title: Directional resolution of synthetic holliday structures by the Cre recombinase.
abstract: The Cre recombinase of bacteriophage P1 cleaves its target site, loxP, in a defined order. Recombination is initiated on one pair of strands to form a Holliday intermediate, which is then resolved by cleavage and exchange of the other pair of strands to yield recombinant products. To investigate the influence of the loxP sequence on the directionality of resolution, we constructed synthetic Holliday (chi) structures containing either wild-type or mutant lox sites. We found that Cre preferentially resolved the synthetic wild-type chi structures on a particular pair of strands. The bias in the direction of resolution was dictated by the asymmetric loxP sequence since the resolution bias was abolished with symmetric lox sites. Systematic substitutions of the loxP site revealed that the bases immediately 5' to the scissile phosphodiester bonds were primarily responsible for the directionality of resolution. Interchanging these base pairs was sufficient to reverse the resolution bias. The Cre-lox co-crystal structures show that Lys(86) makes a base-specific contact with guanine immediately 5' to one of the scissile phosphates. Substituting Lys(86) with alanine resulted in a reduction of the resolution bias, indicating that this amino acid is important for establishing the bias in resolution.
authors.affiliation: Department of Molecular and Medical Genetics, University of Toronto, Toronto, Ontario M5S 1A8, Canada.
authors.forename: L
authors.initials: L
authors.lastname: Lee
authors.forename: P D
authors.initials: PD
authors.lastname: Sadowski
mesh_terms: D001483:Base Sequence; D004247:DNA / Q000096:biosynthesis / Q000737:chemistry*; D019426:Integrases / Q000737:chemistry / Q000502:physiology*; D008969:Molecular Sequence Data; D009154:Mutation; D011995:Recombination, Genetic; D013329:Structure-Activity Relationship; D014764:Viral Proteins / Q000737:chemistry / Q000502:physiology*
chemical_list: D014764:Viral Proteins; D004247:DNA; C045073:Cre recombinase; D019426:Integrases