Coordination and catalysis of the lambda integrase site -specific recombination reaction

Site-specific recombination catalyzed by bacteriophage lambda Integrase (Int) is essential for establishment and termination of the viral lysogenic lifecycle. Int is the archetype of the tyrosine recombinase family whose members are responsible for DNA rearrangement in prokaryotes, eukaryotes, and viruses. Mechanisms regulating catalytic activity during recombination were difficult to interpret because all but one Int mutant to date possessed pleiotropic phenotypes. Studies of tyrosine recombinases bound to their target substrates suggested that the carboxyl termini of the proteins are involved in protein-protein contacts that control the timing of DNA cleavage events during recombination. I investigated an Int truncation mutant (W350) that possesses enhanced topoisomerase activity but greater than 100-fold reduced recombination activity. Synthesis of mutants at the carboxyl terminus indicated that W350A and I353A possess specific deficiencies in site-specific recombination although DNA binding, cleavage and ligation activities are wild-type. Int mutants R346A and R348A are deficient solely in the ability to cleave DNA. To explain these results, a homology-threaded model of the Int structure using a Cre crystal structure was constructed. I propose that residues R346 and R348 are involved in activation of the phosphodiester substrate or orientation of the catalytic tyrosine that cleaves DNA, whereas W350 and I353 control and make intermolecular contacts with other Int proteins in the higher-order recombination structures known as intasomes. These results suggest that Int and the other tyrosine recombinases have evolved regulatory contacts that coordinate site-specific recombination at the carboxyl terminus.;In the course of my doctoral work, I have developed quantitative assays to measure the maximal cleavage and ligation activity of Integrase. From this work, we concluded that para-Nitrophenol tyrosine analogs are optimal for quantitative in vitro measurement of Int ligation. Int could not utilize tyrosine analogs para-Cresol and dimethyl- p-phenylenediamine as ligation substrates under my reaction conditions. However, they may be useful to identify and characterize Int or other tyrosine recombinase proteins with enhanced ligation activities.