Related Experiment Video
Updated: Jun 6, 2026
09:53
Single-Molecule Dwell-Time Analysis of Restriction Endonuclease-Mediated DNA Cleavage
Published on: February 7, 2021
Single-Molecule Visualization of DNase I-Mediated DNA Cleavage by High-Speed Atomic Force Microscopy
Nada Elessawy1, Selma Sinan2, Hongshan Zhang2
1Department of Chemistry, University of Texas at Austin, Austin, Texas 78712, United States.
ACS Chemical Biology
|June 5, 2026
Summary
High-speed atomic force microscopy visualized DNase I enzyme activity. The enzyme binds, slides along DNA, and cleaves both double-stranded DNA (dsDNA) and single-stranded DNA (ssDNA).
Area of Science:
- Biochemistry
- Molecular Biology
- Biophysics
Background:
- DNase I is a nonspecific endonuclease with known structure but poorly understood catalytic mechanism.
- Previous studies relied on indirect methods, lacking direct visualization of DNase I's cleavage process.
Purpose of the Study:
- To directly visualize the dynamic activity of DNase I on various DNA substrates.
- To elucidate the mechanism of DNA cleavage by DNase I at the single-molecule level.
Main Methods:
- High-speed atomic force microscopy (HS-AFM) was used to observe DNase I in real-time.
- Experiments were conducted on linear dsDNA, circular dsDNA plasmids, and circular ssDNA plasmids.
Main Results:
- DNase I was observed as monomers, dimers, and higher-order aggregates.
- The enzyme dynamically bound to and slid along DNA substrates.
- DNase I induced both single-strand and double-strand DNA cleavage events.
- Cleavage efficiency was significantly higher for dsDNA compared to ssDNA.
Conclusions:
- DNase I's catalytic activity is not limited to isolated monomers and involves sliding motion.
- Direct visualization provides mechanistic insights into DNase I's nucleic acid degradation.
- Findings have implications for understanding DNase I's biochemical roles and therapeutic potential.
