Fig 2: Effect of TENT4A and PAXIP1 on the stability of TLS-related mRNAs and level of TLS-related proteins. (A,B) TENT4A and/or PAXIP1 expression was knocked down in MCF-7 cells for 48 h, after which the cells were treated with 5 μg/mL Actinomycin D for up to 8 h. The expression levels of POLH and RAD18 were each determined by qPCR relative to the expression level at time 0. Half-life was calculated using a non-linear fit one-phase decay curve equation. The significance of the differences was calculated using Student’s t-test (one-tailed); Data are presented as mean ± SEM from three independent experiments. ns, non-significant; p > 0.05. (C) MCF-7 cells were transfected with TENT4A and/or PAXIP1 or non-targeting control siRNA for a final combined concentration of 100 nM. At 65 h post-transfection, the cells were UV-irradiated at 20 J/m2 and harvested 1 or 6 h post-irradiation. Whole-cell extracts were fractionated by SDS-PAGE followed by Western blot with indicated antibodies. Amounts are presented relative to those in extracts of unirradiated cells treated with siControl and the values are shown in the corresponding position of the blots.

Fig 3: Effect of TENT4A on binding and stability of TLS-related mRNAs. (A) FLAG-tagged TENT4A expressed in HEK293T cells was immunoprecipitated with an anti-FLAG antibody and RNA bound to it was analyzed by qPCR. The results are shown as fold-change relative to empty vector. The error bars indicate SEM (two independent experiments). (B–D) Representative gel images of RAD18, CYLD and POLH poly(A) tail are shown, each with the poly(A) tail densitometric trace underneath, using Image J. The position of TVN is indicated by an asterisk that predominantly recognizes A12, serving as an internal control. (E–H), TENT4A and/or TENT4B expression was knocked down in MCF-7 cells for 48 h, after which the cells were treated with 5 µg/mL Actinomycin D for up to 8 h. The expression levels of RAD18, CYLD, POLH and POLD3 genes were each determined by qPCR relative to the expression level at time 0. Half-life was calculated by using non-linear fit one-phase decay curve equation. The significance of the differences was calculated using Student’s t-test (one-tailed); Data are presented as mean ± SEM from three independent experiments. ns, non-significant; p > 0.05.

Fig 4: Effect of CYLD on RAD18, DTL, POLH and PCNA monoubiquitination. (A) MCF-7 cells were transfected with CYLD-targeted siRNA (siCYLD) or siControl. At 65 h post-transfection, the cells were irradiated at 20 J/m2 UV and harvested 1 or 6 h post-irradiation. (B) MCF-7 cells were transfected with siRNA against CYLD, RAD18 and DTL or their double or triple combinations. At 65 h post-transfection, the cells were UV-irradiated at 20 J/m2 and harvested 6 h post-irradiation. Whole-cell extracts were fractionated by SDS-PAGE and analyzed by Western blot with indicated antibodies. Protein levels were normalized to GAPDH and presented each relative to its level in unirradiated or UV-irradiated siControl-treated cells.

Fig 5: Pol ? associates with RNAP II and transcription-coupled classical nonhomologous end-joining proteins.A, left panel, nuclear extracts (NEs) (benzonase-treated) from FLAG-POLH HEK293 cells (either mock or Bleo treated) were immunoprecipitated (IP’d) with anti-FLAG Ab (lanes 5 and 6) or mouse IgG (lanes 3 and 4) and tested for the presence of associated proteins with specific Abs as indicated to the left of each row. One percent of NE was used as inputs (Inp) (lanes 1 and 2). IP experiment was repeated at least three times from separate batches of cells, and one representative figure is shown. Right panel, the quantitation of the IP’d bands after normalization with FLAG-Pol ? band intensity. Error bars represent ±SD of the mean. The increase in IP band intensity following Bleo treatment was significant at the level of ***p < 0.005, **p < 0.01, or *p < 0.05. B, left panel, detection of FLAG-Pol ?'s (anti-mouse FLAG Ab) association with PNKP and RNAP II (both anti-rabbit Abs) in mock- or Bleo-treated FLAG-POLH HEK293 cells by proximity ligation assay. Nuclei were counterstained with DAPI (blue). Right panel, average number of proximity ligation assay foci per cell was calculated from 25 randomly selected cells in each sample. Error bars represent ±SD of the mean. The data were significant at ***p < 0.005 between mock and Bleo treatment (n = 3). The scale bar represents 10 µm.