RNF8 and Ubc13 depletion sensitizes cells to UV. (a) UV survival using HeLa cells transiently transfected with siRNA targeting RNF8, Ubc13, XPF, XPG, or control siRNA. The percentage of surviving cells is plotted against the applied UV-C dose (J/m²). RNF8 and Ubc13 depletion sensitizes cells to UV. (b) Gap-filling DNA repair synthesis (UDS) was measured by autoradiography. Wild-type primary fibroblasts (C5RO) were transfected with the indicated siRNA. 48 h after transfection, cells were pulse labeled with [³H]thymidine after exposure to 16 J/m² UV-C. UDS was quantified by counting autoradiographic grains in 50 nuclei, and the number of counts in control-transfected cells was set at 100%. RNF8 and Ubc13 have no effect on UDS. (c) HeLa cells stably expressing Ubc13-GFP or Mrc5 cells expressing RNF8-GFP were locally UV irradiated (60 J/m²) and immunostained for CPD. Both Ubc13 and RNF8 accumulate at LUD. (d) Localization of endogenous RNF8 before and after local UV exposure (60 J/m²). Immunostainings were performed using RNF8 and CPD antibodies. RNF8 accumulation was found up to 24 h after UV damage. (e) RNF8-GFP–expressing cells were exposed to LUD and directly after damage, were incubated for 2 h in medium containing EdU (BrdU analogue). Cells that were in S phase after the UV exposure stain positive for EdU visualized using Alexa Fluor 594. CPD was used as a damage marker. The graph adjacent to the images shows a similar (∼100%) colocalization of RNF8 with CPDs in S phase and non–S phase cells. Arrows indicate local damage sites. Error bars indicate SEM.

NER intermediates trigger RNF8 accumulation in a DNA damage– and ATR-dependent manner. (a) RNF8-GFP stably expressing NER-proficient (MRC5) or NER-deficient (XPC-negative cell lines XP20MA and XP4PA) were locally UV exposed (60 J/m²). 2 h later, damaged cells were immunostained for CPDs. The percentage of cells in which RNF8-GFP colocalizes with CPD after LUD is plotted in the graph. RNF8 does not accumulate in NER-deficient cells. (b) Mouse embryonic fibroblasts expressing both CPD and 6-4PP photolysases were stably transfected with GFP-Ub. 1.5 h after local UV exposure (60 J/m²), cells were cultured either in the dark (left) or were photoreactivated with visible light for 2 h (right). 6 h after initial UV damage, cells were fixed and immunostained for CPDs. After UV lesion removal, GFP-Ub does not accumulate at LUD. (c) ATR hypomorphic human cells (Seckel) and C5RO cells with a wild-type ATR status were locally UV irradiated (25 J/m²) and immunostained after 2 h with antibodies recognizing MDC1 or conjugated Ub (FKII). XPA was used as a damage marker, indicating that MDC1 and Ub accumulate in an ATR-dependent manner. (d) Stably expressing MDC1-GFP (left) or RNF8-GFP (right) cells were incubated for 90 min with ATM and DNA-PK inhibitors (KU-55933 and KU-57788) or with an equal volume of DMSO. Cells were exposed to IR (10 Gy) or UV (60 J/m²), and after 2 h, they were immunostained for γH2AX or CPD. Although the addition of these inhibitors clearly inhibits the IR-induced foci formation of MDC1, MDC1 still accumulates after LUD. (e) Stable MRC5 RNF8-GFP–expressing cells were locally exposed to 20 J/m² UV with or without a pretreatment for 1 h with 1 µg/ml aphidicolin. 1 h after UV damage, cells were fixed and immunostained for CPDs. A strong increase of RNF8 accumulation at the damaged DNA after aphidicolin pretreatment was observed. (f) The percentage of GFP-MDC1, XPB, RPA, γH2AX, GFP-Ub, and XPC-GFP colocalization with a damage marker (either CPD or XPA) at LUD (irradiated with 45 J/m²) is plotted for the different time points at 15 min, 1 h, 4 h, and 8 h after LUD. Although XPC and XPB are colocalizing in almost all cells 15 min after UV damage, the other factor colocalizes significantly with the used damage markers 1 h after LUD. (g) Cells stably expressing the XPC-GFP, GFP-Ub, and GFP-RPAp70 proteins were UV damaged using UV-C (266 nm) laser irradiation. GFP fluorescence intensities at the site of UV damage were measured by real time imaging until they reached a maximum. Assembly kinetic curves were derived from at least six cells for each protein. Relative fluorescence was normalized on 0 (before damage) and 100% (maximum level of accumulation). Arrows and arrowheads indicate local damage sites. Error bars indicate SEM.

RNF8 and Ubc13 knockdown inhibits the continuous UV-induced H2A ubiquitination. (a) GPF-Ub–expressing HeLa cells (left) or nontransfected HeLa cells were locally UV irradiated (60 J/m²) and stained with an antibody-recognizing Ub conjugated to H2A (uH2A). uH2A colocalizes with GFP-Ub (left) and with the damage markers XPC (middle) or XPA (right). (b) HeLa cells stably expressing GFP-Ub were locally UV irradiated (60 J/m²) and fixed after the indicated times. The local UV-irradiated area was visualized using CPD counterstaining. The GFP-Ub accumulation at LUD is visible up to 24 h. (c) The mobility of GFP-Ub in HeLa cells was determined by FRAP. 3.5 h after local UV exposure (60 J/m²), GFP-Ub–expressing HeLa cells were subject to live cell imaging. A nucleus containing a local Ub accumulation was photobleached inside the indicated white box for three iterations at 100% of laser. Pictures were acquired at the indicated times after photobleaching. 9 min after photobleaching, the GFP-Ub was almost completely redistributed. (d) GFP-Ub–expressing HeLa cells were transfected with the indicated siRNA oligonucleotides. 48 h after transfection, the cells were locally UV exposed with 60 J/m² and 3 h later were stained for CPD. Knockdown of RNF8 or Ubc13 results in an almost absence of GFP-Ub accumulation at LUD. The percentage of colocalization of GFP-Ub with CPD is plotted for the different siRNA transfections. (e) HeLa cells were similarly treated as in d and stained for ubiquitinated H2A together with XPA as a damage marker. siRNA-mediated depletion of RNF8 or Ubc13 caused a severely reduced accumulation of uH2A at LUD. The percentage of colocalization of uH2A with XPA after LUD is plotted in the graph for the different siRNA transfections. Arrows and arrowheads indicate local damage sites. Error bars indicate SEM.

MDC1 is essential for Ub accumulation at local damage. (a) U20S cells stably expressing wild-type, *RING (C403S), or *FHA (R42A) forms of GFP-tagged RNF8 were locally UV irradiated followed by CPD immunostaining. The FHA domain of RNF8 is essential for the observed RNF8 accumulation at the damaged area. (b) Wild-type or stably expressing shRNA targeting MDC1 U2OS cells were transiently transfected with GFP-Ub. 36 h after transfection, cells were locally UV exposed and were immunostained for CPDs 2 h later. The percentage of cells in which GFP-Ub colocalizes with CPD after LUD is plotted in the graph. MDC1 is essential for the Ub accumulation at the damaged DNA. (c) HeLa cells were transfected with control siRNA or siRNA targeting MDC1. 36 h after siRNA transfection, cells were UV irradiated (60J/m²) and stained for ubiquitinated H2A and XPA. The percentage of cells in which uH2A colocalizes with XPA after LUD is plotted in the graph. (d) Proliferation status of primary human C5RO fibroblasts was checked using the Ki-67 proliferation marker. Proliferating cells are positive for Ki-67 (right), whereas C5RO cells grown confluent for 7 d are negative for Ki-67 (left), indicating that all cells are in G0. (e) These nonproliferating human primary C5RO fibroblasts were locally UV exposed (60 J/m²), and after 2 h, they were immunostained for endogenous MDC1 and the essential NER protein XPA. MDC1 accumulates at the local damage in nonproliferating cells. Arrows and arrowheads indicate local damage sites. Error bars indicate SEM.

53BP1 and BRCA1 accumulate at LUD in an RNF8- and MDC1-dependent manner. (a and b) U2OS cells conditionally expressing doxycycline (Dox)-inducible shRNA targeting RNF8 were induced or not with doxycycline for 48 h, and cells stably expressing shRNA targeting MDC1 were locally UV damaged and stained for 53BP1 and CPD (a) or for BRCA1 and XPA (b). Both 53BP1 and Brca1 are recruited to the site of UV damage in an RNF8- and MDC1-dependent manner. The percentage of cells in which 53BP1 or BRCA1 colocalize with the used damage marker after LUD is plotted in the graphs. (c) Nonproliferating human primary fibroblasts (C5RO), as determined by a negative Ki-67 staining (not depicted), were stained for endogenous 53BP1 and conjugated Ub accumulation after local UV exposure (60 J/m²). CPD and XPA were used as a damage marker. (d) HeLa cells were transfected with siRNA targeting MDC1 or control siRNA. 36 h after transfection, cells were UV exposed (10 J/m²) and lysed at the indicated time points after UV damage. Chk1 phosphorylation status (Ser317) was analyzed using a phospho-specific antibody, and tubulin staining was used as a loading control. Arrow, phosphorylated Ser317 Chk1; asterisk, α-specific band. The relative amount of phosphorylated Chk1 is plotted in the graph (two blots were quantified, relative phospho-Chk1 levels were normalized, and the highest phospho-Chk1 level was set at 100%), indicating that MDC1 knockdown result is a reduction of the UV-induced Chk1 phosphorylation. (e) Model for the UV-induced DDR. UV-induced lesions are repaired by the core NER machinery, thereby generating single-stranded DNA repair intermediates, which subsequently activate ATR. This results in recruitment of MDC1 at the chromatin, which is essential for the RNF8 accumulation at the DNA damage. In concert with Ubc13, RNF8 ubiquitinates H2A, which triggers the recruitment of 53BP1 and BRCA1. From MDC1 recruitment onwards, the IR- and UV-induced DDR are similar. DSBs are recognized and bound by the Mre11–Rad50–Nbs1 (MRN) complex. Arrows and arrowheads indicate local damage sites. Error bars indicate SEM.