Fig 2: RNAP2 behavior is modified without XAB2.(A) FRAP analysis of RNAP2-GFP expressing WT cells treated or not with UV-C (10 J/m2) after siRNA-mediated knockdown of the indicated factors. Error bars represent the SEM obtained from at least 10 cells. (B, C, D) Quantification of fluorescent signal in the nucleus against the couple RNAP2_6-4PP from PLA experiment (B) or from the IFndone in parallel to PLA assay (C, D). Error bars represent the standard error of the mean (SEM) obtained from at least 80 cells. P-value of Student’s test compared to No UV condition: *<0.05; ***<0.001. Figure 7—source data 1.Source data for Figure 7A: FRAP RNAP2-GFP. Figure 7—source data 2.Source data for Figure 7B–D: quantification of PLA and IF RNAP2_6-4PP.

Fig 3: Model of XAB2 dynamics during DNA damage-dependent transcription inhibition.Considering our results, a hypothetical model of XAB2 roles and dynamics can be sketched. XAB2 is involved in R-loops removal and pre-mRNA splicing, both processes linked to transcription. After DNA damage induction, transcription is blocked and XAB2 (together with some of the proteins involved in the splicing) is massively released from R-loops allowing a subset of XAB2 molecules to interact with UV-stalled RNA polymerase 2 (RNAP2) and participate in the TC-NER process. In the absence of XAB2, TC-NER is defective and as a consequence, RNAP2 remains longer in the proximity of DNA lesions.

Fig 4: XAB2 is involved in DNA repair.(A) Quantification of Unscheduled DNA Synthesis (UDS) assay determined by EdU incorporation after local damage (LD) induction with UV-C (100 J/m2) in WT cells (MRC5 cells) treated with siRNAs against indicated factors. Error bars represent the standard error of the mean (SEM) obtained from at least 30 LDs. (B) Western blot on whole-cell extracts of MRC5 cells treated with siRNA against indicated factors. (C) Quantification of RNA Recovery Synthesis (RRS) assay determined by EU incorporation after UV-C (10 J/m2) exposure in WT cells treated with siRNAs against indicated factors. Error bars represent the SEM obtained from at least 50 cells. (D) Quantification of TCR-UDS assay determined by EdU incorporation after LD induction with UV-C (100 J/m2) in GG-NER-deficient cells (XPC−/− cells) treated with siRNAs against indicated factors. Error bars represent the SEM obtained from at least 15 LDs. For all graphs, p-value of Student’s test compared to siMock condition: ***<0.001. Figure 1—source data 1.Source data for Figure 1A: quantification of UDS siXAB2. Figure 1—source data 2.Source data for Figure 1C: quantification of RRS siXAB2. Figure 1—source data 3.Source data for Figure 1D: quantification of TCR-UDS siXAB2. Figure 1—source data 4.Figures with the uncropped blots and relevant bands clearly labeled for Figure 1B: Western blot siXAB2 efficiency. Figure 1—source data 5.The original files of the full raw unedited gels for Figure 1B: Western blot siXAB2 efficiency.

Fig 5: XAB2 interacts with the UV lesion 6-4 Photoproduct (6-4PP).Quantification of fluorescent signal in the nucleus against the couple XAB2_6-4PP from Proximity Ligation Assay (PLA) experiment (A) or from the immunofluorescence (IF) done in parallel to PLA assay with the same antibodies dilutions (B). Error bars represent the standard error of the mean (SEM) obtained from at least 80 cells. P-value of Student’s test compared to No UV condition: ***<0.001. (C) Representative images of the PLA and IF experiments. Nuclei are delimited by dashed lines. Scale bar: 15 µm. Figure 2—source data 1.Source data for Figure 2A, B: quantification of PLA and IF XAB2_6-4PP.