WT DNMT3A potentiates the glycosylase activity of TDG but not MBD4. (A) Schematic overview of the DNA (de)methylation cascade in humans, including de novo methylation, spontaneous deamination of 5mC, and BER of G-T mismatches. (B) Experimental setup of glycosylase assay. A FAM-labeled double-stranded 32-bp oligo, containing a G-T mismatch, is incubated with TDG (AA 111-348). Functional TDG identifies and excises the mismatch. The subsequent hot alkaline treatment facilitates the oligo to break at the site of the excised base, resulting in an 18-bp product. More functional TDG results in increased levels of product detected on gel. (C-D) Addition of WT DNMT3A to the glycosylase assay stimulates TDG activity in a dose-dependent matter; represented on gel (C) and quantified relative to unstimulated TDG (D). (E-F) Glycosylase activity of MBD4 is not stimulated by the addition of WT DNMT3A to the glycosylase assay; represented on gel (E) and quantified relative to unstimulated MBD4 (F). 5caC, 5-carboxylcytosine; 5fC, 5-formylcytosine; 5hmC, 5-hydroxymethylcytosine; FAM, fluorescein amidite.