Phage lambda integrase (Int) is the archetype of a large family of site-specific DNA recombinases that function in the segregation of plasmids, viral and cellular chromosomes, the regulation of gene expression, and programmed gene rearrangements. These enzymes catalyze DNA cleavage and religation without the addition of high energy cofactors. They are the only enzymes known to both create branched Holliday junction intermediates and to resolve them into recombinant DNA duplexes. Recent crystal structures of the related P 1 Cre recombinase and the yeast Flp protein complexed to Holliday junctions have provided many insights into the recombination reaction. In contrast to these simple recombinases, lambda Int functions in higher order structures comprising multiple DNA sites and accessory factors that bend the DNA into a compact shape. These additional interactions, which are essential for Int-catalyzed recombination in vivo, allosterically regulate the efficiency and fidelity of DNA cleavage and strand transfer. We have determined a crystal structure of a covalent Int-DNA complex that, in comparison to an earlier structure of unbound Int, reveals a DNA-mediated switch in the structure of the enzyme active site. We now propose crystallographic studies of the higher order Int-DNA complexes that will address the physical basis for the allosteric regulation of recombination through interactions of Int's two autonomous DNA binding domains. Diffracting crystals of several of these larger Int-Holliday junction complexes have been grown and isomorphous heavy atom derivatives have been identified. The proposed crystal structure determinations, together with site-directed mutational studies of the protein subunit interfaces in these complexes, will address how DNA cleavage and ligation activities are regulated by the physical organization of the Int-DNA complexes. A physical description of the enzymatic processing of Holliday junctions and other types of DNA recombination joints is a key to mechanistic understanding of a variety of biological processes that maintain chromosome structure or create genetic diversity. The lambda Int recombination system is a model for studying the chemistry of the trans-esterification reactions and the orchestration of pairwise DNA strand exchanges that create the Holliday junction intermediate and then resolve it into recombinant DNA products.
