SPONTANEOUS AND GENOTOXICANT-INDUCED MUTATION MECHANISMS

这项研究的长期目标是了解分子 自发致突变和遗传毒物致突变的机制 有可能采用备用二级结构的DNA序列 结构。自发突变和环境诱发突变 致癌物质会导致人类的遗传性疾病和癌症。在这 我们将使用大肠杆菌来测试某些序列的假设 可以形成二级结构底物，从而促进特定的 突变事件的频率很高。然后我们将测试一下 环境诱变剂增加了这些事件的发生频率。在一个 单独研究我们将应用这个系统(一旦它被清楚地理解) 整个动物系统。用于诱变的二级结构底物 可包括由反向重复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DNA复制过程中形成的滑移错配对结构 在直接重复序列中。可以采用这种替代方案的DNA 构象广泛存在于人类基因组中。我们已经变得敏感起来 体内十字形和Z-DNA检测，并计划确定 体内存在交替构象的关系 DNA的形式和它们的相对诱变潜力。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架的重复序列。这些模型涉及直接 十字臂丧失(可能被切除)，十字形‘溶解’(AS 基因重组的中间体)，并在DNA错配过程中滑落 复制。例如，我们将关联倒置的分数 在体内以十字形形式存在的重复序列具有遗传不稳定性 反向重复。从这样的分析中我们可以确定是否形成了 十字架本身会导致突变，或者如果突变是由 在复制过程中形成稳定的发夹臂。我们还开发了一种 直接重复序列间缺失和复制频率的测定 可以形成确定的二级结构的17-26个碱基对的DNA序列 底物。使用这个系统，我们应该能够识别分子 缺失和复制所涉及的中间体和机制 直接重复。我们还将调查参与的基因在其中的作用， 自发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 定向突变不仅应该用来识别促进 这种类型的事件，但也应该提供对机制的洞察。

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.