Expression of genetic material in all living systems involves highly coordinated interactions between proteins and specific regulatory sites on target DNA and RNA molecules. The long-term objectives of my research are to gain a greater understanding of: (i) the biological roles of DNA-modifications and the enzymes that mediate them; and (ii) the structure/function of the C and Com proteins governing the unusual regulatory mechanisms controlling transcription and translation of the phage Mu mom operon. The high specificity and biological importance of DNA methyltransferases (MTases) make them well-suited for studies of protein-DNA interactions. DNA methylation is known to influence expression of genes for many critical cellular functions; and some studies point to a role in the etiology of certain cancers. This makes it even more critical to understand the structure and function of MTases. Thus, we propose to continue our genetic/biochemical studies delineating functional domains in the T4 Dam [N6-amino]-MTase where we have discovered mutations that increase its catalytic efficiency and lead to ectopic methylation. This has implications for DNA-[C5-cyt]-MTases because analogous somatic mutations in mammals might induce disease; for example, by hypermethylating and abolishing transcription of a tumor suppressor gene. Collaborative efforts on X-ray crystallographic analysis to solve 3-D structures are in progress. These are important to reveal whether flipping out the target base from the double helix, as observed for [C5-cyt]- MTases, is also mediated by a [N6-amino] MTase. A structure will permit modeling of mutant forms, whose biochemical properties we have been defining. We also propose to initiate new research on the [N6-amino] DNA-MTase in the eukaryotic protozoan. Tetrahymena thermophila, to investigate if it has an essential biological function; e.g., in the chromosome processing that occurs during macronuclear biogenesis. Models for C and Com regulation are being investigated. Com, nature's smallest naturally-occurring zinc-finger protein, represents the first known case of a translational activator [it acts by melting a stem-loop structure in its target mRNA]. Their small sizes, Com [62 residues] and C [140, residues], and the ability to do genetic, biochemical and structural analyses, make these proteins exquisite objects for study.
