Current evidence suggest that errors in the establishment of maintenance of genomic methylation patterns contribute to ectopic gene inactivation of reactivation. These errors are likely to be involved in certain human diseases; this is especially true to tumors, whose growth depends on the inactivation of humor suppressor genes. In cultured cells, ectopic methylation and actual mutations are each responsible for approximately half of all cases of gene inactivation, and we make the specific prediction that some portion of human tumors will show inactivation of tumor suppressor genes by ectopic methylation. Also, there exists very intriguing evidence for enzyme-mediated conversion of m5C to T and C to U; these mutations commonly contribute to carcinogenesis and genetic disease. Biochemical and genetic means will be used to test for DNA cytosine deaminases and for DNA 5-methylcytosine deaminases in lysates of mammalian cells. The chemistry of the transmethylation reaction suggests that DNA Methyltransferase should act as a DNA cytosine deaminase in the absence of S-adenosyl L-methionine, and this prediction will also be tested. Sequence-specific methylation patterns are established during gametogenesis and early development. Errors in sequence-specific methylation can be expected to cause developmental defects, and Fragile X syndrome and perhaps Huntington's disease involve defective DNA methylation. Since nothing is known of the regulation of sequence- specific de novo methylation, experiments are proposed that will determine whether sequence-specific de novo methyltransferases are present at developmental stages where methylation patterns are undergoing rapid changes. These experiments will use a very sensitive and specific universal probe for DNA (cytosine-5)-methyltransferases based on the suicide substrate 5-fluoro 2'-deoxycytidine. The specificity and other functions of the known form of DNA Nethyltransferase will be studied in existing mutant cell lines which allow the selection of active mutants of DNA Nethyltransferase. This proposal describes experimental approaches to the role of DNA modification in carcinogenesis and genetic disease.
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