Abstract
DNA double-strand break repair involves phosphorylation of histone variant H2AX ('γH2AX'), which accumulates in foci at sites of DNA damage. In current models, the recruitment of multiple DNA repair proteins to γH2AX foci depends mainly on recognition of this 'mark' by a single protein, MDC1. However, DNA repair proteins accumulate at γH2AX sites without MDC1, suggesting that other 'readers' of this mark exist. Here, we use a quantitative chemical proteomics approach to profile direct, phospho-selective γH2AX binders in native proteomes. We identify γH2AX binders, including the DNA repair mediator 53BP1, which we show recognizes γH2AX through its BRCT domains. Furthermore, we investigate the targeting of wild-type 53BP1, or a mutant form deficient in γH2AX binding, to chromosomal breaks resulting from endogenous and exogenous DNA damage. Our results show how direct recognition of γH2AX modulates protein localization at DNA damage sites, and suggest how specific chromatin mark–reader interactions contribute to essential mechanisms ensuring genome stability.
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Acknowledgements
We are grateful to T. de Lange for the generous gift of 53BP1- and MDC1-deficient MEFs and for helpful discussions. We thank F. Lottersberger for advice and assistance with DNA damage assays. We thank S. Jackson for graciously providing a plasmid encoding MDC1-BRCT cDNA. Live-cell imaging was performed at the Rockefeller University Bio-Imaging Resource Center. We thank P. Ariel for technical assistance with laser scissors. Peptide synthesis was performed by the Rockefeller University Proteomics Resource Center. We thank R. Pisa for assistance with peptide probe synthesis and E. Foley for assistance with pilot experiments. R.E.K. is the Miles S. Nadal Fellow of the Damon Runyon Cancer Research Foundation (DRG-2118-12). The authors acknowledge support from the US National Institutes of Health (GM98579 to T.M.K. and P41 GM103314 and GM109824 to B.T.C.).
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R.E.K. and T.M.K. conceived the project, designed experiments and wrote the paper. R.E.K. designed probes, performed mass spectrometry data collection and analysis, performed biochemical characterization and carried out cellular studies. P.V. validated probes and performed photo-crosslinking experiments. K.R.M. guided mass spectrometry data collection and analysis. B.T.C. directed K.R.M.
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Supplementary Text and Figures
Supplementary Results, Supplementary Figures 1–10 and Supplementary Table 1. (PDF 5165 kb)
Supplementary Dataset 1
Mass spectrometry dataset containing SILAC ratios and protein identifiers from 'affinity filter' experiment. Separate file is attached with the article. (XLSX 158 kb)
Supplementary Dataset 2
Mass spectrometry dataset containing SILAC ratios and protein identifiers from 'selectivity filter' experiment. Separate file is attached with the article. (XLSX 221 kb)
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Kleiner, R., Verma, P., Molloy, K. et al. Chemical proteomics reveals a γH2AX-53BP1 interaction in the DNA damage response. Nat Chem Biol 11, 807–814 (2015). https://doi.org/10.1038/nchembio.1908
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DOI: https://doi.org/10.1038/nchembio.1908
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