Abstract
53BP1 (also called TP53BP1) is a chromatin-associated factor that promotes immunoglobulin class switching and DNA double-strand-break (DSB) repair by non-homologous end joining. To accomplish its function in DNA repair, 53BP1 accumulates at DSB sites downstream of the RNF168 ubiquitin ligase. How ubiquitin recruits 53BP1 to break sites remains unknown as its relocalization involves recognition of histone H4 Lys 20 (H4K20) methylation by its Tudor domain. Here we elucidate how vertebrate 53BP1 is recruited to the chromatin that flanks DSB sites. We show that 53BP1 recognizes mononucleosomes containing dimethylated H4K20 (H4K20me2) and H2A ubiquitinated on Lys 15 (H2AK15ub), the latter being a product of RNF168 action on chromatin. 53BP1 binds to nucleosomes minimally as a dimer using its previously characterized methyl-lysine-binding Tudor domain and a carboxy-terminal extension, termed the ubiquitination-dependent recruitment (UDR) motif, which interacts with the epitope formed by H2AK15ub and its surrounding residues on the H2A tail. 53BP1 is therefore a bivalent histone modification reader that recognizes a histone ‘code’ produced by DSB signalling.
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Acknowledgements
We are grateful to R. Szilard, J. Côté and S. Panier for critically reading the manuscript; to L.-L. Du, A. Nussenzweig, Y. Tong and C. Arrowsmith for plasmids; to M. Cook and A. Rosebrock for centrifugal elutriation; and to B. Sauriol for help with FRAP data analysis. A.F.-T. and A.O. both receive post-doctoral fellowships from the CIHR; C.E.-D. is an Ontario Post-doctoral Fellow; J.K.-L. receives a post-doctoral fellowship from the Leukemia and Lymphoma Society; and C.C.Y.L. and M.-C.L. are post-doctoral fellows of the Canadian Breast Cancer Foundation (Ontario Division). D.D. is the Thomas Kierans Chair in Mechanisms of Cancer Development and a Canada Research Chair (Tier 1) in the Molecular Mechanisms of Genome Integrity. Work in the D.D. laboratory was supported by CIHR grant MOP84297 and grant GL2-01-010 from the Ontario Research Fund.
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A.F.-T. initiated the project, carried out most of the cell biological experiments, found the interaction between ubiquitinated H2A and 53BP1, and contributed to the experimental design and data interpretation. M.D.C. produced the recombinant nucleosomes. A.F.-T. and M.D.C. and carried out the recombinant nucleosome pull-down studies. C.E.-D. examined RIF1 focus formation, the role of the UDR in homologous recombination and helped with some immunofluorescence experiments. A.O. carried out the class switching experiments and the DT40 work. C.C.Y.L. helped with protein binding studies and generated the nucleosome model shown in Supplementary Fig. 12. M.-C.L. helped with the FRAP experiments. J.K.-L. carried out chemical ubiquitination and mass spectrometry to verify histone methylation. H.H. carried out the NMR experiments. S.M.N. carried out mass spectrometry to verify H4K20 methylation. F.S. supervised H.H. D.D. supervised the project and wrote the manuscript with input from the other authors.
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Fradet-Turcotte, A., Canny, M., Escribano-Díaz, C. et al. 53BP1 is a reader of the DNA-damage-induced H2A Lys 15 ubiquitin mark. Nature 499, 50–54 (2013). https://doi.org/10.1038/nature12318
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DOI: https://doi.org/10.1038/nature12318
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