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Updated: May 15, 2026
11:27
Studying DNA Looping by Single-Molecule FRET
Published on: June 28, 2014
The interplay between supercoiling and DNA modifying enzymes at the single-molecule level.
Elise M Wilkinson1, Antoine M van Oijen1, Timothy D Craggs2
1Molecular Horizons and School of Science, University of Wollongong, Wollongong, NSW, Australia.
Scientific Reports
|May 13, 2026
Summary
DNA supercoiling, the twisting of DNA, affects enzyme activity. Studies show supercoiling enhances restriction enzyme cleavage efficiency, revealing DNA topology
Area of Science:
- Molecular Biology
- Biophysics
- Genetics
Background:
- DNA supercoiling is crucial for DNA processes but often ignored in relaxed DNA studies.
- Plectonemes are supercoiled structures formed by DNA overwinding or underwinding.
- Understanding supercoiling's role is vital for comprehending DNA-enzyme interactions.
Purpose of the Study:
- To investigate the effect of DNA supercoiling on enzyme activity at the single-molecule level.
- To visualize and analyze DNA supercoiling dynamics and plectoneme formation.
- To determine how DNA topology influences restriction enzyme function.
Main Methods:
- Construction of topologically constrained 18-kb linear DNA substrates.
- Induction of positive and negative supercoiling using intercalating dyes.
- Single-molecule observation using Total Internal Reflection Fluorescence (TIRF) microscopy.
- Assessing the activity of the restriction enzyme EcoRV on supercoiled DNA.
Main Results:
- Direct visualization of plectonemes diffusing on DNA.
- Plectonemes were observed to be stationary at EcoRV binding sites.
- EcoRV exhibited increased cleavage efficiency on supercoiled DNA, with unaffected binding.
- Supercoiling was found to enhance the kinetic interplay between DNA topology and enzyme activity.
Conclusions:
- DNA supercoiling significantly modulates the activity of DNA modifying enzymes like EcoRV.
- Supercoiling may make DNA cleavage more energetically favorable for enzymes that bend DNA.
- This study highlights the importance of DNA topology in regulating enzymatic processes.
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