Mechanism of the ATP-dependent DNA end-resection machinery from Saccharomyces cerevisiae

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Abstract
When DNA damage introduces double-strand breaks, the ends formed must undergo processing to prepare them for repair. In related studies by the Sung and Kowalczykowski laboratories, this processing reaction has been replicated in vitro using yeast proteins. Processing minimally requires the activities of a helicase, a nuclease and a single-strand binding protein, although the reaction is enhanced by further addition of three factors that help target the core complex and enhance the unwinding activity. When double-strand breaks occur in DNA, the broken ends must undergo processing to prepare them for repair. Here, and in an accompanying study, this processing reaction has now been replicated in vitro using yeast proteins. Processing minimally requires the activities of a helicase, a nuclease and a single-strand-binding protein, although the reaction is enhanced by the addition of three factors that help to target the core complex and stimulate the unwinding activity. If not properly processed and repaired, DNA double-strand breaks (DSBs) can give rise to deleterious chromosome rearrangements, which could ultimately lead to the tumour phenotype1,2. DSB ends are resected in a 5′ to 3′ fashion in cells, to yield single-stranded DNA (ssDNA) for the recruitment of factors critical for DNA damage checkpoint activation and repair by homologous recombination2. The resection process involves redundant pathways consisting of nucleases, DNA helicases and associated proteins3. Being guided by recent genetic studies4,5,6, we have reconstituted the first eukaryotic ATP-dependent DNA end-resection machinery comprising the Saccharomyces cerevisiae Mre11–Rad50–Xrs2 (MRX) complex, the Sgs1–Top3–Rmi1 complex, Dna2 protein and the heterotrimeric ssDNA-binding protein RPA. Here we show that DNA strand separation during end resection is mediated by the Sgs1 helicase function, in a manner that is enhanced by Top3–Rmi1 and MRX. In congruence with genetic observations6, although the Dna2 nuclease activity is critical for resection, the Mre11 nuclease activity is dispensable. By examining the top3 Y356F allele and its encoded protein, we provide evidence that the topoisomerase activity of Top3, although critical for the suppression of crossover recombination2,7, is not needed for resection either in cells or in the reconstituted system. Our results also unveil a multifaceted role of RPA, in the sequestration of ssDNA generated by DNA unwinding, enhancement of 5′ strand incision, and protection of the 3′ strand. Our reconstituted system should serve as a useful model for delineating the mechanistic intricacy of the DNA break resection process in eukaryotes.