Redox Regulation of RAD51 and Homologous Recombination by Peroxiredoxin 1 and Electrophilic Nitro-fatty Acids

Peroxiredoxin 1 (PRDX1) senses and reduces peroxides and coordinates the signaling actions of its binding partners. Loss of Prdx1 function in mice has been shown to enhance susceptibility to various cancers, including breast cancer. Homologous recombination (HR) is a critical process that enables template directed DNA repair to maintain genomic stability. RAD51 is a critical component of HR, which facilitates strand-exchange to repair DNA double-strand breaks (DSB). The role of PRDX1 in DNA damage and RAD51 directed repair was investigated. We show that PRDX1 protects RAD51 from oxidation-induced repression of homologous recombination (HR) repair following γ-irradiation. PRDX1-deficient human breast cancer cells and mouse embryonic fibroblasts display disrupted RAD51 foci formation and decreased HR, resulting in increased DNA damage and sensitization of cells to irradiation. PRDX1 was found to directly bind RAD51 with increasing irradiation and loss of PRDX1 enhanced sulfenylation of RAD51. Site-directed mutagenesis revealed RAD51 Cys319, a thiol located in the SH3 domain targeted by ABL, as a primary target of oxidation. Pharmacological treatment with the thiol reactive electrophilic fatty acid nitroalkene 10-nitro-octadec-9-enoic acid (OA-NO2) targeted the C319 residue of RAD51. Alkylation of RAD51 Cys319 OA-NO2 inhibited RAD51-ABL protein complex formation and downstream Y315 phosphorylation. In breast cancer cells, this reactivity of OA-NO2 was manifested by diminished RAD51 foci formation, enhanced H2AX phosphorylation and sensitization of cells to irradiation. These data establish RAD51 Cys319 as a functionally-significant site for the redox regulation of HR and cellular responses to IR by PRDX1 and soft electrophiles such as OA-NO2.