title: Measurements of DNA-loop formation via Cre-mediated recombination.
abstract: The Cre-recombination system has become an important tool for genetic manipulation of higher organisms and a model for site-specific DNA-recombination mechanisms employed by the λ-Int superfamily of recombinases. We report a novel quantitative approach for characterizing the probability of DNA-loop formation in solution using time-dependent ensemble Förster resonance energy transfer measurements of intra- and inter-molecular Cre-recombination kinetics. Our method uses an innovative technique for incorporating multiple covalent modifications at specific sites in covalently closed DNA. Because the mechanism of Cre recombinase does not conform to a simple kinetic scheme, we employ numerical methods to extract rate constants for fundamental steps that pertain to Cre-mediated loop closure. Cre recombination does not require accessory proteins, DNA supercoiling or particular metal-ion cofactors and is thus a highly flexible system for quantitatively analyzing DNA-loop formation in vitro and in vivo.
authors.affiliation: Department of Molecular and Cell Biology, University of Texas at Dallas, 800 West Campbell Road, Richardson, TX 75080, USA.
authors.forename: Massa J
authors.initials: MJ
authors.lastname: Shoura
authors.forename: Alexandre A
authors.initials: AA
authors.lastname: Vetcher
authors.forename: Stefan M
authors.initials: SM
authors.lastname: Giovan
authors.forename: Farah
authors.initials: F
authors.lastname: Bardai
authors.forename: Anusha
authors.initials: A
authors.lastname: Bharadwaj
authors.forename: Matthew R
authors.initials: MR
authors.lastname: Kesinger
authors.forename: Stephen D
authors.initials: SD
authors.lastname: Levene
mesh_terms: D004247:DNA / Q000737:chemistry*; D031541:Fluorescence Resonance Energy Transfer; D019426:Integrases / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D011995:Recombination, Genetic
chemical_list: D004247:DNA; C045073:Cre recombinase; D019426:Integrases