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Mre11–Rad50–Xrs2 and Sae2 promote 5′ strand resection of DNA double-strand breaks

Nature Structural & Molecular Biology volume 17, pages 1478–1485 (2010)Cite this article

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Abstract

The repair of DNA double-strand breaks (DSBs) by homologous recombination is essential for genomic stability. The first step in this process is resection of 5′ strands to generate 3′ single-stranded DNA intermediates. Efficient resection in budding yeast requires the Mre11–Rad50–Xrs2 (MRX) complex and the Sae2 protein, although the role of MRX has been unclear because Mre11 paradoxically has 3′→5′ exonuclease activity in vitro. Here we reconstitute resection with purified MRX, Sae2 and Exo1 proteins and show that degradation of the 5′ strand is catalyzed by Exo1 yet completely dependent on MRX and Sae2 when Exo1 levels are limiting. This stimulation is mainly caused by cooperative binding of DNA substrates by Exo1, MRX and Sae2. This work establishes the direct role of MRX and Sae2 in promoting the resection of 5′ strands in DNA DSB repair.

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Figure 1: MRX and Sae2 promote 5′ strand degradation at a DNA break.
Figure 2: Characterization of products of cooperative DNA resection by Exo1, MRX and Sae2.
Figure 3: Digestion of linear DNA by Exo1 produces both single-nucleotide and oligonucleotide products.
Figure 4: Mutations in Exo1 and Rad50 prevent DNA end processing.
Figure 5: Mutations in Sae2 reduce the efficiency of Exo1-mediated DSB resection in vitro and in vivo.
Figure 6: MRX and Sae2 promote Exo1 DNA binding.
Figure 7: MRX and Sae2 facilitate Exo1 activity through distinct 'processing' and 'recruitment' pathways.

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References

  1. San Filippo, J., Sung, P. & Klein, H. Mechanism of eukaryotic homologous recombination. Annu. Rev. Biochem. 77, 229–257 (2008).

    Article  CAS  PubMed  Google Scholar 

  2. Aylon, Y., Liefshitz, B. & Kupiec, M. The CDK regulates repair of double-strand breaks by homologous recombination during the cell cycle. EMBO J. 23, 4868–4875 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Ira, G. et al. DNA end resection, homologous recombination and DNA damage checkpoint activation require CDK1. Nature 431, 1011–1017 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Wyman, C. & Kanaar, R. DNA double-strand break repair: all's well that ends well. Annu. Rev. Genet. 40, 363–383 (2006).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  6. Vaze, M.B. et al. Recovery from checkpoint-mediated arrest after repair of a double-strand break requires Srs2 helicase. Mol. Cell 10, 373–385 (2002).

    Article  CAS  PubMed  Google Scholar 

  7. Fishman-Lobell, J., Rudin, N. & Haber, J.E. Two alternative pathways of double-strand break repair that are kinetically separable and independently modulated. Mol. Cell. Biol. 12, 1292–1303 (1992).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Zierhut, C. & Diffley, J.F. Break dosage, cell cycle stage and DNA replication influence DNA double strand break response. EMBO J. 27, 1875–1885 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Chung, W.H., Zhu, Z., Papusha, A., Malkova, A. & Ira, G. Defective resection at DNA double-strand breaks leads to de novo telomere formation and enhances gene targeting. PLoS Genet. 6, e1000948 (2010).

    Article  PubMed  PubMed Central  Google Scholar 

  10. Barlow, J.H., Lisby, M. & Rothstein, R. Differential regulation of the cellular response to DNA double-strand breaks in G1. Mol. Cell 30, 73–85 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Alani, E., Padmore, R. & Kleckner, N. Analysis of wild-type and rad50 mutants of yeast suggests an intimate relationship between meiotic chromosome synapsis and recombination. Cell 61, 419–436 (1990).

    Article  CAS  PubMed  Google Scholar 

  12. Nairz, K. & Klein, F. mre11S—a yeast mutation that blocks double-strand-break processing and permits nonhomologous synapsis in meiosis. Genes Dev. 11, 2272–2290 (1997).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Tsubouchi, H. & Ogawa, H. A novel mre11 mutation impairs processing of double-strand breaks of DNA during both mitosis and meiosis. Mol. Cell. Biol. 18, 260–268 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Clerici, M., Mantiero, D., Lucchini, G. & Longhese, M.P. The Saccharomyces cerevisiae Sae2 protein promotes resection and bridging of double strand break ends. J. Biol. Chem. 280, 38631–38638 (2005).

    Article  CAS  PubMed  Google Scholar 

  16. Paull, T.T. & Gellert, M. The 3′ to 5′ exonuclease activity of Mre 11 facilitates repair of DNA double-strand breaks. Mol. Cell 1, 969–979 (1998).

    Article  CAS  PubMed  Google Scholar 

  17. Connelly, J.C., de Leau, E.S. & Leach, D.R.F. DNA cleavage and degradation by the SbcCD protein complex from Escherichia coli. Nucleic Acids Res. 27, 1039–1046 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Trujillo, K.M. & Sung, P. DNA structure-specific nuclease activities in the Saccharomyces cerevisiae Rad50–Mre11 complex. J. Biol. Chem. 276, 35458–35464 (2001).

    Article  CAS  PubMed  Google Scholar 

  19. Hopfner, K.P. et al. Mre11 and Rad50 from Pyrococcus furiosus: cloning and biochemical characterization reveal an evolutionarily conserved multiprotein machine. J. Bacteriol. 182, 6036–6041 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Llorente, B. & Symington, L.S. The Mre11 nuclease is not required for 5′ to 3′ resection at multiple HO-induced double-strand breaks. Mol. Cell. Biol. 24, 9682–9694 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Usui, T. et al. Complex formation and functional versatility of Mre11 of budding yeast in recombination. Cell 95, 705–716 (1998).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  24. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Budd, M.E. & Campbell, J.L. Interplay of Mre11 nuclease with Dna2 plus Sgs1 in Rad51-dependent recombinational repair. PLoS ONE 4, e4267 (2009).

    Article  PubMed  PubMed Central  Google Scholar 

  26. Hopkins, B.B. & Paull, T.T. The P. furiosus Mre11/Rad50 complex promotes 5′ strand resection at a DNA double-strand break. Cell 135, 250–260 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Lengsfeld, B.M., Rattray, A.J., Bhaskara, V., Ghirlando, R. & Paull, T.T. Sae2 is an endonuclease that processes hairpin DNA cooperatively with the Mre11/Rad50/Xrs2 complex. Mol. Cell 28, 638–651 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Tran, P.T., Erdeniz, N., Dudley, S. & Liskay, R.M. Characterization of nuclease-dependent functions of Exo1p in Saccharomyces cerevisiae. DNA Repair (Amst.) 1, 895–912 (2002).

    Article  CAS  Google Scholar 

  29. Trujillo, K.M., Yuan, S.S., Lee, E.Y. & Sung, P. Nuclease activities in a complex of human recombination and DNA repair factors Rad50, Mre11, and p95. J. Biol. Chem. 273, 21447–21450 (1998).

    Article  CAS  PubMed  Google Scholar 

  30. Yang, S.W. & Nash, H.A. Specific photocrosslinking of DNA-protein complexes: identification of contacts between integration host factor and its target DNA. Proc. Natl. Acad. Sci. USA 91, 12183–12187 (1994).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Thomas, K.R. & Olivera, B.M. Processivity of DNA exonucleases. J. Biol. Chem. 253, 424–429 (1978).

    CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Cejka, P. et al. DNA end resection by Dna2-Sgs1-RPA and its stimulation by Top3-Rmi1 and Mre11-Rad50-Xrs2. Nature 467, 112–116 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Chamankhah, M., Fontanie, T. & Xiao, W. The Saccharomyces cerevisiae mre11(ts) allele confers a separation of DNA repair and telomere maintenance functions. Genetics 155, 569–576 (2000).

    CAS  PubMed  PubMed Central  Google Scholar 

  35. Moreau, S., Morgan, E.A. & Symington, L.S. Overlapping functions of the Saccharomyces cerevisiae Mre11, Exo1 and Rad27 nucleases in DNA metabolism. Genetics 159, 1423–1433 (2001).

    CAS  PubMed  PubMed Central  Google Scholar 

  36. Lewis, L.K., Karthikeyan, G., Westmoreland, J.W. & Resnick, M.A. Differential suppression of DNA repair deficiencies of yeast rad50, mre11 and xrs2 mutants by EXO1 and TLC1 (the RNA component of telomerase). Genetics 160, 49–62 (2002).

    CAS  PubMed  PubMed Central  Google Scholar 

  37. Lee, S.E., Bressan, D.A., Petrini, J.H. & Haber, J.E. Complementation between N-terminal Saccharomyces cerevisiae mre11 alleles in DNA repair and telomere length maintenance. DNA Repair (Amst.) 1, 27–40 (2002).

    Article  CAS  Google Scholar 

  38. Paull, T.T. & Gellert, M. Nbs1 potentiates ATP-driven DNA unwinding and endonuclease cleavage by the Mre11/Rad50 complex. Genes Dev. 13, 1276–1288 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Lee, J.H. & Paull, T.T. ATM activation by DNA double-strand breaks through the Mre11–Rad50–Nbs1 complex. Science 308, 551–554 (2005).

    Article  CAS  PubMed  Google Scholar 

  40. Nimonkar, A.V., Ozsoy, A.Z., Genschel, J., Modrich, P. & Kowalczykowski, S.C. Human exonuclease 1 and BLM helicase interact to resect DNA and initiate DNA repair. Proc. Natl. Acad. Sci. USA 105, 16906–16911 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Milman, N., Higuchi, E. & Smith, G.R. Meiotic DNA double-strand break repair requires two nucleases, MRN and Ctp1, to produce a single size class of Rec12 (Spo11)-oligonucleotide complexes. Mol. Cell. Biol. 29, 5998–6005 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Hartsuiker, E. et al. Ctp1CtIP and Rad32Mre11 nuclease activity are required for Rec12Spo11 removal, but Rec12Spo11 removal is dispensable for other MRN-dependent meiotic functions. Mol. Cell. Biol. 29, 1671–1681 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Rothenberg, M., Kohli, J. & Ludin, K. Ctp1 and the MRN-complex are required for endonucleolytic Rec12 removal with release of a single class of oligonucleotides in fission yeast. PLoS Genet. 5, e1000722 (2009).

    Article  PubMed  PubMed Central  Google Scholar 

  44. Hartsuiker, E., Neale, M.J. & Carr, A.M. Distinct requirements for the Rad32(Mre11) nuclease and Ctp1(CtIP) in the removal of covalently bound topoisomerase I and II from DNA. Mol. Cell 33, 117–123 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Buis, J. et al. Mre11 nuclease activity has essential roles in DNA repair and genomic stability distinct from ATM activation. Cell 135, 85–96 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Lisby, M., Barlow, J.H., Burgess, R.C. & Rothstein, R. Choreography of the DNA damage response: spatiotemporal relationships among checkpoint and repair proteins. Cell 118, 699–713 (2004).

    Article  CAS  PubMed  Google Scholar 

  47. Lee, K., Zhang, Y. & Lee, S.E. Saccharomyces cerevisiae ATM ortholog suppresses break-induced chromosome translocations. Nature 454, 543–546 (2008).

    Article  CAS  PubMed  Google Scholar 

  48. Bhaskara, V. et al. Rad50 adenylate kinase activity regulates DNA tethering by Mre11/Rad50 complexes. Mol. Cell 25, 647–661 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

We are grateful to M. Liskay (Oregon Health and Science University) for yeast Exo1 reagents, to S. Brill (Rutgers University) for RPA antibodies and M. Wold (University of Iowa) for RPA constructs, and to members of the Paull lab for critical comments about this project. This work was supported by US National Institutes of Health grant R01 CA094008 to T.T.P. Studies carried out in the Lee laboratory were supported by R01 GM083010, and S.E.L. is a scholar of the Leukemia and Lymphoma Society.

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Authors and Affiliations

  1. The Howard Hughes Medical Institute and the Department of Molecular Genetics and Microbiology, The University of Texas at Austin, Austin, Texas, USA

    Matthew L Nicolette, Zhi Guo, Julia M Chow & Tanya T Paull

  2. The Department of Molecular Medicine and Institute of Biotechnology, University of Texas Health Science Center at San Antonio, San Antonio, Texas, USA

    Kihoon Lee & Sang Eun Lee

  3. Department of Chemical Engineering, Institute for Cellular and Molecular Biology, The University of Texas at Austin, Austin, Texas, USA

    Mridula Rani

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Contributions

M.L.N. expressed and purified recombinant proteins, performed resection experiments in vitro, analyzed the data and helped to edit the manuscript. K.L. performed resection experiments in vivo in S. cerevisiae. Z.G. carried out quantitative PCR analysis of resection products in vitro and contributed to the editing of the manuscript. M.R. performed the SPR experiment and analyzed the SPR data. J.M.C. expressed and purified Sae2 proteins used in this study. S.E.L. helped to design the in vivo experiments with sae2 mutants and helped in the analysis of the data and editing of the manuscript. T.T.P. performed the DNA binding and some of the resection experiments in vitro, analyzed the data, and wrote and edited the manuscript.

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Correspondence to Tanya T Paull.

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Nicolette, M., Lee, K., Guo, Z. et al. Mre11–Rad50–Xrs2 and Sae2 promote 5′ strand resection of DNA double-strand breaks. Nat Struct Mol Biol 17, 1478–1485 (2010). https://doi.org/10.1038/nsmb.1957

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