Identification of the active site tyrosine of Flp recombinase. Possible relevance of its location to the mechanism of recombination.
A combination of site-directed mutagenesis and amino acid sequence analysis identifies Tyr-343 of Flp recombinase as the residue that covalently attaches to DNA during the strand-cleavage step of recombination. This residue is part of the invariant His-Arg-Tyr triad of the Int family of recombinases. Tyr-343 is located in a highly protease-accessible (and hence "open") region of Flp. This placement may provide the conformational flexibility required for the dual role of Tyr-343 in recombination: nicking of the DNA strands to initiate recombination and joining of the nicked strands across partner substrates to complete recombination. In-frame insertion of a few amino acids close to Tyr-343 (and to its amino-terminal side) does not affect substrate recognition by Flp but abolishes its catalytic function.
Department of Molecular Biology, Research Institute of Scripps Clinic, La Jolla, California 92037.
B R
BR
Evans
J W
JW
Chen
R L
RL
Parsons
T K
TK
Bauer
D B
DB
Teplow
M
M
Jayaram
D000595:Amino Acid Sequence; D001665:Binding Sites; D004252:DNA Mutational Analysis; D004254:DNA Nucleotidyltransferases / Q000737:chemistry / Q000502:physiology* / Q000648:ultrastructure; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000502:physiology / Q000648:ultrastructure; D005656:Fungal Proteins / Q000737:chemistry / Q000648:ultrastructure; D008969:Molecular Sequence Data; D015394:Molecular Structure; D010449:Peptide Mapping; D011994:Recombinant Proteins; D011995:Recombination, Genetic; D014443:Tyrosine
D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D011994:Recombinant Proteins; D014443:Tyrosine; D004254:DNA Nucleotidyltransferases; C045119:FLP recombinase