Fig 2: Chronic elevated glucose inhibits repair of CEdG and cyclobutane pyrimidine dimers by nucleotide excision repair.(A) CEdG in genomic DNA was quantified by LC–MS/MS in 293T WT, CSB, and XPC cells maintained in LG or HG (n = 3; two-way ANOVA with Tukey’s multiple comparisons). (B) 293T WT, XPC, and CSB cells grown in LG or HG were co-transfected with luciferase reporter plasmid containing 695 CEdG/105 dG (pM1-luc; firefly luciferase) and an undamaged transfection control plasmid (pRL-CMV; Renilla luciferase). 24 h post-transfection luminescence was quantified as a marker of repair (two-way ANOVA with Tukey’s multiple comparisons). (C, D) CEdG repair was also measured in (C) XPG+ (XPG complemented) or XP3BR-SV (XPG mutant) human fibroblasts (366 CEdG/105 dG; two-way ANOVA) and (D) MCF10A breast epithelial cells (366 CEdG/105 dG; unpaired t test). (E) Repair of UV-damaged pM1-luc containing 1.33 μM cyclobutane pyrimidine dimer in 293T WT, XPC, and CSB knockout cells grown in LG or HG (two-way ANOVA with Dunnett’s comparison to WT LG). ns, not significant (P > 0.05), *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig 3: XPC modulates TDG-binding dynamics to DNA in vivo. (A) Survival probability of an individual TDG-Halo molecule being bound as a function of time. Individual molecules were tracked using single-molecule imaging over consecutive frames and binned by their total length in cells cotransfected with SNAP-NLS or SNAP-XPC or transduced with lentiviruses containing a control NT shRNA or shRNA against XPC (shXPC). Bold lines represent the mean probability; shaded regions denote the 95% confidence interval. (B) Quantification of TDG DNA-bound residence time using a two-exponential decay model after correction for photobleaching using Halo-H2B (see Supplemental Fig. S7K). Error bars depict the standard deviation of the model fit.

Fig 4: Global DNA methylation is inversely correlated with XPC expression independent of DNA repair activity. Relative global DNA methylation was assayed by 5mC-specific ELISA using genomic DNA from XPC knockdown H9 human ESCs (A) and HDFs overexpressing wild-type (WT) or DNA repair-deficient (W690S) human XPC (B). Relative global DNA methylation in HDFs was also assayed by 5mC dot blot (C) and MeDIP enrichment (D). Methylene blue (MB) staining was used to control for total DNA transferred to the membrane. (E,F) TDG cleavage activity of a 5′-labeled 37mer double-stranded oligonucleotide DNA (0.2 µM) in the presence or absence of decreasing amounts of wild-type or W690S mutant XPC (0.2–0.4 µM). The 37mer dsDNA contains either a 5fC:G (E) or 5caC:G (F) base pair as substrate for TDG. Uncleaved intact 37mer (U) and its cleaved product (C) were separated on a denaturing PAGE gel. Representative gels are shown. The intensity of the cleaved product indicates the efficiency of base excision by TDG and is calculated in the graphs as the percentage of total labeled substrate [=C/(U + C)]. Error bars represent the standard deviation. n = 3. (***) P < 0.001; (**) P < 0.01; (*) P < 0.05, calculated by two-way ANOVA.

Fig 5: TDG is enriched at enhancers and promoters genome-wide and colocalizes with the XPC complex subunit RAD23B. (A) The number of ChIP-seq peaks identified using MACS2 for each data set. Approximately 93% of biotin–TDG (Neri et al. 2015) ChIP-seq peaks overlap with RAD23B in mouse ESCs. (B) Heat map of TDG- and RAD23B-bound regions over a ±5-kb window around the TSSs of genes. Genes in each heat map are sorted according to descending levels of TDG ChIP-seq signal. (C) Integrative Genomics Viewer (IGV)-computed ChIP-seq tracks are plotted as (number of reads) × [1,000,000/(total read count)] for pluripotency genes Nanog and Pou5f1. Mock and normal IgG were used as specificity controls for the TDG and RAD23B ChIP, respectively.