Essigmann J M, Basu A K, Loechler E L
Ann Ist Super Sanita. 1989;25(1):155-61.
This work demonstrates the use of the tools of site-specific mutagenesis to study the mutagenic activity of two DNA adducts, O6-methylguanine and cis-thymine glycol. The former adduct is one of the methylated bases formed by carcinogenic and mutagenic alkylating agents. It was built into the single-stranded genome of bacteriophage M13 and replicated in Escherichia coli (E. coli). The mutation frequency of O6-methylguanine was 0.4% in physiologically normal cells. In cells in which the repair systems for O6-methylguanine were compromised by challenge with an alkylating agent, the mutation frequency rose to approximately 20%. DNA sequencing revealed that O6-methylguanine induced exclusively G----A transitions, which was most consistent with it pairing with thymine during DNA synthesis. The mutagenic effects also were investigated of cis-thymine glycol isomers, which are major, stable products of ionizing radiation and oxidative damage to DNA. By techniques similar to those employed for the study of O6-methylguanine mutagenesis, a single thymine glycol was situated in an M13 phage genome. The genome was replicated in E. coli that were physiologically normal, induced for SOS functions, or deficient in the nth gene product and, in all cases, the mutagenic processing of thymine glycol in vivo yielded mutant progeny phage at a frequency of 0.3-0.4%. All mutations occurred at the site that originally contained thymine glycol, and all were demonstrated by DNA sequencing to have resulted from targeted T----C transitions. These data suggest that thymine glycol pairs with guanine during replication.
这项工作展示了利用位点特异性诱变工具来研究两种DNA加合物——O6-甲基鸟嘌呤和顺式胸腺嘧啶二醇的诱变活性。前一种加合物是致癌和致突变烷基化剂形成的甲基化碱基之一。它被构建到噬菌体M13的单链基因组中，并在大肠杆菌中复制。在生理正常的细胞中，O6-甲基鸟嘌呤的突变频率为0.4%。在用烷基化剂攻击使O6-甲基鸟嘌呤修复系统受损的细胞中，突变频率上升到约20%。DNA测序显示，O6-甲基鸟嘌呤仅诱导G→A转换，这与它在DNA合成过程中与胸腺嘧啶配对最为一致。还研究了顺式胸腺嘧啶二醇异构体的诱变作用，它们是电离辐射和DNA氧化损伤的主要稳定产物。通过与用于研究O6-甲基鸟嘌呤诱变的技术类似的技术，将单个胸腺嘧啶二醇置于M13噬菌体基因组中。该基因组在生理正常、诱导SOS功能或nth基因产物缺陷的大肠杆菌中复制，在所有情况下，胸腺嘧啶二醇在体内的诱变处理产生突变后代噬菌体的频率为0.3-0.4%。所有突变都发生在最初含有胸腺嘧啶二醇的位点，并且通过DNA测序证明所有突变都是由靶向T→C转换导致的。这些数据表明胸腺嘧啶二醇在复制过程中与鸟嘌呤配对。
