PROBING FLP - A NEW APPROACH TO ANALYZE THE STRUCTURE OF A DNA RECOGNIZING PROTEIN BY COMBINING THE GENETIC ALGORITHM, MUTAGENESIS AND NONCANONICAL DNA TARGET SITES

A topological and functional overview of a DNA recognition protein wit h unknown structure can be achieved by combining three different, but complementary approaches: modeling by the genetic algorithm, functiona l analysis of mutated variants, and testing the target DNA using non-c anonical oligonucleotides. As an example we choose the Flp protein, a site-specific recombinase from Saccharomyces cerevisiae. We derive the topological outline including the DNA binding cleft, examine DNA bind ing regions by deletional and mutational analysis, and analyze the DNA binding site using 7-deazaadenine, 7-deazaguanine, inosine and 4-O-me thylthymine as probes. The combined data offer a comprehensive sketch of a plausible protein architecture for Flp. The structure is detailed enough to verify the prediction accuracy for different peptide region s from pre-existing data and by new experimental design. (C) 1997 Else vier Science B.V.