Fig. 5

T457A impairs Polη polyubiqitination at K462. a 293T cells transfected with Flag empty vector, WT, or T457A Flag-Polη and HA-Ub were immunoprecipitated with anti-Flag agarose in a denatured condition followed by quantitative MS analysis as described in “Methods” section. All ubiquitination sites identified in WT and T457A Polη. Error bars: s.e.m. b U2OS cells transfected with WT or mutated (T457A, K462R, and T457AK462R) GFP-Polη were irradiated by UVC (15 J m⁻²) and further incubated for 24 h. The proportion of GFP-Polη-expressing cells with >30 foci was determined. Data represent means ± SEM from three independent experiments. c 293 T cells were transfected with WT or mutated (T457A, K462R, and T457A/K462R) Flag-Polη and HA-Ub. The collected chromatin fractions (CFs) were denatured followed by immunoprecipitation with anti-Flag agarose. The immunoprecipitates were immunoblotted with anti-HA, anti-K48-Ubiquitin, and anti-Flag antibodies. d Mutation frequency in damaged (400 J m⁻² UVC) supF plasmid was determined. Data represent means ± SEM from three independent experiments. ns not significant. e WT, T457A, or K462R GFP-Polη were transfected into 293T cells. The cell lysates were immunoprecipitated with GFP-Trap A, followed by immunoblotting with anti-O-GlcNAc and anti-GFP antibodies. “SE” and “LE” represent short and long exposure, respectively. f WT or T457A GFP-Polη were transfected into siDDB1-, siCDT2-, or siNC-treated 293T cells. The chromatin fractions were immunoprecipitated with GFP-Trap A and analyzed via western blotting with anti-O-GlcNAc and anti-GFP antibodies