We propose to carry out a detailed investigation of the kinetics of the DNA methylation reaction catalyzed by the bacteriophage-encoded T4 Dam DNA [N6-adenine] methyltransferase (MTase), which recognizes the palindromic sites, GATC. T4 Dam transfers a methyl group from donor, S-adenosyl-L-methionine (AdoMet), directly to the free exocyclic amino-nitrogen of adenine, without the formation of a covalent enzyme-AdoMet intermediate, as is the case for [C5]-cytosine MTases. The proposed studies will use both stop-flow fluorescence quench methods and isotopic labeling to measure DNA both pre- and steady- state methylation kinetics. In addition, we propose to investigate T4 Dam mutant enzymic forms that exhibit altered methylation capability depending on the DNA substrate. This study will enable us to deduce the reaction step(s) affected by known single amino acid substitutions in the MTase-target recognition domain, as well as in other conserved sequence motifs. This will provide valuable new insights into the interactions of specific MTase substituents with their substrates. Although the functions of individual residues in certain motifs have been inferred by modeling from 3-D structures of other MTases, we believe that the reality may be more complicated. Because a crystal structure provides a 'snapshot' of these interactions, dynamic changes in protein or DNA conformation may go unnoticed. The power of genetics, coupled with physical-biochemical analysis, will provide invaluable complementary data for understanding such dynamics. Recently, an essential biological role for DNA adenine methylation in determining bacterial virulence was discovered in Salmonella typhimurium, where dam mutants were found to be avirulent. Using the T4 enzyme as a model system for the Dam family of MTases, we hope to gain a better understanding of how these enzymes interact with their substrates. This could facilitate the development of a new class of mechanism-based antibiotics that specifically inhibit Dam MTase activity.

National Institute of Health (NIH)
Fogarty International Center (FIC)
Small Research Grants (R03)
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International and Cooperative Projects 1 Study Section (ICP)
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Katz, Flora N
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University of Rochester
Schools of Arts and Sciences
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