Divergent mechanisms for enzymatic excision of 5-formylcytosine and 5-carboxylcytosine from DNA

Abstract

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 pK(a) 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 (S(N)1) mechanism, and that TDG activity increases with pyrimidine N1 acidity, that is, 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.

Figure 1

Pathway for active DNA demethylation involving TET enzymes and TDG-initiated BER. Details and abbreviations are provided in the main text.

Figure 2

Ionization of N3 and the carboxyl of 5-ca-dC. (a) UV absorbance spectra for 5-ca-dC at pH 0.75 thru 6.0; the absorbance is essentially unchanged for pH 6.0 to 8.0. Isosbestic points are observed for a subset of the scans at 257 nm, 267 nm, and 303 nm; absorbance at these wavelengths depends on only one of the two ionizations. (b) The pH dependence of A₃₀₃ was fitted to eq. 1, giving pK_a = 4.28 ± 0.02. (c) pH dependence of A₂₆₇ was fitted to eq. 1, giving pK_a = 2.49 ± 0.09. (d) pH dependence of A₂₈₆ was fitted to eq. 2, giving pK_a¹ = 4.25 ± 0.04 and pK_a² = 2.41 ± 0.09. (e) Ionization of 5-ca-dC as indicated by our findings.

Figure 3

Shown are the calculated relative stabilities of the amino and imino tautomers of fC and the caC anion, in the gas phase and water. The values are reported as the difference in free energy (∆∆G, kcal mol^-1) with respect to the most stable species (∆∆G = 0).

Figure 4

Acidity for N1 and other sites for pyrimidines in the gas phase and bulk solution. The calculated acidities are reported as the free energy (∆G, kcal mol^-1) required for deprotonation in the gas phase and in water (parenthetical values).

Figure 5

Resonance stabilization of the fC anion.

Figure 6

pH dependence of TDG activity for G·fC (∆) and G·caC (O) substrates. Fitting the G·caC data to a model for ionization of an essential protonated group (eq. 4; dotted line) gives an apparent pK_a = 5.80 ± 0.03 and a limiting k_obs of 4.4 ± 0.1 min^-1, but fitting is poor for pH > 7. Fitting to a model with an essential protonated group and a second, nonessential group (eq. 5, solid line) gives apparent pK_a values of pK_a¹ = 5.75 ± 0.03 and pK_a² = 8.2 ± 0.7, a limiting k_obs of 4.5 ± 0.1 min^-1, and a rate enhancement factor (1 + α) of 4.5 (i.e., the fold increase in k_obs resulting from deprotonation of the second group).

Figure 7

Previously reported structure of TDG (catalytic domain) with a 5-carboxyl-dC analog (non-cleavable) flipped into the active site (PDBID: 3UOB). Hydrogen bonds (dashed lines) and van der Waals contacts (dotted lines, d ≤ 3.7 Å) are shown. Similar interactions are observed for a structure of the N140A-TDG variant (catalytic domain) bound to DNA containing an A·caC mismatch (PDBID: 3UO7).

Figure 8

Effect of the N191A mutation on substrate binding and base excision. (a) Electrophoretic glycosylase assay shows that N191A-TDG has normal G·fC activity, markedly reduced G·T activity, and no significant G·caC activity, even for a reaction time of 3 h. Reactions were performed with 0.20 μM enzyme, 0.10 μM substrate, and were quenched after 5 min or 3 h (as indicated). (b) An electrophoretic mobility shift assay (EMSA) shows that N191A-TDG binds G·caC DNA with high affinity, similar to that observed for N140A-TDG. Previous studies show that N140A-TDG binds G·caC and other substrates with essentially the same affinity and/or enzyme-substrate contacts as native TDG,^, but it does not excise caC (under the EMSA conditions here). DNA (10 nM) was incubated with enzyme (5-50 nM) at 22 °C for 30 min prior to running the EMSA.

Figure 9

Potential mechanisms for TDG excision of fC and caC as suggested by the results here and previous structural studies. (a) Excision of fC does not require a large role for the side chain of any active-site residue examined here, consistent with the robust N1 acidity of fC (Fig. 4). Nevertheless, it seems likely that fC excision involves electrostatic catalysis, where backbone amide groups (139, 140, 152; Fig. 7) stabilize the departing fC anion. (b) In contrast to the results for fC, our findings indicate that excision of the caC anion, the predominant species at neutral pH, requires acid catalysis. Findings that N191 is essential for caC excision but dispensable for fC activity suggest that N191is needed to stabilize an N3-protonated form of caC, which could be the zwitterion 2 or the neutral imino tautomer 4. It seems likely that excision of caC also involves electrostatic catalysis, via the same three backbone amide groups.