Oxidation of DNA bases generates mutagenic and cytotoxic lesions that are implicated in cancer and other diseases. Oxidative base lesions, including 7,8-dihydro-8-oxoguanine, are typically removed through base excision repair. In addition, oxidized deoxynucleotides such as 8-oxo-dGTP are depleted by sanitizing enzymes to preclude DNA incorporation. While pathways that counter threats posed by 7,8-dihydro-8-oxoguanine are well characterized, mechanisms protecting against the major adenine oxidation product, 7,8-dihydro-8-oxoadenine (oxoA), are poorly understood. Human DNA polymerases incorporate dGTP or dCTP opposite oxoA, producing mispairs that can cause A→C or A→G mutations. oxoA also perturbs the activity of enzymes acting on DNA and causes interstrand crosslinks. To inform mechanisms for oxoA repair, we characterized oxoA excision by human thymine DNA glycosylase (TDG), an enzyme known to remove modified pyrimidines, including deaminated and oxidized forms of cytosine and 5-methylcystosine. Strikingly, TDG excises oxoA from G⋅oxoA, A⋅oxoA, or C⋅oxoA pairs much more rapidly than it acts on the established pyrimidine substrates, whereas it exhibits comparable activity for T⋅oxoA and pyrimidine substrates. The oxoA activity depends strongly on base pairing and is 370-fold higher for G⋅oxoA versus T⋅oxoA pairs. The intrinsically disordered regions of TDG contribute minimally to oxoA excision, whereas two conserved residues (N140 and N191) are catalytically essential. Escherichia coli mismatch-specific uracil DNA-glycosylase lacks significant oxoA activity, exhibiting excision rates 4 to 5 orders of magnitude below that of its ortholog, TDG. Our results reveal oxoA as an unexpectedly efficient purine substrate for TDG and underscore the large evolutionary divergence of TDG and mismatch-specific uracil DNA-glycosylase.

DNA 碱基的氧化会产生与癌症和其他疾病有关的诱变和细胞毒性损伤。氧化碱基损伤，包括 7,8-二氢-8-氧代鸟嘌呤，通常通过碱基切除修复去除。此外，氧化脱氧核苷酸如 8-oxo-dGTP 会被消毒酶耗尽，从而阻止 DNA 掺入。虽然对抗 7,8-dihydro-8-oxoguanine 构成的威胁的途径已得到很好的表征，但人们对防止主要腺嘌呤氧化产物 7,8-dihydro-8-oxoadenine (oxoA) 的机制知之甚少。人类 DNA 聚合酶在 oxoA 对面掺入 dGTP 或 dCTP，产生可导致 A→C 或 A→G 突变的错配。oxoA 还会扰乱作用于 DNA 的酶的活性并引起链间交联。告知 oxoA 修复机制，我们通过人胸腺嘧啶 DNA 糖基化酶 (TDG) 对 oxoA 切除进行了表征，TDG 是一种已知可去除修饰嘧啶的酶，包括脱氨和氧化形式的胞嘧啶和 5-甲基胱氨酸。引人注目的是，TDG 从 G⋅oxoA、A⋅oxoA 或 C⋅oxoA 对中切除 oxoA 的速度比它作用于已建立的嘧啶底物的速度快得多，而它对 T⋅oxoA 和嘧啶底物表现出相当的活性。oxoA 活性强烈依赖于碱基配对，G⋅oxoA 的活性高 370 倍 而它对 T⋅oxoA 和嘧啶底物表现出相当的活性。oxoA 活性强烈依赖于碱基配对，G⋅oxoA 的活性高 370 倍 而它对 T⋅oxoA 和嘧啶底物表现出相当的活性。oxoA 活性强烈依赖于碱基配对，G⋅oxoA 的活性高 370 倍与T⋅oxoA 对。TDG 本质上无序的区域对 oxoA 切除的贡献最小，而两个保守残基（N140 和 N191）是催化必需的。大肠杆菌错配特异性尿嘧啶 DNA 糖基化酶缺乏显着的 oxoA 活性，其切除率比其直系同源物 TDG 低 4 至 5 个数量级。我们的结果表明 oxoA 作为 TDG 的一种出乎意料的有效嘌呤底物，并强调了 TDG 和错配特异性尿嘧啶 DNA-糖基化酶的巨大进化差异。