The Rap80-BRCC36 de-ubiquitinating enzyme complex antagonizes RNF8-Ubc13-dependent ubiquitination events at DNA double strand breaks

Abstract

DNA double strand breaks (DSBs) initiate reversible cellular checkpoint and repair activities. Whereas many of the activating events at DSBs have recently been elucidated, the mechanisms used to terminate responses at these sites are largely undefined. Here we report a pathway required to reverse RNF8-Ubc13 dependent ubiquitination events on chromatin flanking DSBs. Inhibition of the Rap80-BRCC36 de-ubiquitinating enzyme complex partially restored DSB-associated ubiquitin levels following RNF8 knockdown or proteasome inhibition. Similarly, BRCC36 knockdown or expression of a BRCC36 de-ubiquitinating enzyme-inactive mutant rescued both 53BP1 recruitment to DSBs and ionizing radiation-induced gammaH2AX ubiquitination following RNF8 depletion, and mitigated ionizing radiation sensitivity resulting from RNF8 deficiency. Thus, concomitant and opposing RNF8-Ubc13 ubiquitin ligase and Rap80-BRCC36 ubiquitin hydrolysis activities are responsible for determining steady-state ubiquitin levels at DNA DSBs. These findings reveal a Rap80-BRCC36 dependent pathway that is required for appropriate DSB recruitment and repair responses.

Fig. 1.

Rap80-BRCC36 K63-DUB activity and DSB responses. (A) Equivalent amounts of Rap80 wt (eRap80) or Rap80Δ233–399 (eΔ233–399) were purified from HeLa nuclear extracts and incubated in DUB buffer with K63-linked hexa-ubiquitin (K63-Ub₆). Higher molecular weight K63-Ub polymers are also present (Left, Top). IB was performed as indicated to detect input and K63-Ub hydrolysis products (Left), and ectopic Rap80 (eRap80) and BRCC36 (B36) (Right). (B) eRap80 complexes were purified from HeLa S3 cells at 48 h after control (Ct) or BRCC36 siRNA and DUB activity assays performed as in A. Mock represents DUB assay performed with Flag IP material from HeLa S3 cells that do not express eRap80. (C) Ectopic BRCC36 (eB36) and ectopic BRCC36 QSQ (eB36 QSQ) are recruited to laser-induced DSBs in U2OS cells 30 min after stripe induction. IF was performed against eBRCC36 with anti-HA antibody. (D) siRNA knockdown of BRCC36 versus control was performed in HeLa S3 cells and ubiquitinated forms of γH2AX analyzed by IB without IR or at 1 h after 10 Gy IR. Non-specific epitope of the BRCC36 antibody was used to indicate similar protein loading. (E) IB was performed for γH2AX in HeLa S3 cells expressing either eB36 wt or eB36 QSQ without IR or at 1 h after 10 Gy IR. (F) 293T cells were transfected with expression constructs for Flag-H2AX and HA-ubiquitin along with either control (Ct) or BRCC36 siRNA. Flag IP followed by IB was performed as indicated. Short and long exposures of the same IB are presented to reveal mono- and di-ubiquitinated Flag-H2AX. IgL indicates IgG light chain.

Fig. 2.

Rap80-BRCC36 regulates DSB associated ubiquitination. (A) eRNF8 and endogenous Rap80 co-localize at IRIF 4 h after 10 Gy IR. IF was performed with a mouse monoclonal antibody against the HA epitope to detect eRNF8 and a rabbit polyclonal antibody that was raised to Rap80 amino acids 233 to 710. (B) Quantification of eRNF8 and endogenous Rap80 at IRIF from 15 min to 4 h after 10 Gy IR. The experiment was performed in duplicate with greater than 200 cells examined for each time point. (C) Laser stripes were performed in HeLa cells 48 h after transfection with the indicated siRNA. MG132 (0.5 μM) was added 30 min after stripe induction. Cells were incubated at 37 °C for the indicated time in minutes following MG132 addition, and subsequently fixed and IF performed for conjugated ubiquitin and γH2AX. The 0 min time point indicates 60 min after stripe induction in the absence of MG132. This experiment was performed in duplicate with a minimum of 100 stripes analyzed for each time point. (D) Quantification of strong (purple) versus weakly detectable (yellow) ubiquitin (FK2) stripes as a percentage of the number of γH2AX stripes is displayed graphically. Total fluorescence intensity was calculated for each stripe to set an intensity threshold limit for both strong and weak stripes using a custom designed macro (Phase 3 Imaging) as described in Materials and Methods. (E) HeLa S3 cells expressing shRNA to luciferase (Luc), or either of two different Rap80 target sequences, were treated with 10 Gy IR, and DMSO or 0.5 μM MG132 was added 30 min later. Histones were acid-extracted 30 min after addition of drug and IB performed with an antibody to γH2AX. γH2AX, mono-ubiquitinated γH2AX (γ-H2AX-Ub), and di-ubiquitinated γH2AX (γ-H2AX-Ub2) are indicated.

Fig. 3.

Rap80-BRCC36 DUB activity reverses RNF8-dependent ubiquitination events at DSBs. (A) HeLa cells were treated with equivalent amounts of siRNA as indicated and then subjected to laser-induced DSBs. Cells were fixed at 30 min following DSB induction and IF performed for γH2AX and conjugated ubiquitin. (B) Quantification of ubiquitin positive stripes (FK2) as performed in Fig. 2. Bars represent the average of at least 3 independent experiments and 150 to 200 stripes. Error bars indicate SEM. P values were calculated by the Student t test. (C) Laser stripes were performed in siRNA-treated U2OS cells that express either BRCC36 wt (B36) or BRCC36 mutant (B36 QSQ). IF for γH2AX and conjugated ubiquitin was performed as indicated. (D) Quantification of strong versus weakly detectable ubiquitin stripes as a percentage of total γH2AX stripes was performed as in Fig. 3B. The displayed figure is a compilation of 3 independent experiments, each done in duplicate. Error bars indicate SEM. P values were calculated by the Student t test. (E) Co-transfection of the indicated siRNA was performed and γH2AX ubiquitination (γH2AX-Ub and γH2AX-Ub2) assessed in HeLa cells 1 h after 10 Gy IR.

Fig. 4.

BRCC36 deficiency restores 53BP1 DSB recruitment and DNA damage responses in RNF8-depleted cells. (A) Laser-induced DSBs were performed 48 h after control versus RNF8 knockdown in U2OS cells expressing either B36 wt or B36 QSQ. IF for 53BP1 and γH2AX was performed 30 min after stripe induction as indicated. Experiments were performed in triplicate with 150 to 200 stripes examined per group. (B) Quantification of strong versus weak 53BP1 stripes from A as a percentage of total γH2AX stripes. The displayed figure is a compilation of 3 independent experiments, with more than 100 stripes analyzed per sample. Error bars indicate SEM. (C) HeLa cells were co-transfected with siRNA to RNF8 and control, or RNF8 and BRCC36. Transfected cells were treated with 10 Gy IR at 48 h after transfection, and IF for 53BP1 and γH2AX was performed 5 h later. (D) Co-transfection of RNF8, Rap80 (R80), or BRCC36 siRNA with control siRNA or co-transfection of RNF8 siRNA with either Rap80 or BRCC36 siRNA was performed in HeLa cells, and the indicated doses of IR were administered 48 h later. Colony formation at 12 days after IR treatment was normalized as a percentage of colony formation at 0 Gy for each point. (E) γH2AX foci formation was quantified in HeLa cells at different times after 10 Gy IR in cells that were transfected with the indicated pairs of siRNA. The percentage of cells containing each number of γH2AX is displayed graphically and was derived from duplicate experiments with more than 200 cells counted each time. (F) Model for opposing regulation of poly-ubiquitination events at DSBs. Equilibrium synthesis and breakdown of poly-ubiquitin on common substrates by RNF8-Ubc13 and Rap80-BRCC36, respectively, is established at early stages of DSB repair. This dynamic equilibrium is predicted to determine steady-state levels of DSB-associated ubiquitin and DSB recruitment for BRCA1 and 53BP1 protein complexes.