5-methylcytosine (mC) is an epigenetic mark that impacts transcription, development, and genome stability, and aberrant DNA methylation contributes to aging and cancer. Active DNA demethylation involves stepwise oxidation of mC to 5-hydroxymethylcytosine, 5-formylcytosine (fC), and potentially 5-carboxylcytosine (caC), excision of fC or caC by thymine DNA glycosylase (TDG), and restoration of cytosine via follow-on base excision repair. Here, we investigate the mechanism for TDG excision of fC and caC. We find that 5-carboxyl-2′-deoxycytidine ionizes with pKa values of 4.28 (N3) and 2.45 (carboxyl), confirming that caC exists as a monoanion at physiological pH. Calculations do not support the proposal that G·fC and G·caC base pairs adopt a wobble structure that is recognized by TDG. Previous studies show that N-glycosidic bond hydrolysis follows a stepwise (SN1) mechanism, and that TDG activity increases with pyrimidine N1 acidity, i.e., leaving-group quality of the target base. Calculations here show that fC and the neutral tautomers of caC are acidic relative to other TDG substrates, but the caC monoanion exhibits poor acidity and likely resists TDG excision. While fC activity is independent of pH, caC excision is acid catalyzed, and the pH profile indicates that caC ionizes in the enzyme-substrate complex with an apparent pKa of 5.8, likely at N3. Mutational analysis reveals that Asn191 is essential for excision of caC but dispensable for fC activity, indicating that N191 may stabilize N3-protonated forms of caC to facilitate acid catalysis, and suggesting that N191A-TDG could potentially be useful for studying DNA demethylation in cells.

Base excision repair (BER) is essential for maintaining genetic integrity and for active DNA demethylation, a central element of epigenetic gene regulation. A key player in both processes is thymine DNA glycosylase (TDG), which excises modified forms of 5‐methylcytosine (mC). TDG excises thymine from mutagenic G/T mispairs arising from mC deamination. TDG also excises 5‐formylcytosine (fC) and 5‐carboxylcytosine (caC), oxidized forms of mC generated by Tet enzymes. TDG is essential for embryonic development, reflecting a crucial function in regulating gene expression that likely involves a key step in active DNA demethylation. We used structural, biochemical, and cell‐based methods to understand how TDG attains specificity for excising select forms of modified mC (i.e., T, fC, and caC). We identified a TDG variant that retains fC activity, but lacks detectible caC activity, indicating a fundamental difference in the excision mechanism for these related bases. We used this variant to study the role of TDG in active DNA demethylation in human cells. The results indicate that TDG excision of fC can account for findings that caC is depleted in cells expressing TDG, consistent with the fact that fC is a precursor for Tet‐mediated formation of caC. The results suggest that TDG excision of fC could be a predominant element in a pathway for active DNA demethylation. Supported by the NIH (R01‐GM072711).