title: Trans complementation of variant Cre proteins for defects in cleavage and synapsis.
abstract: The Cre recombinase is a member of the integrase family of conservative site-specific recombinases. These proteins share five conserved catalytic residues, one of which is a tyrosine that acts as the nucleophile to attack the scissile phosphodiester bond in the DNA target. Recombination by the Cre recombinase takes place in a supramolecular structure called a synapse that consists of four molecules of Cre bound to two DNA target sequences called lox sites. The synapse is held together by an intricate network of protein-protein interactions. They bend the two sites into square planar structure that resembles a Holliday intermediate. We have studied three mutant Cre proteins that appear to have defects in synapsis (Cre A36V, Cre T41F, and Cre G314R). We found that they were unable to carry out strand cleavage but that cleavage occurred if they were mixed with a cleavage-defective Cre protein that lacks the catalytic nucleophilic tyrosine residue. The three variant proteins could also be complemented for the formation of a novel structure ("complexV"), which may be a cleaved synaptic intermediate. We suggest that these three mutant proteins have a defect in DNA bending and discuss the relationship between bending, synapsis, and cleavage.
authors.affiliation: Department of Molecular and Medical Genetics, University of Toronto, Toronto M5S 1A8, Canada.
authors.forename: A C
authors.initials: AC
authors.lastname: Shaikh
authors.forename: P D
authors.initials: PD
authors.lastname: Sadowski
mesh_terms: D004247:DNA / Q000737:chemistry; D005816:Genetic Complementation Test; D019426:Integrases / Q000235:genetics* / Q000378:metabolism*; D008957:Models, Genetic; D009154:Mutation; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011995:Recombination, Genetic; D014764:Viral Proteins
chemical_list: D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; D004247:DNA; C045073:Cre recombinase; D019426:Integrases