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


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.

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Figure 1: Reconstitution and 5′ polarity of DNA end resection.
Figure 2: Requirement for Sgs1 helicase and Dna2 nuclease activities.
Figure 3: Role of MRX and Top3–Rmi1 in DNA resection and unwinding.
Figure 4: Role of Top3 and RPA in resection.


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We thank J. Campbell, S. Brill and L. Symington for providing materials, X. Xue for the double Holliday junction substrate and S. Kowalczykowski for communicating results. This work was supported by grants from the US National Institutes of Health and by a postdoctoral fellowship from the Susan G. Komen for the Cure Foundation.

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Authors and Affiliations



H.Y.N, G.I. and P.S. designed the experiments and wrote the paper. H.Y.N., W.-H.C., Z.Z., Y.H.K., P.C., W.X.Z. and R.P. conducted the experiments. L.L. and D.L. provided key materials and technical expertise.

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Correspondence to Grzegorz Ira or Patrick Sung.

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The authors declare no competing financial interests.

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Niu, H., Chung, WH., Zhu, Z. et al. Mechanism of the ATP-dependent DNA end-resection machinery from Saccharomyces cerevisiae. Nature 467, 108–111 (2010).

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