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Related Concept Videos

DNA Topoisomerases02:02

DNA Topoisomerases

33.0K
Topoisomerases are enzymes that relax overwound DNA molecules during various cell processes, including DNA replication and transcription. These enzymes regulate positive and negative DNA supercoiling without changing the nucleotide sequence. DNA overwinding in a clockwise direction results in positively supercoiled DNA, whereas underwinding in a counterclockwise direction produces negatively supercoiled DNA.
Types and Mechanism of action
Topoisomerases are divided into two main types. ...
33.0K

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Related Experiment Video

Updated: Oct 18, 2025

Simple and Fast Rolling Circle Amplification-Based Detection of Topoisomerase 1 Activity in Crude Biological Samples
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Simple and Fast Rolling Circle Amplification-Based Detection of Topoisomerase 1 Activity in Crude Biological Samples

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Topoisomerase Assays.

John L Nitiss1, Kostantin Kiianitsa2, Yilun Sun3

  • 1Pharmaceutical Sciences Department, University of Illinois College of Pharmacy, Rockford, Illinois.

Current Protocols
|October 4, 2021
PubMed
Summary
This summary is machine-generated.

This study provides protocols to measure DNA topoisomerase activity and their inhibitors, crucial for developing anti-cancer therapies. These methods assess enzyme function and DNA-protein complexes for broader applications in characterizing DNA adducts.

Keywords:
DNA cleavageICE assayRADAR enrichmentcamptothecinetoposideprotein-DNA adducttopoisomerasetopoisomerase Itopoisomerase IItopoisomerase poison

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Enzymology

Background:

  • Topoisomerases are essential enzymes regulating DNA topology during replication, transcription, and segregation.
  • Type I and Type II topoisomerases perform distinct DNA cleavage and ligation functions.
  • These enzymes are validated targets for anti-cancer drug development.

Purpose of the Study:

  • To present detailed protocols for assessing DNA topoisomerase activities and their inhibitors.
  • To describe methods for quantifying topoisomerase-DNA covalent complexes.
  • To explore the utility of these approaches for characterizing other DNA-protein adducts.

Main Methods:

  • Assays for topoisomerase I (DNA relaxation) and topoisomerase II (DNA decatenation) activity.
  • In vivo complex of enzyme (ICE) assay for determining topoisomerase covalent complexes.
  • Rapid approach to DNA adduct recovery (RADAR) combined with ELISA or Western blot for quantification.
  • DNA cleavage assays, including plasmid linearization and gel electrophoresis.

Main Results:

  • Established protocols enable robust measurement of topoisomerase I and II activities.
  • Methods for enriching and quantifying topoisomerase-DNA covalent complexes were detailed.
  • Assays for DNA cleavage by topoisomerases were optimized.
  • The described techniques are adaptable for characterizing diverse DNA-protein adducts.

Conclusions:

  • The provided protocols offer comprehensive tools for studying topoisomerase function and inhibition.
  • These methods facilitate the discovery and development of novel anti-cancer agents targeting topoisomerases.
  • The adaptable techniques can advance the characterization of various DNA-protein interactions and therapeutic targets.