Fig 2: Loss of SIRT6 leads to the retention of DDB2 on chromatin in response to UV irradiation. (A) Changes in DDB2 bound to chromatin in the absence of SIRT6 in response to UV irradiation were evaluated. Control and SIRT6-depleted HCA2-hTERT cells were irradiated with UVC (20 J/m2), harvested at the indicated time points and subjected to cellular protein fractionation, followed by western blot analysis of DDB2 in the chromatin fractions. (B) Depleting SIRT6 abolished the increased ubiquitination of DDB2 upon UV irradiation. Control and SIRT6-depleted HEK293 cells were transfected with a plasmid encoding DDB2-Flag and treated with MG132 at 10 μM. At 16 h post transfection, the cells were harvested for immunoprecipitation with an antibody against Flag, followed by western blot analysis with an antibody recognizing ubiquitin. (C) The ubiquitination level of the DDB2 2KR mutant was lower than that of DDB2 WT. HEK293 cells were transfected with a DDB2-Flag or DDB2-2KR-Flag plasmid. After 16 h, the cells were harvested for immunoprecipitation with an antibody against Flag, followed by western blot analysis with an antibody recognizing ubiquitin. (D) DDB2 2KR partially abolished the interaction between DDB2 and p97. HEK293 cells were transfected with a pControl, DDB2-GFP or DDB2-2KR-GFP plasmid. After 16 h, the cells were irradiated with UVC or left untreated, and at 5 min post UV irradiation, the cells were harvested for immunoprecipitation with an antibody against GFP and immunoblotted with the indicated antibodies. IPed, immunoprecipitated. (E) SIRT6 greatly diminished the interaction between DDB2 and p97. Control and SIRT6-depleted HEK293 cells were transfected with a plasmid encoding DDB2-Flag. After 16 h, the cells were harvested for immunoprecipitation with an antibody against Flag and immunoblotted with the indicated antibodies. IPed, immunoprecipitated. (F) Immunoblot analysis of changes in XPC hyperubiquitination in the absence or presence of SIRT6 was performed. Control and SIRT6-depleted HCA2-hTERT cells were UVC irradiated (20 J/m2) and harvested at the indicated time points, followed by western blot analysis.

Fig 3: P2RY6 deletion inhibited UVB‐induced DNA damage repair. (A) P2ry6 knockdown inhibited the phosphorylation of CHK1 and CHK2. The phosphorylation of CHK1 and CHK2 in normal (ShNC) and P2RY6 knockdown (ShP2RY6) HaCaT cells at 0.5 and 1 h post‐UVB (20 mJ/cm2) was detected by western blots. (B) P2ry6 knockdown inhibited the ubiquitination of XPC. The level of XPC ubiquitination in the extracts (0.15 mg) of ShNC and ShP2RY6 HaCaT cells at 0.5 and 1 h post‐UVB (20 mJ/cm2) was compared using western blot assays. (C, D) Immunoblot analysis of XPC, XPA, XPB and α‐tubulin in ShNC and ShP2RY6 ShNC and ShP2RY6 HaCaT cells at 0.5, 1, 6 and 12 h post‐UVB (20 mJ/cm2). All the experiments were repeated three times.

Fig 4: DOT1L/methylated H3K79 is involved in NER on UVB-induced DNA damage through interacting with XPC. a Melanomas from Queensland, Australia were irradiated with different doses of UVB. Cell viability was measured by MTT assay 24 h after UVB irradiation. b HPMs with stable shDOT1L or shControl were irradiated with different doses of UVB as indicated. Cell viability was measured by MTT assay 24 h after UVB irradiation. c WT or different DOT1L mutants as indicated was introduced into HPMs with stable shDOT1L expression. The resulting cells were irradiated with different doses of UVB as indicated. Cell viability was measured by MTT assay 24 h after UVB irradiation. d–f Melanomas from Queensland, Australia (d), HPMs with stable shDOT1L expression (e), or HPMs with stable shDOT1L expression and WT or different mutant DOT1L reintroduction (f) were irradiated with 100 J m−2 UVB and then collected at different time points as indicated after UVB irradiation. Genomic DNA was extracted and photoproducts were detected. g, h HPM cells treated with EPZ-5676 or vehicle control (g) or DOT1L depletion or shControl (h) were subjected to 100 J m−2 UVB under 5 µm micropore filter and were co-stained for CPD and XPC after 0.5 h. Scale bar, 20 μm. i The whole-cell extracts from HPM subjected to 100 J m−2 UVB were prepared for Co-IP assay to test the interaction of DOT1L with XPC. j The whole-cell extracts from HPM subjected to 100 J m−2 UVB were prepared for Co-IP assay to test the interaction of XPC with H3K79me2. k HPMs with DOT1L depletion or shControl were exposed to 100 J m−2 UVB. After 0.5 h, the chromatin fraction was prepared for western blot. *p < 0.05, **p < 0.01, ***p < 0.001, unpaired Student’s t-test. Error bars represent ± s.d.

Fig 5: hSSB2 depletion delays XPC recruitment, impairs RPA recruitment to UV damage sites and increases sensitivity to UVB exposure. (a) Representative images of HeLa cells probed for CPD (green, left panels) and RPA32 (red, middle panels) without UV exposure or 1 h and 3 h after UVC irradiation with a 5 µm filter. Cells were irradiated 48 h after negative control (si Control) and hSSB2 (si hSSB2) siRNAs transfection with UVC covered with 5 µm filter. DNA is stained with DAPI (blue, right panels). Average RPA32 fluorescent staining intensity at the CPD sites (± sem, n = 3) has been reported in graph (b). (c) Representative images of HeLa cells probed for CPD (green, left panels) and XPC (red, middle panels) without UV exposure or 15 min and 1 h after UVC irradiation. DNA is stained with DAPI (blue, right panels). Average XPC fluorescent staining intensity at the CPD sites (± sem) has been reported in graph (d). Scale bar represents 5 µm. (e,f) Measure of the average live cells (± sem, n = 3) 48 h after UVB exposure of HeLa cells transfected with negative control (si Control) or hSSB2 (si hSSB2) siRNAs (e) and U-2OS cells (f). Analysis was carried out with the Cytell imaging system 20 min after Hoechst and Propidium Iodide staining (****P-value < 0.0001 in a Student test).