Cleavage-dependent ligation by the FLP recombinase. Characterization of a mutant FLP protein with an alteration in a catalytic amino acid.
The FLP recombinase of the 2 microM plasmid of Saccharomyces cerevisiae belongs to the integrase family of recombinases whose members have in common four absolutely conserved residues (Arg-191, His-305, Arg-308, and Tyr-343). We have studied the mutant protein FLP R308K in which the arginine residue at position 308 has been replaced by lysine. Although FLP R308K was previously reported to be defective in ligation of certain substrates (Pan, G., Luetke, K., and Sadowski, P.D., Mol. Cell. Biol. 13, 3167-3175, 1993b), we show in this work that the protein is able to ligate those substrates that it can cleave (cleavage-dependent ligation activity). FLP R308K is defective in in vitro recombination and in strand exchange. It is able to carry out strand exchange at one of the two cleavage sites of the FLP recognition target site (FRT site), but is defective in strand exchange at the other cleavage site. These results are consistent with a model in which wild-type FLP initiates recombination only at one of the two cleavage sites. FLP R308K may be defective in the initiation of recombination.
Zhu
X D
XD
Department of Molecular and Medical Genetics, University of Toronto, Ontario, Canada.
Sadowski
P D
PD
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D017124:Conserved Sequence; D004254:DNA Nucleotidyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005656:Fungal Proteins / Q000378:metabolism; D008025:Ligases / Q000378:metabolism; D008957:Models, Genetic; D008961:Models, Structural; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis / Q000737:chemistry; D010957:Plasmids; D017354:Point Mutation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D011995:Recombination, Genetic; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D013379:Substrate Specificity
D005656:Fungal Proteins; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D004254:DNA Nucleotidyltransferases; C045119:FLP recombinase; D008025:Ligases