Histone H2A Phosphorylation Controls Crb2 Recruitment at DNA Breaks, Maintains Checkpoint Arrest, and Influences DNA Repair in Fission Yeast

Abstract

Mammalian ATR and ATM checkpoint kinases modulate chromatin structures near DNA breaks by phosphorylating a serine residue in the carboxy-terminal tail SQE motif of histone H2AX. Histone H2A is similarly regulated in Saccharomyces cerevisiae. The phosphorylated forms of H2AX and H2A, known as γ-H2AX and γ-H2A, are thought to be important for DNA repair, although their evolutionarily conserved roles are unknown. Here, we investigate γ-H2A in the fission yeast Schizosaccharomyces pombe. We show that formation of γ-H2A redundantly requires the ATR/ATM-related kinases Rad3 and Tel1. Mutation of the SQE motif to AQE (H2A-AQE) in the two histone H2A genes caused sensitivity to a wide range of genotoxic agents, increased spontaneous DNA damage, and impaired checkpoint maintenance. The H2A-AQE mutations displayed a striking synergistic interaction with rad22Δ (Rad52 homolog) in ionizing radiation (IR) survival. These phenotypes correlated with defective phosphorylation of the checkpoint proteins Crb2 and Chk1 and a failure to recruit large amounts of Crb2 to damaged DNA. Surprisingly, the H2A-AQE mutations substantially suppressed the IR hypersensitivity of crb2Δ cells by a mechanism that required the RecQ-like DNA helicase Rqh1. We propose that γ-H2A modulates checkpoint and DNA repair through large-scale recruitment of Crb2 to damaged DNA. This function correlates with evidence that γ-H2AX regulates recruitment of several BRCA1 carboxyl terminus domain-containing proteins (NBS1, 53BP1, MDC1/NFBD1, and BRCA1) in mammals.

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Histone H2A Phosphorylation Controls Crb2 Recruitment at DNA Breaks, Maintains Checkpoint Arrest, and Influences DNA Repair in Fission Yeast

SUPPLEMENTAL MATERIAL

We thank T. Carr, T. Enoch, S. Forsberg, and A. Pastink for S. pombe strains, K. Tanaka for the purified Crb2 preparation, S. Saitoh and H. Zhao for the Rad26 construct, the Yeast Resource Center at the University of Washington for the YFP vector, and J. Bailis for tips on γ-H2AX immunofluorescence microscopy. We also thank C. McGowan, B. Moser, M. Rodríguez, and P.-H. Gaillard for critical reading of the manuscript and members of the Scripps Cell Cycle groups for discussions.

T.M.N. was supported in part by the Damon Runyon Cancer Research Foundation (DRG-1565). L.-L.D. is supported by the Leukemia and Lymphoma Society. This work was funded by National Institutes of Health grants CA77325 and GM59447 awarded to P.R.