Unveiling the Holliday junction conformational dynamics of upon binding with DNA architectural proteins IHF; A Single molecule FRET approach

Abstract

AbstractIntegration host factor (IHF) ofE. coliis a nucleoid-associated protein with diverse roles in DNA packaging, viral DNA integration, and recombination. IHF binds to duplex DNA containing a 13 bp consensus sequence with nanomolar affinity and induces a significant bend of approximately 160° upon binding. While the Wild type IHF (WtIHF) is involved in DNA bending, thereby facilitating the integration of foreign DNA into the host genome, its engineered version, Single chain IHF (ScIHF) was designed for specific genetic engineering and biotechnological applications. We investigated interaction of the two IHF variants with Holliday junctions (HJ), crucial intermediates in DNA repair and homologous recombination. Our finding demonstrate that both variant of IHF binds to HJs with high affinity in presence of the consensus sequence, indicating a structure-based recognition mechanism. HJs are dynamic structures that can adopt open or stacked conformation. The open conformation facilitates processes like branch migration and strand exchange. Through quantitative binding studies using microscale thermophoresis, we determined the binding of IHF to four-way DNA junctions that harboured two specific binding sequences H’ & H1. Circular dichroism (CD) experiments revealed the protein’s impact on the junction conformation. This was further confirmed by Single molecule Förster resonance energy transfer (smFRET) technique that was used to examine the binding of IHF to the junction and its effect on the dynamicity of junction conformation. We also probed the population distribution of junction conformations. Interestingly, our results revealed that binding of both WtIHF & ScIHF shifts the population towards the open conformation of the junction and stabilised it in that conformation. In summary, our findings demonstrate that IHF binds HJs with a strong affinity and has a stabilizing effect on maintaining the junction’s open conformation.