Fig. 3
CSB recruits RAD52 via an acidic domain. a Schematic diagram of CSB fragments. GFP-RAD52 foci frequency is compared in U2OS TRE WT, CSB KO cells, and CSB fragment stable expression cells constructed by lenti-virus infection in CSB KO cells (n = 3, 50 cells per replicate). b RAD51 foci frequency at TA-KR in WT, CSB KO cells, and CSB-AD transiently transfected CSB KO cells with or without siRAD52 treatment (n = 3, 50 cells per replicate). c Survival of U2OS TRE WT, CSB KO and CSB fragment stable expression cell lines under 2 Gy IR in a colony-formation assay (n = 3). d The recruitment of GFP-CSB fragments to TA-KR sites. The CSB fragments showing colocalization with TA-KR are marked with +; the foci relative intensity was quantified (n = 10 cells in one experiment, scale bar: 2 μm). e Interaction of Myc-RAD52 and GFP-CSB-AD by anti-GFP Co-IP in Flp-in 293 cells. f Cross-linking between GST-CSB-AD and RAD52 protein analyzed by mass spectrometry. Red and gray lines indicate inter- and intra-molecule crosslinks, respectively. g Schematic diagram of CSB-AD10A and 22A. h Interactions between Myc-RAD52 and GFP-CSB-AD WT, 10A, and 22A were tested by anti-GFP Co-IP in Flp-in 293 cells. The relative foci intensity of GFP-tagged CSB-AD WT and the 22A mutant to TA-KR in CSB KO cells is quantified (n = 10 cells in one experiment, scale bar: 2 μm). i GFP-RAD52 and j RAD51 foci frequency at TA-KR in CSB KO cells transfected with Myc-tagged CSB-AD WT, 10A, and 22A (n = 3, 50 cells per replicate). k γH2AX foci frequency at TA-KR at early (1 h) and late (36 h) time points after damage induction in CSB KO cells transfected with CSB-AD WT, 10A, and 22A (n = 3, 50 cells per replicate). Unpaired t-test, error bars represent SEM. *P < 0.05, **P < 0.01, ***P < 0.001
