Since the discovery of sequence-specific DNA binding proteins there has been considerable speculation regarding """"""""codes"""""""" that might control such interactions. This idea has been applied to zinc finger motifs, formulated as a set of rules that correlate the sequence of amino acids in the recognition region of a zinc finger domain with the triplet of base pairs to which it is capable of binding. This set of rules serves as a starting point for a more general set of rules which incorporate context dependent information. Structures of individual zinc finger domains in solution, as determined by NMR spectroscopy, are strikingly similar to the structure of zinc fingers bound to DNA determined by X-ray diffraction. We have shown that Fpg protein contains a single zinc finger, the enzyme requires this motif to bind duplex DNA containing 8-oxoguanine, revealing a 5 base foot print on the modified strand. This protein provides an unusual example of a zinc finger protein that does not bind to an undamaged DNA sequence but that may enjoy the zinc finger in recognition of DNA damage. We propose to explore the structural biology of this interaction. Experiments to date have shown that four cysteine residues which constitute the putative zinc finger of Fpg protein are required for binding to duplex DNA containing 8-oxodG or abasic sites. We will extend these site directed mutagenesis studies by modifying selected amino acid residues within the zinc finger domain. Binding and catalytic properties of the altered proteins will be determined. Positions to be modified will be guided initially by a model built by Dr. Klein in the CGL, applying a rule that has emerged from recent analyses of zinc fingers; namely, that the position just prior to the beginning of the alpha helix and positions 2, 3, and 6 of the alpha helix [residues 5, 7, 8, and 11 in the Fpg alpha helix] were critical for DNA binding. Our model was subjected to molecular dynamics (>200 psec) and serves as a basis for site directed mutagenesis studies.
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