phenocopies PARPi sensitivity caused by BRCA2 loss in the ovarian cell line SKOV3, while the same is not true for the other BCDX2 complex component RAD51B, it is tempting to speculate that there is a more important function of the CX3 RAD51 paralog complex in mediating PARPi responses.…”

“…Defects in core HRR factors phenocopy BRCA deficiency PALB2 and the RAD51 paralogs are key proteins in the HRR pathway and we identified their loss as a cause of sensitivity to PARPi in our CRISPR screen analyses (Fig 1B; Supp TableS2). Mammalian cells with mutations in these factors display increased spontaneous chromosomal abnormalities and sensitivity to DNA damaging agents(32,33). Mutations in PALB2, RAD51B or RAD51C have been explored in clinical trials as biomarkers of sensitivity to PARPi(7,10,11).…”

Cancer-Associated SF3B1 Mutations Confer a BRCA-Like Cellular Phenotype and Synthetic Lethality to PARP Inhibitors

“…However, there are a number of DNA repair genes that are misspliced and/or inefficiently exported for translation in SF3B1 K700E cells, including EME1 and XRCC3, which both exhibit increased alternative 3' splice site usage (Figure S5B-C). Notably, both genes have been previously shown to be important for HR downstream of Rad51 loading (34,35) although, it remains to be determined whether the DSB repair abnormalities observed in cells expressing SF3B1 K700E arise directly from aberrant splicing of either of these genes.…”