Relationship between DNA methylation and mutational patterns induced by a sequence selective minor groove methylating agent
Relationship between DNA methylation and mutational patterns induced by a sequence selective minor groove methylating agent
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DOI：
10.1074/jbc.274.26.18327
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发表时间：
1999-06-25
影响因子：
4.8
通讯作者：
Fronza, G
中科院分区：
文献类型：
--
作者：
Kelly, JD;Inga, A;Fronza, G
Me-lex 是一种附加到中性 N-甲基吡咯甲酰胺基二肽上的甲基磺酸酯，其被合成以优先生成 N-3-甲基腺嘌呤 (3-MeA) 加合物，预计该加合物具有细胞毒性而不是致突变性 DNA 损伤。在本研究中，通过将加合位点转化为单链断裂并进行测序凝胶分析，在 p53 cDNA 中确定了 Me-lex 对 DNA 烷基化的序列特异性。为了确定 Me-lex 损伤的诱变和致死特性，在体外用 Me-lex 处理含有人类野生型 p53 cDNA 的酵母表达载体，并转染到含有由 p53 响应启动子调控的 ADE2 基因的酵母菌株中。结果表明：1）Me-lex引起的病变99%以上是3-MeA； 2)同时添加偏端霉素定量抑制所有小沟位点的甲基化； 3) Me-lex选择性甲基化位于或紧邻lex平衡结合位点的A； 4) 33个独立突变中除了6个之外的所有突变都是碱基对替换，其中大多数（17/33；52%）是AT靶向的； 5) AT --> TA 颠换是观察到的主要突变（13/33；39%）； 6) 38 个独立突变中的 13 个 (39%) 涉及包含位置 A(600-602) 的单个 lex 结合位点，其中 9 个发生在位置 602，这是真正的 Me-lex 突变热点（n = 9，p < 10(-6)，泊松正态分布）。提出了解释该位点突变事件的假设模型。目前的工作是关于 Me-lex 突变特性的第一份报告。我们的结果表明，3-MeA 不仅具有细胞毒性，而且还是一种诱变前病变，它以严格的序列依赖性方式发挥这种意想不到的特性。
Me-lex, a methyl sulfonate ester appended to a neutral N-methylpyrrolecarboxamide-based dipeptide, was synthesized to preferentially generate N-3-methyladenine (3-MeA) adducts which are expected to be cytotoxic rather than mutagenic DNA lesions. In the present study, the sequence specificity for DNA alkylation by Me-lex was determined in the p53 cDNA through the conversion of the adducted sites into single strand breaks and sequencing gel analysis. In order to establish the mutagenic and lethal properties of Me-lex lesions, a yeast expression vector harboring the human wild-type p53 cDNA was treated in vitro with Me-lex, and transfected into a yeast strain containing the ADE2 gene regulated by a p53-responsive promoter. The results showed that: 1) more than 99% of the lesions induced by Me-lex are 3-MeA; 2) the co-addition of distamycin quantitatively inhibited methylation at all minor groove sites; 3) Me-lex selectively methylated A's that are in, or immediately adjacent to, the lex equilibrium binding sites; 4) all but 6 of the 33 independent mutations were base pair substitutions, the majority of which (17/33; 52%) were AT-targeted; 5) AT --> TA transversions were the predominant mutations observed (13/33; 39%); 6) 13 out of 38 (39%) independent mutations involved a single lex-binding site encompassing positions A(600-602), and 9 occurred at position 602 which is a real Me-lex mutation hotspot (n = 9, p < 10(-6), Poisson's normal distribution). A hypothetical model for the interpretation of mutational events at this site is proposed. The present work is the first report on mutational properties of Me-lex. Our results suggest that 3-MeA is not only a cytotoxic but also a premutagenic lesion which exerts this unexpected property in a strict sequence-dependent manner.