Elucidation of Interactions Between Integration Host Factor and a DNA Four-Way Junction

Integration host factor (IHF) is an E. coli nucleoid-associated protein that was initially discovered in the integration of bacterial phage DNA. IHF binds with nanomolar affinity to duplex DNA containing a 13 bp consensus sequence and induces a bend of ~160° upon binding. Our previous results have shown that IHF binds to DNA 4WJ that do not contain the consensus sequence with high affinity. DNA Four-Way Junctions (4WJ) are important intermediates in double-strand break repair and homologous recombination. These junctions can adopt either an open or stacked conformation, in which the open conformation facilitates the process of branch migration and strand exchange. In this study, we investigated the IHF-induced changes in the population distribution of junction conformations, using ensemble and single molecule Förster resonance energy transfer (FRET) methods. Our steady state and time-resolved FRET data indicated that under conditions stabilizing the stacked junction conformation, upon binding, IHF induces the open conformation of junction. Single-molecule FRET (smFRET) experiments, performed with total internal reflection fluorescence microscopy, measured population distributions of the junction and showed clear opening of the junctions upon IHF binding. Further analysis of smFRET dynamics revealed that even in the presence of protein, the junctions remain dynamic as fast transitions are observed for the protein-bound open state. Cross correlation analyses reveal that the protein slows down the transition rate at 1 mM Mg2+ but accelerates the transition rate at 10 mM Mg2+, consistent with stabilization of the open conformation. These findings which show that IHF binds to 4WJ with high affinity, induces the open conformation and alters conformational dynamics, suggest that IHF may play multiple roles in the processes of integration and recombination.