Abstract
One of the earliest marks of a double-strand break (DSB) in eukaryotes is serine phosphorylation of the histone variant H2AX at the carboxy-terminal SQE motif to create γH2AX-containing nucleosomes1. Budding-yeast histone H2A is phosphorylated in a similar manner by the checkpoint kinases Tel1 and Mec1 (ref. 2; orthologous to mammalian ATM and ATR, respectively) over a 50-kilobase region surrounding the DSB3. This modification is important for recruiting numerous DSB-recognition and repair factors to the break site, including DNA damage checkpoint proteins4,5, chromatin remodellers6 and cohesins7,8. Multiple mechanisms for eliminating γH2AX as DNA repair completes are possible, including removal by histone exchange followed potentially by degradation, or, alternatively, dephosphorylation. Here we describe a three-protein complex (HTP-C, for histone H2A phosphatase complex) containing the phosphatase Pph3 that regulates the phosphorylation status of γH2AX in vivo and efficiently dephosphorylates γH2AX in vitro. γH2AX is lost from chromatin surrounding a DSB independently of the HTP-C, indicating that the phosphatase targets γH2AX after its displacement from DNA. The dephosphorylation of γH2AX by the HTP-C is necessary for efficient recovery from the DNA damage checkpoint.
This is a preview of subscription content, access via your institution
Access options
Similar content being viewed by others
References
Rogakou, E. P., Pilch, D. R., Orr, A. H., Ivanova, V. S. & Bonner, W. P. DNA double-strand breaks induce H2AX phosphorylation on serine 139. J. Biol. Chem. 273, 5858–5868 (1998)
Downs, J. A., Lowndes, N. F. & Jackson, S. P. A role for Saccharomyces cerevisiae histone H2A in DNA repair. Nature 408, 1001–1004 (2000)
Shroff, R. et al. Distribution and dynamics of chromatin modification at a defined DNA double strand break. Curr. Biol. 14, 1703–1711 (2004)
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)
Fernandez-Capetillo, O. et al. DNA-damage induced G2-M checkpoint activation by histone H2AX and 53BP1. Nature Cell Biol. 4, 993–997 (2002)
Thiriet, C. & Hayes, J. J. Chromatin in need of a fix: phosphorylation of H2AX connects chromatin to DNA repair. Mol. Cell 18, 617–622 (2005)
Ström, L., Lindroos, H. B., Shirahige, K. & Sjögren, C. Postreplicative recruitment of cohesin to double-strand breaks is required for DNA repair. Mol. Cell 16, 1003–1015 (2004)
Ünal, 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)
Huh, W.-K. et al. Global analysis of protein localization in budding yeast. Nature 425, 686–691 (2003)
Rigaut, G. et al. A generic protein purification method for protein complex characterisation and proteome exploration. Nature Biotechnol. 17, 1030–1032 (1999)
Krogan, N. J. et al. RNA polymerase II elongation factors of Saccharomyces cerevisiae: a targeted proteomics approach. Mol. Cell. Biol. 22, 6979–6992 (2002)
Gingras, A. C. et al. A novel, evolutionarily conserved protein phosphatase complex involved in cisplatin sensitivity. Mol. Cell Proteomics ahead of print publication, 2005 (doi:10.1074/mcp.M500231-MCP200).
Chowdhury, D. et al. γH2AX dephosphorylation by protein phosphatase 2A facilitates DNA double strand break repair. Mol. Cell (in the press)
Tong, A. H. Y. et al. Systematic genetic analysis with ordered arrays of yeast deletion mutants. Science 294, 2364–2368 (2001)
Wu, H. I., Brown, J. A., Dorie, M. J., Lazzeroni, L. & Brown, J. M. Genome-wide identification of genes conferring resistance to the anticancer agents Cisplatin, Oxaliplatin and Mitomycin C. Cancer Res. 64, 3940–3948 (2004)
Haber, J. E. Mating-type gene switching in Saccharomyces cerevisiae. Annu. Rev. Genet. 32, 561–599 (1998)
Pâques, F. & Haber, J. E. Multiple pathways of recombination induced by double strand breaks in Saccharomyces cerevisiae. Microbiol. Mol. Biol. Rev. 63, 349–404 (1999)
Moore, J. K. & Haber, J. E. Cell-cycle and genetic requirements of two pathways of nonhomologous end-joining repair of double-strand breaks in Saccharomyces cerevisiae. Mol. Cell. Biol. 16, 2164–2173 (1996)
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)
White, C. I. & Haber, J. E. Intermediates of recombination during mating type switching in Saccharomyces cerevisiae. EMBO J. 9, 663–673 (1990)
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)
Kusch, T. et al. Acetylation by Tip60 is required for selective histone variant exchange at DNA lesions. Science 306, 2084–2087 (2004)
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)
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)
Vialard, J. E., Gilbert, C. S., Green, C. M. & Lowndes, N. F. The budding yeast Rad9 checkpoint protein is subjected to Mec1/Tel1-dependent hyperphosphorylation and interacts with Rad53 after DNA damage. EMBO J. 17, 5679–5688 (1998)
Matsuoka, S., Huang, M. & Elledge, S. J. Linkage of ATM to cell cycle regulation by the Chk2 protein kinase. Science 282, 1893–1897 (1998)
Blasina, A. et al. A human homologue of the checkpoint kinase Cds1 directly inhibits Cdc25 phosphatase. Curr. Biol. 9, 1–10 (1999)
Sandell, L. L. & Zakian, V. A. Loss of a yeast telomere: arrest, recovery and chromosome loss. Cell 75, 729–739 (1993)
Leroy, C. et al. PP2C phosphatases Ptc2 and Ptc3 are required for DNA checkpoint inactivation after a double-strand break. Mol. Cell 11, 827–835 (2003)
Pellicioli, A., Lee, S. E., Lucca, C., Foiani, M. & Haber, J. E. Regulation of Saccharomyces Rad53 checkpoint kinase during adaptation from DNA damage-induced G2/M arrest. Mol. Cell 7, 293–300 (2001)
Acknowledgements
We thank C. J. Ingles for critically reading the manuscript and for discussion; C. Redon, W. Hofer and N. Lowndes for antibodies; E. O'Shea and J. Weissman for yeast strains; and X. Wu, G. Zhong and X. Guo for technical assistance. N.J.K. was supported by a Doctoral Fellowship from the Canadian Institutes of Health Research (CIHR). J.C.H. and D.C. are fellows of the Leukemia and Lymphoma Society. D.D. is a recipient of the Hitchings-Elion Fellowship of the Burroughs-Wellcome Fund and a Canada Research Chair (tier II) in Proteomics, Bioinformatics and Functional Genomics. This research was supported by grants to J.F.G. from the CIHR and the Ontario Genomics Institute with funds from Genome Canada, to D.D. by the CIHR, to J.E.H. by the National Institutes of Health (NIH), and to S.B. by the NIH. Author Contributions M.C.K., J.A.K. and M.D. contributed equally to this work. N.J.K. was responsible for data in Figs 1b (with mass spectrometry help from A.E.) and 2, and Supplementary Figs 3 and 4; M.C.K. in Fig. 1d and Supplementary Fig. 1b–d (with substrate provided by M.O.), Fig. 3c (with D.C. and J.L.), d, and Supplementary Figs 6 and 9; J.A.K. in Fig. 3a, b, and Supplementary Figs 2 and 5; M.D. and D.D. in Fig. 4 and Supplementary Fig. 8a, c; J.F. in Fig. 1a; J.C.H. in Supplementary Fig. 7; S.G. in Supplementary Fig. 1a; N.D. in Supplementary Fig. 8b; and X.S. in Fig. 1c. M.C.K., S.B., J.E.H., D.D., J.F.G. and N.J.K. wrote the paper.
Author information
Authors and Affiliations
Corresponding authors
Ethics declarations
Competing interests
Reprints and permissions information is available at npg.nature.com/reprintsandpermissions. The authors declare no competing financial interests.
Supplementary information
Supplementary Notes
Additional details on the methods used in this study and Supplementary Figure Legends. (DOC 71 kb)
Supplementary Table 1
Yeast strains. (DOC 58 kb)
Supplementary Table 2
Oligos. (DOC 100 kb)
Supplementary Notes 2
Gene list. (DOC 52 kb)
Supplementary Figure 1
Specificity of Pph3 for γH2AX and optimization of Pph3 reaction conditions. (PDF 542 kb)
Supplementary Figure 2
The HTP-C interacts with DNA repair and replication factors. (PDF 148 kb)
Supplementary Figure 3
The HTP-C associates with its histone H2A substrate (PDF 693 kb)
Supplementary Figure 4
The HTP-C associates with its histone H2A substrate. (PDF 128 kb)
Supplementary Figure 5
γH2AX is lost from the chromatin surrounding a DSB in rad54δ cells, even though this mutant is unable to complete DSB repair. (PDF 440 kb)
Supplementary Figure 6
The lower levels of γH2AX immediately adjacent to a DSB are independent of Pph3. (PDF 93 kb)
Supplementary Figure 7
Pph3 is not required for checkpoint adaptation. (PDF 37 kb)
Supplementary Figure 8
Pph3 specificity for γH2AX. (PDF 518 kb)
Supplementary Figure 9
The majority of γH2AX in cells is chromatin-associated. (PDF 261 kb)
Rights and permissions
About this article
Cite this article
Keogh, MC., Kim, JA., Downey, M. et al. A phosphatase complex that dephosphorylates γH2AX regulates DNA damage checkpoint recovery. Nature 439, 497–501 (2006). https://doi.org/10.1038/nature04384
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/nature04384
Comments
By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.
