1. Department of Biological Chemistry and Molecular Pharmacology,
2. Department of Pediatrics, and
3. Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115, USA
4. Rosenstiel Center and Department of Biology, Brandeis University, Waltham, Massachusetts 02454, USA
5. Departments of Medical Genetics and Microbiology, and
6. The Banting and Best Department of Medical Research, University of Toronto, Toronto, Ontario M5G 1L6, Canada
7. Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario M5G 1X5, Canada
8. CBR Institute for Biomedical Research, Boston, Massachusetts 02115, USA
9. Department of Carcinogenesis, MD Anderson Cancer Center, Smithville, Texas 78957, USA
10. Present address: Department of Cellular and Molecular Pharmacology, UCSF, San Francisco, California 94143, USA
11. These authors contributed equally to this work

Correspondence to: Daniel Durocher^5,7Jack F. Greenblatt^5,6 Correspondence and requests for materials should be addressed to D.D. (Email: durocher@mshri.on.ca) or J.F.G. (Email: jack.greenblatt@utoronto.ca).

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 H2AX-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 proteins^{4, 5}, chromatin remodellers⁶ and cohesins^{7, 8}. Multiple mechanisms for eliminating H2AX 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 H2AX in vivo and efficiently dephosphorylates H2AX in vitro. H2AX is lost from chromatin surrounding a DSB independently of the HTP-C, indicating that the phosphatase targets H2AX after its displacement from DNA. The dephosphorylation of H2AX by the HTP-C is necessary for efficient recovery from the DNA damage checkpoint.

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