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

Alison C. Carley, Manisha Jalan, Shyamal Subramanyam, Rohini Roy, Gloria E.O. Borgstahl, Simon N. Powell

Research output: Contribution to journalArticlepeer-review

1 Scopus citations

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.

Original languageEnglish (US)
Article numbere00524
JournalMolecular and cellular biology
Volume42
Issue number2
DOIs
StatePublished - Feb 2022

Keywords

  • BRCA deficient
  • DNA repair
  • Double-strand-break repair
  • Homologous recombination
  • RAD52
  • RPA phosphorylation

ASJC Scopus subject areas

  • Molecular Biology
  • Cell Biology

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