SPONTANEOUS AND GENOTOXICANT-INDUCED MUTATION MECHANISMS

这项研究的长期目标是了解 自发和遗传毒性诱发突变的机制 DNA序列有可能采用交替的次级 结构. 自然突变和环境诱发突变 致癌物质可导致人类遗传疾病和癌症。 在这 建议我们使用E。大肠杆菌来检验某些序列 可以形成二级结构底物， 高频率的突变事件。 然后我们将测试 环境诱变剂增加了这些事件的频率。 中 我们将单独研究这个系统（一旦它被清楚地理解） 一个完整的动物系统。 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，形成于嘌呤/嘧啶交替区域 序列;以及在DNA复制过程中形成的滑动错配结构 直接重复的序列。 能够采用这种交替的DNA 构象在人类基因组中广泛存在。 我们已经发展出敏感的 十字形和Z-DNA的体内试验，并计划确定 体内存在的交替构象之间的关系 DNA的形式及其相对的致突变潜力。 我们可以测试各种 序列定向缺失的模型，使用我们的许多系列的反向 在体内可以形成十字形的重复序列。 这些模型直接涉及 十字形臂的丢失（可能切除），十字形的“消退”（如 基因重组的中间体），以及DNA重组过程中的滑动错配 复制的 例如，我们将把反转的分数 重复序列在体内以十字形存在， 反向重复。 通过这种分析，我们可以确定， 十字形本身会导致突变，或者如果突变是 在复制过程中形成稳定的发夹臂。 我们还开发了一个 同向重复序列之间缺失和重复频率的测定 17-26 bp的DNA序列，可以形成确定的二级结构 印刷受体. 利用这个系统我们应该能够识别 中间体和机制参与删除和复制之间 直接重复。 我们还将研究基因的作用， 在自发的DNA定向中的重组、DNA修复和复制 诱变 环境诱变剂对DNA频率的影响 定向突变不仅可以用来识别促进 这种类型的事件，但也应该提供深入了解机制。

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.