title: Real-time single-molecule tethered particle motion analysis reveals mechanistic similarities and contrasts of Flp site-specific recombinase with Cre and λ Int.
abstract: Flp, a tyrosine site-specific recombinase coded for by the selfish two micron plasmid of Saccharomyces cerevisiae, plays a central role in the maintenance of plasmid copy number. The Flp recombination system can be manipulated to bring about a variety of targeted DNA rearrangements in its native host and under non-native biological contexts. We have performed an exhaustive analysis of the Flp recombination pathway from start to finish by using single-molecule tethered particle motion (TPM). The recombination reaction is characterized by its early commitment and high efficiency, with only minor detraction from 'non-productive' and 'wayward' complexes. The recombination synapse is stabilized by strand cleavage, presumably by promoting the establishment of functional interfaces between adjacent Flp monomers. Formation of the Holliday junction intermediate poses a rate-limiting barrier to the overall reaction. Isomerization of the junction to the conformation favoring its resolution in the recombinant mode is not a slow step. Consistent with the completion of nearly every initiated reaction, the chemical steps of strand cleavage and exchange are not reversible during a recombination event. Our findings demonstrate similarities and differences between Flp and the mechanistically related recombinases λ Int and Cre. The commitment and directionality of Flp recombination revealed by TPM is consistent with the physiological role of Flp in amplifying plasmid DNA.
authors.affiliation: Department of Life Sciences and Institute of Genome Sciences, National Yang-Ming University, Taipei 112, Taiwan. hffan2@ym.edu.tw
authors.forename: Hsiu-Fang
authors.initials: HF
authors.lastname: Fan
authors.forename: Chien-Hui
authors.initials: CH
authors.lastname: Ma
authors.forename: Makkuni
authors.initials: M
authors.lastname: Jayaram
mesh_terms: D010582:Bacteriophage lambda / Q000201:enzymology; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D053837:DNA Cleavage; D004254:DNA Nucleotidyltransferases / Q000378:metabolism*; D019426:Integrases / Q000378:metabolism; D007700:Kinetics; D009038:Motion; D011995:Recombination, Genetic; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism; D013816:Thermodynamics
chemical_list: D029701:Saccharomyces cerevisiae Proteins; D004247:DNA; C045073:Cre recombinase; D004254:DNA Nucleotidyltransferases; C045119:FLP recombinase; D019426:Integrases