Duran H L, Wani A A
Biochim Biophys Acta. 1987 Jan 28;908(1):60-9. doi: 10.1016/0167-4781(87)90022-4.
The mutagenicity of the DNA base O-alkylation adduct, O4-ethylthymine, specifically incorporated into the plasmid vector pUC8 at the unique SalI and HincII recognition sites, was studied in vivo. Escherichia coli, Micrococcus luteus and AMV DNA polymerases catalyze the incorporation of O4-ethylTMP against template adenine and guanine residues, resulting in DNA sequence alteration during subsequent replication in the host E. coli K-12 strain JM83. The greatest mutation frequency was observed with error-prone AMV DNA polymerase. High levels of cognate restriction endonuclease-resistant mutant plasmid isolates were obtained by gap replication repair in the presence of O4-ethylTTP. The yields of mutant isolates were dependent upon the relative concentration of the competing pyrimidine deoxynucleoside triphosphates, TTP and dCTP, in the misreplication reaction. Repair of incorporated O4-ethylTMP of plasmid DNA by in vitro treatment with specific alkyltransferase, prior to transformation in the host, effectively increases the mutagenic efficiency of the adduct. The results obtained are consistent with the high miscoding potential O4-ethylthymine observed in in vitro studies and its ability to base-pair with noncomplementary guanine residues in DNA.
对特异性掺入质粒载体pUC8的独特SalI和HincII识别位点的DNA碱基O-烷基化加合物O4-乙基胸腺嘧啶的诱变性进行了体内研究。大肠杆菌、藤黄微球菌和禽成髓细胞瘤病毒（AMV）DNA聚合酶催化O4-乙基胸苷三磷酸（O4-ethylTMP）掺入模板腺嘌呤和鸟嘌呤残基，导致在宿主大肠杆菌K-12菌株JM83随后的复制过程中发生DNA序列改变。在易出错的AMV DNA聚合酶作用下观察到最大的突变频率。在存在O4-乙基三磷酸胸腺嘧啶（O4-ethylTTP）的情况下，通过缺口复制修复获得了高水平的同源限制性内切酶抗性突变体质粒分离物。突变体分离物的产量取决于错配复制反应中竞争性嘧啶脱氧核苷三磷酸TTP和dCTP的相对浓度。在宿主中转化之前，用特异性烷基转移酶进行体外处理修复质粒DNA中掺入的O4-乙基胸腺嘧啶单磷酸（O4-ethylTMP），可有效提高加合物的诱变效率。所得结果与体外研究中观察到的高错配编码潜力的O4-乙基胸腺嘧啶及其与DNA中非互补鸟嘌呤残基碱基配对的能力一致。
