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Updated: Jun 25, 2025
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Single-Molecule Förster Resonance Energy Transfer Methods for Real-Time Investigation of the Holliday Junction Resolution by GEN1
Published on: September 18, 2019
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Molecular Insight into the Structural Dynamics of Holliday Junctions Modulated by Integration Host Factor.
Farhana Islam1,2, Padmaja Prasad Mishra1,2
1Single Molecule Biophysics Lab, Chemical Sciences Division, Saha Institute of Nuclear Physics, 1/AF Bidhannagar, Kolkata 700064, India.
The Journal of Physical Chemistry. B
|May 29, 2024
Summary
Integration host factor (IHF) binds strongly to Holliday junctions (HJs), stabilizing their open conformation. Both wild-type IHF (WtIHF) and engineered single-chain IHF (ScIHF) recognize HJs based on their structure, impacting DNA repair and recombination.
Area of Science:
- Molecular Biology
- Structural Biology
- Biochemistry
Background:
- Integration host factor (IHF) from Escherichia coli is a nucleoid-associated protein crucial for DNA packaging, viral integration, and recombination.
- IHF binds specific DNA sequences, inducing significant DNA bending.
- Engineered single-chain IHF (ScIHF) offers biotechnological applications.
Purpose of the Study:
- To investigate the interaction between IHF variants (WtIHF and ScIHF) and Holliday junctions (HJs).
- To elucidate the structural and dynamic effects of IHF binding on HJs.
Main Methods:
- Microscale thermophoresis to quantify IHF-HJ binding affinity.
- Circular dichroism (CD) spectroscopy to assess conformational changes.
- Single-molecule Förster resonance energy transfer (smFRET) to study junction dynamics.
Main Results:
- Both WtIHF and ScIHF exhibit high affinity for HJs, indicating structure-based recognition.
- IHF binding influences the conformation and dynamics of HJs.
- IHF binding shifts the equilibrium towards and stabilizes the open conformation of HJs.
Conclusions:
- IHF possesses a robust affinity for Holliday junctions.
- IHF binding stabilizes the open conformation of HJs, impacting DNA recombination and repair processes.
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