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.
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