Binding and Bending Parameters of Integration Host Factor to Four-Way Holliday Junction

Abstract

Binding and Bending Parameters of Integration Host Factor to Four-Way Holliday JunctionIntegration 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.