The Quantitative DNA Relaxation Assay is one of the most important biochemical assays used in molecular biology to evaluate the activity of human DNA topoisomerase I. This enzyme plays a critical role in maintaining DNA topology during replication, transcription, and other cellular processes. In this article, we explore in depth how to optimize detection conditions for supercoiled substrates, and how to design a reproducible, high-sensitivity assay for topoisomerase activity analysis in research labs.
What Is Human Topoisomerase I?
Human Topoisomerase I is a type IB enzyme that relaxes negative and positive DNA supercoils by making a transient single-strand break in the DNA backbone. It does not require ATP, and its activity can be easily observed by its ability to convert supercoiled DNA into relaxed forms. The gene encoding this enzyme, TOP1, has been thoroughly studied and is cataloged in resources like NCBI Gene and GeneCards.
It is widely expressed in human cells and used in laboratories for:
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Studying DNA relaxation mechanisms.
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Analyzing nucleic acid-protein interactions.
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Screening enzyme inhibitors for academic biochemical studies.
DNA Substrates for the Relaxation Assay
The assay relies on supercoiled plasmid DNA, which is commonly prepared from Escherichia coli using anion-exchange columns or silica-based kits. Supercoiling is necessary for sensitivity, as the conversion from supercoiled to relaxed forms is what the assay detects.
Preparation involves:
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Growing E. coli with a high-copy plasmid (e.g., pUC19).
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Performing plasmid DNA extraction via alkaline lysis (Harvard University).
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Purifying DNA using a spin column or CsCl gradient (Stanford.edu).
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Verifying the purity via A260/A280 UV spectrophotometry.
Check DNA form using agarose gel electrophoresis with ethidium bromide or GelRed, visualized on a UV transilluminator (Cold Spring Harbor Laboratory).
Standard Assay Setup
The typical setup for a topoisomerase I relaxation assay includes the following components:
| Reagent | Final Concentration |
|---|---|
| Supercoiled DNA | 0.5 µg per 20 µL |
| Recombinant Topoisomerase I | 1–5 units |
| 10X Relaxation Buffer | 10 mM Tris-HCl, 1 mM EDTA, 0.1% BSA |
| DTT (optional) | 1 mM |
| MgCl₂ | 5 mM (optimize from 2–10 mM) |
| Incubation Time | 30 min |
| Temperature | 37°C |
Terminate the reaction with 0.5% SDS and proteinase K, then load on a 1% agarose gel. Run at 100V for 60–90 minutes in TAE buffer, and visualize the shift from supercoiled to relaxed DNA bands.
Detailed protocol available via NEB Educational Resources.
Detection and Quantification
DNA topological forms migrate differently in agarose gel:
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Supercoiled DNA moves fastest.
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Relaxed open circular DNA migrates slower.
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Nicked linear DNA (a damage control) moves between the two.
Quantify the percentage of relaxation using ImageJ or NIH ImageJ/Fiji:
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Load gel image.
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Draw boxes around bands.
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Measure intensity using gel analysis plugin.
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Normalize total lane intensity.
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Report %Relaxed = (Relaxed Band / Total DNA) × 100.
More guidance is available from NIH Training Materials.
Optimization Strategy
1. DNA Quality
Use highly supercoiled DNA only. Degraded DNA or nicked substrates reduce sensitivity. Freshly purified plasmids yield best results.
2. Enzyme Titration
Start with 1 unit of topoisomerase and increase until full relaxation is achieved. Avoid excess enzyme which may cause smearing or relaxed band overloading.
3. Mg²⁺ Titration
Test MgCl₂ concentrations between 2–10 mM. This ion stabilizes DNA-enzyme interaction and is necessary for activity.
4. Time Course Study
Relaxation often completes within 30 min, but for kinetic analysis, run parallel reactions at 5, 10, 20, and 30 minutes.
5. Negative and Positive Controls
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No enzyme control (to confirm no endogenous activity).
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Heat-inactivated enzyme.
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Known Topo I enzyme lot (positive control).
More design details at UCSF Core Facilities.
Applications in Molecular Biology
The Topo I Relaxation Assay is used in:
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Enzyme characterization (via buffer condition screening).
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Kinetics studies (using Michaelis-Menten models).
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Chemical inhibitor evaluation (e.g., with camptothecin, PubChem).
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DNA supercoiling dynamics in chromatin (NCBI PMC).
It is also relevant in quality control for DNA production processes in labs and biotech manufacturing settings.
Data Example
To provide accurate interpretation, the following mock results can be used for standardization:
| Condition | % Relaxed DNA |
|---|---|
| 0 U Enzyme | 0% |
| 1 U Enzyme | 25% |
| 3 U Enzyme | 65% |
| 5 U Enzyme | 95% |
| 5 U + Inhibitor | 10% |
Statistical analysis with standard deviation and t-tests (using Excel or R) can validate consistency between batches (NIST.gov).
Additional Resources
Summary
The Quantitative DNA Relaxation Assay using Human Topoisomerase I is a robust and essential tool for researchers studying DNA enzyme activity. The proper optimization of:
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Substrate purity
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Enzyme concentration
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Ion balance (Mg²⁺)
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Time and temperature
…allows reproducible and sensitive detection of topoisomerase-mediated relaxation of DNA supercoils.
This assay is easy to implement, requires no advanced instrumentation, and is widely compatible with standard gel documentation systems. With correct planning and high-purity reagents, this method is a gold standard for evaluating topological changes in DNA in a wide range of research fields.