title: Asymmetry in Flp-mediated cleavage.
abstract: Flp is a member of the integrase family of site-specific recombinases. Members of the integrase family mediate DNA strand cleavage via a transesterification reaction involving an active site tyrosine residue. The first step of the reaction results in covalent linkage of the protein to the 3'-phosphoryl DNA terminus, leaving a 5'-hydroxyl group at the site of the nick. We have used Flp recognition target (FRT) sites containing a 5'-bridging phosphorothioate linkage at the site of Flp cleavage to accumulate intermediates in which Flp is covalently bound at a cleavage site. We have probed these intermediates with dimethylsulfate using methylation protection and find that Flp-mediated cleavage is associated with protection of two adenine residues that are opposite the sites of cleavage and covalent attachment by Flp. Methylation interference studies showed that cleavage and covalent attachment are also accompanied by differences in the contacts of Flp with each of the two cleavage sites and with the surrounding symmetry elements. Therefore, we provide evidence that Flp-mediated cleavage and covalent attachment result in changes to the conformation of the Flp-FRT complex. These changes may be required for Flp-mediated strand exchange activity.
authors.affiliation: Department of Medical Genetics and Microbiology, University of Toronto, MSB, Toronto, Ontario M5S 1A8, Canada.
authors.forename: K H
authors.initials: KH
authors.lastname: Luetke
authors.forename: B P
authors.initials: BP
authors.lastname: Zhao
authors.forename: P D
authors.initials: PD
authors.lastname: Sadowski
mesh_terms: D000225:Adenine; D004247:DNA / Q000378:metabolism*; D019175:DNA Methylation; D004254:DNA Nucleotidyltransferases / Q000378:metabolism*; D006147:Guanine; D006358:Hot Temperature; D009838:Oligodeoxyribonucleotides; D013379:Substrate Specificity; D013873:Thionucleotides
chemical_list: D009838:Oligodeoxyribonucleotides; D013873:Thionucleotides; D006147:Guanine; D004247:DNA; D004254:DNA Nucleotidyltransferases; C045119:FLP recombinase; D000225:Adenine