Pol β gap filling, DNA ligation and substrate-product channeling during base excision repair opposite oxidized 5-methylcytosine modifications.
Pol β gap filling, DNA ligation and substrate-product channeling during base excision repair opposite oxidized 5-methylcytosine modifications.
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DOI：
10.1016/j.dnarep.2020.102945
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发表时间：
2020-11
期刊：
影响因子：
3.8
通讯作者：
Çağlayan M
中科院分区：
文献类型：
--
作者：
Çağlayan M
CpG 岛胞嘧啶上的 DNA 甲基化生成 5-甲基胞嘧啶 (5mC)，5mC 的进一步修饰可产生氧化变体 5-羟甲基 (5hmC)、5-甲酰基 (5fC) 和 5-羧基 (5caC)。碱基切除修复 (BER) 对于基因组维护和修饰胞嘧啶产物的主动 DNA 去甲基化至关重要，并且涉及从 DNA 聚合酶 (pol) β 的核苷酸插入到后续连接步骤的底物产物通道。在这里，我们报道，与 pol β 错配插入产物（dCTP、dATP 和 dTTP）相比，当 5mC 氧化修饰存在于体外模板位置相反时，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。 Pol β K280A 突变会扰乱活性位点内这些碱基修饰的稳定性，从而阻碍 BER 连接酶。此外，模拟pol β错配插入产物的切口修复中间体，即与3'-预插入的dGMP或dTMP相对模板5hmC、5fC或5caC，可以有效地连接，而预插入的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了解 pol β 与不同胞嘧啶碱基形式相反的插入倾向、DNA 连接酶 I 和 DNA 连接酶 III/XRCC1 复合物在含有缺口和切口损伤的修复中间体背景下的连接特性，以及在模板链中涉及氧化 5mC 碱基修饰的情况下 BER 最后步骤中底物通道的效率和保真度。
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.
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