Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine , Baltimore, Maryland 21201, United States.
J Am Chem Soc. 2013 Oct 23;135(42):15813-22. doi: 10.1021/ja406444x. Epub 2013 Oct 7.
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.
5- 甲基胞嘧啶（mC）是一种影响转录、发育和基因组稳定性的表观遗传标记，异常的 DNA 甲基化导致衰老和癌症。活性 DNA 去甲基化涉及 mC 逐步氧化为 5-羟甲基胞嘧啶、5- 甲酰胞嘧啶（fC）和潜在的 5- 羧基胞嘧啶（caC）、胸腺嘧啶 DNA 糖基化酶（TDG）切除 fC 或 caC 以及通过后续碱基切除修复恢复胞嘧啶。在这里，我们研究了 TDG 切除 fC 和 caC 的机制。我们发现 5- 羧基-2'-脱氧胞苷离子化，pK(a) 值分别为 4.28（N3）和 2.45（羧基），证实了 caC 在生理 pH 下以单阴离子形式存在。计算不支持 fC 和 caC 碱基对采用 wobble 结构的建议，该结构被 TDG 识别。先前的研究表明，N-糖苷键水解遵循逐步（S(N)1）机制，并且 TDG 活性随嘧啶 N1 酸度增加，即靶碱基的离去基团质量增加。这里的计算表明，fC 和 caC 的中性互变异构体相对于其他 TDG 底物呈酸性，但 caC 单阴离子表现出较差的酸度，可能抵抗 TDG 切除。虽然 fC 活性与 pH 无关，但 caC 切除受酸催化，pH 曲线表明 caC 在酶-底物复合物中离子化，表观 pKa 为 5.8，可能在 N3。突变分析表明，Asn191 对于 caC 的切除是必需的，但对于 fC 活性是可有可无的，表明 N191 可能稳定 caC 的 N3-质子化形式，以促进酸催化，并表明 N191A-TDG 可能有助于研究细胞中的 DNA 去甲基化。
