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Updated: May 21, 2026
Simple and Fast Rolling Circle Amplification-Based Detection of Topoisomerase 1 Activity in Crude Biological Samples
Published on: December 2, 2022
Topoisomerase assays
John L Nitiss1, Eroica Soans, Anna Rogojina
1Molecular Pharmacology Department, St. Jude Children's Research Hospital, Memphis, Tennessee, USA.
Abstract:
Topoisomerases are nuclear enzymes that play essential roles in DNA replication, transcription, chromosome segregation, and recombination. All cells have two major forms of topoisomerases: type I enzymes, which make single-stranded cuts in DNA, and type II enzymes, which cut and pass double-stranded DNA. DNA topoisomerases are important targets of approved and experimental anti-cancer agents. The protocols described in this unit are for assays used to assess new chemical entities for their ability to inhibit both forms of DNA topoisomerase. Included are an in vitro assay for topoisomerase I activity based on relaxation of supercoiled DNA, and an assay for topoisomerase II based on the decatenation of double-stranded DNA. The preparation of mammalian cell extracts for assaying topoisomerase activity is described, along with a protocol for an ICE assay to examine topoisomerase covalent complexes in vivo, and an assay for measuring DNA cleavage in vitro.
Insights
This study details assays for evaluating new anti-cancer agents targeting DNA topoisomerases (type I and II). These methods assess inhibition of enzymes crucial for DNA replication and repair.
Area of Science:
- Biochemistry
- Molecular Biology
- Pharmacology
Background:
- Topoisomerases are essential nuclear enzymes regulating DNA topology during vital cellular processes.
- Type I and II topoisomerases, differing in DNA cutting mechanisms, are validated targets for anti-cancer therapies.
Purpose of the Study:
- To describe protocols for assessing the inhibitory potential of novel chemical entities against DNA topoisomerases.
- To provide methods for evaluating both type I and type II topoisomerase activity.
Main Methods:
- In vitro assay for Topoisomerase I activity using supercoiled DNA relaxation.
- In vitro assay for Topoisomerase II activity based on double-stranded DNA decatenation.
- Preparation of mammalian cell extracts, in vivo ICE assay for covalent complexes, and in vitro DNA cleavage assays.
Main Results:
- Established protocols allow for the assessment of topoisomerase inhibition by new chemical entities.
- Methods cover in vitro enzyme activity, in vivo complex formation, and DNA cleavage.
Conclusions:
- The described assays are valuable tools for the discovery and development of novel anti-cancer drugs targeting DNA topoisomerases.
- These protocols facilitate the comprehensive evaluation of potential therapeutic agents affecting DNA topology and integrity.
