title: Mechanism of active site exclusion in a site-specific recombinase: role of the DNA substrate in conferring half-of-the-sites activity.
abstract: The Flp site-specific recombinase assembles its active site by recruiting the catalytic tyrosine (Tyr-343) from one Flp monomer into the pro-active site containing a triad of Arg-191, His-305, and Arg-308 from a second monomer. In principle, two active sites may be assembled from a Flp dimer by simultaneous, reciprocal contribution of the shared amino acids by its constituent monomers. In practice, only one of the two active sites is assembled at a time, as would be consistent with a recombination mechanism involving two steps of single-strand exchanges. By using substrates containing strand-specific base bulges, we demonstrate that the relative disposition of their DNA arms can account for this active site exclusion. We also show that the exclusion mechanism operates only at the level of positioning Tyr-343 with respect to the pro-active site, and not at the level of orienting the labile phosphodiester bond within the DNA chain. It is not negative cooperativity of substrate binding but, rather, the substrate-induced negative cooperativity in protein orientation that accomplishes half-of-the-sites activity in the Flp system.
authors.affiliation: Department of Microbiology and Institute of Cell and Molecular Biology, University of Texas at Austin, Austin, Texas 78712, USA.
authors.forename: J
authors.initials: J
authors.lastname: Lee
authors.forename: T
authors.initials: T
authors.lastname: Tonozuka
authors.forename: M
authors.initials: M
authors.lastname: Jayaram
mesh_terms: D001665:Binding Sites; D004247:DNA / Q000737:chemistry; D004254:DNA Nucleotidyltransferases / Q000737:chemistry*; D004268:DNA-Binding Proteins / Q000737:chemistry*; D046911:Macromolecular Substances; D009690:Nucleic Acid Conformation; D011995:Recombination, Genetic; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D014443:Tyrosine / Q000737:chemistry
chemical_list: D004268:DNA-Binding Proteins; D046911:Macromolecular Substances; D014443:Tyrosine; D004247:DNA; D004254:DNA Nucleotidyltransferases; C045119:FLP recombinase