Touati E, Phillips D H, Quillardet P, Hofnung M
Unité de Programmation Moléculaire et de Toxicologie Génétique, CNRS URA 1444, Institut Pasteur, Paris, France.
Mutagenesis. 1993 Mar;8(2):149-54. doi: 10.1093/mutage/8.2.149.
The characterization of target nucleotides involved in the binding to DNA of 7-methoxy-2-nitro-naphtho[2,1-b]furan (R7000), a very potent genotoxic nitrofuran derivative, was investigated. Since R7000 undergoes metabolic activation prior to interacting with DNA, plasmids containing AT-rich and GC-rich sequences were devised and treated by R7000 in bacterial cells presenting nitroreductase activity. The nucleotide modifications to these homogeneous fragments that resulted from R7000 treatment were analyzed using the 'postlabeling' method. A preferential binding to the GC segment was demonstrated. Using a modification of the Maxam-Gilbert sequencing technique, it was demonstrated that activated R7000 creates alkali-labile phosphodiester bonds at the positions of guanines. In addition, the analysis of DNA replication-blocking properties of R7000 lesions was performed using avian myeloblastosis virus (AMV) reverse transcriptase as DNA polymerase. The termination of DNA replication occurred preferentially at the sites of guanine residues in the template strand, indicating that one nucleotide was inserted opposite a lesion. All these results indicate that guanine residues are the preferential sites of formation of R7000-DNA adducts.
研究了一种极具遗传毒性的硝基呋喃衍生物7-甲氧基-2-硝基萘并[2,1-b]呋喃（R7000）与DNA结合所涉及的靶核苷酸的特征。由于R7000在与DNA相互作用之前会经历代谢活化，因此设计了含有富含AT和富含GC序列的质粒，并在具有硝基还原酶活性的细菌细胞中用R7000进行处理。使用“后标记”方法分析了R7000处理导致的这些均匀片段的核苷酸修饰。结果表明R7000优先与GC片段结合。通过对Maxam-Gilbert测序技术进行改进后表明，活化的R7000在鸟嘌呤位置形成碱不稳定的磷酸二酯键。此外，使用禽成髓细胞瘤病毒（AMV）逆转录酶作为DNA聚合酶，对R7000损伤的DNA复制阻断特性进行了分析。DNA复制的终止优先发生在模板链中鸟嘌呤残基的位点，这表明在损伤位点对面插入了一个核苷酸。所有这些结果表明，鸟嘌呤残基是R7000-DNA加合物形成的优先位点。
