Protection against methylation-induced cytotoxicity by DNA polymerase β-dependent long patch base excision repair
Protection against methylation-induced cytotoxicity by DNA polymerase β-dependent long patch base excision repair
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DOI：
10.1074/jbc.275.3.2211
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发表时间：
2000-01-21
影响因子：
4.8
通讯作者：
Wilson, SH
中科院分区：
文献类型：
--
作者：
Horton, JK;Prasad, R;Wilson, SH
使用基于质粒的含尿嘧啶的 DNA 底物，我们发现野生型小鼠成纤维细胞提取物的长补丁碱基切除修复 (BER) 活性被 DNA 聚合酶 β (β-pol) 的抗体部分抑制，这表明除了单核苷酸 BER 之外，β-pol 也参与长补丁 BER。在单核苷酸 BER 中，脱碱基位点中的脱氧核糖磷酸 (dRP) 被 β-pol 的裂解酶活性去除。甲氧胺 (MX) 可以与脱碱基位点的醛反应，使其难以抵抗 dRP 裂解酶机制的 β-消除步骤，从而阻断单核苷酸 BER。 MX 暴露使野生型小鼠胚胎成纤维细胞对甲磺酸甲酯 (MMS) 和甲基亚硝基脲的细胞毒性作用敏感，但对 β-pol 缺失小鼠胚胎成纤维细胞不敏感。无效细胞中 beta-pol 的表达恢复了 MX 调节 MMS 敏感性的能力。已知 beta-pol 无效细胞对 MMS 和甲基亚硝基脲高度敏感，并且在 MX 存在的情况下（即在单核苷酸 BER 被阻断的条件下），无效细胞仍然比野生型更敏感。这些数据与 β-pol 在长补丁 BER 中的作用一致，有助于保护细胞免受甲基化损伤引起的细胞毒性。
Using a plasmid-based uracil-containing DNA substrate, we found that the long patch base excision repair (BER) activity of a wild-type mouse fibroblast extract was partially inhibited by an antibody to DNA polymerase beta (beta-pol), This suggests that beta-pol participates in long patch BER, in addition to single-nucleotide BER. In single-nucleotide BER, the deoxyribose phosphate (dRP) in the abasic site is removed by the lyase activity of beta-pol. Methoxyamine (MX) can react with the aldehyde of an abasic site, making it refractory to the beta-elimination step of the dRP lyase mechanism, thus blocking single-nucleotide BER. MX exposure sensitizes wildtype, but not beta-pol null mouse embryonic fibroblasts, to the cytotoxic effects of methyl methanesulfonate (MMS) and methylnitrosourea. Expression of beta-pol in the null cells restores the ability of MX to modulate sensitivity to MMS. The beta-pol null cells are known to be hypersensitive to MMS and methylnitrosourea, and in the presence of MX (i.e. under conditions where single-nucleotide BER is blocked) the null cells are still considerably more sensitive than wild-type. The data are consistent with a role of beta-pol in long patch BER, which helps protect cells against methylation damage-induced cytotoxicity.