The deubiquitylating enzyme UCHL3 regulates Ku80 retention at sites of DNA damage.

Abstract

Non-homologous end-joining (NHEJ), which can promote genomic instability when dysfunctional, is a major DNA double-strand break (DSB) repair pathway. Although ubiquitylation of the core NHEJ factor, Ku (Ku70-Ku80), which senses broken DNA ends, is important for its removal from sites of damage upon completion of NHEJ, the mechanism regulating Ku ubiquitylation remains elusive. We provide evidence showing that the ubiquitin carboxyl-terminal hydrolase L3 (UCHL3) interacts with and directly deubiquitylates one of the Ku heterodimer subunits, Ku80. Additionally, depleting UCHL3 resulted in reduced Ku80 foci formation, Ku80 binding to chromatin after DSB induction, moderately sensitized cells to ionizing radiation and decreased NHEJ efficiencies. Mechanistically, we show that DNA damage induces UCHL3 phosphorylation, which is dependent on ATM, downstream NHEJ factors and UCHL3 catalytic activity. Furthermore, this phosphorylation destabilizes UCHL3, despite having no effect on its catalytic activity. Collectively, these data suggest that UCHL3 facilitates cellular viability after DSB induction by antagonizing Ku80 ubiquitylation to enhance Ku80 retention at sites of damage.This work was funded by Grant-in-Aid for Research Activity start-up 15H06738 (R.N.), Grant-in Aid for Young Scientists (A) 16H05888 (R.N.), Daiichi Sankyo Foundation of Life Science (R.N.), Mochida Memorial Foundation for Medical and Pharmaceutical Research (R.N.), Cancer Research UK (CRUK) Grant C6/A11224 and C6/A18796 (P.W.), CRUK Project Grant C6/A14831 (R.N.). T.L.B. and Q.W. are funded by Wellcome Trust Investigator Award (200814_Z_16_Z). Research in the B.M.K. laboratory is supported by a John Fell Fund 133/075, the Wellcome Trust (097813/Z/11/Z) and the Engineering and Physical Sciences Research Council (EP/N034295/1). Research in the S.P.J. laboratory is funded by CRUK Program Grant C6/A18796, and Wellcome Trust Grant WT206388/Z/17/Z. Cancer Research UK Grant C6946/A24843 and Wellcome Trust Grant WT203144 provided core infrastructure funding