Extended Data Fig. 5: The poly(ADP-ribose) binding activity of XRCC1 promotes transcriptional recovery following oxidative damage.

a, Quantification of the levels of global transcription (EU immunofluorescence) shown in Fig. 4b. Data are means (±s.e.m.) of three independent experiments, and statistically significant differences were determined by two-way ANOVA with Sidak’s multiple comparisons test (p values are indicated). b, Representative images of the RNAPI (RPA194) and XRCC1 immunofluorescence in WT U2OS cells, XRCC1^−/− U2OS cells, and XRCC1^−/− U2OS cells transiently transfected with empty vector (EV) or expression construct encoding the indicated XRCC1 proteins, following mock-treatment or 2 h after treatment with 1 mM H₂O₂ for 20 min. Scale bars, 10 μm. c, Quantification of the RNAPI foci (RPA194) shown in (b). Data are means (±s.e.m.) of three independent experiments, and statistically significant differences were determined by two-way ANOVA with Sidak’s multiple comparisons test (p values are indicated). d, Immunoblot of XRCC1 in XRCC1^−/− U2OS cells stably expressing the indicated XRCC1 proteins. A representative blot from three independent experiments is shown. e, Immunoblot of RNAPII hyperphosphorylation in XRCC1^−/− U2OS cells stably expressing the indicated XRCC1 proteins, following mock-treatment or 2 h after treatment with 1 mM H₂O₂ for 20 min. A representative image from one of three independent experiments is shown. f and g, Quantification of DNA strand breaks measured by alkaline comet assays in XRCC1^−/− U2OS cells stably expressing the indicated XRCC1 proteins, following mock treatment (not treated; ‘NT’) or at the indicated times after treatment with either 50 μM H₂O₂ for 10 min on ice (conditions to measure SSB repair kinetic; panel f)or with 100 μM H₂O₂ for 20 min (refreshed every 10 min) in serum-free medium at 37 °C followed by recovery for 2 h (conditions, at which transcription recovery is perturbed in XRCC1^−/− cells; panel g). Data show individual tail moments (an arbitrary measure of DNA breaks) from 50 cells per sample for three independent experiments. For each sample, the fifty tail moments are stacked vertically and the three experiments are positioned side-by-side (exp1, exp2, exp3) with the tick mark centred on exp2. Statistical significance was determined by one way ANOVA with Sidak’s two tailed Student’s t-test (p values are indicated). h, Quantification of the levels of global transcription (EU immunofluorescence) shown in Fig. 4d. Data are means (±s.e.m.) of three independent experiments, and statistically significant differences were determined by two-way ANOVA with Tukey’s multiple comparisons test (p values are indicated). i, Immunoblot of APLF in the XRCC1^−/− U2OS cells stably expressing the indicated APLF proteins. Single validation of SDS–PAGE mobility shift. j, Immunoblot of PARP1 auto-ADP-ribosylation in an in vitro ADP-ribosylation assay. Human recombinant PARP1 (100 nM) was incubated for 30 min in the presence of ssDNA (100 nM), NAD⁺(2.5 μM) and with or without 1.5 μM of wild type or Zn-finger double mutant APLF (APLF^WT; APLF^ZFD respectively). A representative blot from one of three independent experiments is shown. k, Binding of full length XRCC1 and APLF, as well as their PAR binding mutants denoted as XRCC1^RK and APLF^ZFD respectively, to the indicated mock-ribosylated (-NAD⁺) or ribosylated (50 μM NAD⁺) histones. Data are means (±s.e.m.) of three independent experiments, and statistically significant differences were determined by two-way ANOVA with Tukey’s multiple comparisons test (p values are indicated).

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