Characterization of IHF Binding to DNA Four-Way Junctions and Forks

Abstract

Integration Host Factor (IHF) is an architectural protein that binds and bends DNA, facilitating the formation of protein-DNA complexes important for gene regulation. IHF binds with high affinity to a specific consensus sequence and induces a 160° bend upon binding. We have shown that IHF binds DNA four way junctions (4WJ) that do not contain the consensus sequence with nanomolar affinity and 1:1 stoichiometry for the specific interaction. We have also observed that IHF binds DNA forks with nanomolar affinity. The binding to junctions and forks is in direct contrast to IHF binding to linear duplex DNA, which is typically 1000-fold weaker. In this study we investigate whether the presence of the IHF consensus sequence influences IHF binding to DNA junctions and forks. We utilized gel shift and fluorescence binding assays to measure affinity and have observed that the high affinity for these non-native structures is independent of the presence of the consensus sequence. We are further exploring how IHF binding influences junction conformation. Junction conformation is modulated by ion concentration where high concentrations of ions induces pairwise stacking of the helical arms resulting in quasi-continuous helices. In our investigations IHF binding to the non-consensus junction induces an open conformation. We are specifically examining the distortion of the junction and DNA fork substrates upon IHF binding using Forster Resonance Energy Transfer. Through these measurements, we are also exploring whether the mechanism of recognition differs between junctions and forks.