Global and transcription-coupled repair of 8-oxoG is initiated by nucleotide excision repair proteins

Abstract

UV-DDB, consisting of subunits DDB1 and DDB2, recognizes UV-induced photoproducts during global genome nucleotide excision repair (GG-NER). We recently demonstrated a noncanonical role of UV-DDB in stimulating base excision repair (BER) which raised several questions about the timing of UV-DDB arrival at 8-oxoguanine (8-oxoG), and the dependency of UV-DDB on the recruitment of downstream BER and NER proteins. Using two different approaches to introduce 8-oxoG in cells, we show that DDB2 is recruited to 8-oxoG immediately after damage and colocalizes with 8-oxoG glycosylase (OGG1) at sites of repair. 8-oxoG removal and OGG1 recruitment is significantly reduced in the absence of DDB2. NER proteins, XPA and XPC, also accumulate at 8-oxoG. While XPC recruitment is dependent on DDB2, XPA recruitment is DDB2-independent and transcription-coupled. Finally, DDB2 accumulation at 8-oxoG induces local chromatin unfolding. We propose that DDB2-mediated chromatin decompaction facilitates the recruitment of downstream BER proteins to 8-oxoG lesions.

Fig. 1. DDB2 facilitates 8-oxoG repair and is rapidly recruited to sites of 8-oxoG within telomeric DNA.

a, b Immunofluorescence and quantification of 8-oxoG in cells transfected with control, DDB2 or OGG1 siRNA. c Schematic of the repair enzyme-based assay for 8-oxoG quantification in DNA. Genomic DNA containing 8-oxoG is treated with FPG to convert 8-oxoG to one nucleotide gaps. Treating with S1 nuclease converts the gaps to double stranded breaks (DSBs). The cleaved DNA is subjected to pulse field gel electrophoresis (PFGE) to track repair, as damaged DNA migrates faster than repaired DNA. d Quantification of 8-oxoG repair in U2OS cells transfected with control or DDB2 siRNA and treated with KBrO3. e Clonogenic cell survival curves in U2OS WT and DDB2 knockout (KO) cells treated with a range of concentrations of KBrO3. f Schematic of dye plus light treatment. Cells stably expressing FAP-TRF1 were treated with dye (100 nM, 15 min) plus light (660 nm, 10 min) to introduce 8-oxoG lesions at telomeres. g (left) Recruitment of DDB2-mCherry to 8-oxoG sites at telomeres in untreated, dye alone, light alone, and dye plus light treated cells. (right) Percentage telomeres colocalized with DDB2-mCherry. h Proximity ligation assay (PLA) for DDB2-mCherry and TRF1 in untreated cells and cells treated with dye (100 nM, 15 min) plus light (660 nm, 10 min). Data (a, b, d, g, h) represent mean ± SEM from two to three independent experiments. “n” represents the number of cells scored for each condition. Data (e) shows one representative experiment (performed in triplicate) from three independent experiments, mean ± SD. One-way ANOVA (Sidak multiple comparison test) (b, g), Student’s two-tailed Student’s t-test (h) and two-way ANOVA (Sidak multiple comparison test) (d, e) were performed for statistical analysis: *p < 0.05, **p < 0.01, ****p < 0.0001, ns Not significant. Scale: 5 µm. Source data are provided as a Source Data file. (See also Supplementary Fig. 1 and 2).

Fig. 2. DDB2 is required for efficient OGG1 recruitment to 8-oxoG.

a DDB2-mCherry and OGG1-GFP associate at 8-oxoG sites as shown by PLA after dye (100 nM, 15 min) plus light (660 nm, 10 min) treatment, over a period of 3 h. Antibodies against mCherry and GFP were used. b Quantification of PLA. c Accumulation of DDB2-mCherry at telomeric 8-oxoG 30 min post dye plus light treatment in U2OS-FAP-TRF1 cells transfected with control or OGG1 siRNA. d Percent telomeres colocalized with DDB2-mCherry as shown in (c). e Recruitment of OGG1-GFP at damaged telomeres in cells transfected with control or DDB2 siRNA. f Percent telomeres colocalized with OGG1-GFP as shown in (e). Data (a–f) represents mean ± SEM from two independent experiments. “n” represents the number of cells scored for each condition. One-way ANOVA (Sidak multiple comparison test): *p < 0.05, **p < 0.01, ****p < 0.0001. Scale: 5 µm. Source data are provided as a Source Data file. (See also Supplementary Fig. 3).

Fig. 3. DDB2 recruits XPC to telomeric 8-oxoG, while XPA recruitment is transcription-coupled and independent of DDB2.

a, c Representative images showing recruitment of GFP-XPC (a) or GFP-XPA (c) to 8-oxoG at telomeres after dye (100 nM, 15 min) plus light (660 nm, 10 min) treatment in U2OS WT and DDB2 KO cells, 30 min post treatment. b, d Percentage telomeres colocalized with GFP-XPC (b) or GFP-XPA (d) after treatment, over a period of 3 h. e, g Representative images of GFP-XPC (e) or GFP-XPA (g) accumulation at damaged telomeres 30 min after dye plus light treatment in cells pretreated with transcription inhibitors α-amanitin and THZ1. f, h Quantification of e (f) and g (h). i, j Colocalization of GFP-XPC with telomeres after dye plus light treatment in U2OS-FAP-TRF1 cells transfected with control or OGG1 siRNA. k, l Colocalization of GFP-XPA with telomeres after dye plus light treatment in U2OS-FAP-TRF1 cells transfected with control or OGG1 siRNA. Data (a–l) represents mean ± SEM from two independent experiments. “n” represents the number of cells scored for each condition. One-way ANOVA (Sidak multiple comparison test) (b, d, f, h) and Student’s two-tailed t-test (j, l): **p < 0.01; ***p < 0.001; ****p < 0.0001. Scale: 5 µm. Source data are provided as a Source Data file. (See also Supplementary Fig. 4).

Fig. 4. DDB2 binds sparse telomeric 8-oxoG independently of the DDB1-Cul4A-RBX1 E3 ligase.

a Representative images showing recruitment of DDB2-mCherry to telomeric 8-oxoG in cells transfected with control, DDB1 or Cul4A siRNA. b Quantification of a. c, e DDB2-mCherry and GFP-DDB1 (c) or DDB2-mCherry and GFP-Cul4A (e) accumulation at 8-oxoG sites after dye (100 nM, 15 min) plus light (660 nm, 10 min) treatment. d, f Quantification of c and e respectively. g Western blot for DDB2 in U2OS-FAP-TRF1 cells treated with UVC, potassium bromate (KBrO₃) or dye plus light at indicated doses. Independent experiments are represented by black circles. h Colocalization of DDB2-mCherry and GFP-Cul4A at damaged telomeres in U2OS-FAP-TRF1 cells transfected with control or OGG1 siRNA. i Quantification of h. Data (a–h) represents mean ± SEM from two independent experiments. ‘n’ represents the number of cells scored for each condition. One-way ANOVA (Sidak multiple comparison test) (b, i) was performed for statistical analysis: *p < 0.05, **p < 0.01, ***p < 0.001, ns Not significant. Scale: 5 µm. Source data are provided as a Source Data file. (See also Supplementary Fig. 5).

Fig. 5. DDB2 stimulates OGG1 recruitment to densely clustered 8-oxoG sites.

a Representative time-lapse pictures of OGG1-GFP and GFP-DDB2 accumulation at micro-irradiated (405 nm laser) sub-nuclear area, indicated by arrows, in the presence of 50 μM Ro 19-8022 photosensitizer. b Quantification of accumulation kinetics of OGG1-GFP and GFP-DDB2 (as shown in a). c Schematic overview of the molecular interactions of DDB2 within the CUL4A-DDB1-RBX1 E3 ubiquitin ligase complex (CRL), which is required for the successive molecular interactions by ubiquitylation and subsequent DNA repair. The activation of CRL is mediated by covalent attachment of the ubiquitin-like activator NEDD8 on CUL4A and its proteolytic removal leads to the deactivation of ubiquitin ligase function. These crucial events can be fine-tuned by specific inhibitors MLN4924 (NAE1i) and SB-58-SN29 (CSN5i), acting on NEDD8-activating enzyme NAE1 and CSN5, respectively. d Representative time-lapse pictures of OGG1-GFP accumulation at micro-irradiated (405 nm laser) sub-nuclear area, indicated by arrows, in the presence of 10 μM Ro 19-8022 photosensitizer. Cells were pretreated with DMSO (CTR), NEDDylation inhibitor (NAE1i) or de-NEDDylation inhibitor (CSN5i) for 1.5 h. e Quantification of accumulation kinetics of OGG1-GFP (as shown in d). f Immunoblot analysis for DDB2, CUL4A, CSA and AQR (loading control) in MRC-5 expressing OGG1-GFP. Cells were treated with inhibitors as indicated in d. Scale bars: 5 µm. Data were normalized to the background and represent mean ± SEM from three independent experiments. Total number of cells “n” measured are indicated in figure legends. ****P  <  0.001, analyzed by ROC curve analysis. Source data are provided as a Source Data file. (See also Supplementary Fig. 6).

Fig. 6. DDB2 mediates chromatin decompaction at sites of telomeric 8-oxoG.

a, b Distribution of the largest 20% telomeres in untreated and dye plus light treated U2OS-FAP-TRF1 WT and DDB2 KO cells. Cells were fixed 30 min post treatment. c, d Distribution of the largest 20% telomeres in untreated and dye plus light treated RPE-FAP-TRF1 WT and DDB2 KO cells. Cells were fixed 30 min post treatment. Source data are provided as a Source Data file. (See also Supplementary Fig. 7, Supplementary Movie 1 and 2).

Fig. 7. Unified working model: role of NER proteins in 8-oxoguanine repair.

Treatment of cells expressing FAP-TRF1 with dye (100 nM, 15 min) plus light (660 nm, 10 min) introduces 8-oxoG lesions at telomeres. In the DDB2-dependent repair pathway, DDB2 recognizes 8-oxoG lesions and facilitates chromatin relaxation through chromatin decompaction allowing the recruitment of XPC and OGG1 to the damage site. OGG1 recruitment facilitates the dissociation of DDB2. In the absence of downstream repair, DDB2 is retained longer at 8-oxoG sites requiring DDB1-Cul4A-RBX1 (CRL) mediated DDB2 dissociation. At actively transcribed regions, OGG1 can access the lesion independent of DDB2. 8-oxoG processing can lead to toxic BER intermediates that can act as a transcription block. Transcription-coupled repair (TCR) proteins, including XPA, participate in the repair of these BER intermediates. (See also Supplementary Movie 3).