Collaborative roles of γH2AX and the Rad51 paralog Xrcc3 in homologous recombinational repair

Abstract One of the earliest events in the signal transduction cascade that initiates a DNA damage checkpoint is the phosphorylation on serine 139 of histone H2AX (γH2AX) at DNA double-strand breaks (DSBs). However, the role of γH2AX in DNA repair is poorly understood. To address this question, we generated chicken DT40 cells carrying a serine to alanine mutation at position 139 of H2AX ( H2AX −/S139A ) and examined their DNA repair capacity. H2AX −/S139A cells exhibited defective homologous recombinational repair (HR) as manifested by delayed Rad51 focus formation following ionizing radiation (IR) and hypersensitivity to the topoisomerase I inhibitor, camptothecin (CPT), which causes DSBs at replication blockage. Deletion of the Rad51 paralog gene, XRCC3 , also delays Rad51 focus formation. To test the interaction of Xrcc3 and γH2AX, we disrupted XRCC3 in H2AX −/S139A cells. XRCC3 −/− / H2AX −/S139A mutants were not viable, although this synthetic lethality was reversed by inserting a transgene that conditionally expresses wild-type H2AX. Upon repression of the wild-type H2AX transgene, XRCC3 −/− / H2AX −/S139A cells failed to form Rad51 foci and exhibited markedly increased levels of chromosomal aberrations after CPT treatment. These results indicate that H2AX and XRCC3 act in separate arms of a branched pathway to facilitate Rad51 assembly.