The goal of the investigation is to gain a greater understanding of the mechanism and regulation of post-replicative methylation of DNA catalyzed by T4 and T2 Dam methylases. The methylation of specific residues was shown to be involved in regulation of the expression of the variety of genes in higher eukaryotes as well as in regulation of DNA transcription and repair in prokaryote systems. Although methyltransferases were proposed to act as monomers while interacting with DNA, there is some evidence (largely supported by the work of foreign collaborator's laboratory) that the general structure of methyltransferase-DNA complexes involves dimerization of the enzyme. According to the proposed hypothesis, methyltransferase binds to DNA non- specifically as a monomer and scans the sequence. The specific interaction of the enzyme with the recognition site then requires the binding of the second monomer. Initiation of the methylation reaction can cause dissociation of the enzyme subunit participation in the process of recognition but not methyl transfer. To examine this hypothesis two types of experiments will be carried out: 1) determination of precise enzyme-substrate stoichiometry in catalytically competent complexes and elucidation of the thermodynamic characteristics of the interaction stages and structural parameters of the methyltransferase-substrate complexes and 2)investigation of the subunit state of methyltransferase in catalytically competent enzyme-substrate complexes. Oligonucleotides- enzyme complexes will be examined by gel-chromatography, sucrose density ultracentrifugation, gel mobility shift assay, small-angle X- ray scattering and fluorescence spectroscopy. Recombinant T2 and T4 methyltransferases as well as mutant forms of the enzymes expressed and purified in the principal investigator's laboratory will be used in the experiments.

Agency
National Institute of Health (NIH)
Institute
Fogarty International Center (FIC)
Type
Small Research Grants (R03)
Project #
5R03TW000529-03
Application #
2520100
Study Section
International and Cooperative Projects 1 Study Section (ICP)
Project Start
1995-09-01
Project End
1999-08-31
Budget Start
1997-09-01
Budget End
1999-08-31
Support Year
3
Fiscal Year
1997
Total Cost
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