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.
Highlights
The Cre recombinase is one of the members of the integrase family of site-specific recombinases
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
Two Cre variants, Cre A36V and Cre T41F are recombination-defective. These Cre proteins bind to the lox site, but fail to generate higher order complexes indicative of synapsis [17, 22]
Summary
V36A ϩ CNS F41T ϩ CNS RG314 ϩ CTX TA312 ϩ CTX a Primers used to construct variant Cre genes. b In CTX, the underlined nucleotides indicate the XhoI site. V36A ϩ CNS F41T ϩ CNS RG314 ϩ CTX TA312 ϩ CTX a Primers used to construct variant Cre genes. B In CTX, the underlined nucleotides indicate the XhoI site. The bold letters indicate the sites of the mutations. C The primer pairs used to amplify the PCR1 and PCR2 fragments are shown for each of the mutations The bold letters indicate the sites of the mutations. c The primer pairs used to amplify the PCR1 and PCR2 fragments are shown for each of the mutations
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