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 涉及自发和遗传毒性诱导诱变的机制 有可能采用替代次级的DNA序列的 结构。 环境引起的自发突变和突变 致癌物可以导致人类​​的遗传疾病和癌症。 在这个 提案我们将使用大肠杆菌测试某些序列的假设 DNA可以形成二级结构底物，以促进特定 高频突变事件。 然后我们将测试是否 环境诱变剂增强了这些事件的频率。 在 单独的研究我们将应用此系统（一旦清楚地理解了该系统） 到整个动物系统。 诱变的二级结构底物 可以包括由反复重复DNA形成的十字形结构 序列； Z-DNA，在交替嘌呤/嘧啶的区域形成 顺序;并在DNA复制期间形成的错误配合结构滑倒 在直接重复序列中。 可以采用这种替代的DNA 构象在人类基因组中广泛发生。 我们已经发展了敏感 在体内分析和Z-DNA的体内测定，并计划确定 体内存在的替代构象存在之间的关系 DNA的形式及其相对诱变的潜力。 我们可以测试各种 使用我们的众多倒置的序列删除模型 可以在体内形成十字形的重复。 这些模型涉及直接 十字形臂的损失（可能是切除），十字形的“分辨率”（如 基因重组中的中间体），在DNA期间滑倒了 复制。 例如，我们将关联倒置的分数 在体内重复作为十字形，具有遗传不稳定性 倒重复。 通过这种分析，我们可以确定是否形成 十字形本身会导致突变或突变是a的结果 在复制过程中形成稳定的发夹臂。 我们还开发了一个 分析直接重复之间的删除频率和重复 可以形成定义的二级结构的17-26 bp DNA序列 基材。 使用此系统，我们应该能够识别分子 删除和重复涉及的中间体和机制 直接重复。 我们还将研究涉及的基因的作用， 定向自发的DNA中的重组，DNA修复和复制 诱变。 环境诱变剂对DNA频率的影响 定向突变不仅应用于识别促进的代理 这种类型的事件，但也应提供对机制的洞察力。