Servius Hardler W, Drohat Alexander C
Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, Maryland, USA.
Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, Maryland, USA; Molecular and Structural Biology Program, University of Maryland Marlene and Stewart Greenebaum Comprehensive Cancer Center, Baltimore, Maryland, USA.
J Biol Chem. 2025 Jun 16;301(7):110363. doi: 10.1016/j.jbc.2025.110363.
Oxidation of DNA yields mutagenic and cytotoxic lesions that threaten genomic integrity, cause cancer and other diseases, and contribute to aging. Oxidative damage is countered by base excision repair, a pathway initiated by DNA glycosylases, which cleave bases through N-glycosyl bond hydrolysis. The major adenine oxidative lesion, 7,8-dihydro-8-oxoadenine (oxoA), is mutagenic in mammalian cells, but repair mechanisms are poorly understood. Thymine DNA glycosylase (TDG) removes T from mutagenic G⋅T mispairs arising through 5-methylcytosine deamination and mediates active DNA demethylation by excising 5-formylcytosine and 5-carboxylcytosine (caC). TDG excises oxoA from G⋅oxoA, A⋅oxoA, or C⋅oxoA pairs with remarkably high activity and from T⋅oxoA pairs with lower activity, comparable to that for established pyrimidine substrates. To further characterize TDG excision of oxoA, single-turnover experiments were collected with varying enzyme concentration, revealing vast differences in catalytic efficiency (k/K) among oxoA pairs, reflecting large variances in both substrate affinity (K) and maximal activity (k). TDG excision of oxoA depends strongly on the 3' base, as seen for excision of T from G⋅T pairs. Unlike MutY excision of adenine or TDG excision of caC, TDG excision of oxoA is not acid catalyzed, indicating that TDG stabilizes an anionic oxoA leaving group. A conserved TDG residue, H151, strongly promotes oxoA excision, whereas it antagonizes excision of T and uracil. The hydroxyl of Y152 catalyzes excision of oxoA and T, but not uracil, 5-formylcytosine, or caC, whereas its aromatic ring is essential for all substrates. Our results inform the catalytic requirements for enzymatic excision of oxoA from DNA.
DNA的氧化会产生诱变和细胞毒性损伤，这些损伤会威胁基因组完整性、引发癌症和其他疾病，并导致衰老。碱基切除修复可对抗氧化损伤，这是一条由DNA糖基化酶启动的途径，DNA糖基化酶通过N-糖苷键水解来切割碱基。主要的腺嘌呤氧化损伤产物7,8-二氢-8-氧代腺嘌呤（oxoA）在哺乳动物细胞中具有诱变作用，但对其修复机制了解甚少。胸腺嘧啶DNA糖基化酶（TDG）可从因5-甲基胞嘧啶脱氨产生的诱变G⋅T错配中去除T，并通过切除5-甲酰基胞嘧啶和5-羧基胞嘧啶（caC）介导活性DNA去甲基化。TDG以极高的活性从G⋅oxoA、A⋅oxoA或C⋅oxoA碱基对中切除oxoA，从T⋅oxoA碱基对中切除oxoA的活性较低，与既定的嘧啶底物相当。为了进一步表征TDG对oxoA 的切除作用，我们在不同酶浓度下进行了单周转实验，结果显示oxoA碱基对之间的催化效率（k/K）存在巨大差异，这反映了底物亲和力（K）和最大活性（k）的巨大差异。TDG对oxoA的切除强烈依赖于3'端碱基，这与从G⋅T碱基对中切除T的情况类似。与腺嘌呤的MutY切除或caC的TDG切除不同，oxoA的TDG切除不是酸催化的，这表明TDG可稳定阴离子oxoA离去基团。一个保守的TDG残基H151强烈促进oxoA的切除，而它会拮抗T和尿嘧啶的切除。Y152的羟基催化oxoA和T的切除，但不催化尿嘧啶、5-甲酰基胞嘧啶或caC的切除，而其芳香环对所有底物都至关重要。我们的结果为从DNA中酶促切除oxoA的催化要求提供了信息。
