Ceccotti S, Aquilina G, Macpherson P, Yamada M, Karran P, Bignami M
Istituto Superiore di Sanita, Rome, Italy.
Curr Biol. 1996 Nov 1;6(11):1528-31. doi: 10.1016/s0960-9822(96)00758-0.
Human cell extracts perform an aberrant form of DNA synthesis on methylated plasmids [1], which represents processing of O6-methylguanine (O6-meG). Here, we show that extracts of colorectal carcinoma cells with defects in the mismatch repair proteins that normally correct replication errors do not carry out this synthesis. hMSH2-defective LoVo cell extracts (hMSH for human MutS homologue) performed O6-meG-dependent DNA synthesis only after the addition of the purified hMutS alpha mismatch recognition complex. Processing of O6-meG by mismatch correction requires PCNA and therefore probably DNA polymerase delta and/or epsilon. Mismatch repair-defective cells withstand O6-meG in their DNA [2], making them tolerant to methylating agents. Methylation-tolerant HeLaMR clones, with a mutator phenotype and a defect in either mismatch recognition or correction in vitro, also performed little O6-meG-dependent DNA synthesis. Assays of pairwise combinations of tolerant and colorectal carcinoma cell extracts identified hMLH1 as the missing mismatch repair function in a group of tolerant clones. The absence of processing by extracts of methylation-tolerant cells provides the first biochemical evidence that lethality of DNA O6-meG derives from its interaction with mismatch repair.
人细胞提取物对甲基化质粒进行异常形式的DNA合成[1]，这代表了O6-甲基鸟嘌呤（O6-meG）的处理过程。在此，我们表明，在通常纠正复制错误的错配修复蛋白存在缺陷的结肠癌细胞提取物中，这种合成过程无法进行。hMSH2缺陷的LoVo细胞提取物（hMSH表示人MutS同源物）仅在添加纯化的hMutSα错配识别复合物后才进行O6-meG依赖性DNA合成。通过错配修复处理O6-meG需要增殖细胞核抗原（PCNA），因此可能还需要DNA聚合酶δ和/或ε。错配修复缺陷的细胞能够耐受其DNA中的O6-meG[2]，这使得它们对甲基化剂具有耐受性。具有突变表型且在体外错配识别或校正存在缺陷的甲基化耐受HeLaMR克隆，也几乎不进行O6-meG依赖性DNA合成。对耐受细胞和结肠癌细胞提取物的两两组合分析表明，hMLH1是一组耐受克隆中缺失的错配修复功能。甲基化耐受细胞提取物无法进行处理，这首次提供了生化证据，证明DNA O6-meG的致死性源于其与错配修复的相互作用。
