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.

Figure 1

UCHL3 interacts with Ku80 and is recruited to sites of damage in a catalytic activity dependent manner. (A) Summary of mass spectrometry analysis of immunoprecipitate with an anti-GFP antibody from GFP-UCHL3 expressing cells. The hits obtained from GFP-UCHL3 over-expressing cells, but not detected with GFP-expressing cells (control) are shown with the number of peptide matches. Only proteins related to DNA damage responses are listed. (B,C) U2OS cells were transfected with a plasmid expressing GFP-UCHL3 (B) or GFP-Ku80 (C). The soluble fraction was subjected to immunoprecipitation with an anti-GFP antibody with or without EtBr followed by immunoblotting with the indicated antibodies. Transfection with the plasmid expressing GFP was used as a negative control. (D) The soluble fraction of U2OS cells was subjected to immunoprecipitation with an anti-UCHL3 antibody (Ab) or rabbit IgG (IgG) followed by immunoblotting with the indicated antibodies. (E) U2OS cells were transfected with a plasmid expressing GFP-UCHL3 (wild-type: WT) or catalytically inactive mutant (C95A) and then treated with 10 Gy IR or mock treated. The soluble fraction was subjected to immunoprecipitation with an anti-GFP antibody followed by immunoblotting with the indicated antibodies. Transfection with the plasmid expressing GFP was used as a negative control. Full-length blots are presented in Supplementary Fig. S8.

Figure 2

UCHL3 promotes cellular survival after IR and DSB repair. (A,B) U2OS cells transfected with the indicated siRNAs were processed for immunoblotting (A) or were subjected to clonogenic survival assay after IR (B) (Mean ± SEM, n = 3). (C,D) U2OS (wild-type: WT) or U2OS UCHL3 KO (KO#1 and KO#2) cells were processed for immunoblotting (C) or were subjected to clonogenic survival assay after IR (D) (Mean ± SEM, n = 3). (E) U2OS cells transfected with the indicated siRNAs were subjected to neutral comet assay. Efficiency of DSB repair is measured as the tail moment ratio between 2 hours after phleomycin removal and immediately after treatment (Mean ± SEM, n = 3). (F) U2OS cells stably expressing FLAG-UCHL3 or FLAG (empty vector: EV) were transfected with the indicated siRNAs and then subjected to neutral comet assay (Mean ± SEM, n = 3). *p < 0.05, **p < 0.01, ***p < 0.001. The tail moments measured immediately after phleomycin treatment, which indicate the amount of generated DSB, are presented in Supplementary Fig. S3. Full-length blots are presented in Supplementary Fig. S9.

Figure 3

UCHL3 facilitates classical NHEJ. (A) Schematic representation of a fluorescent reporter assay measuring c-NHEJ efficiency. I-SceI digestion sites and inserted stop codon are indicated by arrow heads and red characters, respectively. (B) U2OS cells transfected with the indicated siRNAs were subjected to c-NHEJ assay. The efficiency of c-NHEJ was normalized to control siRNA-transfected cells and set to 100% (Mean ± SEM, n = 3). (C) U2OS or UCHL3 KO#2 cells transfected with the indicated plasmid coding FLAG (empty vector: EV) or FLAG-UCHL3 were subjected to c-NHEJ assay. The efficiency of c-NHEJ was normalized to EV transfected U2OS cells and set to 100% (Mean ± SEM, n = 3). (D) U2OS cells transfected with the indicated siRNAs were subjected to direct-repeat GFP assay. The efficiency of homology mediated repair was normalized to control siRNA-transfected cells and set to 100% (Mean ± SEM, n = 3). (E,F) U2OS (WT) or UCHL3 KO#1 cells transfected with the indicated siRNAs were processed for immunoblotting analysis (E) or subjected to clonogenic survival assay after IR (F) (Mean ± SEM, n = 3). (G,H) U2OS cells stably expressing FLAG-UCHL3 (wild-type: WT) (G) or catalytically inactive mutant (C95A) (H) were transfected with the indicated siRNAs and subjected to clonogenic survival assay after IR (Mean ± SEM, n = 3). *p < 0.05, ***p < 0.001, N. S.; not significant. Full-length blots are presented in Supplementary Fig. S10.

Figure 4

UCHL3 enhances Ku80 chromatin binding upon DSB induction. (A) U2OS cells transfected with the indicated siRNAs were exposed to 10 Gy IR and further cultured for various time periods. Immunofluorescent staining with anti-Ku80 antibody and anti-γH2AX antibody was carried out. (B) Quantitative analysis of the data shown in (A). The number of Ku80 foci per nucleus was plotted (Mean ± SEM, siCtrl: n = 4, siUCHL3#3: n = 6). (C) U2OS cells or UCHL3 KO cell lines (#1 and #2) were subjected to chromatin fractionation assay with phleomycin. Whole cell lysates and chromatin fractions were subjected to immunoblotting with the indicated antibodies. (D) U2OS cells or UCHL3 KO cell line (#2) transfected with the indicated plasmids were subjected to chromatin fractionation assay with phleomycin. Whole cell lysates and chromatin fractions were subjected to immunoblotting with the indicated antibodies. For the detection with an anti-UCHL3 antibody, short exposure (short exp.) and long exposure (long exp.) of the film are shown. The exogenously expressed UCHL3 (FLAG-UCHL3) and endogenous UCHL3 (End. UCHL3) are indicated. The immunoblotting with an anti-γH2AX antibody was used as a control for the DNA damage induced.

Figure 5

UCHL3 directly deubiquitylates Ku80 ubiquitylation that is dependent on DSB and downstream NHEJ factors. (A) Purified GST or GST-UCHL3 was subjected to in vitro deubiquitylating enzyme activity assay, verifying purified UCHL3 is enzymatically active. An asterisk indicates HA-Ub-Vs bound UCHL3. (B) Silver staining of purified Ku used for electrophoretic mobility shift assay. (C) Electrophoretic mobility shift assay was carried out with purified Ku and UCHL3 proteins. The fluorescein labelled double-strand DNA (dsDNA, 50 nM) was incubated with the purified Ku (100 nM) in combination either with GST (600 nM) or GST-UCHL3 (300 or 600 nM). Free dsDNA and dsDNA bound with Ku are indicated. (D) GFP-Ku80 was immune-purified from GFP-Ku80 expressing-U2OS cells treated with phleomycin. Purified GFP-Ku80 was subjected to in vitro deubiquitylation assay. GFP-Ku80 bound beads were incubated either with GST or GST-UCHL3 and analysed by immunoblotting with the indicated antibodies. The bracket indicates ubiquitylated Ku80. (E) U2OS cells stably expressing GFP or GFP-Ku80 were subjected to in vivo ubiquitylation assay. Cells were transfected with siRNA targeting XRCC4 or control siRNA and further treated with phleomycin and then subjected to immunoprecipitation (IP) with an anti-GFP antibody. Input and GFP-IPed fractions were subjected to immunoblotting with the indicated antibodies. The bracket indicates ubiquitylated Ku80. Full-length blots are presented in Supplementary Fig. S11.

Figure 6

UCHL3 phosphorylation requiring its catalytic activity and downstream NHEJ factors regulates UCHL3 stability. (A) U2OS cells transfected with a plasmid expressing FLAG-UCHL3 were treated with phleomycin. Immunoprecipitation with an anti-FLAG antibody was carried out, followed by immunoblotting analysis with the indicated antibodies. Transfection with the plasmid coding FLAG was used as a negative control. (B) U2OS cells transfected with a plasmid expressing either FLAG-UCHL3 (wild-type: WT) or catalytically inactive mutant (C95A) were treated with phleomycin or mock treated. Immunoprecipitation with an anti-FLAG antibody was carried out, followed by immunoblotting analysis with the indicated antibodies. Transfection with the plasmid coding FLAG was used as a negative control. (C) U2OS cells transfected with a plasmid expressing either FLAG or FLAG-UCHL3 were treated with phleomycin for 1 hour and further cultured for the indicated time periods after removal of phleomycin. Cells were processed for immunoprecipitation with anti-FLAG antibody followed by immunoblotting analyses with the indicated antibodies. (D) U2OS cells transfected with the indicated siRNAs were further transfected with a plasmid expressing either GFP-UCHL3 or GFP. Following phleomycin treatment, cell extracts were subjected to immunoprecipitation with an anti-GFP antibody and ensuing immunoblotting analysis with the indicated antibodies. The plasmid expressing GFP was used as a negative control. (E) U2OS cells stably expressing FLAG-UCHL3 (WT, C95A, S75A or S75E) were transfected with siRNA targeting endogenous UCHL3. Cells were incubated with cycloheximide (100 μg/ml) for 1 hour prior to IR (10 Gy) and cultured for the indicated time periods after IR. Immunoblotting analyses were performed with the indicated antibodies. For anti-FLAG antibody detection, short exposure (short exp.) and long exposure (long exp.) of films are shown. Full-length blots are presented in Supplementary Fig. S12.

Figure 7

A proposed model for classical NHEJ. UCHL3 could regulate classical NHEJ by antagonizing Ku80 ubiquitylation in its catalytic activity and phosphorylation dependent manner. See main text for detailed description of the proposed model.