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.
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