DNA methylation on cytosine in CpG islands generates 5-methylcytosine (5mC), and further modification of 5mC can result in the oxidized variants 5-hydroxymethyl (5hmC), 5-formyl (5fC), and 5-carboxy (5caC). Base excision repair (BER) is crucial for both genome maintenance and active DNA demethylation of modified cytosine products and involves substrate-product channeling from nucleotide insertion by DNA polymerase (pol) β to the subsequent ligation step. Here, we report that, in contrast to the pol β mismatch insertion products (dCTP, dATP, and dTTP), the nicked products after pol β dGTP insertion can be ligated by DNA ligase I or DNA ligase III/XRCC1 complex when a 5mC oxidation modification is present opposite in the template position in vitro. A Pol β K280A mutation, which perturbates the stabilization of these base modifications within the active site, hinders the BER ligases. Moreover, the nicked repair intermediates that mimic pol β mismatch insertion products, i.e., with 3′-preinserted dGMP or dTMP opposite templating 5hmC, 5fC or 5caC, can be efficiently ligated, whereas preinserted 3′-dAMP or dCMP mismatches result in failed ligation reactions. These findings herein contribute to our understanding of the insertion tendencies of pol β opposite different cytosine base forms, the ligation properties of DNA ligase I and DNA ligase III/XRCC1 complex in the context of gapped and nicked damage-containing repair intermediates, and the efficiency and fidelity of substrate channeling during the final steps of BER in situations involving oxidative 5mC base modifications in the template strand.

CpG 岛胞嘧啶上的 DNA 甲基化生成 5-甲基胞嘧啶 (5mC)，5mC 的进一步修饰可导致氧化变体 5-羟甲基 (5hmC)、5-甲酰基 (5fC) 和 5-羧基 (5caC)。碱基切除修复 (BER) 对基因组维护和修饰的胞嘧啶产物的活性 DNA 去甲基化至关重要，并且涉及从 DNA 聚合酶 (pol) β 插入核苷酸到后续连接步骤的底物-产物通道。在这里，我们报告说，与 pol β 错配插入产物（dCTP、dATP 和 dTTP）相比，pol β dGTP 插入后的切口产物可以通过 DNA 连接酶 I 或 DNA 连接酶 III/XRCC1 复合物连接，当 5mC 氧化时修饰在体外模板位置相反存在. Pol β K280A 突变会扰乱活性位点内这些碱基修饰的稳定性，阻碍了 BER 连接酶。此外，模拟 pol β 错配插入产物的切口修复中间体，即.，使用 3'-预插入 dGMP 或 dTMP 与模板 5hmC、5fC 或 5caC 相反，可以有效连接，而预插入 3'-dAMP 或 dCMP 错配会导致连接反应失败。本文的这些发现有助于我们理解 pol β 与不同胞嘧啶碱基形式相反的插入趋势、DNA 连接酶 I 和 DNA 连接酶 III/XRCC1 复合物在含缺口和缺口的修复中间体的背景下的连接特性，以及效率在涉及模板链中氧化 5mC 碱基修饰的情况下，BER 最终步骤中底物通道的保真度。