DNA methylation is essential for normal mammalian development, and inhibition of the enzyme responsible for DNA methylation [DNA cytosine C(5) methyltransferase, DCMTase] aids in alleviating oncogenesis. The long-term goal of the proposed characterization of the mammalian DCMTase is the development of novel inhibitors with potential applications as anticancer drugs. 1) The first specific aim is to determine the mechanism of cytosine methylation by the mammalian DCMTase and the structurally characterized bacterial DCMTase, M.Hhal. The goal is to provide a detailed kinetic description of the events starting with substrate addition, and ending with the methyl transfer step. This information is essential for the evaluation of inhibitor potency and for a mechanistic understanding of inhibitor action. This kinetic analysis will also form the basis for a quantitative assignment of the effects of designated DCMTase mutants.
This specific aim will be addressed using pre-steady state kinetic methods, partition analysis, steady-and pre- steady state fluorescence spectroscopy, and mutagenesis of active site residues. 2) The second specific aim is to characterize the mechanism of mammalian DCMTase inhibition observed with single-stranded DNA. The DNA structural features that are essential for this potent inhibition will be identified. The hypothesis that the large, N-terminal domain of the DCMTase, is involved in binding the inhibitor will be tested. 3) The third specific aim is to determine if the mammalian DCMTse can catalyze the deamination of 5-methylcytosine to generate thymine. Others have proposed that this mutagenic reaction may account for many human genetic diseases including cancer. However, the reaction has only been demonstrated with bacterial DCMTases.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM056289-02
Application #
2734836
Study Section
Special Emphasis Panel (ZRG3-BIO (02))
Project Start
1997-07-01
Project End
2001-06-30
Budget Start
1998-07-01
Budget End
1999-06-30
Support Year
2
Fiscal Year
1998
Total Cost
Indirect Cost
Name
University of California Santa Barbara
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
City
Santa Barbara
State
CA
Country
United States
Zip Code
93106
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Svedruzic, Zeljko M; Reich, Norbert O (2005) DNA cytosine C5 methyltransferase Dnmt1: catalysis-dependent release of allosteric inhibition. Biochemistry 44:9472-85
Flynn, J; Reich, N (1998) Murine DNA (cytosine-5-)-methyltransferase: steady-state and substrate trapping analyses of the kinetic mechanism. Biochemistry 37:15162-9