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

Nature volume 467, pages 108–111 (2010)Cite this article

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Abstract

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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References

  1. Sung, P. & Klein, H. Mechanism of homologous recombination: mediators and helicases take on regulatory functions. Nature Rev. Mol. Cell Biol. 7, 739–750 (2006)

    CAS  Google Scholar 

  2. Krogh, B. O. & Symington, L. S. Recombination proteins in yeast. Annu. Rev. Genet. 38, 233–271 (2004)

    CAS  Google Scholar 

  3. Mimitou, E. P. & Symington, L. S. Nucleases and helicases take center stage in homologous recombination. Trends Biochem. Sci. 34, 264–272 (2009)

    CAS  Google Scholar 

  4. Gravel, S., Chapman, J. R., Magill, C. & Jackson, S. P. DNA helicases Sgs1 and BLM promote DNA double-strand break resection. Genes Dev. 22, 2767–2772 (2008)

    CAS  Google Scholar 

  5. Mimitou, E. P. & Symington, L. S. Sae2, Exo1 and Sgs1 collaborate in DNA double-strand break processing. Nature 455, 770–774 (2008)

    CAS  Google Scholar 

  6. Zhu, Z., Chung, W. H., Shim, E. Y., Lee, S. E. & Ira, G. Sgs1 helicase and two nucleases Dna2 and Exo1 resect DNA double-strand break ends. Cell 134, 981–994 (2008)

    CAS  Google Scholar 

  7. Ira, G., Malkova, A., Liberi, G., Foiani, M. & Haber, J. E. Srs2 and Sgs1-Top3 suppress crossovers during double-strand break repair in yeast. Cell 115, 401–411 (2003)

    CAS  Google Scholar 

  8. Zou, L. & Elledge, S. J. Sensing DNA damage through ATRIP recognition of RPA-ssDNA complexes. Science 300, 1542–1548 (2003)

    CAS  Google Scholar 

  9. Budd, M. E., Choe, W. C. & Campbell, J. L. DNA2 encodes a DNA helicase essential for replication of eukaryotic chromosomes. J. Biol. Chem. 270, 26766–26769 (1995)

    CAS  Google Scholar 

  10. Bae, S. H., Bae, K. H., Kim, J. A. & Seo, Y. S. RPA governs endonuclease switching during processing of Okazaki fragments in eukaryotes. Nature 412, 456–461 (2001)

    CAS  Google Scholar 

  11. Ivanov, E. L., Sugawara, N., White, C. I., Fabre, F. & Haber, J. E. Mutations in XRS2 and RAD50 delay but do not prevent mating-type switching in Saccharomyces cerevisiae . Mol. Cell. Biol. 14, 3414–3425 (1994)

    CAS  Google Scholar 

  12. Bae, S. H. & Seo, Y. S. Characterization of the enzymatic properties of the yeast Dna2 helicase/endonuclease suggests a new model for Okazaki fragment processing. J. Biol. Chem. 275, 38022–38031 (2000)

    CAS  Google Scholar 

  13. Trujillo, K. M. et al. Yeast Xrs2 binds DNA and helps target Rad50 and Mre11 to DNA ends. J. Biol. Chem. 278, 48957–48964 (2003)

    CAS  Google Scholar 

  14. Furuse, M. et al. Distinct roles of two separable in vitro activities of yeast Mre11 in mitotic and meiotic recombination. EMBO J. 17, 6412–6425 (1998)

    CAS  Google Scholar 

  15. Moreau, S., Ferguson, J. R. & Symington, L. S. The nuclease activity of Mre11 is required for meiosis but not for mating type switching, end joining, or telomere maintenance. Mol. Cell. Biol. 19, 556–566 (1999)

    CAS  Google Scholar 

  16. Bressan, D. A., Olivares, H. A., Nelms, B. E. & Petrini, J. H. Alteration of N-terminal phosphoesterase signature motifs inactivates Saccharomyces cerevisiae Mre11. Genetics 150, 591–600 (1998)

    CAS  Google Scholar 

  17. Paull, T. T. & Gellert, M. The 3′ to 5′ exonuclease activity of Mre11 facilitates repair of DNA double-strand breaks. Mol. Cell 1, 969–979 (1998)

    CAS  Google Scholar 

  18. Lu, J. et al. Human homologues of yeast helicase. Nature 383, 678–679 (1996)

    CAS  Google Scholar 

  19. Liu, Y., Kao, H. I. & Bambara, R. A. FLAP endonuclease 1: a central component of DNA metabolism. Annu. Rev. Biochem. 73, 589–615 (2004)

    CAS  Google Scholar 

  20. Budd, M. E. & Campbell, J. L. A yeast replicative helicase, Dna2 helicase, interacts with yeast FEN-1 nuclease in carrying out its essential function. Mol. Cell. Biol. 17, 2136–2142 (1997)

    CAS  Google Scholar 

  21. Chiolo, I. et al. Srs2 and Sgs1 DNA helicases associate with Mre11 in different subcomplexes following checkpoint activation and CDK1-mediated Srs2 phosphorylation. Mol. Cell. Biol. 25, 5738–5751 (2005)

    CAS  Google Scholar 

  22. Chen, L., Trujillo, K., Ramos, W., Sung, P. & Tomkinson, A. E. Promotion of Dnl4-catalyzed DNA end-joining by the Rad50/Mre11/Xrs2 and Hdf1/Hdf2 complexes. Mol. Cell 8, 1105–1115 (2001)

    CAS  Google Scholar 

  23. Chen, C. F. & Brill, S. J. Binding and activation of DNA topoisomerase III by the Rmi1 subunit. J. Biol. Chem. 282, 28971–28979 (2007)

    CAS  Google Scholar 

  24. Wu, L. & Hickson, I. D. The Bloom’s syndrome helicase suppresses crossing over during homologous recombination. Nature 426, 870–874 (2003)

    CAS  Google Scholar 

  25. Masuda-Sasa, T., Polaczek, P., Peng, X. P., Chen, L. & Campbell, J. L. Processing of G4 DNA by Dna2 helicase/nuclease and replication protein A (RPA) provides insights into the mechanism of Dna2/RPA substrate recognition. J. Biol. Chem. 283, 24359–24373 (2008)

    CAS  Google Scholar 

  26. Bae, K. H. et al. Bimodal interaction between replication-protein A and Dna2 is critical for Dna2 function both in vivo and in vitro . Nucleic Acids Res. 31, 3006–3015 (2003)

    CAS  Google Scholar 

  27. Cejka, P. et al. DNA end resection by Dna2–Sgs1–RPA and its stimulation by Top3–Rmi1 and Mre11–Rad50–Xrs2. Nature 10.1038/nature09355 (this issue)

  28. Niu, H., Raynard, S. & Sung, P. Multiplicity of DNA end resection machineries in chromosome break repair. Genes Dev. 23, 1481–1486 (2009)

    CAS  Google Scholar 

  29. Zheng, L. et al. Human DNA2 is a mitochondrial nuclease/helicase for efficient processing of DNA replication and repair intermediates. Mol. Cell 32, 325–336 (2008)

    CAS  Google Scholar 

  30. Duxin, J. P. et al. Human Dna2 is a nuclear and mitochondrial DNA maintenance protein. Mol. Cell. Biol. 29, 4274–4282 (2009)

    CAS  Google Scholar 

Download references

Acknowledgements

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

  1. Department of Molecular Biophysics and Biochemistry, Yale University School of Medicine, New Haven, 06520, Connecticut, USA

    Hengyao Niu, Youngho Kwon, Weixing Zhao, Peter Chi, Rohit Prakash, Changhyun Seong, Dongqing Liu, Lucy Lu & Patrick Sung

  2. Department of Molecular and Human Genetics, Baylor College of Medicine, One Baylor Plaza, Houston, Texas 77030, USA,

    Woo-Hyun Chung, Zhu Zhu & Grzegorz Ira

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  1. Hengyao Niu
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Contributions

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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The file contains Supplementary Methods, Supplementary Figures 1-12 with legends, a Supplementary Table and additional references. (PDF 4343 kb)

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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). https://doi.org/10.1038/nature09318

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