Eco RI DNA Methyltransferase: Catalytic and Recognition Mechanisms

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9412078 Reich The information content of DNA is expanded in almost all organisms by methylation of adenine (N6) or cytosine (C5 and N4). DNA methylation is involved in the control of tissue specific and developmentally regulated gene expression; the protection of host (bacterial) DNA against endonuclease cleavage; DNA repair; aging; and genetic imprinting. The study of two bacterial DNA methyltransferases is proposed: EcoRI N6 adenine methyltransferase, which methylates the second adenine in GAATTC, and BamHI N4 cytosine methyltransferase, which methylates the first cytosine in GGATCC. The ready availability of the genes and large quantities of homogeneous protein provides the basis for the structure-function analyses to be performed. The general goal is to understand the molecular details of sequence-specific DNA methylation sufficiently to design novel biocatalysts, enzyme "mimics", and/or modify the sequence-specificity of the target enzyme. Two specific aims are detailed. The first is to identify structural features of the EcoRI methyltransferase or its substrate, and to a lesser extent the BamHI methyltransferase, that are essential for catalysis. The second aim is to determine the three-dimensional structure of the EcoRI or BamHI methyltransferase and their corresponding protein-cofactor as well as protein DNA-cofactor complexes using X-ray crystallographic analyses of the wild type proteins as well as point and deletion mutants. This structural information will aid our understanding of past structure-function studies with the EcoRI methyltransferase as well as provide the basis for future studies aimed at elucidating sequence-specific DNA modification. %%% These experiments are designed to characterize bacterial DNA methyltransferases. These enzymes modify unique DNA sequences by methylating adenine and cytosine. Structural characterization using X-ray crystallography and nuclear magnetic resonance is proposed. Also portions of the enzymes which contact the DNA will be identified using laser cross linking and mass spectrometry. Functional characterization will largely be through protein engineering and protein modification experiments. The goal of this research is to obtain a molecular understanding of how these enzymes modify unique sites within DNA. Sequence-specific DNA modification is a universal biological process which has practical applications in the field of molecular biology. ***