EXPRESSION OF BACTERIAL GENES FOR THE REPAIR OF ALKYLATION DAMAGE

建议的实验旨在加深我们对 自发诱变的基本分子机制。 他们特别关注SOS突变体背后的机制 在大肠杆菌中的作用及胞嘧啶甲基转移酶在 S-腺苷蛋氨酸催化胞嘧啶脱氨反应 (SAM)限制。 在表达SOS突变体的大肠杆菌细胞中观察到的突变体效应 SOS功能是由通常有助于 自发突变的光谱。的威力和便利性 CDCE/hifiPCR技术获得突变谱，已被 由Thilly和他的同事开发的，将被利用来执行 SOS突变子遗传基础的系统研究 表型。例如，不同的宪法表达方式 将对SOS系统进行检查，以确定它们是否产生相同的SOS 我们将研究突变子的表型和复制子的作用。这个 鸡传染性支气管炎引起的自发突变对dinB功能的要求 我们将研究SOS突变器效应。错配修复的影响 以及SOS突变体表型的其他功能将被研究。这个 DinB基因产物与SOS突变子表型有关，可能 发挥可能在确定频率方面发挥作用 移码突变发生在重复碱基的运行中。DinB基因将会 被克隆和测序。DinB蛋白的生化作用将是 研究及其与SOS突变体效应和自发效应的相关性 已确定有诱变。 最近的研究表明，如果SAM是限制性的，DNA胞嘧啶 甲基转移酶可以催化胞嘧啶的脱氨基反应。 形成具有饱和C5-C6键的中间体。此外，它还 最近有人提出，这种自发的机制 突变可能会导致癌症的进展。为了理解 进一步说明这一现象的生理相关性，即 限制SAM水平对自发突变发生频率的影响 大肠杆菌中的DCM甲基化位点将使用两个独立的 接近了。排除两种潜在的尿嘧啶修复对患者的影响 SAM限制条件下ung和VSR突变的机制 将会被调查。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. 大肠杆菌导致CpG位点自发突变的频率增加 在SAM限制条件下。拟议的研究具有 可能有助于定义与癌症相关的新基因，类似于 分析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.