Waters T R, Swann P F
Department of Biochemistry and Molecular Biology, University College London, U.K.
Biochemistry. 1997 Mar 4;36(9):2501-6. doi: 10.1021/bi9621573.
It has been suggested that the cytotoxicity of 6-thioguanine depends upon (1) incorporation of 6-thioguanine into DNA, (2) methylation by S-adenosylmethionine (SAM) of the thio group to give S6-methylthioguanine, (3) miscoding during DNA replication to give [SMeG] x T base pairs, and (4) recognition of these base pairs by proteins of the postreplicative mismatch repair system. Here we have investigated systematically the ability of proteins present in human cell extracts to bind to DNA containing S6-methylthioguanine. We found that [SMeG] x T base mismatches were bound by the mismatch binding complex, hMutS alpha, and that the level of binding was dependent upon the base 5' to the S6-methylthioguanine in the order G > C = A > T. Extracts from cells that lack either hMSH2 (LoVo cells) or GTBP (HCT-15 cells), two components of the hMutS alpha complex, were unable to bind the [SMeG] x T base pair. We also found that hMutS alpha was able to bind to [SMeG] x C base pairs when the S6-methylthioguanine was in the sequence 5'-Cp[SMeG]. This suggests that miscoding by S6-methylthioguanine residues in DNA during DNA synthesis may not be an absolutely required step in the mechanism of cytotoxicity. Also, since CpG sequences are so important in gene regulation, this result may be of considerable significance.
有人提出，6-硫鸟嘌呤的细胞毒性取决于：（1）6-硫鸟嘌呤掺入DNA；（2）硫基团被S-腺苷甲硫氨酸（SAM）甲基化生成S6-甲基硫鸟嘌呤；（3）DNA复制过程中的错配编码生成[SMeG]×T碱基对；（4）复制后错配修复系统的蛋白质对这些碱基对的识别。在此，我们系统地研究了人细胞提取物中存在的蛋白质与含有S6-甲基硫鸟嘌呤的DNA结合的能力。我们发现，[SMeG]×T碱基错配被错配结合复合物hMutSα结合，且结合水平取决于S6-甲基硫鸟嘌呤5'端的碱基，顺序为G＞C = A＞T。缺乏hMutSα复合物的两个组分hMSH2（LoVo细胞）或GTBP（HCT-15细胞）的细胞提取物无法结合[SMeG]×T碱基对。我们还发现，当S6-甲基硫鸟嘌呤位于5'-Cp[SMeG]序列中时，hMutSα能够结合[SMeG]×C碱基对。这表明DNA合成过程中DNA中S6-甲基硫鸟嘌呤残基的错配编码可能不是细胞毒性机制中绝对必需的步骤。此外，由于CpG序列在基因调控中非常重要，这一结果可能具有相当重要的意义。
