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A phosphatase complex that dephosphorylates γH2AX regulates DNA damage checkpoint recovery

A Corrigendum to this article was published on 04 May 2006


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.

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Figure 1: The HTP-C regulates H2A in vivo and dephosphorylates γH2AX in vitro.
Figure 2: The HTP-C interacts genetically with DNA repair factors.
Figure 3: The HTP-C is not required for DSB repair and dephosphorylates displaced γH2AX rather than chromatin-associated γH2AX at a DSB.
Figure 4: γH2AX dephosphorylation by the HTP-C is required for DNA damage checkpoint recovery.


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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.

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Correspondence to Daniel Durocher or Jack F. Greenblatt.

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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)

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Keogh, MC., Kim, JA., Downey, M. et al. A phosphatase complex that dephosphorylates γH2AX regulates DNA damage checkpoint recovery. Nature 439, 497–501 (2006).

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