The Binding Parameters of Integration Host Factor to the Lambda Phage Cosi1 Site and Holliday Junction DNA

Abstract

Integration Host Factor (IHF) is a heterodimeric DNA-binding protein produced by E.coli. IHF interacts with the minor groove and phosphodiester backbone of the DNA to create sharp bends in the DNA and is often referred to as an architectural protein. Although the protein binds sequence-specifically, it also recognizes DNA conformation. Prokaryotic cellular processes involving IHF include viral integration, replication and activation of gene expression. We have recently shown that IHF exhibits high binding affinity for the Holliday or four-way junction DNA (4WJ), an intermediate in homologous recombination where strands from different chromosomes cross over. Förster Resonance Energy Transfer (FRET), where the 5'-ends of DNA strands are labeled with fluorophores, is used to determine the conformational changes of the 4WJ when interacting with DNA-binding proteins. These measurements have shown that the 4WJ opens upon binding with IHF, which is consistent with data promoting the protein's involvement in recombinase action. Subsequent FRET experiments performed confirm this observation, and provide a value for the efficiency of binding and the degree of distortion induced. Interactions between IHF and the cos I1 site of DNA from the lambda bacteriophage will also be explored. It is known that IHF induces bending at cos sites. The affinity of binding to Δ cos DNA is compared to that between IHF and its consensus sequence (H1) in E. coli DNA. Gel mobility shift assay (GMSA) and solutions experiments will measure the binding and affinity of IHF with the cos I1 site. FRET experiments with labeled cosI1 DNA will reveal the distortion induced upon IHF binding and these results will be compared to those with IHF binding to the H1 site.