[en] Cells respond to DNA double-strand breaks by recruiting factors such as the DNA-damage mediator protein MDC1, the p53-binding protein 1 (53BP1), and the breast cancer susceptibility protein BRCA1 to sites of damaged DNA. Here, we reveal that the ubiquitin ligase RNF8 mediates ubiquitin conjugation and 53BP1 and BRCA1 focal accumulation at sites of DNA lesions. Moreover, we establish that MDC1 recruits RNF8 through phosphodependent interactions between the RNF8 forkhead-associated domain and motifs in MDC1 that are phosphorylated by the DNA-damage activated protein kinase ataxia telangiectasia mutated (ATM). We also show that depletion of the E2 enzyme UBC13 impairs 53BP1 recruitment to sites of damage, which suggests that it cooperates with RNF8. Finally, we reveal that RNF8 promotes the G2/M DNA damage checkpoint and resistance to ionizing radiation. These results demonstrate how the DNA-damage response is orchestrated by ATM-dependent phosphorylation of MDC1 and RNF8-mediated ubiquitination.
Disciplines :
Life sciences: Multidisciplinary, general & others
Author, co-author :
Kolas, Nadine K; Samuel Lunenfeld Research Institute, Mount Sinai Hospital, 600 University Avenue, Toronto M5G1X5, Ontario, Canada
Chapman, J Ross; Wellcome Trust and Cancer Research UK Gurdon Institute, Department of Zoology, University of Cambridge, Cambridge CB2 1QN, United Kingdom
Nakada, Shinichiro; Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ont. M5G 1X5, Canada
Ylanko, Jarkko; Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ont. M5G 1X5, Canada ; Department of Molecular Genetics, University of Toronto, Toronto, Ont., Canada
CHAHWAN, Richard ; University of Luxembourg > Faculty of Science, Technology and Medicine (FSTM) > Department of Health, Medicine and Life Sciences (DHML) ; Wellcome Trust and Cancer Research UK Gurdon Institute, Department of Zoology, University of Cambridge, Cambridge CB2 1QN, United Kingdom
Sweeney, Frédéric D; Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ont. M5G 1X5, Canada ; Department of Molecular Genetics, University of Toronto, Toronto, Ont., Canada
Panier, Stephanie; Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ont. M5G 1X5, Canada
Mendez, Megan; Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ont. M5G 1X5, Canada
Wildenhain, Jan; Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ont. M5G 1X5, Canada
Thomson, Timothy M; Department of Molecular and Cellular Biology, Instituto de Biología Molecular de Barcelona, 08034 Barcelona, Spain
Pelletier, Laurence; Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ont. M5G 1X5, Canada ; Department of Molecular Genetics, University of Toronto, Toronto, Ont., Canada
Jackson, Stephen P; Wellcome Trust and Cancer Research UK Gurdon Institute, Department of Zoology, University of Cambridge, Cambridge CB2 1QN, United Kingdom
Durocher, Daniel; Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ont. M5G 1X5, Canada ; Department of Molecular Genetics, University of Toronto, Toronto, Ont., Canada
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We thank members of the Durocher and Jackson laboratories for input on the manuscript and M. Vojvodic for experimental assistance. We also thank A. C. Gingras for the stable YFP-RNF8 line, KuDOS Pharmaceuticals for providing inhibitors and reagents, and Abcam for the MDC1 pT719 antibody. This work was supported by grants from the Canadian Institutes of Health Research (CIHR) to D.D. and by funding from Cancer Research UK and the European Union (S.P.J.). N.K.K. is a CIHR postdoctoral fellow and an alumnus of the Excellence in Radiation Research for the 21st Century training program; F.D.S. holds a Terry-Fox studentship from the National Cancer Institute of Canada; S.N. is a Gail-Posluns Fellow and is supported by the Mitsubishi Pharma Research Foundation. D.D. is a Canada Research Chair (Tier II) in Proteomics, Functional Genomics, and Bioinformatics.
