The DNA adenine methylase, specified by the dam gene of E. coli, has a role in spontaneous mutability. Spontaneous mutability increases substantially if the enzyme is absent or if it is overproduced. This poses two questions: (i) How is the dam gene regulated?, and (ii) What is the mutation spectrum in cells overproducing the enzyme or in which it is not produced at all? This proposal seeks to answer these questions through a combined in vitro and in vivo approach. In vitro manipulations and mutations generated in vivo will be used to identify the promoter(s) and other regulatory elements which control the expression of the dam gene. In vivo generated mutations in a small repressor gene will be analyzed by in vitro DNA sequencing to identify mutations at the nucleotide level. An analysis of the spectrum will test the model that methylation dependent mismatch repair is a major mechanism for removal of potential frameshift mutations. This proposal also seeks to determine which genes of E. coli can have their expression altered by DNA methylation. This will be done by examing expression of fused genes in the presence or absence of DNA adenine methylase. The studies with DNA adenine methylase function in DNA repair and gene expression in the E. coli model systems offers a new approach to the elucidation of the origin and maintenance of mutations and, thus, may contribute significantly to a more thorough understanding of the process linking carcinogenesis and spontaneous congenital abnormalities in man.
