| RAD52 deficiency is synthetically lethal in BRCA1 and BRCA2 deficient tumors. RAD52 is therefore a potential therapeutic target for breast cancer patients with BRCA mutations, but not much is known about its role in humans. RAD52 and the BRCA proteins are involved in the homologous recombination (HR) pathway of DNA double-strand break (DSB) repair. In HR, DSBs are processed to generate single-stranded DNA (ssDNA) overhangs, which are then bound by the RPA complex. RAD51 is then recruited and performs homology search and strand invasion. S. cerevisiae RAD52 and hBRCA2 mediate the exchange of RPA for RAD51 and stimulate RAD51 strand invasion. Recent publications show that hRAD52 provides an alternative mediator pathway in cells that lack the BRCA pathway. RPA hyperphosphorylation and dephosphoprylation after DNA damage are important for HR, but its effect on RAD52 function is not well understood. Here, we show that phosphorylation of RPA is important for the alternative RAD52 pathway. Using BRCA2-depleted human cells, in which the only available mediator pathway is RAD52-dependent, expressing non-phosphorylatable (RPA2-A) and mock phosphorylated (RPA2-D) RPA2, we show that HR is reduced in the RPA2-phosphomutant cells compared to RPA2-WT cells, measured by the DR-GFP recombination assay and RAD51 focus formation. Furthermore, RPA-phosphomutant cells have reduced association of RAD52 and RAD51 by colocalization. Interestingly, there is no effect of RPA phosphorylation on RAD52 recruitment to repair foci in RPA-mutant cells after treatment with camptothecin. However, the RPA-phosphomutants do not colocalize with RAD52 as well as the RPA-WT protein and more RAD52 immunoprecipitates with RPA-WT than RPA-A after camptothecin treatment. Finally, using biochemical assays we show that RPA phosphorylation does not affect RAD51 strand exchange, RAD52-mediation of RAD51 strand exchange, and RAD52-dependent ssDNA annealing, suggesting there are factors in cells not present in these assays that allow RPA phosphorylation to promote RAD52 function, or that cycling of phosphorylation and dephosphorylation is needed. Thus, although RAD52 is able to be recruited regardless of RPA phosphorylation status, RPA phosphorylation improves RAD52's association with RPA, and subsequently promotes RAD52-HR. RPA phosphorylation is therefore important for both BRCA2-directed and RAD52-directed HR. |
