Fig 2: Polη and Rad18 act in the same pathway in response to replication stress and this requires Rad18 UBZ but not its ubiquitin ligase activity.(a) MRC5-V1 cells were transfected with siRNAs directed against polη (siPOLH) and/or Rad18 (siRAD18) mRNAs 48 h before exposure to 0.15 μM APH for 24 h. The percentage of anaphases with chromosome fragments was scored (mean±s.d. of three independent experiments, n=50 for each experiment). See Supplementary Fig. 10a for siRNA efficiency. (b) The percentage of anaphases with chromosome fragments was scored in polη-depleted WT or RAD18−/− HCT116 cells 24 h after 0.15 μM APH (mean±s.d. of three independent experiments, n=50 for each experiment). See Supplementary Fig. 10c for siRNA efficiency. (c,d) MRC5-V1 cell populations expressing GFP, GFP-Rad18WT, GFP-Rad18C28F or GFP-Rad18C207F were depleted for endogenous Rad18 using a siRNA directed against the 3′-UTR of the mRNA (siR18 3′-UTR). Cells were then treated for 24 h with 0.15 μM APH and anaphases were analysed in GFP-positive cells. Data are the mean±s.d. of four (c) or three (d) independent experiments. See Supplementary Fig. 10d for siRNA efficiency. (e) The impact of Rad18 or PIAS1 depletion on the chromosome fragments in anaphase was determined in XPV, polηWT and polηSUMO cells 24 h after 0.3 μM APH. Data are the mean±s.d. of three independent experiments (n=50 for each experiment). ns: not significant, *P<0.05, **P<0.01, ***P<0.001 (t-test). (f) Western blot showing the efficiency of the siRNAs used in e.

Fig 3: Polη is SUMOylated in a PIAS1-dependent manner.(a) 293FT cells were transfected with pcDNA-POLH and Flag-PIAS1 or Flag-PIAS4 and co-immunoprecipitations were performed using an anti-polη antibody. (b) Reversed immunoprecipitations were performed with an anti-Flag antibody. (c) 293FT cells were transfected with the indicated siRNAs 24 h before co-transfection of plasmids expressing polη and His-SUMO3. Denaturing Ni pull-down was carried out as described in Fig. 2. (d) WT or K163R (KR) polη was co-expressed in 293FT cells with His-SUMO3 and Flag or Flag-PIAS1 and cells were processed as in c. (e) XPV, polηWT and polηSUMO cells were transfected with non-targeting or PIAS1 siRNAs. Nascent DNA was pulse-labelled with EdU for 5 min and cells were pre-extracted and fixed. Biotin was conjugated to EdU by click chemistry in order to perform an in situ PLA between polη and EdU-biotin. EdU-biotin was then counterstained using a fluorescent secondary antibody. The distribution of the number of PLA spots per EdU-positive cells was assessed in two independent experiments. One representative experiment is shown in box-plot with 10–90 percentile whiskers (n>150, ns: not significant, ***P<0.001, Mann–Whitney test). (f) Western blot showing the efficiency of PIAS1 depletion. (g) Representative images of the PLA experiment quantified in (e) (magnification × 63).

Fig 4: K163R and SUMO polη retain full catalytic activity in vitro.(a) Schematic representations of the polη mutants used in the study. In addition to WT and K163R polη, a constitutively SUMOylated polη (SUMO) was constructed by inserting the sequence of SUMO2 in place of K163. A track of 7 glycines (G) was added before SUMO2 to confer flexibility. The C-terminal di-G motif of SUMO2 was mutated to alanines to prevent cleavage by the SENPs. Two constructs were generated: SUMOa contains the atg of SUMO2, whereas it was mutated to G in SUMOb. A catalytically inactive polη (polDEAD) was used as a negative control. (b) The indicated proteins were produced in rabbit reticulocytes and their catalytic activity was analysed by primer extension on a circular single-stranded undamaged template (representative gel). (c) Polymerase activity is shown as a percentage of the activity measured for WT polη in 10-min reaction (mean±s.d. of three independent experiments). (d) For analysis of TLS efficiency, equal amounts of the in vitro translated proteins were introduced in an XPV cell extract. Bypass of a single TT-CPD (TLS) was assessed as described in Methods. L-1: fragment elongated up to one nucleotide before the lesion.

Fig 5: RAD51 limits RF uncoupling in UV-irradiated XPV cells. (A) iPOND was performed in XPV and XPVpolη cells as described in Figure 1A with a 1-hour thymidine chase when indicated. Immunoblotting was done with the indicated antibodies. (B) DNA fiber assay was performed in XPV cells pre-treated or not with 20 μM B02 for 8 h. The distributions of replication track lengths are shown (n > 200, Kruskal–Wallis followed by Dunn's multiple comparisons test, ns: not significant; ****P < 0.0001). (C) TEM analysis was performed 30 min after UVC exposure (25 J m–2) in XPV cells pre-treated or not with 20 μM B02 for 8 h in two independent experiments. At least 50 RIs were analysed per condition in each experiment. Upper and lower left panels: distributions of all the ssDNA stretches measured at and behind the elongation point, respectively (dot plots with median and interquartile range, pool of two experiments). Upper and lower right panels: number of uncoupled and gapped forks, respectively, expressed as a percentage of all the forks analysed (mean ± SD of two independent experiments, t-test, **P < 0.01, ***P < 0.001). (D) XPV cells were depleted or not for PrimPol and pre-treated with 20 μM B02 for 8 h prior to UVC exposure (25 J m–2). TEM analysis was performed 30 min after irradiation. Values are the mean ± SD of two independent experiments with 50 RFs analysed per condition and per experiment. siPRIMPOL (siPP) efficiency was assessed by western blot.