Mutagenesis, genotoxicity, and repair of 1-methyladenine, 3-alkylcytosines, 1-methylguanine and 3-methylthymine, in alkB Escherichia coli
Mutagenesis, genotoxicity, and repair of 1-methyladenine, 3-alkylcytosines, 1-methylguanine and 3-methylthymine, in alkB Escherichia coli
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DOI：
10.1073/pnas.0403489101
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发表时间：
2004-09-28
影响因子：
11.1
通讯作者：
Essigmann, JM
中科院分区：
文献类型：
--
作者：
Delaney, JC;Essigmann, JM
AlkB通过直接逆转碱基损伤来修复DNA中的1-烷基腺嘌呤和3-甲基胞嘧啶损伤。尽管已经进行了随机烷基化底物的修复研究，但尚未探索这些和相关的单独烷基化碱基的错误编码性质以及细胞内AlkB的诱变抑制。在这里，我们通过合成含有每种烷基化碱基的单链载体，然后通过大肠杆菌传递载体，解决了1-甲基脱氧腺苷（m1A）， 3-甲基脱氧胞苷（m3C）， 3-乙基脱氧胞苷（e3C）， 1-甲基脱氧鸟苷（m1G）和3-甲基脱氧胸苷（m3T）的错误编码潜力。在SOS-、alkb缺陷细胞中，m1A的致突变性仅为1%；而m3C和e3C的致突变性为30%，在SOS+细胞中达到70%。相反，当表达SOS聚合酶时，m1G和m3T在AlkB（-）细胞中的诱变性略有下降（m1G从80%降至66%，m3T从60%降至53%）。在野生型（AlkB(+)）细胞中，m1A、m3C和e3C的致突变性被消除，而m3T的致突变性仅部分降低。值得注意的是，m1G诱变性在AlkB+细胞中也被消除，这表明它是一种天然的AIkB底物。所有病变都阻断了alkb缺陷细胞的复制。在野生型细胞中，m1A、m3C和e3C阻断蛋白被完全去除；m1G阻断被部分去除，m3T阻断不受AlkB存在的影响。当诱导SOS聚合酶时，所有病变都表现出增强的旁路。这项工作提供了直接证据，表明AIkB通过生理上实际的低剂量1-烷基嘌呤和3-烷基嘧啶DNA损伤在体内抑制遗传毒性和诱变。
AlkB repairs 1-alkyladenine and 3-methylcytosine lesions in DNA by directly reversing the base damage. Although repair studies with randomly alkylated substrates have been performed, the miscoding nature of these and related individually alkylated bases and the suppression of mutagenesis by AlkB within cells have not yet been explored. Here, we address the miscoding potential of 1-methyldeoxyadenosine (m1A), 3-methyldeoxycytidine (m3C), 3-ethyldeoxycytidine (e3C), 1-methyldeoxyguanosine (m1G), and 3-methyldeoxythymidine (m3T) by synthesizing single-stranded vectors containing each alkylated base, followed by vector passage through Escherichia coli. In SOS-, AlkB-deficient cells, m1A was only 1% mutagenic; however, m3C and e3C were 30% mutagenic, rising to 70% in SOS+ cells. In contrast, the mutagenicity of m1G and m3T in AlkB(-) cells dropped slightly when SOS polymerases were expressed (m1G from 80% to 66% and m3T from 60% to 53%). Mutagenicity was abrogated for m1A, m3C, and e3C in wild-type (AlkB(+)) cells, whereas m3T mutagenicity was only partially reduced. Remarkably, m1G mutagenicity was also eliminated in AlkB+ cells, establishing it as a natural AIkB substrate. All lesions were blocks to replication in AlkB-deficient cells. The m1A, m3C, and e3C blockades were completely removed in wild-type cells; the m1G blockade was partially removed and that for m3T was unaffected by the presence of AlkB. All lesions demonstrated enhanced bypass when SOS polymerases were induced. This work provides direct evidence that AIkB suppresses both genotoxicity and mutagenesis by physiologically realistic low doses of 1-alkylpurine and 3-alkylpyrimidine DNA damage in vivo.