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.

Holliday or DNA four-way junctions are important intermediates in recombination and repair processes. These structures can change conformation rapidly in solution interconverting from an open, four-fold symmetrical structure, with no central base stacking to one in which coaxial stacking of the helical arms has been observed. The open structure, which is capable of branch migration, is functionally relevant; however, many proteins have been observed to bind to and stabilize the stacked form. Our investigations have focused on elucidating the conformational changes induced by the binding of ions and the architectural proteins, HU and IHF, to improve understanding of the stacked form of the junction. Using fluorescence methods, we have examined the ion-binding site and have investigated the coordination of ions in the central region of the junction. FÅrster resonance energy transfer (FRET) experiments have revealed that the degree of stacking or interduplex angle (IDA) of the junction is modulated by ion size, where larger ionic radii lead to larger IDAs. Ion luminescence measurements indicate that minimally two ions are needed to induce junction stacking. The proteins, HU and IHF, stabilize the junction in the stacked conformation and induce a greater degree of stacking upon binding. In contrast, the repair protein, Msh2-Msh6, induces the junction to adopt an open conformation. All three proteins recognize and bind to the junction structure with nanomolar affinity and interestingly, Msh2-Msh6 binds the junction with higher affinity than a mismatch site. A FRET-mapping approach has been employed to determine the location of HU on the junction and indicates that HU binds to the central region of the junction. We have utilized molecular dynamics simulations coupled with the fluorescence information to generate several models for the protein-junction complex and propose a novel structural model for the interaction.

Binding and Bending Parameters of Integration Host Factor to Four-Way Holliday Junction Integration host factor (IHF) is a small heterodimeric protein that sequence specifically binds the minor groove of DNA and facilitates a bend of nearly 180 degrees. This bending is crucial for cellular processes such as recombination, replication and transcription. Previous work in the Mukerji lab characterized the binding properties of the structurally similar but sequence non-specific DNA-binding protein HU, to duplex and Holliday junction DNA. This research demonstrated that HU binds to the central region of the junction with nanomolar affinity and prefers the stacked form of the Holliday junction. Given the similarities in structure and function of HU and IHF, we elected to study the binding capability of IHF to the Holliday junction. We have compared the binding affinity of IHF for Holliday junction DNA with its binding affinity to duplex DNA with and without a consensus sequence. Binding measurements were performed using florescence intensity and anisotropy methods and confirmed with the gel mobility shift assay. All binding assays established that IHF binds to the Holliday junction lacking a consensus sequence with high affinity (∼3 nanomolar Kd) similar to HU, suggesting that both proteins might recognize and bind similar structural aspects of the Holliday junction. Moreover, anisotropy affinity measurements demonstrated that IHF binds the Holliday junction with similar affinity as it does to duplex DNA containing its consensus sequence. The span of binding capabilities exhibited by IHF indicate that it can function both as a specific and non-sequence specific DNA binding protein.