Analysis of Cre-loxP interaction by surface plasmon resonance: influence of spermidine on cooperativity.
To study target site selectivity of one important class of DNA-binding proteins, site-specific DNA recombinases, we developed an automated real-time kinetic assay based on surface plasmon resonance (BIACORE) and formulated a curve-fitting model that takes into account cooperative interactions. Monitoring the interaction between the Cre DNA recombinase and its specific target site loxP by BIACORE, we found that Cre associates with loxP tightly and highly cooperatively. We observed that the cooperative moment of the Cre-loxP interaction is strongly dependent on the concentration of spermidine, a small polyamine influencing DNA conformation. Thus, DNA conformation can have a profound impact on substrate recognition and subsequent recombination.
Rüfer
Andreas
A
Oklahoma Medical Research Foundation, Oklahoma City 73104, USA.
Neuenschwander
Pierre F
PF
Sauer
Brian
B
D001483:Base Sequence; D001665:Binding Sites; D003198:Computer Simulation; D019426:Integrases / Q000378:metabolism*; D007700:Kinetics; D008024:Ligands; D008954:Models, Biological; D009690:Nucleic Acid Conformation; D011995:Recombination, Genetic; D012091:Repetitive Sequences, Nucleic Acid; D013095:Spermidine / Q000494:pharmacology*; D020349:Surface Plasmon Resonance; D014764:Viral Proteins / Q000378:metabolism*
D008024:Ligands; D014764:Viral Proteins; C045073:Cre recombinase; D019426:Integrases; D013095:Spermidine