Replication protein A (RPA), the eukaryotic single-strand deoxyribonucleic acid (DNA [ss-DNA])–binding protein, is involved in DNA replication, nucleotide damage repair, mismatch repair, and DNA damage checkpoint response, but its function in DNA double-strand break (DSB) repair is poorly understood. We investigated the function of RPA in homology-dependent DSB repair using Xenopus laevis nucleoplasmic extracts as a model system. We found that RPA is required for single-strand annealing, one of the homology-dependent DSB repair pathways. Furthermore, RPA promotes the generation of 3′ single-strand tails (ss-tails) by stimulating both the Xenopus Werner syndrome protein (xWRN)–mediated unwinding of DNA ends and the subsequent Xenopus DNA2 (xDNA2)–mediated degradation of the 5′ ss-tail. Purified xWRN, xDNA2, and RPA are sufficient to carry out the 5′-strand resection of DNA that carries a 3′ ss-tail. These results provide strong biochemical evidence to link RPA to a specific DSB repair pathway and reveal a novel function of RPA in the generation of 3′ ss-DNA for homology-dependent DSB repair.

Werner syndrome is associated with premature aging and increased risk of cancer. Werner syndrome protein (WRN) is a RecQ-type DNA helicase, which seems to participate in DNA replication, double-strand break (DSB) repair, and telomere maintenance; however, its exact function remains elusive. Using Xenopus egg extracts as the model system, we found that Xenopus WRN (xWRN) is recruited to discrete foci upon induction of DSBs. Depletion of xWRN has no significant effect on nonhomologous end-joining of DSB ends, but it causes a significant reduction in the homology-dependent single-strand annealing DSB repair pathway. These results provide the first direct biochemical evidence that links WRN to a specific DSB repair pathway. The assay for single-strand annealing that was developed in this study also provides a powerful biochemical system for mechanistic analysis of homology-dependent DSB repair.