Department of Genetics and Biochemistry, Clemson University, Room 049 Life Sciences Facility, 190 Collings Street, Clemson, SC 29634, USA.
Department of Genetics and Biochemistry, Clemson University, Room 049 Life Sciences Facility, 190 Collings Street, Clemson, SC 29634, USA.
DNA Repair (Amst). 2022 Nov;119:103408. doi: 10.1016/j.dnarep.2022.103408. Epub 2022 Sep 22.
5-Methylcytosine (mC) is an epigenetic mark that impacts transcription, development, diseases including cancer and aging. The demethylation process involves Tet-mediated stepwise oxidation of mC to hmC, fC, or caC, excision of fC or caC by thymine-DNA glycosylase (TDG), and subsequent base excision repair. Thymine-DNA glycosylase (TDG) belongs to uracil-DNA glycosylase (UDG) superfamily, which is a group of enzymes that are initially found to be responsible for excising the deaminated bases from DNA and generating apurinic/apyrimidinic (AP) sites. mC oxidative derivatives may also be generated from Fenton chemistry and γ-irradiation. In screening DNA glycosylase activity in UDG superfamily, we identified new activity on fC- and caC-containing DNA in family 2 MUG/TDG and family 6 HDG enzymes. Surprisingly, we found a glycosylase SMUG2 from bacterium Pedobacter heparinus (Phe), a subfamily of family 3 SMUG1 DNA glycosylase, displayed catalytic activity towards not only DNA containing uracil, but also fC and caC. Given the sequence and structural differences between the family 3 and other family enzymes, we investigated the catalytic mechanism using mutational, enzyme kinetics and molecular modeling approaches. Mutational analysis and kinetics measurements identified I62, N63 and F76 of motif 1, and H205 of motif 2 in Phe SMUG2 as important catalytic residues, of which H205 of motif 2 played a critical role in catalyzing the removal of fC and caC. A catalytic model underlying the roles of these residues was proposed. The structural and catalytic differences between Phe SMUG2 and human TDG were compared by molecular modeling and molecular dynamics simulations. This study expands our understanding of DNA glycosylase capacity in UDG superfamily and provides insights into the molecular mechanism of fC and caC excision in Phe SMUG2.
5- 甲基胞嘧啶（mC）是一种影响转录、发育、包括癌症和衰老在内的疾病的表观遗传标记。去甲基化过程涉及 Tet 介导的 mC 逐步氧化为 hmC、fC 或 caC、胸腺嘧啶 -DNA 糖基化酶（TDG）切除 fC 或 caC，以及随后的碱基切除修复。胸腺嘧啶 -DNA 糖基化酶（TDG）属于尿嘧啶 -DNA 糖基化酶（UDG）超家族，该酶家族最初被发现负责从 DNA 中切除脱氨碱基，并产生无嘌呤/无嘧啶（AP）位点。mC 氧化衍生物也可能来自芬顿化学和 γ 辐射。在筛选 UDG 超家族中的 DNA 糖基化酶活性时，我们在家族 2 MUG/TDG 和家族 6 HDG 酶中鉴定了含有 fC 和 caC 的 DNA 的新活性。令人惊讶的是，我们发现来自细菌 Pedobacter heparinus（Phe）的家族 3 SMUG1 DNA 糖基化酶的亚家族 SMUG2 糖苷酶不仅对含有尿嘧啶的 DNA 具有催化活性，而且对 fC 和 caC 也具有催化活性。鉴于家族 3 和其他家族酶之间的序列和结构差异，我们使用突变、酶动力学和分子建模方法研究了催化机制。突变分析和动力学测量确定了 Phe SMUG2 中 motif1 的 I62、N63 和 F76 以及 motif2 的 H205 是重要的催化残基，其中 motif2 的 H205 在催化 fC 和 caC 的去除中起关键作用。提出了一个催化模型，说明了这些残基的作用。通过分子建模和分子动力学模拟比较了 Phe SMUG2 和人 TDG 之间的结构和催化差异。这项研究扩展了我们对 UDG 超家族中 DNA 糖苷酶能力的理解，并为 Phe SMUG2 中 fC 和 caC 切除的分子机制提供了深入了解。
