Tests for the fractional active-site model in Flp site-specific recombination. Assembly of a functional recombination complex in half-site and full-site strand transfer.
The Arg191-His305-Arg308 (the RHR triad) and Tyr343 of Flp site-specific recombinase correspond to the invariant tetrad residues of the integrase family of proteins. Flp mutants altered at these positions are blocked at the strand cleavage or the strand exchange step of recombination. Hybrid half-site-recombinase complexes formed by step-arrest mutants of Flp have revealed that an Flp monomer occupying a half-site does not cleave that half-site but rather cleaves a half-site occupied by a second Flp monomer. This trans-DNA cleavage is neatly accommodated by a model in which an Flp active site is assembled by contribution of amino acid residues from at least two protein monomers. Using a combination of wild type Flp, single, double, and triple step-arrest Flp mutants, critical predictions of the fractional active-site model have been verified. First, a wild type monomer paired with an RHR triad-Tyr343 double mutant is a catalytically inactive combination. Second, each pairwise combination of a single, double, or triple RHR mutant with Flp (Y343F) yields approximately equivalent levels of catalytic complementation. Half-site to half-site and half-site to full-site crosses suggest that execution of a strand transfer event within a half-site and between a half-site and a full site requires dimeric and tetrameric Flp configurations, respectively.
Chen
J W
JW
Department of Microbiology, University of Texas, Austin 78712.
Yang
S H
SH
Jayaram
M
M
D000595:Amino Acid Sequence; D001482:Base Composition; D001483:Base Sequence; D001665:Binding Sites; D004254:DNA Nucleotidyltransferases / Q000378:metabolism*; D005656:Fungal Proteins / Q000378:metabolism; D046911:Macromolecular Substances; D008957:Models, Genetic; D008961:Models, Structural; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis / Q000378:metabolism; D011487:Protein Conformation; D011995:Recombination, Genetic; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D013379:Substrate Specificity
D005656:Fungal Proteins; D046911:Macromolecular Substances; D009838:Oligodeoxyribonucleotides; D004254:DNA Nucleotidyltransferases; C045119:FLP recombinase