Figure 1.
K63RUb expression induces genomic instability.
(A) Schematic overview of WTUb and K63RUb expression constructs. Expression of a 6xHis-tagged WT or K63R ubiquitin B-GFP fusion protein is driven by the UBC promoter. (B) mRNA expression levels of endogenous ubiquitin B and expressed 6xHis-tagged WT or K63R ubiquitin B determined by real-time PCR. (C) Impaired K63-linked ubiquitin chain formation, in untreated or CPT (1h, 100nM) treated A549 K63RUb cells, confirmed by comparison to K63 ubiquitin chain abundance in A549 WTUb cells by Western blot using a K63-chain specific antibody. Ponceau S staining indicates equal loading. (D) Cell cycle distribution of normal cycling A549 WTUb and K63RUb overexpressing cells determined by FACS analysis. Data are mean of 3 independent exp's ± sd. (E-F) Mutations at the HPRT locus analysed in WTUb and K63RUb HPRT mutant-free HAT-selected cells after (E) 0, 7 or 14 days proliferation (spontaneous), data are mean ± s.e.m. of 7 (7 days growth) and 2 (14 days growth) independent exp's (n = 5-50 per exp). (F) 6 days continuous treatment with 1 µM PARPi, 5 or 20 nM CPT, 1 and 4 Gy IR, Ctrl is 7 days spontaneous. Data are mean ± s.e.m. of 3 independent exps (n = 5 per exp). (G) A549 K63RUb cells were treated as described in (e) to obtain spontaneous, CPT- and IR-induced HPRT mutated clones. RNA and genomic DNA were isolated from individual colonies and mutations were assessed by sequence analysis of HPRT cDNA and/or exon1-9 presence was scored by exon-specific PCR amplification. (H) Metaphases of untreated or CPT-treated (20 nM, 24 h) WTUb and K63RUb cells were harvested. Chromosomal aberrations were scored using giemsa-staining in 50 metaphases per treatment. Data represents average ± s.e.m. *P<0.05, **P<0.01, ***P<0.001.
Table 1.
Spontaneous, IR- and CPT-induced mutation spectra in K63R ubiquitin expressing cells.
Figure 2.
K63RUb expression sensitizes cells to replication-associated DNA damage.
(A-C) Clonogenic survival of A549 cells expressing empty vector (EV), WTUb or K63RUb was determined after (A) CPT (24h) treatment started following cell attachment. Data are mean ± sd. of 2 independent exp's (n = 3 per exp). (B) Continuous PARPi treatment, data are mean ± sd. of 3 independent exp's (n = 3 per exp). (C) IR, data are mean ± sd. of 3 independent exp's (n = 3 per exp) *P<0.05, **P<0.01, ***P<0.001.
Figure 3.
K63RUb expression induces S-phase specific repair defects.
(A) WT and K63R Ub cells were fixed and immuno-stained for γH2AX and 53BP1 foci at the indicated time points following 2 Gy IR or untreated (Ctrl). Mean values ± sd. of 2 independent exp's, >150 cells were analysed per time-point (B) 30 min EdU (10 µM) incorporation before 2 Gy IR, cells were fixed 30 min or 24 hrs post IR, EdU and 53BP1 foci were visualized by fluorescent staining and quantified. Mean values ± s.e.m. of 2 independent exp's (n>100 per treatment). (C) 30 min EdU (10 µM) incorporation alone (untreated) or before 2.5 h 100 nM CPT treatment. Cells were fixed and immuno-stained for EdU and γH2AX 3 h post treatment. (D) Quantification of 53BP1 foci. Cells were fixed and immuno-stained for 53BP1 directly or 24 h after 1 h 100 nM CPT treatment, >80 cells were analysed per sample. (E) Quantification of γH2AX immuno-staining of untreated (Ctrl) or 1 h 100 nM CPT treated cells. Mean values ± s.e.m. Data is representative exp (n>130) of 3 independent exp's, *P<0.05.
Figure 4.
K63RUb phenotype is not due to NHEJ.
(A-C) Clonogenic survival of WT and K63R Ub cells after treatment with (A) 1 µM DNA-PKi alone for indicated times, (B) 1 µM DNA-PKi for 24 or 48 hrs, started 1 h before combined 24 hrs treatment with 50 nM CPT or (C) 1 µM DNA-PKi for 24 hrs, started 1 h before 2 Gy IR (A-C) mean ± s.d. (n = 3). (D) Mutations at the HPRT locus were determined after continuous treatment with 1 µM DNA-PKi started 1 h before combined with additional treatments either spontaneous or 20 nM CPT for 6 days total, mean ± s.d. (n = 5).
Figure 5.
RNF8 depletion does not reproduce K63RUb phenotype.
(A-C) WT and K63R Ub cells were transfected with siRNA against RNF8 or RAP80 on day 1 and 3. (A) Knockdown of RNF8 and RAP80 was assessed by mRNA expression levels determined by real-time PCR. Clonogenic survival was assessed after (B) 4 Gy IR and (C) PARPi (continuous) (B-C) Data are mean ± s.d. of 2 independent exp's (n = 3 per exp). (D) Spontaneous and IR-induced (4 Gy) mutations were determined at the HPRT locus, mean ± s.e.m. of 3 independent exp's (n = 10 per exp), *P<0.05, **P<0.01, ***P<0.001.
Figure 6.
Dual inhibition of PCNA and RNF8 signalling pathways does not mimic the K63RUb phenotype.
(A) Depletion of HLTF or SHPRH in combination with knock-down of RNF8 was achieved using lentiviral shRNA's and was confirmed using real-time PCR. (B) Spontaneous or CPT (20 nM) induced mutations were determined at the HPRT locus, mean ± s.d. (n = 5) per exp). (C) Clonogenic survival of A549 cells after double knock-down of HLTF and RNF8 or SHPRH and RNF8 was determined after treatment with 100 nM CPT (24 h). Data are mean ± s.d. (n = 3), *P<0.05, **P<0.01, ***P<0.001.
Figure 7.
K63RUb expression sensitizes RNF8-/- MEFs to CPT and PARPi.
(A-C) Clonogenic survival of RNF8-/- MEFs uninfected or infected with K63RUb or empty vector (EV) after (A) IR, (B) CPT (24 h) or (C) PARPi (continuous). Data are mean ± s.d. of 2 independent exp's (n = 3 per exp), *P<0.05.