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The Rad1-Rad10 nuclease promotes chromosome translocations between dispersed repeats

Nature Structural & Molecular Biology volume 19, pages 964–971 (2012)Cite this article

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Abstract

Holliday junctions can be formed during homology-dependent repair of DNA double-strand breaks, and their resolution is essential for chromosome segregation and generation of crossover products. The Mus81-Mms4 and Yen1 nucleases are required for mitotic crossovers between chromosome homologs in Saccharomyces cerevisiae; however, crossovers between dispersed repeats are still detected in their absence. Here we show that the Rad1-Rad10 nuclease promotes formation of crossover and noncrossover recombinants between ectopic sequences. Crossover products were not recovered from the mus81Δ rad1Δ yen1Δ triple mutant, indicating that all three nucleases participate in processing recombination intermediates that form between dispersed repeats. We suggest a new mechanism for crossovers that involves Rad1-Rad10 clipping and resolution of a single Holliday junction–containing intermediate by Mus81-Mms4 or Yen1 cleavage or by replication. Consistent with the model, we show accumulation of Rad1-dependent joint molecules in the mus81Δ yen1Δ mutant.

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Figure 1: Rad1 is required for plasmid integration.
Figure 2: Rad1 and Mus81 cooperate to generate crossovers between dispersed repeats.
Figure 3: Absence of crossovers from survivors of the mus81Δ rad1Δ yen1Δ mutant.
Figure 4: Joint molecules accumulate in the mus81Δ yen1Δ double mutant.
Figure 5: Rad1 is not required for crossovers between chromosome homologs.
Figure 6: Model for the role of Rad1-Rad10 cleavage in formation of recombinants between dispersed repeats.

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References

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

    Article  CAS  Google Scholar 

  2. Barbera, M.A. & Petes, T.D. Selection and analysis of spontaneous reciprocal mitotic cross-overs in Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. USA 103, 12819–12824 (2006).

    Article  CAS  Google Scholar 

  3. Chua, P. & Jinks-Robertson, S. Segregation of recombinant chromatids following mitotic crossing over in yeast. Genetics 129, 359–369 (1991).

    CAS  PubMed  PubMed Central  Google Scholar 

  4. Hagstrom, S.A. & Dryja, T.P. Mitotic recombination map of 13cen-13q14 derived from an investigation of loss of heterozygosity in retinoblastomas. Proc. Natl. Acad. Sci. USA 96, 2952–2957 (1999).

    Article  CAS  Google Scholar 

  5. Bachrati, C.Z. & Hickson, I.D. RecQ helicases: suppressors of tumorigenesis and premature aging. Biochem. J. 374, 577–606 (2003).

    Article  CAS  Google Scholar 

  6. Whitby, M.C. The FANCM family of DNA helicases/translocases. DNA Repair (Amst.) 9, 224–236 (2010).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  8. Szostak, J.W., Orr-Weaver, T.L., Rothstein, R.J. & Stahl, F.W. The double-strand-break repair model for recombination. Cell 33, 25–35 (1983).

    Article  CAS  Google Scholar 

  9. Bzymek, M., Thayer, N.H., Oh, S.D., Kleckner, N. & Hunter, N. Double Holliday junctions are intermediates of DNA break repair. Nature 464, 937–941 (2010).

    Article  CAS  Google Scholar 

  10. Collins, I. & Newlon, C.S. Meiosis-specific formation of joint DNA molecules containing sequences from homologous chromosomes. Cell 76, 65–75 (1994).

    Article  CAS  Google Scholar 

  11. Schwacha, A. & Kleckner, N. Identification of joint molecules that form frequently between homologs but rarely between sister chromatids during yeast meiosis. Cell 76, 51–63 (1994).

    Article  CAS  Google Scholar 

  12. Schwacha, A. & Kleckner, N. Identification of double Holliday junctions as intermediates in meiotic recombination. Cell 83, 783–791 (1995).

    Article  CAS  Google Scholar 

  13. Ferguson, D.O. & Holloman, W.K. Recombinational repair of gaps in DNA is asymmetric in Ustilago maydis and can be explained by a migrating D-loop model. Proc. Natl. Acad. Sci. USA 93, 5419–5424 (1996).

    Article  CAS  Google Scholar 

  14. Nassif, N., Penney, J., Pal, S., Engels, W.R. & Gloor, G.B. Efficient copying of nonhomologous sequences from ectopic sites via P-element-induced gap repair. Mol. Cell. Biol. 14, 1613–1625 (1994).

    Article  CAS  Google Scholar 

  15. Bernstein, K.A., Gangloff, S. & Rothstein, R. The RecQ DNA helicases in DNA repair. Annu. Rev. Genet. 44, 393–417 (2010).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  17. Dayani, Y., Simchen, G. & Lichten, M. Meiotic recombination intermediates are resolved with minimal crossover formation during return-to-growth, an analogue of the mitotic cell cycle. PLoS Genet. 7, e1002083 (2011).

    Article  CAS  Google Scholar 

  18. Cejka, P., Plank, J.L., Bachrati, C.Z., Hickson, I.D. & Kowalczykowski, S.C. Rmi1 stimulates decatenation of double Holliday junctions during dissolution by Sgs1-Top3. Nat. Struct. Mol. Biol. 17, 1377–1382 (2010).

    Article  CAS  Google Scholar 

  19. Schwartz, E.K. & Heyer, W.D. Processing of joint molecule intermediates by structure-selective endonucleases during homologous recombination in eukaryotes. Chromosoma 120, 109–127 (2011).

    Article  CAS  Google Scholar 

  20. Blanco, M.G., Matos, J., Rass, U., Ip, S.C. & West, S.C. Functional overlap between the structure-specific nucleases Yen1 and Mus81-Mms4 for DNA-damage repair in S. cerevisiae. DNA Repair (Amst.) 9, 394–402 (2010).

    Article  CAS  Google Scholar 

  21. Muñoz-Galvan, S. et al. Distinct roles of Mus81, Yen1, Slx1-Slx4, and Rad1 nucleases in the repair of replication-born double-strand breaks by sister chromatid exchange. Mol. Cell. Biol. 32, 1592–1603 (2012).

    Article  Google Scholar 

  22. Boddy, M.N. et al. Mus81-Eme1 are essential components of a Holliday junction resolvase. Cell 107, 537–548 (2001).

    Article  CAS  Google Scholar 

  23. Ho, C.K., Mazon, G., Lam, A.F. & Symington, L.S. Mus81 and Yen1 promote reciprocal exchange during mitotic recombination to maintain genome integrity in budding yeast. Mol. Cell 40, 988–1000 (2010).

    Article  CAS  Google Scholar 

  24. Ashton, T.M., Mankouri, H.W., Heidenblut, A., McHugh, P.J. & Hickson, I.D. Pathways for Holliday junction processing during homologous recombination in Saccharomyces cerevisiae. Mol. Cell. Biol. 31, 1921–1933 (2011).

    Article  CAS  Google Scholar 

  25. De Muyt, A. et al. BLM helicase ortholog Sgs1 is a central regulator of meiotic recombination intermediate metabolism. Mol. Cell 46, 43–53 (2012).

    Article  CAS  Google Scholar 

  26. Zakharyevich, K., Tang, S., Ma, Y. & Hunter, N. Delineation of joint molecule resolution pathways in meiosis identifies a crossover-specific resolvase. Cell 149, 334–347 (2012).

    Article  CAS  Google Scholar 

  27. Agmon, N., Yovel, M., Harari, Y., Liefshitz, B. & Kupiec, M. The role of Holliday junction resolvases in the repair of spontaneous and induced DNA damage. Nucleic Acids Res. 39, 7009–7019 (2011).

    Article  CAS  Google Scholar 

  28. Fagbemi, A.F., Orelli, B. & Scharer, O.D. Regulation of endonuclease activity in human nucleotide excision repair. DNA Repair (Amst.) 10, 722–729 (2011).

    Article  CAS  Google Scholar 

  29. Sekelsky, J.J., McKim, K.S., Chin, G.M. & Hawley, R.S. The Drosophila meiotic recombination gene mei-9 encodes a homologue of the yeast excision repair protein Rad1. Genetics 141, 619–627 (1995).

    CAS  PubMed  PubMed Central  Google Scholar 

  30. Dowling, E.L., Maloney, D.H. & Fogel, S. Meiotic recombination and sporulation in repair-deficient strains of yeast. Genetics 109, 283–302 (1985).

    CAS  PubMed  PubMed Central  Google Scholar 

  31. Lyndaker, A.M. & Alani, E. A tale of tails: insights into the coordination of 3′ end processing during homologous recombination. Bioessays 31, 315–321 (2009).

    Article  CAS  Google Scholar 

  32. Langston, L.D. & Symington, L.S. Opposing roles for DNA structure-specific proteins Rad1, Msh2, Msh3, and Sgs1 in yeast gene targeting. EMBO J. 24, 2214–2223 (2005).

    Article  CAS  Google Scholar 

  33. Schiestl, R.H. & Prakash, S. RAD1, an excision repair gene of Saccharomyces cerevisiae, is also involved in recombination. Mol. Cell. Biol. 8, 3619–3626 (1988).

    Article  CAS  Google Scholar 

  34. Niedernhofer, L.J. et al. The structure-specific endonuclease Ercc1-Xpf is required for targeted gene replacement in embryonic stem cells. EMBO J. 20, 6540–6549 (2001).

    Article  CAS  Google Scholar 

  35. Bärtsch, S., Kang, L.E. & Symington, L.S. RAD51 is required for the repair of plasmid double-stranded DNA gaps from either plasmid or chromosomal templates. Mol. Cell. Biol. 20, 1194–1205 (2000).

    Article  Google Scholar 

  36. Fishman-Lobell, J. & Haber, J.E. Removal of nonhomologous DNA ends in double-strand break recombination: the role of the yeast ultraviolet repair gene RAD1. Science 258, 480–484 (1992).

    Article  CAS  Google Scholar 

  37. Symington, L.S., Kang, L.E. & Moreau, S. Alteration of gene conversion tract length and associated crossing over during plasmid gap repair in nuclease-deficient strains of Saccharomyces cerevisiae. Nucleic Acids Res. 28, 4649–4656 (2000).

    Article  CAS  Google Scholar 

  38. Welz-Voegele, C. & Jinks-Robertson, S. Sequence divergence impedes crossover more than noncrossover events during mitotic gap repair in yeast. Genetics 179, 1251–1262 (2008).

    Article  CAS  Google Scholar 

  39. Aylon, Y. & Kupiec, M. The checkpoint protein Rad24 of Saccharomyces cerevisiae is involved in processing double-strand break ends and in recombination partner choice. Mol. Cell. Biol. 23, 6585–6596 (2003).

    Article  CAS  Google Scholar 

  40. Nicholson, A., Fabbri, R.M., Reeves, J.W. & Crouse, G.F. The effects of mismatch repair and RAD1 genes on interchromosomal crossover recombination in Saccharomyces cerevisiae. Genetics 173, 647–659 (2006).

    Article  CAS  Google Scholar 

  41. Pâques, F. & Haber, J.E. Two pathways for removal of nonhomologous DNA ends during double-strand break repair in Saccharomyces cerevisiae. Mol. Cell. Biol. 17, 6765–6771 (1997).

    Article  Google Scholar 

  42. Inbar, O., Liefshitz, B., Bitan, G. & Kupiec, M. The relationship between homology length and crossing over during the repair of a broken chromosome. J. Biol. Chem. 275, 30833–30838 (2000).

    Article  CAS  Google Scholar 

  43. Roseaulin, L. et al. Mus81 is essential for sister chromatid recombination at broken replication forks. EMBO J. 27, 1378–1387 (2008).

    Article  CAS  Google Scholar 

  44. Al-Minawi, A.Z., Saleh-Gohari, N. & Helleday, T. The ERCC1/XPF endonuclease is required for efficient single-strand annealing and gene conversion in mammalian cells. Nucleic Acids Res. 36, 1–9 (2008).

    Article  CAS  Google Scholar 

  45. Osman, F., Dixon, J., Doe, C.L. & Whitby, M.C. Generating crossovers by resolution of nicked Holliday junctions: a role for Mus81-Eme1 in meiosis. Mol. Cell 12, 761–774 (2003).

    Article  CAS  Google Scholar 

  46. Bastin-Shanower, S.A., Fricke, W.M., Mullen, J.R. & Brill, S.J. The mechanism of Mus81-Mms4 cleavage site selection distinguishes it from the homologous endonuclease Rad1-Rad10. Mol. Cell. Biol. 23, 3487–3496 (2003).

    Article  CAS  Google Scholar 

  47. Ehmsen, K.T. & Heyer, W.D. A junction branch point adjacent to a DNA backbone nick directs substrate cleavage by Saccharomyces cerevisiae Mus81-Mms4. Nucleic Acids Res. 37, 2026–2036 (2009).

    Article  CAS  Google Scholar 

  48. Mitchel, K., Zhang, H., Welz-Voegele, C. & Jinks-Robertson, S. Molecular structures of crossover and noncrossover intermediates during gap repair in yeast: implications for recombination. Mol. Cell 38, 211–222 (2010).

    Article  CAS  Google Scholar 

  49. 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).

    Article  CAS  Google Scholar 

  50. Orr-Weaver, T.L. & Szostak, J.W. Yeast recombination: the association between double-strand gap repair and crossing-over. Proc. Natl. Acad. Sci. USA 80, 4417–4421 (1983).

    Article  CAS  Google Scholar 

  51. Tay, Y.D. & Wu, L. Overlapping roles for Yen1 and Mus81 in cellular Holliday junction processing. J. Biol. Chem. 285, 11427–11432 (2010).

    Article  CAS  Google Scholar 

  52. Wardrope, L., Okely, E. & Leach, D. Resolution of joint molecules by RuvABC and RecG following cleavage of the Escherichia coli chromosome by EcoKI. PLoS ONE 4, e6542 (2009).

    Article  Google Scholar 

  53. Sargent, R.G. et al. Recombination-dependent deletion formation in mammalian cells deficient in the nucleotide excision repair gene ERCC1. Proc. Natl. Acad. Sci. USA 94, 13122–13127 (1997).

    Article  CAS  Google Scholar 

  54. Lupski, J.R. & Stankiewicz, P. Genomic disorders: molecular mechanisms for rearrangements and conveyed phenotypes. PLoS Genet. 1, e49 (2005).

    Article  Google Scholar 

  55. Matos, J., Blanco, M.G., Maslen, S., Skehel, J.M. & West, S.C. Regulatory control of the resolution of DNA recombination intermediates during meiosis and mitosis. Cell 147, 158–172 (2011).

    Article  CAS  Google Scholar 

  56. Aylon, Y., Liefshitz, B., Bitan-Banin, G. & Kupiec, M. Molecular dissection of mitotic recombination in the yeast Saccharomyces cerevisiae. Mol. Cell. Biol. 23, 1403–1417 (2003).

    Article  CAS  Google Scholar 

  57. Hyppa, R.W. & Smith, G.R. Using Schizosaccharomyces pombe meiosis to analyze DNA recombination intermediates. Methods Mol. Biol. 557, 235–252 (2009).

    Article  CAS  Google Scholar 

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Acknowledgements

This study was supported by grants from the US National Institute of General Medical Sciences of the National Institutes of Health under award numbers R01GM041784 and R01GM094386 to L.S.S., and from the Israeli Ministry of Science and Technology and the Israel Science Foundation to M.K.

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  1. Chu Kwen Ho

    Present address: Present address: Massachusetts General Hospital Cancer Center, Harvard Medical School, Charlestown, Massachusetts, USA.,

Authors and Affiliations

  1. Department of Microbiology and Immunology, Columbia University Medical Center, New York, New York, USA

    Gerard Mazón, Alicia F Lam, Chu Kwen Ho & Lorraine S Symington

  2. Department of Molecular Microbiology and Biotechnology, Tel Aviv University, Ramat Aviv, Israel

    Martin Kupiec

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Contributions

G.M., C.K.H., M.K. and L.S.S. designed experiments, and L.S.S. wrote the paper. Experiments in Figure 1 and Supplementary Figure 1 were carried out by A.F.L. and C.K.H., in Figures 2, 3 and 5, and Supplementary Figure 2 by G.M. and A.F.L., and in Figure 4 and Supplementary Figure 3 by G.M.

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Correspondence to Lorraine S Symington.

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Mazón, G., Lam, A., Ho, C. et al. The Rad1-Rad10 nuclease promotes chromosome translocations between dispersed repeats. Nat Struct Mol Biol 19, 964–971 (2012). https://doi.org/10.1038/nsmb.2359

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