EXPRESSION OF BACTERIAL GENES FOR THE REPAIR OF ALKYLATION DAMAGE

所提出的实验旨在加深我们对 负责自发突变的基本分子机制。 他们特别关注SOS突变子的潜在机制 在E.和胞嘧啶甲基转移酶在 在S-腺苷甲硫氨酸条件下催化胞嘧啶脱氨 (SAM)限制. 在E.大肠杆菌细胞表达 SOS功能产生于通常有助于 自发突变谱。的强大功能和便利性， CDCE/hifiPCR技术获得突变谱， 由蒂利和他的同事开发，将被利用来进行一个 SOS突变基因遗传基础的系统研究 表型。例如，不同的方式组成的表达， 将检查SOS系统，以确定它们是否产生等效SOS 将研究增变子表型和复制子的作用。的 在自发突变中对dinB功能的要求 将检验SOS突变子效应。错配修复的影响 和其他功能的SOS增变表型将被调查。的 dinB基因产物与SOS突变体表型有关， 可能在决定 移码突变发生在重复碱基的运行中。dinB基因将 进行克隆和测序DinB蛋白的生化作用将是 研究及其与SOS增变因子效应和自发 确定诱变。 最近已经表明，如果SAM是限制性的，DNA胞嘧啶 甲基转移酶可以通过甲基转移酶催化胞嘧啶的脱氨基作用。 形成具有饱和C5-C6键的中间体。而且 最近有人提出，这种自发的机制 诱变可能有助于癌症进展。为了解 进一步研究这种现象的生理相关性， 限制SAM水平对发生自发突变的频率， Dcm甲基化位点。将使用两个独立的 接近。消除两种潜在尿嘧啶修复的影响 SAM限制条件下ung和vsr突变的机制 将被调查。最后将进行实验以确定 哺乳动物DNA胞嘧啶甲基转移酶在E. 大肠杆菌引起CpG位点自发突变频率增加 在SAM限制条件下。拟议的研究包括 可能有助于确定与癌症相关的新基因， 对E.大肠杆菌错配修复基因mutS和 mutL，辅助鉴定人HNPCC癌 易感基因

The proposed experiments are designed to further our understanding of the fundamental molecular mechanisms responsible for spontaneous mutagenesis. They focus particularly on the mechanisms underlying the SOS mutator effect in E. coli and on the role of cytosine methyltransferases in catalyzing cytosine deamination under conditions of S-adenosylmethionine (SAM) limitation. The SOS mutator effects that are observed in E. coli cells expressing the SOS functions result from mechanisms that normally contribute to the spectrum of spontaneous mutations. The power and convenience of the CDCE/hifiPCR technology to obtain mutational spectra, which has been developed by Thilly and his colleagues, will be exploited to carry out a systematic investigation of the genetic basis of the SOS mutator phenotype. For example, different ways of constitutively expressing the SOS system will be examined to determine whether they yield equivalent SOS mutator phenotypes and the role of the replicon will be investigated. The requirement for dinB function in spontaneous mutagenesis resulting from the SOS mutator effect will be examined. The influence of mismatch repair and other functions on the SOS mutator phenotype will be investigated. The dinB gene product been implicated in the SOS mutator phenotype and may play possibly play a role in determining the frequency with which frameshift mutations occur in runs of repeated bases. The dinB gene will be cloned and sequenced. The biochemical role of the DinB protein will be investigated and its relevance to the SOS mutator effect and spontaneous mutagenesis determined. Recently it has been shown that, if SAM is limiting, DNA cytosine methyltransferases can catalyze the deamination of cytosine through the formation of an intermediate with a saturated C5-C6 bond. Furthermore, it has recently been suggested that such a mechanism of spontaneous mutagenesis may contribute to cancer progression. In order to understand further the physiological relevance of this phenomenon, the influence of limiting SAM levels on the frequency of spontaneous mutations occurring at Dcm methylation sites in E. coli will be examined using two independent approaches. The influence of eliminating two potential uracil repair mechanisms by ung and vsr mutations under conditions of SAM limitation will be investigated. Finally experiments will be performed to determine whether expression of a mammalian DNA cytosine methyltransferase in E. coli causes an increased frequency of spontaneous mutations at CpG sites under conditions of SAM limitation. The proposed studies have the potential to help define new genes relevant to cancer similar to the fashion in which analyses of the E. coli mismatch repair genes, mutS and mutL, assisted in the identification of the human HNPCC cancer susceptibility genes.