Functional roles of individual recombinase monomers in strand breakage and strand union during site-specific DNA recombination.
The site-specific recombinase Flp from Saccharomyces cerevisiae accomplishes recombination between two target DNA sites by executing a pair of strand exchanges at either end of the strand exchange region. One round of recombination requires the cooperative action of four recombinase monomers. We demonstrate here that, in the presence of the appropriate nucleophiles, a single Flp monomer associated with its binding element can mediate strand cleavage and strand joining at the exchange site phosphate adjacent to it. Our results support a model of recombination in which pairs of Flp monomers reverse catalytic roles to mediate the first and second sets of strand breakage/union reactions. They disfavor a model that involves a relay of recombinase monomers between binding elements to assemble separate active sites for strand cleavage and strand joining. Our data are consistent with the breakage and joining reactions being carried out by a single composite active site in which some residues contribute to both reactions while others contribute to one of the two reactions.
Lee
J
J
Department of Microbiology, University of Texas, Austin 78712, USA.
Jayaram
M
M
D001665:Binding Sites; D002846:Chromatography, Affinity; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D004254:DNA Nucleotidyltransferases / Q000302:isolation & purification / Q000378:metabolism*; D005656:Fungal Proteins / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D046911:Macromolecular Substances; D009838:Oligodeoxyribonucleotides; D011995:Recombination, Genetic; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013379:Substrate Specificity
D005656:Fungal Proteins; D046911:Macromolecular Substances; D009838:Oligodeoxyribonucleotides; D004247:DNA; D004254:DNA Nucleotidyltransferases; C045119:FLP recombinase