Comparative kinetic analysis of FLP and cre recombinases: mathematical models for DNA binding and recombination.
The integrase class site specific recombinases FLP from Saccharomyces cerevisiae, and Cre from bacteriophage P1, have been extensively used to direct DNA rearrangements in heterologous organisms. Although their reaction mechanisms have been relatively well characterised, little comparative analysis of the two enzymes has been published. We present a comparative kinetic analysis of FLP and Cre, which identifies important differences. Gel mobility shift assays show that Cre has a higher affinity for its target, loxP (7. 4x10(10) M-1), than FLP for its target, FRT (8.92x10(8) M-1). We show that both recombinases bind the two halves of their target sites cooperatively, and that Cre shows approximately threefold higher cooperativity than FLP. Using a mathematical model describing the sequential binding of recombinase monomers to DNA, we have determined values for the association and dissociation rate constants for FLP and Cre.FLP and Cre also showed different characteristics in in vitro recombination assays. In particular, approximately tenfold more active FLP was required than Cre to optimally recombine a given quantity of excision substrate. FLP was able to reach maximum excision levels approaching 100%, whilst Cre-mediated excision did not exceed 75%. To investigate possible reasons for these differences a mathematical model describing the excision recombination reaction was established. Using measured DNA binding parameters for FLP and Cre in the model, and comparing simulated and experimental recombination data, the values of the remaining unknown parameters were determined. This analysis indicates that the synaptic complex is more stable for Cre than for FLP.
Ringrose
L
L
EMBL, Gene Expression Programme, Meyerhofstr. 1, Heidelberg, 69117, Germany.
Lounnas
V
V
Ehrlich
L
L
Buchholz
F
F
Wade
R
R
Stewart
A F
AF
D000494:Allosteric Regulation; D017101:Bacteriophage P1 / Q000201:enzymology; D002474:Cell-Free System; D004254:DNA Nucleotidyltransferases / Q000378:metabolism*; D005656:Fungal Proteins / Q000378:metabolism; D019426:Integrases / Q000378:metabolism*; D007700:Kinetics; D008962:Models, Theoretical; D011485:Protein Binding; D011995:Recombination, Genetic; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D014764:Viral Proteins / Q000378:metabolism
D005656:Fungal Proteins; D014764:Viral Proteins; C045073:Cre recombinase; D004254:DNA Nucleotidyltransferases; C045119:FLP recombinase; D019426:Integrases