Integration host factor (IHF) is a nucleoid-associated protein involved in DNA packaging, integration of viral DNA and recombination. IHF binds with nanomolar affinity to duplex DNA containing a 13 bp consensus sequence, inducing a bend of ~160° upon binding. We determined that IHF binds to DNA Four-way or Holliday junctions (HJ) with high affinity regardless of the presence of the consensus sequence, signifying a structure-based mechanism of recognition. Junctions, important intermediates in DNA repair and homologous recombination, are dynamic and can adopt either an open or stacked conformation, where the open conformation facilitates branch migration and strand exchange. Using ensemble and single molecule Förster resonance energy transfer (FRET) methods, we investigated IHF-induced changes in the population distribution of junction conformations and determined that IHF binding shifts the population to the open conformation. 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. Protein binding alters junction conformational dynamics, as cross correlation analyses reveal the protein slows the transition rate at 1 mM Mg2+ but accelerates the transition rate at 10 mM Mg2+. Stopped flow kinetic experiments provide evidence for two binding steps, a rapid, initial binding step followed by a slower step potentially associated with a conformational change. These measurements also confirm that the protein remains bound to the junction during the conformer transitions and further suggest that the protein forms a partially dissociated state that allows junction arms to be dynamic. These findings, which demonstrate that IHF binds HJs with high affinity and stabilizes junctions in the open conformation, suggest that IHF may play multiple roles in the processes of integration and recombination in addition to stabilizing bacterial biofilms.
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.
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