The first step of homology-dependent DNA double-strand break (DSB) repair is the 5′ strand-specific processing of DNA ends to generate 3′ single-strand tails. Despite extensive effort, the nuclease(s) that is directly responsible for the resection of 5′ strands in eukaryotic cells remains elusive. Using nucleoplasmic extracts (NPE) derived from the eggs of Xenopus laevis as the model system, we have found that DNA processing consists of at least two steps: an ATP-dependent unwinding of ends and an ATP-independent 5′→3′ degradation of single-strand tails. The unwinding step is catalyzed by DNA helicases, the major one of which is the Xenopus Werner syndrome protein (xWRN), a member of the RecQ helicase family. In this study, we report the purification and identification of the Xenopus DNA2 (xDNA2) as one of the nucleases responsible for the 5′→3′ degradation of single-strand tails. Immunodepletion of xDNA2 resulted in a significant reduction in end processing and homology-dependent DSB repair. These results provide strong evidence that xDNA2 is a major nuclease for the resection of DNA ends for homology-dependent DSB repair in eukaryotes.

The first step of homology-dependent repair of DNA doublestrand breaks is the strand-specific processing of DNA ends to generate 3 single-strand tails. Despite its importance, the molecular mechanism underlying end processing is poorly understood in eukaryotic cells. We have taken a biochemical approach to investigate DNA end processing in nucleoplasmic extracts derived from the unfertilized eggs of Xenopus laevis. We found that double-strand DNA ends are specifically degraded in the 5 3 3 direction in this system. The reaction consists of two steps: an ATP-dependent unwinding of doublestrand ends and an ATP-independent 5 3 3 degradation of single-strand tails. We also found that the Xenopus Werner syndrome protein, a member of the RecQ helicase family, plays an important role in DNA end processing. Mechanistically, Xenopus Werner syndrome protein (xWRN) is required for the unwinding of DNA ends but not for the degradation of singlestrand tails. The xWRN-mediated end processing is remarkably similar to the end processing that has been proposed for the Escherichia coli RecQ helicase and RecJ single-strand nuclease, suggesting that this mechanism might be conserved in prokaryotes and eukaryotes.

The single-strand DNA–binding protein RPA promotes 5′-strand resection to generate 3′ single strands for homology-dependent DNA double-strand 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.

The processing of DNA double-strand breaks (DSBs) into 3′ single-stranded tails is the first step of homology-dependent DSB repair. A key player in this process is the highly conserved eukaryotic exonuclease 1 (EXO1), yet its precise mechanism of action has not been rigorously determined. To address this issue, we reconstituted 5′-strand resection in cytosol derived from unfertilized interphase eggs of the frog Xenopus laevis. Xenopus EXO1 (xEXO1) was found to display strong 5′→3′ dsDNA exonuclease activity but no significant ssDNA exonuclease activity. Depletion of xEXO1 caused significant inhibition of 5′ strand resection. Co-depletion of xEXO1 and Xenopus DNA2 (xDNA2) showed that these two nucleases act in parallel pathways and by distinct mechanisms. While xDNA2 acts on ssDNA unwound mainly by the Xenopus Werner syndrome protein (xWRN), xEXO1 acts directly on dsDNA. Furthermore, xEXO1 and xWRN are required for both the initiation stage and the extension stage of resection. These results reveal important novel information on the mechanism of 5′-strand resection in eukaryotes.

The resection of DNA double-strand breaks (DSBs) into 3′ single-strand tails is the initiating step of homology-dependent repair pathways. A key player in this process is the MRE11-RAD50-NBS1 complex, but its contribution to and mechanistic role in resection are not well understood. In this study, we took advantage of the Xenopus egg extract system to address these questions. We found that depletion of MRE11 caused a dramatic inhibition of 5′-resection, even for the first nucleotide at the 5′-end. Depletion of Xenopus CtIP also inhibited 5′-strand resection, but this inhibition could be alleviated by excess MRN. Both MRE11 and CtIP could be bypassed by a DNA that carried a 3′-ss-tail. Finally, using purified proteins, we found that MRN could stimulate both the WRN-DNA2-RPA pathway and the EXO1 pathway of resection. These findings provide important insights into the function of MRE11 in 5′-strand resection.