Structure-Function Analysis of the EcoRI DNA Methylase

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This proposal addresses a fundamental problem in biochemistry-the nature of specific protein-DNA interactions. The general goal is to elucidate how the EcoRI methylase catalyzes transfer of methyl group from S-adenosylmethionine (AdoMet) to the second adenine in the double stranded sequence: 5' GAATTC3'. Specific goals are to characterize: (1) the kinetic mechanism, (2) the chemical mechanism, (3) the contributions toward specificity deriving from individual enzyme-substrate interactions, (4) the structure of the methylase-DNA complex. These goals will be addressed with steady and presteady state kinetics, pH analyses, and modified DNA substrates. Specific chenical modification and genetic substitution of amino acids will be used with the kinetic studies to implicate specific residues in catalysis and substrate binding. The applicability of DNA-footprinting assays to determine the methylase-DNA complex topology and binding affinity will be tested. Collaborative crystallographic and 1H N.M.R. analyses of the methylase-DNA complex are ongoing. DNA methylases are essential for the in vitro manipulation of DNA and play a critical role in the regulation of gene expression in eukaryotes. Yet no structural and little function information is available for any DNA methylase. The proposed work is aimed at obtaining this information for the EcoRI methylase, a member of this important class of enzymes.