Neddylation promotes ubiquitylation and release of Ku from DNA-damage sites.
The activities of many DNA-repair proteins are controlled through reversible covalent modification by ubiquitin and ubiquitin-like molecules. Nonhomologous end-joining (NHEJ) is the predominant DNA double-strand break (DSB) repair pathway in mammalian cells and is initiated by DSB ends being recognized by the Ku70/Ku80 (Ku) heterodimer. By using MLN4924, an anti-cancer drug in clinical trials that specifically inhibits conjugation of the ubiquitin-like protein, NEDD8, to target proteins, we demonstrate that NEDD8 accumulation at DNA-damage sites is a highly dynamic process. In addition, we show that depleting cells of the NEDD8 E2-conjugating enzyme, UBE2M, yields ionizing radiation hypersensitivity and reduced cell survival following NHEJ. Finally, we demonstrate that neddylation promotes Ku ubiquitylation after DNA damage and release of Ku and Ku-associated proteins from damage sites following repair. These studies provide insights into how the NHEJ core complex dissociates from repair sites and highlight its importance for cell survival following DSB induction.
The Wellcome Trust and Cancer Research UK Gurdon Institute, University of Cambridge, Cambridge 2 1QN, UK.
Jessica S
JS
Brown
The Wellcome Trust and Cancer Research UK Gurdon Institute, University of Cambridge, Cambridge 2 1QN, UK.
Natalia
N
Lukashchuk
The Wellcome Trust and Cancer Research UK Gurdon Institute, University of Cambridge, Cambridge 2 1QN, UK.
Matylda
M
Sczaniecka-Clift
The Wellcome Trust and Cancer Research UK Gurdon Institute, University of Cambridge, Cambridge 2 1QN, UK; Institut de Pharmacologie et de Biologie Structurale, CNRS, Université de Toulouse-Université Paul Sabatier, Equipe Labellisée Ligue contre le Cancer, 31077 Toulouse, France.
Sébastien
S
Britton
The Wellcome Trust and Cancer Research UK Gurdon Institute, University of Cambridge, Cambridge 2 1QN, UK.
Carlos
C
le Sage
Institut de Pharmacologie et de Biologie Structurale, CNRS, Université de Toulouse-Université Paul Sabatier, Equipe Labellisée Ligue contre le Cancer, 31077 Toulouse, France.
Patrick
P
Calsou
Institute of Molecular Biology (IMB), 55128 Mainz, Germany. Electronic address: p.beli@imb-mainz.de.
Petra
P
Beli
The Wellcome Trust and Cancer Research UK Gurdon Institute, University of Cambridge, Cambridge 2 1QN, UK. Electronic address: y.galanty@gurdon.cam.ac.uk.
Yaron
Y
Galanty
The Wellcome Trust and Cancer Research UK Gurdon Institute, University of Cambridge, Cambridge 2 1QN, UK. Electronic address: s.jackson@gurdon.cam.ac.uk.
Stephen P
SP
Jackson
D034961:Antigens, Nuclear / Q000737:chemistry / Q000378:metabolism*; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D003517:Cyclopentanes / Q000633:toxicity; D004249:DNA Damage / Q000187:drug effects / Q000528:radiation effects; D059766:DNA End-Joining Repair; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D006657:Histones / Q000378:metabolism; D006801:Humans; D000072200:Ku Autoantigen; D000075687:NEDD8 Protein; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D040901:Proteomics; D011743:Pyrimidines / Q000633:toxicity; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism; D011839:Radiation, Ionizing; D044763:Ubiquitin-Conjugating Enzymes / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D054875:Ubiquitination / Q000187:drug effects; D014452:Ubiquitins / Q000037:antagonists & inhibitors / Q000378:metabolism*
D034961:Antigens, Nuclear; D003517:Cyclopentanes; D004268:DNA-Binding Proteins; C468783:H2AX protein, human; D006657:Histones; D000075687:NEDD8 Protein; C082860:NEDD8 protein, human; D011743:Pyrimidines; D034741:RNA, Small Interfering; D014452:Ubiquitins; D044763:Ubiquitin-Conjugating Enzymes; C000606509:Xrcc6 protein, human; D000072200:Ku Autoantigen; C000596156:UBE2M protein, human; C539933:pevonedistat