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Chemical proteomics reveals a γH2AX-53BP1 interaction in the DNA damage response

Nature Chemical Biology volume 11, pages 807–814 (2015)Cite this article

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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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Figure 1: Chemical probes to capture direct γH2AX interaction partners.
Figure 2: Proteomic profiling of high-affinity direct binders of γH2AX.
Figure 3: Proteomic profiling of phospho-selective direct binders of γH2AX.
Figure 4: Biochemical characterization of the 53BP1-γH2AX interaction.
Figure 5: Live-cell analysis of 53BP1 localization at DNA damage produced by 'laser scissors'.
Figure 6: Comparative analysis of WT and K1814M 53BP1 and γH2AX foci generated in response to TRF2 depletion or ionizing radiation (IR).

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References

  1. Ciccia, A. & Elledge, S.J. The DNA damage response: making it safe to play with knives. Mol. Cell 40, 179–204 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Lord, C.J. & Ashworth, A. The DNA damage response and cancer therapy. Nature 481, 287–294 (2012).

    Article  CAS  PubMed  Google Scholar 

  3. Jenuwein, T. & Allis, C.D. Translating the histone code. Science 293, 1074–1080 (2001).

    Article  CAS  PubMed  Google Scholar 

  4. Rogakou, E.P., Pilch, D.R., Orr, A.H., Ivanova, V.S. & Bonner, W.M. DNA double-stranded breaks induce histone H2AX phosphorylation on serine 139. J. Biol. Chem. 273, 5858–5868 (1998).

    Article  CAS  PubMed  Google Scholar 

  5. Stucki, M. et al. MDC1 directly binds phosphorylated histone H2AX to regulate cellular responses to DNA double-strand breaks. Cell 123, 1213–1226 (2005).

    Article  CAS  PubMed  Google Scholar 

  6. Spycher, C. et al. Constitutive phosphorylation of MDC1 physically links the MRE11–RAD50–NBS1 complex to damaged chromatin. J. Cell Biol. 181, 227–240 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Jackson, S.P. & Durocher, D. Regulation of DNA damage responses by ubiquitin and SUMO. Mol. Cell 49, 795–807 (2013).

    Article  CAS  PubMed  Google Scholar 

  8. Pei, H. et al. MMSET regulates histone H4K20 methylation and 53BP1 accumulation at DNA damage sites. Nature 470, 124–128 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Lou, Z. et al. MDC1 maintains genomic stability by participating in the amplification of ATM-dependent DNA damage signals. Mol. Cell 21, 187–200 (2006).

    Article  CAS  PubMed  Google Scholar 

  10. Nakamura, A.J., Rao, V.A., Pommier, Y. & Bonner, W.M. The complexity of phosphorylated H2AX foci formation and DNA repair assembly at DNA double-strand breaks. Cell Cycle 9, 389–397 (2010).

    Article  CAS  PubMed  Google Scholar 

  11. Taverna, S.D., Li, H., Ruthenburg, A.J., Allis, C.D. & Patel, D.J. How chromatin-binding modules interpret histone modifications: lessons from professional pocket pickers. Nat. Struct. Mol. Biol. 14, 1025–1040 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Li, X. et al. Quantitative chemical proteomics approach to identify post-translational modification-mediated protein-protein interactions. J. Am. Chem. Soc. 134, 1982–1985 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Li, X. et al. Examining post-translational modification-mediated protein-protein interactions using a chemical proteomics approach. Protein Sci. 22, 287–295 (2013).

    Article  PubMed  Google Scholar 

  14. Campbell, S.J., Edwards, R.A. & Glover, J.N.M. Comparison of the structures and peptide binding specificities of the BRCT domains of MDC1 and BRCA1. Structure 18, 167–176 (2010).

    Article  CAS  PubMed  Google Scholar 

  15. Yang, Y.Y., Grammel, M., Raghavan, A.S., Charron, G. & Hang, H.C. Comparative analysis of cleavable azobenzene-based affinity tags for bioorthogonal chemical proteomics. Chem. Biol. 17, 1212–1222 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Orth, J.D., Loewer, A., Lahav, G. & Mitchison, T.J. Prolonged mitotic arrest triggers partial activation of apoptosis, resulting in DNA damage and p53 induction. Mol. Biol. Cell 23, 567–576 1 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. DiTullio, R.A. Jr. et al. 53BP1 functions in an ATM-dependent checkpoint pathway that is constitutively activated in human cancer. Nat. Cell Biol. 4, 998–1002 (2002).

    Article  CAS  PubMed  Google Scholar 

  18. Fernandez-Capetillo, O. et al. DNA damage-induced G2-M checkpoint activation by histone H2AX and 53BP1. Nat. Cell Biol. 4, 993–997 (2002).

    Article  CAS  PubMed  Google Scholar 

  19. Schultz, L.B., Chehab, N.H., Malikzay, A. & Halazonetis, T.D. p53 binding protein 1 (53BP1) is an early participant in the cellular response to DNA double-strand breaks. J. Cell Biol. 151, 1381–1390 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Wang, B., Matsuoka, S., Carpenter, P.B. & Elledge, S.J. 53BP1, a mediator of the DNA damage checkpoint. Science 298, 1435–1438 (2002).

    Article  CAS  PubMed  Google Scholar 

  21. Wood, J.L., Singh, N., Mer, G. & Chen, J. MCPH1 functions in an H2AX-dependent but MDC1-independent pathway in response to DNA damage. J. Biol. Chem. 282, 35416–35423 (2007).

    Article  CAS  PubMed  Google Scholar 

  22. Dobbin, M.M. et al. SIRT1 collaborates with ATM and HDAC1 to maintain genomic stability in neurons. Nat. Neurosci. 16, 1008–1015 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Hu, B., Li, S., Zhang, X. & Zheng, X. HSCARG, a novel regulator of H2A ubiquitination by downregulating PRC1 ubiquitin E3 ligase activity, is essential for cell proliferation. Nucleic Acids Res. 42, 5582–5593 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Oberdoerffer, P. et al. SIRT1 redistribution on chromatin promotes genomic stability but alters gene expression during aging. Cell 135, 907–918 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Bunting, S.F. et al. 53BP1 inhibits homologous recombination in Brca1-deficient cells by blocking resection of DNA breaks. Cell 141, 243–254 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Dimitrova, N., Chen, Y.C., Spector, D.L. & de Lange, T. 53BP1 promotes non-homologous end joining of telomeres by increasing chromatin mobility. Nature 456, 524–528 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Zimmermann, M., Lottersberger, F., Buonomo, S.B., Sfeir, A. & de Lange, T. 53BP1 regulates DSB repair using Rif1 to control 5′ end resection. Science 339, 700–704 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Huyen, Y. et al. Methylated lysine 79 of histone H3 targets 53BP1 to DNA double-strand breaks. Nature 432, 406–411 (2004).

    Article  CAS  PubMed  Google Scholar 

  29. Botuyan, M.V. et al. Structural basis for the methylation state-specific recognition of histone H4–K20 by 53BP1 and Crb2 in DNA repair. Cell 127, 1361–1373 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Fradet-Turcotte, A. et al. 53BP1 is a reader of the DNA-damage-induced H2A Lys 15 ubiquitin mark. Nature 499, 50–54 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Manke, I.A., Lowery, D.M., Nguyen, A. & Yaffe, M.B. BRCT repeats as phosphopeptide-binding modules involved in protein targeting. Science 302, 636–639 (2003).

    Article  CAS  PubMed  Google Scholar 

  32. Yu, X., Chini, C.C., He, M., Mer, G. & Chen, J. The BRCT domain is a phospho-protein binding domain. Science 302, 639–642 (2003).

    Article  CAS  PubMed  Google Scholar 

  33. Ward, I.M., Minn, K., Jorda, K.G. & Chen, J. Accumulation of checkpoint protein 53BP1 at DNA breaks involves its binding to phosphorylated histone H2AX. J. Biol. Chem. 278, 19579–19582 (2003).

    Article  CAS  PubMed  Google Scholar 

  34. Joo, W.S. et al. Structure of the 53BP1 BRCT region bound to p53 and its comparison to the Brca1 BRCT structure. Genes Dev. 16, 583–593 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Clapperton, J.A. et al. Structure and mechanism of BRCA1 BRCT domain recognition of phosphorylated BACH1 with implications for cancer. Nat. Struct. Mol. Biol. 11, 512–518 (2004).

    Article  CAS  PubMed  Google Scholar 

  36. Huen, M.S. et al. RNF8 transduces the DNA-damage signal via histone ubiquitylation and checkpoint protein assembly. Cell 131, 901–914 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Kolas, N.K. et al. Orchestration of the DNA-damage response by the RNF8 ubiquitin ligase. Science 318, 1637–1640 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Rogakou, E.P., Boon, C., Redon, C. & Bonner, W.M. Megabase chromatin domains involved in DNA double-strand breaks in vivo. J. Cell Biol. 146, 905–916 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Cotsiki, M. et al. Simian virus 40 large T antigen targets the spindle assembly checkpoint protein Bub1. Proc. Natl. Acad. Sci. USA 101, 947–952 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Murray, J.M., Stiff, T. & Jeggo, P.A. DNA double-strand break repair within heterochromatic regions. Biochem. Soc. Trans. 40, 173–178 (2012).

    Article  CAS  PubMed  Google Scholar 

  41. Hammet, A., Magill, C., Heierhorst, J. & Jackson, S.P. Rad9 BRCT domain interaction with phosphorylated H2AX regulates the G1 checkpoint in budding yeast. EMBO Rep. 8, 851–857 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Sanders, S.L., Arida, A.R. & Phan, F.P. Requirement for the phospho-H2AX binding module of Crb2 in double-strand break targeting and checkpoint activation. Mol. Cell. Biol. 30, 4722–4731 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Bothmer, A. et al. Regulation of DNA end joining, resection, and immunoglobulin class switch recombination by 53BP1. Mol. Cell 42, 319–329 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Callen, E. et al. 53BP1 mediates productive and mutagenic DNA repair through distinct phosphoprotein interactions. Cell 153, 1266–1280 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Ward, I. et al. The tandem BRCT domain of 53BP1 is not required for its repair function. J. Biol. Chem. 281, 38472–38477 (2006).

    Article  CAS  PubMed  Google Scholar 

  47. Cann, K.L. & Dellaire, G. Heterochromatin and the DNA damage response: the need to relax. Biochem. Cell Biol. 89, 45–60 (2011).

    Article  CAS  PubMed  Google Scholar 

  48. Noon, A.T. et al. 53BP1-dependent robust localized KAP-1 phosphorylation is essential for heterochromatic DNA double-strand break repair. Nat. Cell Biol. 12, 177–184 (2010).

    Article  CAS  PubMed  Google Scholar 

  49. Schoeftner, S. & Blasco, M.A.A. 'Higher order' of telomere regulation: telomere heterochromatin and telomeric RNAs. EMBO J. 28, 2323–2336 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Ruthenburg, A.J., Li, H., Patel, D.J. & Allis, C.D. Multivalent engagement of chromatin modifications by linked binding modules. Nat. Rev. Mol. Cell Biol. 8, 983–994 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Cox, J. & Mann, M. MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification. Nat. Biotechnol. 26, 1367–1372 (2008).

    CAS  PubMed  Google Scholar 

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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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Authors and Affiliations

  1. Laboratory of Chemistry and Cell Biology, The Rockefeller University, New York, New York, USA

    Ralph E Kleiner, Priyanka Verma & Tarun M Kapoor

  2. Laboratory of Mass Spectrometry and Gaseous Ion Chemistry, The Rockefeller University, New York, New York, USA

    Kelly R Molloy & Brian T Chait

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Contributions

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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Correspondence to Tarun M Kapoor.

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Supplementary information

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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