Comparison of mutagenesis by O6-methyl- and O6-ethylguanine and O4-methylthymine in Escherichia coli using double-stranded and gapped plasmids.
Comparison of mutagenesis by O6-methyl- and O6-ethylguanine and O4-methylthymine in Escherichia coli using double-stranded and gapped plasmids.
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使用双链和缺口质粒比较大肠杆菌中 O6-甲基-、O6-乙基鸟嘌呤和 O4-甲基胸腺嘧啶的诱变。
DOI：
10.1093/carcin/19.3.457
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发表时间：
1998
期刊：
影响因子：
4.7
通讯作者：
R. Moschel
中科院分区：
文献类型：
--
作者：
G. Pauly;Stephen H. Hughes;R. Moschel
为了比较 O6-甲基鸟嘌呤 (m6G)、O4-甲基胸腺嘧啶 (m4T) 和 O6-乙基鸟嘌呤 (e6G) 的诱变，并评估它们在大肠杆菌中的遗传毒性，构建了在 lacZ' 基因的起始密码子 (ATG) 中含有单个 m6G、e6G 或 m4T 的双链缺口质粒。修饰碱基诱导的突变通过含有 X-gal 的培养基上 lacZ' 活性的丧失进行评分，导致形成白色或扇形（突变体）而不是蓝色（非突变体）集落。使用含有修饰碱基的缺口质粒进行的基因毒性实验表明，m4T 产生的细菌菌落数量比 m6G 或 e6G 多。当掺入双链质粒时，在修复能力（w.t.）大肠杆菌中，m4T 比 m6G（6%）或 e6G（11%）更具诱变性（45% 突变菌落）。在缺口质粒中，m4T 在两种 w.t. 中产生 99% 的突变菌落（如之前在 e6G 中观察到的那样）。大肠杆菌或缺乏 O6-烷基鸟嘌呤-DNA 烷基转移酶以及甲基化定向错配修复 (ada(-)-ogt(-)-mutS[-]）的大肠杆菌。缺口质粒中的 m6G 在 w.t. 中产生了 62% 的突变菌落。大肠杆菌，但在 ada(-)-ogt(-)-mutS(-) 菌株中，这一百分比增加至 94%。在双链质粒中，m4T 和 m6G 在 ada(-)-ogt(-)-mutS(-) 菌株中产生非常相似的突变体和非突变体集落分布。这些观察结果得出这样的结论：m6G 和 m4T 在 w.t. 中的致突变性差异。与 m4T 相比，大肠杆菌是通过烷基转移酶和错配修复机制优先修复 m6G 的结果，并且没有反映它们各自编码效率的差异或它们对 DNA 合成的固有阻碍性（如 e6G 所观察到的）。结论是烷基转移酶和错配修复的组合是双链质粒中 m6G 与 m4T 相比明显遗传毒性的主要原因。
To compare mutagenesis by O6-methylguanine (m6G), O4-methylthymine (m4T) and O6-ethylguanine (e6G), and assess their genotoxicity in Escherichia coli, double-stranded and gapped plasmids were constructed containing a single m6G, e6G or m4T in the initiation codon (ATG) of a lacZ' gene. Modified base induced mutations were scored by the loss of lacZ' activity on X-gal-containing media resulting in formation of white or sectored (mutant) rather than blue (non-mutant) colonies. Genotoxicity experiments with gapped plasmids containing the modified bases indicated that m4T produced a greater number of bacterial colonies than m6G or e6G. m4T was more mutagenic (45% mutant colonies) than m6G (6%) or e6G (11%) in repair competent (w.t.) E. coli when incorporated in double-stranded plasmids. In gapped plasmids, m4T produced 99% mutant colonies (as was observed previously for e6G) in both w.t. E. coli or E. coli deficient in both O6-alkylguanine-DNA alkyltransferases as well as methylation-directed mismatch repair (ada(-)-ogt(-)-mutS[-]). m6G in gapped plasmids produced 62% mutant colonies in w.t. E. coli, but this percentage increased to 94% in the ada(-)-ogt(-)-mutS(-) strain. In double-stranded plasmids both m4T and m6G produced very similar distributions of mutant and non-mutant colonies in the ada(-)-ogt(-)-mutS(-) strain. These observations led to the conclusion that differences in the mutagenicity of m6G and m4T in w.t. E. coli were a result of preferential repair of m6G compared to m4T by alkyltransferase and mismatch repair mechanisms, and did not reflect differences in their respective coding efficiency or their inherent obstructiveness to DNA synthesis as was observed with e6G. The combination of alkyltransferase and mismatch repair was concluded to be primarily responsible for the apparent genotoxicity of m6G compared to m4T in double-stranded plasmids.
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