MECHANISMS AND REGULATION OF SPONTANEOUS MUTAGENESIS AND DNA REPAIR

本研究旨在从分子水平上阐明 自发突变的机制及其相关的DNA修复系统 以及它们是如何在大肠杆菌中受到调控的。我们的 方法是利用大量具有缺陷的突变大肠杆菌菌株进行遗传 一种特定的致突变或DNA修复途径。最近的突破 涉及到人类基因的发现，这些基因增加了患癌症的风险 患上各种癌症是这些信息的直接后果 在过去30年里收集的关于细菌诱变和修复的研究 小路。这些所谓的“癌症基因”具有家族特征。 容易患上各种癌症，其中一些已被证明会导致 错配修复和DNA复制保真度提高 其他致癌基因的突变频率。 我们希望探索诱变和保真度之间的关系 使用在某些方面有缺陷的突变株进行DNA复制 精确的DNA复制。我们最近分离出三个新的突变等位基因 在gyra基因座，DNA旋转酶A亚单位的结构基因。这是 DNA旋转酶参与DNA复制的第一个迹象 富达。我们希望通过找出是否 DNA旋转酶通过改变程度间接影响复制的准确性 或直接影响复制时的DNA聚合酶 叉子。我们还计划确定gyraA的突变谱 变种人等位基因。 人们普遍认为，由活性氧引起的氧化损伤 呼吸过程中氧气不完全还原产生的物种是 对导致基因突变的自发突变负有大部分责任 疾病和癌症。我们将测量突变频率在一个 几个已知的大肠杆菌突变体等位基因的厌氧室 在DNA复制保真度的某些方面有缺陷。通过比较这些 与有氧运动的价值，对氧化损伤程度的估计 对变种人活动的贡献是可以做出的。 我们正在使用抗突变等位基因，它可以减少自发突变。 频率，以表明氧化损伤是 野生型细胞的诱变。两个这样的大肠杆菌抗突变等位基因， RecA56和umuC122可降低需氧突变频率，但未显示 厌氧条件下测得的野生型值与野生型值的差异 密室。我们计划对这种依赖氧气的突变进行表征 野生型细胞中的途径。

This research is intended to elucidate at the molecular level the mechanisms of spontaneous mutagenesis and associated DNA repair systems and how they are regulated in the bacterium Escherichia coli. Our approach is genetic using the numerous mutant E. coli strains defective in a particular mutagenic or DNA repair pathway. Recent breakthroughs involving the discovery of human genes that confer an increased risk of getting various cancers are a direct consequence of the information gathered over the past 30 years on bacterial mutagenic and repair pathways. These so-called "cancer genes" were characterized in families prone to various cancers and some have been shown to cause defects in mismatch repair and DNA replication fidelity which lead to increased mutation frequencies in other genes causing cancers. We hope to explore the relationship between mutagenesis and fidelity of DNA replication using mutator strains that are defective in some aspect of accurate DNA replication. We recently isolated three new mutator alleles at the gyrA locus, the structural gene for DNA gyrase A subunit. This is the first indication that DNA gyrase is involved in DNA replication fidelity. We wish to characterize this involvement by finding out whether DNA gyrase affects replication accuracy indirectly by altering the degree of supercoiling or directly affects the DNA polymerase at the replication fork. We also plan to determine the mutational spectrum of the gyraA mutator alleles. It is widely believed that oxidative damage caused by reactive oxygen species generated by incomplete reduction of oxygen during respiration is responsible for much of spontaneous mutagenesis that leads to genetic diseases and cancers. We will measure mutation frequencies in an anaerobic chamber for several E. coli mutator alleles that are known to be defective in some aspect of DNA replication fidelity. By comparing these values with aerobic ones, an estimation of how much oxidative damage contributes to the mutator activity can be made. We are using antimutator alleles, which decrease spontaneous mutation frequencies, to show that oxidative damage is an important source of mutagenesis in wild-type cells. Two such E. coli antimutator alleles, recA56 and umuC122, decrease aerobic mutation frequencies but show no differences compared to wild-type values measured in the anaerobic chamber. We plan to characterize this oxygen-dependent mutagenesis pathway in wild-type cells.