The cleavage mode of the Cre recombinase

grantor: University of TorontoThe Cre protein, encoded by the bacteriophage P1, and the Flp protein, encoded by the 2 [mu]M plasmid found in budding yeast, belong to the Integrase family of site-specific recombinases. Cre and Flp recombinases each bind specifically to recombination targets comprised of inverted repeats of their cognate DNA binding sites, the 'lox' and FRT symmetry elements, respectively, that flank a central 8 bp spacer region. Like all integrases, both recombinases cleave one strand of the target site through a nucleophilic attack of the scissile phosphate by a conserved catalytic tyrosine residue. Cleavage generates a covalent attachment of the tyrosine to the 3'-phosphate and a free 5'-OH end. Association of two recombinase-bound target sites that have been cleaved into a synaptic complex provides the framework for the exchange of the crossing (cleaved) strands. The 3'-covalent linkage is subsequently destroyed by the nucleophilic attack of the incoming 5'-end and the DNA strands made continuous through a recombinase-mediated ligation that results in the formation of a Holliday intermediate. This Holliday junction is resolved into linear DNA products through a second set of recombinase-mediated cleavages, strand exchanges and ligations. An important issue in the chemistry of the recombination reaction is the location of the recombinase molecule that provides the catalytic nucleophile in the synapse. The mode of cleavage by a recombinase has been denoted as either occurring in 'cis,' where the cleaving monomer is bound adjacent to the scissile phosphate, or in 'trans,' where the cleaving monomer is bound elsewhere in the synaptic complex. Studies of a number of integrases have shown that the [lambda]Int and XerC/D proteins cleave in 'cis,' while the Flp recombinase cleaves in 'trans. ' I used half-site complementation to show that Cre cleaves its ' lox' target site in 'trans.' Following publication of this report, the crystal structure of the Cre synapse showed 'cis '-cleavage by Cre. To resolve this discrepancy and to answer whether my use of conditionally active sites and Cre proteins biased the results in favor of 'trans'-cleavage, I constructed novel recombinases, Fre and Clp, that were functional chimeras of the Cre and Flp proteins. I showed that these chimeric proteins had altered binding specificities compared to their respective native recombinase and designed novel specific target sites for these chimeric proteins. I used hybrid recombination sites that combined the target sites of a chimeric and native recombinase to test the mode of cleavage by Cre and Flp in conditions that did not exclude either ' cis'- or 'trans'-cleavage from occurring. I found that consistent with previous reports, Cre cleaved in 'cis' and Flp cleaved in 'trans.'Ph.D