Fig 2: RNF111 is required for XPC release.(a) Relative XPC–GFP accumulation at sites of LUD in control and RNF111-depleted cells. GFP fluorescence intensity at UV-C laser induced LUD was measured over time using live-cell confocal imaging and quantified to predamage intensity set at 1 at t=0 (n>15 cells per sample, measured in two independent experiments; error bars are the mean±2 × s.e.m.). (b) Top panel: representative immunofluorescence pictures of co-localization of XPC with CPD at LUD in WT and Rnf111−/−MEFs at the indicated time points after UV irradiation (60 J m−2) are shown. Scale bars, 5 μm. Lower panel: quantification of the XPC co-localization with CPD (n>50 cells with LUD were analysed per sample in three independent experiments; error bars are the mean±s.d.). (c) Top panel: FRAP analysis of XPC–GFP in mock treated or global UV-irradiated (10 J m−2) XP4PA (XPC deficient) cells, on transfection with the indicated siRNA's. XPC–GFP was bleached in a small strip within the nucleus and fluorescence recovery was measured over 45 s and normalized to prebleach intensity (n=40; from two independent experiments error bars are the mean±2 × s.e.m.). The immobilized fraction (%)=1−((average fluorescence intensity UV-irradiated cells−the first data point after bleaching)/(average fluorescence intensity unchallenged cells−the first data point after bleaching)), is plotted in the lower panel. The immobilized fraction was calculated over the last 10 s. (d) Inverse FRAP (iFRAP) analysis of XPC–GFP at LUD. XP4PA cells stably expressing XPC–GFP were transfected with the indicated siRNA's. Seventy-two hours after transfection, cells were locally exposed to a 266-nm UV-C laser. After the accumulation plateau was reached (5 min after exposure) the undamaged part of the nucleus was continuously bleached and fluorescence in the damaged area was monitored. Fluorescence was normalized to prebleach intensity (n>15 cells per sample, measured in two independent experiments; error bars are the mean±s.e.m.).

Fig 3: RNF111 is required for binding of XPG and XPF/ERCC1 to the NER complex.(a) U2OS cells expressing CPD–photolyase–mCherry were transfected with the indicated siRNA's 3 days before the immunofluorescence experiment. Cells were local UV irradiated (60 J m−2) and immunostained for the indicated proteins 30 min later. The percentage of co-localization with the damage marker CPD–photolyase–mCherry at LUD is plotted in the graph (n>50 cells containing a LUD were scored in at least three independent experiments; error bars are the mean±s.d.). (b) The immobilized fraction of XPB–GFP, GFP–XPA, XPG–GFP and ERCC1–GFP as determined by FRAP analysis in mock or UV-treated (10 J m−2) cells on transfection with the indicated siRNA's (n>32 cells from at least two independent experiments; error bars are the mean±2 × s.e.m.). (c) Cells stably expressing XPG–GFP and ERCC1–GFP transfected with the indicated siRNA's were locally irradiated using a 266-nm UV-C laser. GFP fluorescence intensity at LUD was monitored for 6 min, with 10 s intervals and normalized to predamage values. (n=24 cells from three independent experiments; error bars are the mean±s.e.m.).

Fig 4: RNF111 is necessary for efficient GG-NER.(a) Representative pictures of unscheduled DNA synthesis (UDS) of the indicated MEFs, determined by 5-ethynyl-2′-deoxyuridine (EdU) incorporation over 3 h after UV irradiation (16 J m−2). Scale bar, 25 μm. (b) Quantification of UDS levels in MEFs, as determined by EdU incorporation over a time period of 3 or 9 h after UV irradiation (16 J m−2). UDS levels in WT MEFs were set at 100% (n>100 cells per sample, in at least two independent experiment; error bars are the mean±s.d.). (c) 6-4PP removal assayed by immunofluorescence, using a 6-4PP specific antibody. The indicated MEFs were UV-irradiated (10 J m−2) and allowed to repair 6-4PPs for the indicated time points. Relative fluorescence directly after UV exposure was set at 100%. (n>70 cells, three independent experiments; error bars are the mean±s.d.).

Fig 5: UBXN7 inhibits RNF111 auto-ubiquitylation in a UAS-dependent manner. a UBXN7 inhibits RNF111 auto-ubiquitylation in cells. Lysates (input) from U2OS UBXN7-KO clone #1 transiently transfected with Flag-RNF111-WT or Flag-RNF111-CA together with HA-tagged empty vector ( −), UBXN7-WT (WT), UBXN7-∆UAS (∆UAS), or UBXN7-UAS (UAS) were immunoprecipitated with the UB pan selector resin and subsequently analyzed by western blotting. b UBXN7 inhibits RNF111 auto-ubiquitylation in vitro. Recombinant RNF111-Cter-CA or WT were incubated at 37 °C for 1 h in the presence of UBE1 (E1), UBE2D2 (E2), ubiquitin (UB), and recombinant UBXN7-WT (WT), UBXN7-∆UAS (∆UAS), or UBXN7-UAS (UAS). RNF111 ubiquitylation was revealed by western blotting using an antibody targeting the RNF111 C-terminal domain. c The UAS domain inhibits free ubiquitin chains formation induced by RNF111-RING domain in vitro. Recombinant RNF111-RING was incubated at 37 °C for 10 min in the presence of UBE1 (E1), UBE2D2 (E2), ubiquitin (UB) and 1 to 10 molar excess of recombinant UBXN7-UAS. Ubiquitin chains were revealed by western blotting using an anti-UB antibody