Mutagenesis of a conserved region of the gene encoding the FLP recombinase of Saccharomyces cerevisiae. A role for arginine 191 in binding and ligation.
The FLP recombinase from the 2 microns plasmid of Saccharomyces cerevisiae contains a region from amino acid 185 to 203 that is conserved among several FLP-like proteins from different yeasts. Using site-directed mutagenesis, we have made mutations in this region of the FLP gene. Five of twelve mutations in the region yielded proteins that were unable to bind to the FLP recombination target (FRT) site. A change of arginine at position 191 to lysine resulted in a protein (FLP-R191K) that could bind to the FRT site but could not catalyze recombination. This mutant protein accumulated as a stable protein-DNA complex in which one of the two bound FLP proteins was covalently attached to the DNA. FLP-R191K was defective in strand exchange and ligation and was unable to promote protein-protein interaction with half-FRT sites. The conservation of three residues in all members of the integrase family of site-specific recombinases (His305, Arg308, Tyr343 in FLP) implies a common mechanism of recombination. The conservation of arginine 191 and the properties of the FLP-R191K mutant protein suggest that this arginine also plays an important role in the mechanism of FLP-mediated site-specific recombination.
Friesen
H
H
Department of Molecular and Medical Genetics, University of Toronto, Ontario, Canada.
Sadowski
P D
PD
D000595:Amino Acid Sequence; D001120:Arginine / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D004254:DNA Nucleotidyltransferases / Q000235:genetics* / Q000378:metabolism; D004271:DNA, Fungal / Q000378:metabolism; D005656:Fungal Proteins / Q000235:genetics* / Q000378:metabolism; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D016296:Mutagenesis / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D011995:Recombination, Genetic / Q000502:physiology; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics
D004271:DNA, Fungal; D005656:Fungal Proteins; D046911:Macromolecular Substances; D001120:Arginine; D004254:DNA Nucleotidyltransferases; C045119:FLP recombinase