Mechanical conversion of low-affinity Integration Host Factor binding sites into high-affinity sites

TL;DR

This study reveals how mechanical bending forces on DNA significantly enhance the binding affinity of the IHF protein, especially at weak binding sites, highlighting the importance of DNA mechanics in protein-DNA interactions.

Contribution

It demonstrates that small DNA bending forces can substantially increase IHF binding affinity, even at non-consensus sites, providing new insights into DNA-protein interaction regulation.

Findings

Small bending forces increase IHF binding affinity.

Bending beyond ~3 pN saturates the effect.

Mechanically bent weak sites bind more tightly than linear high-affinity sites.

Abstract

Although DNA is often bent in vivo, it is unclear how DNA-bending forces modulate DNA-protein binding affinity. Here, we report how a range of DNA-bending forces modulates the binding of the Integration Host Factor (IHF) protein to various DNAs. Using solution fluorimetry and electrophoretic mobility shift assays, we measured the affinity of IHF for DNAs with different bending forces and sequence mutations. Bending force was adjusted by varying the fraction of double-stranded DNA in a circular substrate, or by changing the overall size of the circle (1). DNA constructs contained a pair of Forster Resonance Energy Transfer dyes that served as probes for affinity assays, and read out bending forces measured by optical force sensors (2). Small bending forces significantly increased binding affinity; this effect saturated beyond ~3 pN. Surprisingly, when DNA sequences that bound IHF only weakly were mechanically bent by circularization, they bound IHF more tightly than the linear "high-affinity" binding sequence. These findings demonstrate that small bending forces can greatly augment binding at sites that deviate from a protein's consensus binding sequence. Since cellular DNA is subject to mechanical deformation and condensation, affinities of architectural proteins determined in vitro using short linear DNAs may not reflect in vivo affinities.