Deriving kinetic constants for Holliday junction resolution by phage lambda Integrase and implications for the mechanism of site-specific recombination

Bacteriophage lambda uses site specific recombination to integrate its DNA into the chromosome of its bacterial host. Site-specific recombination is performed by a phage-encoded enzyme, Integrase, and proceeds via an intermediate known as a Holliday junction. First, the two recombining DNA substrates, which we collectively call A, undergo one round of catalytic events – DNA cleavage, strand exchange, and ligation – to generate the first Holliday junction isomer, which we call B. This initial Holliday junction isomerizes to a different conformation, which we call C. Finally, the C Holliday junction undergoes another round of catalytic events to generate the two products, which we collectively call E.  Our hypothesis is that isomerization of the junction determines the efficiency of recombination. We used three Holliday junctions to follow the time course and extent of resolution: the wild type version and two whose conformation is biased towards either the B or the C conformation. We also designed a system of four differential equations to fit to the experimental data. Parameters in the model are kinetic constants and their values are selected by minimizing the sum of squared errors between the measurements and the predictions of concentrations of four chemical species (A, B, C, and E) as a function of time. We extracted kinetic constants and obtained a ratio of C to B isomers for the three different Holliday Junctions. We are now developing ways to distinguish between the C and B isomers, in order to test the predictions of our model.