Fig 2: RPA loading is partially abrogated by AQR knockdown. (a) DSB responses in AQR-knockdown cells. U2OS cells were treated with NCS (20 ng/ml, 4 h) or CPT (1 μM, 2 h) 48 h after siRNA transfection. Cells were lysed with SDS sample buffer and the indicated proteins were detected by western blotting. NT, no treatment. (b) Percentage of RPA foci (>5)-positive cells. HCT116 cells were treated with NCS (20 ng/ml, 4 h) 48 h after siRNA transfection. Cells were pre-extracted with 0.2% Triton X-100/PBS prior to fixation and immunostained with anti-RPA32 antibody. Data represent mean ± SD from three independent experiments. n.s., not significant. (c) Number of RPA foci per cell. After RPA immunostaining, shown in (b), the numbers of RPA foci were counted in at least 60 cells and are shown as a boxplot. ***p < 0.005. n.s., not significant.

Fig 3: XAB2 and AQR are released from R-loops during DNA repair.(A) Immunoprecipitation (IF) of XAB2 in nuclear extract from different cell lines treated with 10 J/m2 of UV-C at different times. Bound proteins were revealed by Western blotting with antibodies against XAB2. INPUT, 10% of the lysate used for IP reaction. (B) IP of R-loops in non-crosslinked chromatin extract from WT cells treated or not with Benzonase. XAB2 bounds to R-loops was revealed by Western blotting. INPUT, 10% of the lysate used for IP reaction. (C) Quantification of IF against R-loops in WT cells treated with siRNAs against indicated factors. Quantification of fluorescent signal in the nucleus against the couple XAB2_R-loops or AQR_R-loops from PLA experiments (D) or from the IF done in parallel to PLA assays (E). Error bars represent the standard error of the mean (SEM) obtained from at least 50 cells. P-value of Student’s test compared to No UV or siMock condition: **<0.01; ***<0.001. Figure 5—source data 1.Source data for Figure 5C: quantification of IF R-loops in silenced cells. Figure 5—source data 2.Source data for Figure 5D, E: quantification of PLA and IF XAB2_R-loops and AQR_R-loops. Figure 5—source data 3.Figures with the uncropped blots and relevant bands clearly labeled for Figure 5A: Western blot of IP XAB2. Figure 5—source data 4.The original files of the full raw unedited gels for Figure 5A: Western blot of IP XAB2. Figure 5—source data 5.Figures with the uncropped blots and relevant bands clearly labeled for Figure 5B: Western blot of IP R-loops. Figure 5—source data 6.The original files of the full raw unedited gels for Figure 5B: Western blot of IP R-loops.

Fig 4: DDB2 stimulates OGG1 recruitment to densely clustered 8-oxoG sites.a Representative time-lapse pictures of OGG1-GFP and GFP-DDB2 accumulation at micro-irradiated (405 nm laser) sub-nuclear area, indicated by arrows, in the presence of 50 µM Ro 19-8022 photosensitizer. b Quantification of accumulation kinetics of OGG1-GFP and GFP-DDB2 (as shown in a). c Schematic overview of the molecular interactions of DDB2 within the CUL4A-DDB1-RBX1 E3 ubiquitin ligase complex (CRL), which is required for the successive molecular interactions by ubiquitylation and subsequent DNA repair. The activation of CRL is mediated by covalent attachment of the ubiquitin-like activator NEDD8 on CUL4A and its proteolytic removal leads to the deactivation of ubiquitin ligase function. These crucial events can be fine-tuned by specific inhibitors MLN4924 (NAE1i) and SB-58-SN29 (CSN5i), acting on NEDD8-activating enzyme NAE1 and CSN5, respectively. d Representative time-lapse pictures of OGG1-GFP accumulation at micro-irradiated (405 nm laser) sub-nuclear area, indicated by arrows, in the presence of 10 µM Ro 19-8022 photosensitizer. Cells were pretreated with DMSO (CTR), NEDDylation inhibitor (NAE1i) or de-NEDDylation inhibitor (CSN5i) for 1.5 h. e Quantification of accumulation kinetics of OGG1-GFP (as shown in d). f Immunoblot analysis for DDB2, CUL4A, CSA and AQR (loading control) in MRC-5 expressing OGG1-GFP. Cells were treated with inhibitors as indicated in d. Scale bars: 5 µm. Data were normalized to the background and represent mean ± SEM from three independent experiments. Total number of cells “n” measured are indicated in figure legends. ****P < 0.001, analyzed by ROC curve analysis. Source data are provided as a Source Data file. (See also Supplementary Fig. 6).

Fig 5: AQR knockdown suppresses HR. (a) Representative images of 53BP1 and Rad51 foci formation in AQR-knockdown cells. HCT116 cells were treated with or without MMC (200 ng/ml, 4 h) 48 h after siRNA transfection, and immunostained with anti-53BP1 or anti-Rad51 antibody. (b) Percentage of 53BP1 foci (>5)-positive cells in AQR-knockdown cells shown in (a). Data represent mean ± SD from three independent experiments. (c) Percentage of Rad51 foci (>10)-positive cells in AQR-knockdown cells shown in (a). (d and e) Percentage of γH2AX foci (>10)- and FANCD2 foci (>10)-positive cells in AQR-knockdown cells in response to MMC (200 ng/ml, 4 h). (f) Percentage of Rad51 foci-positive cells in AQR-knockdown cells in response to NCS (20 ng/ml, 4 h) or CPT (0.5 μM, 4 h). NT, no treatment. (g) DR-GFP assay for measuring HR frequency. DR-GFP U2OS cells were transfected with siRNA and then transfected with I-SceI expression plasmid 24 h after siRNA transfection. Percentages of GFP-positive cells were measured by flow cytometry. *p < 0.05; ***p < 0.005. n.s., not significant.