SPONTANEOUS AND GENOTOXICANT INDUCED MUTATION MECHANISMS

自发和基因毒性诱导的突变机制

• 批准号: 3253817
• 负责人: RICHARD Rankin SINDEN
• 金额: $ 14.7万
• 依托单位: TEXAS A&M AGRILIFE RESEARCH
• 依托单位国家: 美国
• 财政年份: 1990
• 资助国家: 美国
• 起止时间: 1990-12-14 至 1995-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3253817
• 关键词: DNA; DNA repair; DNA replication; Escherichia coli; Z DNA; chemical stability; conformation; environmental toxicology; gene deletion mutation; gene duplication; gene mutation; genetic recombination; mutagens; nucleic acid repetitive sequence; nucleic acid sequence; nucleic acid structure; site directed mutagenesis; toxicant interaction

The long term goal of this research is to understand the molecular mechanisms involved in the spontaneous and genotoxicant induced mutagenesis of DNA sequences that have the potential to adopt alternate secondary structures. Spontaneous mutation and mutations induced by environmental carcinogens can lead to genetic disease and cancer in humans. In this proposal we will use E. coli to test the hypothesis that certain sequences of DNA can form secondary structure substrates which promote specific mutation events at a high frequency. We will then test whether environmental mutagens enhance the frequency of these events. In a separate study we will apply this system (once it is clearly understood) to a whole animal system. Secondary structure substrates for mutagenesis can include cruciform structures, formed from inverted repeated DNA sequences; Z-DNA, formed in regions of alternating purine/pyrimidine sequence; and slipped mis-paired structures, formed during DNA replication in direct repeated sequences. DNA that can adopt such alternate conformations occurs widely in human genomes. We have developed sensitive in vivo assays for cruciforms and Z-DNA and plan to determine the relationship between the in vivo existence of alternate conformational forms of DNA and their relative mutagenic potential. We can test various models for sequence directed deletion using our numerous series of inverted repeats that can form cruciforms in vivo. These models involve direct loss (possibly excision) of cruciform arms, 'resolution" of cruciforms (as intermediates in genetic recombination), and slipped mispairing during DNA replication. For example, we will correlate the fraction of inverted repeats existing as cruciforms in vivo with the genetic instability of the inverted repeat. From such analysis we can determine if the formation of cruciforms itself leads to mutation or if mutation is the result of a stable hairpin arm formed during replication. We have also developed an assay for the frequency of deletion and duplication between direct repeats of 17-26 bp DNA sequences that can form defined secondary structure substrates. Using this system we should be able to identify the molecular intermediates and mechanisms involved in deletion and duplication between direct repeats. We will also investigate the role of genes involved in, recombination, DNA repair, and replication in spontaneous DNA directed mutagenesis. The effect of environmental mutagens on the frequency of DNA directed mutation should not only serve to identify agents that facilitate this type of event, but should also provide insight into mechanisms.

这项研究的长期目标是了解分子 参与自发和遗传毒物诱导突变的机制 有可能采用替代二级的 DNA 序列 结构。 自发突变和环境诱导突变 致癌物质可导致人类遗传病和癌症。 在这个 建议我们将使用大肠杆菌来测试某些序列的假设 DNA 可以形成二级结构底物，促进特定的 突变事件发生频率较高。 然后我们将测试是否 环境诱变剂增加了这些事件的发生频率。 在一个 我们将应用这个系统进行单独的研究（一旦清楚地理解） 到整个动物系统。 用于诱变的二级结构底物 可以包括由反向重复 DNA 形成的十字形结构 序列； Z-DNA，在嘌呤/嘧啶交替区域形成 顺序;以及 DNA 复制过程中形成的错配结构 直接重复序列。 可以采用这种替代的DNA 构象广泛存在于人类基因组中。 我们已经发展出敏感的 十字形和 Z-DNA 的体内测定并计划确定 体内交替构象存在与否之间的关系 DNA 的形式及其相对诱变潜力。 我们可以测试各种 使用我们众多的倒排序列进行序列定向删除的模型 可以在体内形成十字形的重复序列。 这些模型涉及直接 十字形臂的损失（可能切除），十字形的“分辨率”（如 基因重组的中间体），并在DNA过程中发生错配 复制。 例如，我们将关联倒数的分数 由于遗传不稳定性，在体内以十字形重复存在 倒转重复。 从这样的分析我们可以确定是否形成 十字形本身会导致突变，或者如果突变是某种原因的结果 复制过程中形成稳定的发夹臂。 我们还开发了一个 测定直接重复之间的缺失和重复频率 17-26 bp DNA 序列，可形成明确的二级结构 基材。 使用这个系统我们应该能够识别分子 参与删除和复制的中间体和机制 直接重复。 我们还将研究涉及的基因的作用， 自发 DNA 引导下的重组、DNA 修复和复制 诱变。 环境诱变剂对DNA频率的影响 定向突变不仅应该用于识别促进 此类事件，但还应该提供对机制的深入了解。