Fig 2: Methylated USP11 regulates PRMT1 activity towards MRE11.a USP11 regulates MRE11 methylation. Knockdown of USP11 was induced by doxycycline treatment of HeLa-pTRIPZ-shCTRL and shUSP11 cells, and asymmetric dimethylation of MRE11 was detected by PLA (antibodies directed towards myc-tagged MRE11 and ADMA). Data are from three independent biological experiments (median PLA foci for shUSP11 (+dox) and shUSP11 (-dox) were 19 and 34, respectively; Mann–Whitney n1 = 203; n2 = 207; p < 0.0001; two-tailed). The middle and right panels display representative PLA images and immunoblots. Scale bar represents 5 µm. b Methyl-USP11 regulates MRE11 methylation. HeLa cells stably expressing myc-MRE11 and USP11 wildtype or R433K were transfected with siUSP11 to deplete endogenous protein, and levels of MRE11 methylation were detected by PLA. Data are from three independent experiments (median PLA foci for USP11 WT and USP11-R433K were 36 and 16, respectively; Mann–Whitney n1 = 209; n2 = 207; p < 0.0001; two-tailed). The middle and right panels display representative PLA images and immunoblots. Scale bar represents 5 µm. c Depletion of USP11 reduced PRMT1 and MRE11 interactions as determined by co-immunoprecipitation. HeLa-myc-MRE11/pTRIPZ-shCTRL or HeLa-myc-MRE11/pTRIPZ-shUSP11 cells were treated with doxycycline and MRE11 immunoprecipitated with anti-myc antibodies. Associated PRMT1 was detected by immunoblotting (representative image of n = 3 independent biological experiments). d Expression of USP11-R433K decreases PRMT1 and MRE11 interactions in HeLa cells as determined by co-immunoprecipitation. HeLa-myc-MRE11 cells were generated to stably express USP11 or USP11-R433K, and MRE11 was immunoprecipitated with anti-myc antibodies. Associated PRMT1 was detected by immunoblotting (representative image of n = 3 independent biological experiments). e PRMT1-mediated methylation of USP11 and MRE11 is epistatic for RPA foci formation after ionising radiation in late S/G2 cells. HeLa cells stably expressing USP11, USP11-R433K, MRE11 and MRE11-R/K (MRE11 mutated in all PRMT1-methyl acceptor sites) were transfected with siUSP11, exposed to 3 Gy IR, and harvested 6 h later. CENPF-positive cells were scored for RPA foci formation (mean ± SD; n = 3 independent biological experiments; one-way ANOVA and Tukey post hoc test: ****p < 0.0001). Uncropped blots, raw and processed graphical data provided as a Source Data file.

Fig 3: Methyl-USP11 regulates end-resection, ATR signalling and G2/M checkpoint.a USP11 and MRE11 are epistatic in the regulation of RPA foci formation after IR (3 Gy) in late S/G2 HeLa cells. CENPF-positive cells were scored for RPA foci formation (mean ± SD; n = 3 independent biological repeats; ****p < 0.0001; one-way ANOVA and Tukey post hoc test). b USP11 is not epistatic with CtIP in the regulation of RPA foci formation after IR (3 Gy) in late S/G2 HeLa cells (mean ± SD; n = 3 independent biological repeats; Student’s t test (two-sided, equal variance): ***p = 0.0096). c Methylated USP11 is epistatic with MRE11 in the regulation of RPA foci formation after IR in late S/G2 MCF7 cells. CENPF-positive cells were scored for RPA foci formation (mean ± SD; n = 3 independent biological repeats; one-way ANOVA and Tukey post hoc test: groups A and B *p = 0.031; groups A–C **p = 0.0046; groups A–D **p = 0.0022; groups A–E *p = 0.0162; groups A–F ***p = 0.0006). d Methylated USP11 is epistatic with MRE11 in the regulation of RPA foci formation after IR in late S/G2 HeLa cells. CENPF-positive cells were scored for RPA foci formation (mean ± SD; n = 3 independent biological repeats; one-way ANOVA and Tukey post hoc test: ****p < 0.0001). e HeLa-USP11-R433K cells display defective ATR signalling. HeLa-USP11 or HeLa-USP11-R433K cells were transfected with siUSP11, exposed to 10 Gy IR and then harvested at the time-points indicated for immunoblot analysis. Representative image of n = 3 independent biological repeats. f HeLa-USP11-R433K cells exhibit a G2/M checkpoint deficiency. Cells were treated with 5 Gy IR and harvested at the time points indicated. Proportion of mitotic cells was determined by phospho-Histone H3 (S10) staining (mean ± SEM; n = 3 biological repeats; Student’s t test (two-sided, equal variance): **p = 0.0023). Data on bar graph underneath represents the 90 min time point (mean ± SEM; n = 3 biological repeats; Student’s t test (two-sided, equal variance): **p = 0.0023). Uncropped blots, raw and processed graphical data provided as a Source Data file.