The ability to site-specifically methylate DNA has numerous potential uses including 1) a tool for the study of DNA methylation patterns, 2) as a tool to silence gene of interest, and 3) as a potential gene therapy device to correct conditions caused by hypomethylation. Current approaches include linking methyltransferases to DNA binding domains to localize enzymes next to a target site. This approach has achieved site-biased methylation, however the engineered methyltransferases are still active in the absence of binding their intended target and methylate non-target sites. What is necessary to make a truly site-specific methyltransferase is to require binding to the target site for catalytic activity. We propose to use non-associating fragments of natural heterodimeric methyltransferases as a platform for building such enzymes, with DNA at the target site acting as a template for enzyme assembly. The naturally occurring C5-methylcytosine methyltransferase M.EcoHK31I has alpha and beta peptide chains that associate to create a functional enzyme. We have made non-associating fragments by creating truncated versions of the beta fragment. We have evaluated these truncated fragments as a platform for creating site-specific methyltransferases by creating small combinatorial libraries of fusions with zinc finger proteins and screening these libraries for site-specific methylation activity. We have found that a high degree of specificity can be achieved at a target site flanked by zinc finger binding sites. Specificity appears to be dependent on the distance of DNA binding sites from the target methylation site as well as the length of the flexible linker connecting the fragments to the zinc finger proteins.

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