Spontaneous mutagenesis contributes significantly to genome instability in all organisms. It is essential to understand the mechanisms of spontaneous mutagenesis both for this reason and in order to be able to distinguish spontaneous mutations from those induced by exogenous agents. The goal of this project is to investigate mechanisms for GC->AT mutations observed at sites in genomes containing 5-methylcytosine (m5C), which are generally regarded to be due to "spontaneous" events. It has been known for almost 2 decades that m5C sites can be mutagenic hotspots in E.coli, and it appears that mutagenesis at m5C in human cells can be an important pathway that contributes to both tumorigenesis and germ line mutagenesis. One important component of this process appears to be m5C deamination to thymine to give a G:T mispair, which might ultimately give rise to a m5C:G->T:A mutation. In organisms which have m5C in their genomes there are (or appear to be) specific DNA repair pathways solely devoted to solving the problem of m5C deamination to T. The main question posed in this proposal is: why--in spite of a repair system specifically designed to combat this problem--does m5C:G->T:A remain an unusually prevalent pathway of mutagenesis? In this regard two lines of questions are pursued: (l) does the presence of other pathways of DNA repair that compete with the repair of m5C->T deaminations frustrate the effectiveness of the latter; and (2) are there alternatives to m5C deamination that might contribute and perhaps even dominate m5C mutagenesis in some contexts? In this regard one specific aim is directed toward evaluating whether other mismatch repair pathways might prevent effective repair of m5C deamination. A second specific aim is devoted to evaluating whether other pathways may contribute significantly to mutagenesis from m5C. For example, misreplication to give a m5C:A mispair might be unusually frequent; alternatively, this mispair might be repaired ineffectively. Finally, if any of the projects in this Program Project generates data to suggest that a particular lesion is important to spontaneous mutagenesis, a site-specific study of that lesion will be conducted. Most of the studies proposed herein will be done in E. coli. The studies are only feasible given the development of the enabling technology, CDCE/hifi PCR, which significantly reduces the time and effort required to determine a mutational spectrum.