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

• Published in: Scientific reports Vol. 8; no. 1; pp. 17891 - 15
• Main Authors: Nishi, Ryotaro, Wijnhoven, Paul W G, Kimura, Yusuke, Matsui, Misaki, Konietzny, Rebecca, Wu, Qian, Nakamura, Keisuke, Blundell, Tom L, Kessler, Benedikt M
• Format: Journal Article
• Language: English
• Published: England Nature Publishing Group 17.12.2018; Nature Publishing Group UK
• Subjects: Antigens, Nuclear - genetics; Cell Line; Cell Survival - genetics; Chromatin; Chromatin - genetics; Cysteine Endopeptidases - genetics; Deoxyribonucleic acid; DNA; DNA - genetics; DNA Breaks, Double-Stranded; DNA damage; DNA Damage - genetics; DNA End-Joining Repair - genetics; DNA repair; DNA Repair - genetics; DNA-Binding Proteins - genetics; Double-strand break repair; Genomic instability; Humans; Hydrolase; Ionizing radiation; Ku Autoantigen - genetics; Non-homologous end joining; Phosphorylation; Phosphorylation - genetics; Protein Binding - genetics; Ubiquitin; Ubiquitin Thiolesterase; Ubiquitination - genetics
• ISSN: 2045-2322; 2045-2322
• DOI: 10.1038/s41598-018-36235-0

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.