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.

Agency
National Institute of Health (NIH)
Institute
Fogarty International Center (FIC)
Type
Small Research Grants (R03)
Project #
1R03TW005755-01
Application #
6440826
Study Section
International and Cooperative Projects 1 Study Section (ICP)
Program Officer
Katz, Flora N
Project Start
2001-12-14
Project End
2004-11-30
Budget Start
2001-12-14
Budget End
2002-11-30
Support Year
1
Fiscal Year
2002
Total Cost
$38,705
Indirect Cost
Name
University of Rochester
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
208469486
City
Rochester
State
NY
Country
United States
Zip Code
14627
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Zinoviev, Victor V; Evdokimov, Alexey A; Malygin, Ernst G et al. (2007) Differential methylation kinetics of individual target site strands by T4Dam DNA methyltransferase. Biol Chem 388:1199-207
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Zinoviev, Victor V; Evdokimov, Alexey A; Malygin, Ernst G et al. (2003) Bacteriophage T4 Dam DNA-(N6-adenine)-methyltransferase. Processivity and orientation to the methylation target. J Biol Chem 278:7829-33
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Malygin, Ernst G; Lindstrom Jr, William M; Zinoviev, Victor V et al. (2003) Bacteriophage T4Dam (DNA-(adenine-N6)-methyltransferase): evidence for two distinct stages of methylation under single turnover conditions. J Biol Chem 278:41749-55