Direct Observation of DNA Bending/Unbending Kinetics in Complex with DNA-Bending Protein IHF

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 103; no. 49; pp. 18515 - 18520
• Main Authors: Kuznetsov, Serguei V., Sugimura, Sawako, Vivas, Paula, Crothers, Donald M., Ansari, Anjum
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences 05-12-2006; National Acad Sciences
• Subjects: Activation energy; Bacterial proteins; Bending; Binding sites; Biological Sciences; Deoxyribonucleic acid; DNA; DNA, Bacterial - chemistry; DNA, Bacterial - metabolism; DNA, Single-Stranded - chemistry; DNA, Single-Stranded - metabolism; E coli; Emission spectra; Escherichia coli; Escherichia coli Proteins - chemistry; Escherichia coli Proteins - metabolism; Fluorescence; Fluorescence Resonance Energy Transfer; Free energy; Integration Host Factors - chemistry; Integration Host Factors - metabolism; Kinetics; Lasers; Oligomers; Reaction kinetics; Temperature measurement; Thermodynamics
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.0608394103

Regulation of gene expression involves formation of specific protein-DNA complexes in which the DNA is often bent or sharply kinked. Kinetics measurements of DNA bending when in complex with the protein are essential for understanding the molecular mechanism that leads to precise recognition of specific DNA-binding sites. Previous kinetics measurements on several DNA-bending proteins used stopped-flow techniques that have limited time resolution of few milliseconds. Here we use a nanosecond laser temperature-jump apparatus to probe, with submillisecond time resolution, the kinetics of bending/unbending of a DNA substrate bound to integration host factor (IHF), an architectural protein from Escherichia coli. The kinetics are monitored with time-resolved FRET, with the DNA substrates end-labeled with a FRET pair. The temperature-jump measurements, in combination with stopped-flow measurements, demonstrate that the binding of IHF to its cognate DNA site involves an intermediate state with straight or, possibly, partially bent DNA. The DNA bending rates range from ≈2 ms⁻¹ at ≈37°C to ≈40 ms⁻¹ at ≈10°C and correspond to an activation energy of ≈14 ± 3 kcal/mol. These rates and activation energy are similar to those of a single A:T base pair opening inside duplex DNA. Thus, our results suggest that spontaneous thermal disruption in base-paring, nucleated at an A:T site, may be sufficient to overcome the free energy barrier needed to partially bend/kink DNA before forming a tight complex with IHF.