This project seeks to dissect the biochemical mechanism by which bacteriophage lambda integrates its DNA into the chromosome of its E. coli host. A hallmark of lambda integration is that it is site-specific. One element that contributes to this specificity is the requirement for perfect homology between attachment site partners. We constructed an attachment site in which the two strands of DNA are covalently held together by a crosslink. This substrate initiates recombination normally but is blocked precisely at the step at which homology is important. The result distinguishes between plectonemic and paranemic mechanisms for homologous interactions. We have also investigated the structure and function of IHF, a protein that assists recombination. Experiments in which artificial bends are introduced in place of binding sites for the protein have implicated deformation of DNA as an important function for IHF. In order to rule out adventitious binding of IHF to bend-swap chimeras and to test for roles of IHF in addition to DNA bending, we have developed an IHF-independent recombination system. The ability of the chimeras to function in the absence of IHF proves that a major role of this protein is to bend DNA. To identify amino acid residues of IHF that contact DNA, we isolated mutants that have lost IHF function but retained the basic elements of structure. These mutants are defective in binding for DNA and cluster in a small segment of the protein.
