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Chromatin remodelling at a DNA double-strand break site in Saccharomyces cerevisiae

Abstract

The repair of DNA double-strand breaks (DSBs) is crucial for maintaining genome stability. Eukaryotic cells repair DSBs by both non-homologous end joining and homologous recombination. How chromatin structure is altered in response to DSBs and how such alterations influence DSB repair processes are important issues. In vertebrates, phosphorylation of the histone variant H2A.X occurs rapidly after DSB formation1, spreads over megabase chromatin domains, and is required for stable accumulation of repair proteins at damage foci2. In Saccharomyces cerevisiae, phosphorylation of the two principal H2A species is also signalled by DSB formation, which spreads 40 kb in either direction from the DSB3. Here we show that near a DSB phosphorylation of H2A is followed by loss of histones H2B and H3 and increased sensitivity of chromatin to digestion by micrococcal nuclease; however, phosphorylation of H2A and nucleosome loss occur independently. The DNA damage sensor MRX4 is required for histone loss, which also depends on INO80, a nucleosome remodelling complex5. The repair protein Rad51 (ref. 6) shows delayed recruitment to DSBs in the absence of histone loss, suggesting that MRX-dependent nucleosome remodelling regulates the accessibility of factors directly involved in DNA repair by homologous recombination. Thus, MRX may regulate two pathways of chromatin changes: nucleosome displacement for efficient recruitment of homologous recombination proteins; and phosphorylation of H2A, which modulates checkpoint responses to DNA damage2.

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Figure 1: Chromatin changes at the MATα DSB.
Figure 2: MRX is involved in histone loss at the MATα DSB.
Figure 3: The INO80 complex is required for histone eviction at the MATα DSB.
Figure 4: MRX and INO80 are required for recruiting Rad51 to the MATα DSB.

References

  1. Rogakou, E. P., Pilch, D. R., Orr, A. H., Ivanova, V. S. & Bonner, W. M. DNA double-stranded breaks induce histone H2AX phosphorylation on serine 139. J. Biol. Chem. 273, 5858–5868 (1998)

    CAS  Article  Google Scholar 

  2. Arkady, C. et al. Histone H2AX phosphorylation is dispensable for the initial recognition of DNA breaks. Nature Cell Biol. 5, 675–679 (2003)

    Article  Google Scholar 

  3. Shroff, R. et al. Distribution and dynamics of chromatin modification induced by a defined DNA double-strand break. Curr. Biol. 14, 1703–1711 (2004)

    CAS  Article  Google Scholar 

  4. Usui, T., Ogawa, H. & Petrini, J. H. A DNA damage response pathway controlled by Tel1 and the Mre11 complex. Mol. Cell 7, 1255–1266 (2001)

    CAS  Article  Google Scholar 

  5. Shen, X., Mizuguchi, G., Hamiche, A. & Wu, C. A chromatin remodelling complex involved in transcription and DNA processing. Nature 406, 541–544 (2000)

    ADS  CAS  Article  Google Scholar 

  6. Sugawara, N., Wang, X. & Haber, J. E. In vivo roles of Rad52, Rad54, and Rad55 proteins in Rad51-mediated recombination. Mol. Cell 12, 209–219 (2003)

    CAS  Article  Google Scholar 

  7. Lee, S. E. et al. Saccharomyces Ku70, Mre11/Rad50 and RPA proteins regulate adaptation to G2/M arrest after DNA damage. Cell 94, 399–409 (1998)

    CAS  Article  Google Scholar 

  8. Downs, J. A., Lowndes, N. F. & Jackson, S. P. A role for Saccharomyces cerevisiae histone H2A in DNA repair. Nature 408, 1001–1004 (2000)

    ADS  CAS  Article  Google Scholar 

  9. Weiss, K. & Simpson, R. T. High-resolution structural analysis of chromatin at specific loci: Saccharomyces cerevisiae silent mating type locus HMLα. Mol. Cell. Biol. 18, 5392–5403 (1998)

    CAS  Article  Google Scholar 

  10. Reinke, H. & Horz, W. Histones are first hyperacetylated and then lose contact with the activated PHO5 promoter. Mol. Cell 11, 1599–1607 (2003)

    CAS  Article  Google Scholar 

  11. Kristjuhan, A. & Svejstrup, J. Q. Evidence for distinct mechanisms facilitating transcript elongation through chromatin in vivo. EMBO J. 23, 4243–4252 (2004)

    CAS  Article  Google Scholar 

  12. Schwabish, M. A. & Struhl, K. Evidence for eviction and rapid deposition of histones upon transcriptional elongation by RNA polymerase II. Mol. Cell. Biol. 24, 10111–10117 (2004)

    CAS  Article  Google Scholar 

  13. Uziel, T. et al. Requirement of the MRN complex for ATM activation by DNA damage. EMBO J. 22, 5612–5621 (2003)

    CAS  Article  Google Scholar 

  14. 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)

    CAS  Article  Google Scholar 

  15. Shen, X., Ranallo, R., Choi, E. & Wu, C. Involvement of actin-related proteins in ATP-dependent chromatin remodeling. Mol. Cell 12, 147–155 (2003)

    CAS  Article  Google Scholar 

  16. Morrison, A. J. et al. INO80 and γ-H2AX interaction links ATP-dependent chromatin remodeling to DNA damage repair. Cell 119, 767–775 (2004)

    CAS  Article  Google Scholar 

  17. van Attikum, H., Fritsch, O., Hohn, B. & Gasser, S. M. Recruitment of the INO80 complex by H2A phosphorylation links ATP-dependent chromatin remodeling with DNA double-strand break repair. Cell 119, 777–788 (2004)

    CAS  Article  Google Scholar 

  18. Downs, J. A. et al. Binding of chromatin-modifying activities to phosphorylated histone H2A at DNA damage sites. Mol. Cell 16, 979–990 (2004)

    CAS  Article  Google Scholar 

  19. Frank-Vaillant, M. & Marcand, S. Transient stability of DNA ends allows nonhomologous end joining to precede homologous recombination. Mol. Cell 10, 1189–1199 (2002)

    CAS  Article  Google Scholar 

  20. Palter, K. B., Foe, V. E. & Alberts, B. M. Evidence for the formation of nucleosome-like histone complexes on single-stranded DNA. Cell 18, 451–467 (1979)

    CAS  Article  Google Scholar 

  21. Mizuguchi, G. et al. ATP-driven exchange of histone H2AZ variant catalyzed by SWR1 chromatin remodeling complex. Science 303, 343–348 (2004)

    ADS  CAS  Article  Google Scholar 

  22. Wang, X. & Haber, J. E. Role of Saccharomyces single-stranded DNA-binding protein RPA in the strand invasion step of double-strand break repair. PLoS Biol. 2, 0104–0112 (2004)

    CAS  Article  Google Scholar 

  23. Kantake, N., Sugiyama, T., Kolodner, R. D. & Kowalczykowski, S. C. The recombination-deficient mutant RPA (rfa1-t11) is displaced slowly from single-stranded DNA by Rad51 protein. J. Biol. Chem. 278, 23410–23417 (2003)

    CAS  Article  Google Scholar 

  24. Unal, E. et al. DNA damage response pathway uses histone modification to assemble a double-strand break-specific cohesin domain. Mol. Cell 16, 991–1002 (2004)

    Article  Google Scholar 

  25. Chai, B., Huang, J., Cairns, B. & Laurent, B. C. Distinct roles for the Rsc and Swi/Snf ATP-dependent chromatin remodelers in DNA double-strand break repair. Genes Dev. 19, 1656–1661 (2005)

    CAS  Article  Google Scholar 

  26. Miyazaki, T., Bressan, D. A., Shinohara, M., Haber, J. E. & Shinohara, A. In vivo assembly and disassembly of Rad51 and Rad52 complexes during double-strand break repair. EMBO J. 23, 939–949 (2004)

    CAS  Article  Google Scholar 

  27. Nakamura, T. M., Du, L. L., Redon, C. & Russell, P. Histone H2A phosphorylation controls Crb2 recruitment at DNA breaks, maintains checkpoint arrest, and influences DNA repair in fission yeast. Mol. Cell. Biol. 24, 6215–6230 (2004)

    CAS  Article  Google Scholar 

  28. Kuo, M. H. & Allis, C. D. In vivo cross-linking and immunoprecipitation for studying dynamic protein:DNA associations in a chromatin environment. Methods 19, 425–433 (1999)

    CAS  Article  Google Scholar 

  29. Lichten, W. Data and Error Analysis in the Introductory Physics Laboratory (Allyn and Bacon, Newton, MA, 1988)

    MATH  Google Scholar 

  30. Fleming, A. B. & Pennings, S. Antagonistic remodelling by Swi-Snf and Tup1-Ssn6 of an extensive chromatin region forms the background for FLO1 gene regulation. EMBO J. 20, 5219–5231 (2001)

    CAS  Article  Google Scholar 

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Acknowledgements

We thank J. Haber, X. Shen, J. Downs, M. Christman and V. Zakian for strains or plasmids; W.-D. Heyer for antibodies against RPA and Rad51; C. Hillyer and N. Clark for technical assistance; and C.-F. Kao for comments. This work was supported by grants from the NIH. (to M.A.O. and to J.A.N.).

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Correspondence to Mary Ann Osley.

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Supplementary information

Supplementary Methods

Additional details of the methods used in this study (DOC 33 kb)

Supplementary Figure Legends

Legends to accompany the below Supplementary Figures. (DOC 29 kb)

Supplementary Figure 1

Densitometry traces of MNase ladders at MATα. (PDF 61 kb)

Supplementary Figure 2

MNase digestion of bulk chromatin. (PDF 554 kb)

Supplementary Figure 3

γ-H2A levels and histone eviction profiles in mre11δ and arp8δ mutants. (PDF 107 kb)

Supplementary Figure 4a

MATα DNA is cleaved and resected in an arp8δ mutant. (PDF 5895 kb)

Supplementary Figure 4b,c

MATα DNA is cleaved and resected in an arp8δ mutant. (PDF 57 kb)

Supplementary Figure 5

Role of γ-H2A in recruitment of Ino80 to the MATα DSB. (PDF 68 kb)

Supplementary Figure 6

DNA repair phenotypes of an arp8δ mutant. (PDF 130 kb)

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Tsukuda, T., Fleming, A., Nickoloff, J. et al. Chromatin remodelling at a DNA double-strand break site in Saccharomyces cerevisiae. Nature 438, 379–383 (2005). https://doi.org/10.1038/nature04148

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