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.

Extracellular matrix stiffness determines DNA repair efficiency and cellular sensitivity to genotoxic agents

“…Chromatin fractionation was performed largely as described previously but with several modifications ( 43 ). Cells were washed twice with ice-cold phosphate-buffered saline (PBS) and collected with an appropriate volume of ice-cold PBS followed by centrifugation at 1000 g for 1 min.…”

Mouse Models of Inherited Retinal Degeneration with Photoreceptor Cell Loss

“…Repair of such damage is essential for DNA replication and, of particular importance for long-lived post-mitotic neuronal cells, transcription [386][387][388]. Proteins encoded by Bmi1, Dgcr8, Dicer1, Elp1, Ercc1, Ercc6, Msi1, Sirt6, Top2b, Ubb, and Uchl3 are known to participate in the DNA damage response [387,[389][390][391][392][393][394][395][396][397][398], some in transcription-coupled DNA repair. For example, BMI1 represses transcription at sites of UV-induced DNA damage to allow repair [389]; ELP1 is a required component of the Elongator complex [399], which couples RNA polymerase II to an alkyladenine glycosylase that initiates base excision repair [392]; ERCC6 promotes DSB repair in actively transcribed regions by displacing RNA polymerase from the lesion site [387], and DGCR8 interacts with both RNA polymerase II and ERCC6 to mediate transcription-coupled nucleotide excision repair of UV-induced DNA lesions [390].…”

Deubiquitylase UCHL3 drives error correction at kinetochores and chromosome segregation independent of spindle assembly checkpoint

“…3K-M). It is unlikely, that observed alignment defects are due to misregulation of DNA repair by the NHEJ factor Ku80, a direct deubiquitylation substrate of UCHL3 (38), as Ku80-deficient cells display no differences in number of misaligned chromosomes relative to control cells in contrast to UCHL3-downregulated cells ( Fig. S3A-C).…”

“…3F, 6E). Interestingly, downregulation of Ku80, reported direct deubiquitylation substrate of UCHL3 (38) and the core non-homologous end-joining (NHEJ) factor ( Fig. S3A), did not result in defects in chromosome alignment as compared to UCHL3 siRNA and relative to control siRNA treated cells (Fig.…”