Site-specific recombination of asymmetric lox sites mediated by a heterotetrameric Cre recombinase complex.
Previous reports have demonstrated that new Cre recombinase specificities can be developed for symmetrically designed lox mutants through directed evolution. The development of Cre variants that allow the recombination of true asymmetric lox mutant sites has not yet been addressed, however. In the present study, we demonstrate that a mixture of two different site-specific Cre recombinase molecules (wt Cre and a mutant Cre) catalyzes efficient recombination between two asymmetric lox sites in vitro, presumably via formation of a functionally active heterotetrameric complex. The results may broaden the application of site-specific recombination in basic and applied research, including the custom-design of recombinases for natural, asymmetric, and lox-related target sequences present in the genome. Future applications may potentially include genomic manipulations, for example, site-specific integrations, deletions or substitutions within precise regions of the genomes of mammalians and other organisms.
Saraf-Levy
Talia
T
Institute of Plant Sciences, A.R.O, The Volcani Center, PO Box 6, Bet-Dagan 50250, Israel.
Santoro
Stephen W
SW
Volpin
Hanne
H
Kushnirsky
Tzvika
T
Eyal
Yoram
Y
Schultz
Peter G
PG
Gidoni
David
D
Carmi
Nir
N
D000818:Animals; D001483:Base Sequence; D003198:Computer Simulation; D004254:DNA Nucleotidyltransferases / Q000235:genetics* / Q000378:metabolism*; D006801:Humans; D019426:Integrases / Q000235:genetics* / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D011485:Protein Binding; D051381:Rats; D013379:Substrate Specificity; D014764:Viral Proteins / Q000235:genetics* / Q000378:metabolism*
D014764:Viral Proteins; C045073:Cre recombinase; D004254:DNA Nucleotidyltransferases; D019426:Integrases; C077570:Site-specific recombinase