9603567 Reich Part 1-Technical The proposed work focuses on the bacterial DNA methyltransferases, M.EcoRI and M.BamHI, which methylate the underlined N6 and N4 cytosine in GAATTC and GGATCC, respectively. M.EcoRI was recently shown to bend DNA by 50 degrees and to flip out the adenine prior to methylation. Base-flipping was detected by a novel, dynamic assay which uses the fluorescent base analog, 2-aminopurine. The first goal uses M.EcoRI and various methods to investigate base-flipping and DNA bending processes. Presteady state fluorescence experiments will be used to determine if base-flipping is enzyme-assisted or if the enzyme simple stabilizes the extrahelical base. Time-resolved fluorescence measurements will be used to determine the orientation and mobility of the extrahelical base within the enzyme-DNA complex. The combined DNA fluorescence measurements will be compared to similar changes in protein (tryptophan) fluorescence so that a complete kinetic description of early steps in the catalytic cycle should be provided. This analysis will provide detailed kinetic information about the enzyme-mediated deformation of DNA conformation. Amino acid residues implicated in catalysis and adenine stabilization by structural homology to the related M. TaqI enzyme will investigated to assess their functional role (s). The second goal is to determine the structures of M.EcoRI and M. BamHI using X-ray diffraction methods. Small crystals of an M.EcoRI-DNA complex and crystals of M.BamHI which diffract to 2.7 angstrom have been obtained. Further crystallization trials are proposed. Crystallization trials of various M.EcoRI mutants are proposed. Part 2-Non technical Many enzymes which alter the ahemical structure of DNA, do so by stabilizing large scale conformational changes within the DNA molecule. These include significant DNA "bends" of up to 100 degrees and movement of individual bases outside of the normal double-helix of DNA. How these enzyme-mediated changes in DNA conf ormation contribute to the enzyme's function is not understood. This proposal will measure changes in DNA structure involving bending and "base flipping" using the bacterial DNA methyltransferase, M.EcoRI. This enzyme transfers a methyl group to the second adenine in GAATTC. We have developed a novel fluorescence-based assay to measure base-flipping in this enzyme, and propose to extend these experiments to measure how this occurs over time. Related experiments will be done to measure how the enzyme changes the flexibility and orientation of the base once it is removed from the double helix. The proposal also seeks to identify which amino acids within the protein are responsible for these conformational changes. A second long term goal of the proposal is to determine the three-dimensional structure of the enzyme using X-ray crystallographic methods.
