Mutations of the FLP recombinase gene that cause a deficiency in DNA bending and strand cleavage.
We have used site-directed mutagenesis to change several amino acids of the C-terminal portion of the FLP recombinase of Saccharomyces cerevisiae. These residues are absolutely conserved among the six FLP-like proteins from various yeast strains. We have examined the ability of the altered proteins to catalyze recombination in vivo and in vitro and to perform various partial steps of the reaction in vitro. Two of the mutations produced a partial defect in DNA binding but the remainder resulted in normal binding. All of these mutations caused impairment of the ability of the protein to induce the type II bend of the FRT site, and some of these proteins were also defective in DNA strand cleavage. None of the mutations affected the ability of the proteins to perform synapsis between two FRT sites, but some were defective in strand ligation. Interestingly, some mutant proteins showed impairment of the initial stages of the recombination reaction on a linear substrate and yet they maintained the ability to resolve a Holliday intermediate in the reaction. We conclude that this conserved region of the FLP protein is important for the early stage(s) of the recombination reaction.
Kulpa
J
J
Department of Molecular and Medical Genetics, University of Toronto, Ontario, Canada.
Dixon
J E
JE
Pan
G
G
Sadowski
P D
PD
D000595:Amino Acid Sequence; D001665:Binding Sites; D004247:DNA / Q000378:metabolism*; D004254:DNA Nucleotidyltransferases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D005800:Genes, Fungal; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009690:Nucleic Acid Conformation; D010957:Plasmids; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D011995:Recombination, Genetic; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics
D011994:Recombinant Proteins; D004247:DNA; D004254:DNA Nucleotidyltransferases; C045119:FLP recombinase