Fig 1: TDG interacts with R-loops in mESCs. (a) Heatmap representations on a window of ±3 kb around the center of TDG ChIP-seq peaks. Reads were parsed based on the overlap of TDG peaks with MapR peaks and ordered based on TDG ChIP-Seq signal. (b) Pie charts comparing the genome-wide distribution of all TDG peaks (n = 71,772) to those that overlap with R-loops (n = 21,602). Promoter-TSS: +/− 1 kb from TSS; Extended promoter: −10 kb to −1 kb from TSS. (c) Percent of TDG peaks at R-loops. (d) Box plot of the percentage of total 5fC/5caC at TDG/R-loop promoters in TDG knockdown (shTDG) versus control mESCs. **p < 0.01. (e) TDG is associated with cellular R-loops. Lysates from TDG-expressing HeLa cells were immunoprecipitated with the S9.6 antibody and co-precipitated TDG was visualized by Western blot. Where indicated, the S9.6 antibody was treated with competitor (comp.) prior to the immunoprecipitation step. Quantification of precipitated TDG from three independent DRIP experiments is shown below. Data are mean ± S.D. normalized to the input (10%).
Fig 2: TDG is catalytically active on DNA/RNA hybrids. (a) Single-turnover kinetics of TDG (1000 nM) acting on the indicated G•U containing substrate (100 nM). (b) Single-turnover kinetics of TDG or TDGA145G (1000 nM) acting on the indicated G•T containing substrate (100 nM). The data for H1-T is obscured by that for H2-T. The data for H1-T and H2-T were fitted to a linear equation. (c) Single-turnover kinetics of TDG (1000 nM) acting on the indicated 5fC/5caC containing substrate (100 nM). All reactions contained 100 mM NaCl, 2.5 mM MgCl2, and 10 mM Tris-HCl (pH 7.5) and were carried out at 30 °C. Data are mean ± S.D. (n = 3).
Fig 3: TDG functions on authentic R-loop structures. (a) DNA used in this work. The sequence is derived from the TCF21 gene promoter (−29 to +36 relative to the TSS). The blue line indicates the position of the endogenous R-loop, whereas the red line indicates the position of the R-loop used herein. Individual CpG dinucleotides are numbered and the position of dU incorporation is indicated by blue text. See Figure S6 for sequence details. (b) Schematic illustration of the TCF21-derived substrates. Black and red colors denote DNA and RNA, respectively. (c) Single-turnover kinetics of TDG (1000 nM) acting on the indicated substrate (100 nM). Reaction conditions are identical to those described in Figure 3. Data are mean ± S.D. (n = 3).
Fig 4: Processing of symmetrically modified R-loops by TDG. (a,b) Single-turnover kinetics of TDG (1000 nM) acting on either (a) symGU or (b) symGU-R (100 nM). Substrates were labelled on the top strand with Cy5 (red) and on the bottom strand with Cy3 (green). Reaction conditions are identical to those described in Figure 3. Data are mean ± S.D. (n = 3). (c) Representative native PAGE gel showing the formation of DSBs. The indicated substrate (100 nM) was treated with TDG (200 nM) and/or APE1 (20 nM) in a buffer containing 100 mM NaCl, 2.5 mM MgCl2, and 10 mM Tris-HCl (pH 7.5) for 30 minutes at 30 °C. Asterisks indicated the nicked duplex. The diamond indicates a DSB control product generated via the treatment of symGU with restriction enzyme Hpy188I.
Fig 5: TDG binds to DNA/RNA hybrid duplexes. (a) Sequences used in this study. Black and red colors denote DNA and RNA, respectively. See also Table S1. (b,c) Representative EMSA data and corresponding saturation plots for binding of TDG to either D1/H1 (b) or D2/H2 (c). For each reaction, the indicated substrate (5 nM) was incubated with TDG (0 – 300 nM) in a buffer containing 100 mM NaCl, 2.5 mM MgCl2, 10 mM Tris-HCl (pH 7.5), and 5% glycerol for 30 minutes at 30 °C. Data are mean ± S.D. (n = 3).
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