Replication Protein A Phosphorylation Facilitates RAD52-Dependent Homologous Recombination in BRCA-Deficient Cells

ABSTRACT

Loss of RAD52 is synthetically lethal in BRCA-deficient cells, owing to its role in backup homologous recombination (HR) repair of DNA double-strand breaks (DSBs). In HR in mammalian cells, DSBs are processed to single-stranded DNA (ssDNA) overhangs, which are then bound by replication protein A (RPA). RPA is exchanged for RAD51 by mediator proteins: in mammals, BRCA2 is the primary mediator; however, RAD52 provides an alternative mediator pathway in BRCA-deficient cells. RAD51 stimulates strand exchange between homologous DNA duplexes, a critical step in HR. RPA phosphorylation and dephosphorylation are important for HR, but its effect on RAD52 mediator function is unknown. Here, we show that RPA phosphorylation is required for RAD52 to salvage HR in BRCA-deficient cells. In BRCA2-depleted human cells, in which the only available mediator pathway is RAD52 dependent, the expression of a phosphorylation-deficient RPA mutant reduced HR. Furthermore, RPA-phosphomutant cells showed reduced association of RAD52 with RAD51. Interestingly, there was no effect of RPA phosphorylation on RAD52 recruitment to repair foci. Finally, we show that RPA phosphorylation does not affect RAD52-dependent ssDNA annealing. Thus, although RAD52 can be recruited independently of RPA’s phosphorylation status, RPA phosphorylation is required for RAD52’s association with RAD51 and its subsequent promotion of RAD52-mediated HR.

KEYWORDS:

• BRCA deficient
• DNA repair
• RAD52
• RPA phosphorylation
• double-strand-break repair
• homologous recombination

Declaration of Interests

The authors declare no conflict of interest.

ACKNOWLEDGMENTS

We thank James A. Borowiec for the RPA-WT and RPA-A expression plasmids, Melody Di Bona for helping develop the analysis pipeline for quantifying the immunofluorescence images, Ryan Jensen and Carol Kolar for technical assistance, and Qingwen Zhou and William Holloman for their helpful discussions during the course of the experiments. Last, we thank the members of the Powell lab for helpful discussions. Figures were created with the aid of BioRender.

This work was supported by the National Institutes of Health (CA169306 to S.N.P.).

We declare no conflict of interest. We declare that we have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.