Abstract
SummaryThe 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.
Highlights
The DNA-damage response (DDR), comprising the sensing, signaling, and repair of damaged DNA, requires recruitment and post-translational modification (PTM) of many proteins at DNA-damage sites (Polo and Jackson, 2011)
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
Summary
The DNA-damage response (DDR), comprising the sensing, signaling, and repair of damaged DNA, requires recruitment and post-translational modification (PTM) of many proteins at DNA-damage sites (Polo and Jackson, 2011). Classical NHEJ requires binding of the Ku70/ Ku80 heterodimer to DNA ends, with ensuing recruitment of DNA-PKcs, PAXX, and end-processing factors leading to repair by the DNA ligase IV/XRCC4/XLF complex (Davis and Chen, 2013; Grundy et al, 2014; Wang and Lees-Miller, 2013; Ochi et al, 2015; Xing et al, 2015). Activated NEDD8 is conjugated to substrates, predominantly by the E2/ E3 enzyme complexes UBE2M/RBX1 or UBE2F/RBX2 (Huang et al, 2009). The best-characterized NEDD8 substrates, cullins (CUL1, 2, 3, 4A, 4B, 5, and 7 and PARC in human cells), serve as molecular scaffolds for cullin-RING ubiquitin ligases (CRLs; Lydeard et al, 2013; Sarikas et al, 2011). We establish that neddylation is crucial for cell survival after DSB induction, and that it promotes Ku ubiquitylation and release from DSB sites
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
