(242g) Non-Associating Heterodimeric DNA Methyltransferases as a Platform for Developing Site-Specific Methyltransferases

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 methyltransferases M. AquI and M. EcoHK31I each have 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 fragments. We have evaluated these 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.