MECHANISMS AND REGULATION OF SPONTANEOUS MUTAGENESIS AND DNA REPAIR

这项研究旨在从分子水平上阐明 自发突变的机制和相关的DNA修复系统 以及它们在大肠杆菌中是如何被调节的。 我们 方法是使用大量的突变体E.大肠杆菌菌株， 一种特殊的诱变或DNA修复途径。 最近的突破 包括发现人类基因， 患上各种癌症的直接后果 在过去30年里收集的关于细菌诱变和修复的 途径。 这些所谓的“癌症基因”在家庭中表现出特征， 易患各种癌症，有些已被证明会导致缺陷， 错配修复和DNA复制保真度，这导致增加 其他致癌基因的突变频率。 我们希望探讨突变和保真度之间的关系， 使用在某些方面有缺陷的突变菌株进行DNA复制， 精确的DNA复制 我们最近分离出了三个新的增变基因等位基因 在gyrA位点，DNA促旋酶A亚基的结构基因。 这是 DNA旋转酶参与DNA复制的第一个迹象 忠诚 我们希望通过查明这种参与是否 DNA旋转酶通过改变DNA旋转酶的活性， 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， 增变基因等位基因 人们普遍认为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质， 负责许多自发突变，导致遗传 疾病和癌症。 我们将测量突变频率， 厌氧室中培养数株E.大肠杆菌突变体等位基因， 在DNA复制保真度的某些方面有缺陷。 通过比较这些 值与有氧值，估计有多少氧化损伤 可以对增变因子活性做出贡献。 我们使用的是抗突变等位基因， 频率，以表明氧化损伤是一个重要的来源， 在野生型细胞中的诱变。 两个这样的E。大肠杆菌抗突变体等位基因， recA56和umuC122降低了需氧突变频率，但没有显示 与在厌氧条件下测量的野生型值相比的差异 室。 我们计划表征这种氧依赖性诱变 在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.