A RADAR method to measure DNA topoisomerase covalent complexes

Alice Meroni, Alessandro Vindigni

Research output: Chapter in Book/Report/Conference proceedingChapterpeer-review

Abstract

DNA topoisomerases resolve topological stress by introducing transient single- or double-strand breaks into the DNA duplex. This reaction requires the covalent binding of topoisomerases to DNA while the topological stress is being released. This transient intermediate is known as topoisomerase-covalent complex and represents the target of many anti-cancer drugs. Here, we describe a protocol to quantitatively detect topoisomerase-covalent complexes in vivo, called RADAR (rapid approach to DNA adduct recovery). DNA and protein-DNA covalent complexes are rapidly isolated from cells through chaotropic extraction. After normalization, samples are loaded on a slot blot, and the covalent complexes are detected using specific topoisomerase antibodies. In addition to being fast and robust, this assay produces quantitative results. Consequently, the RADAR assay can be applied to investigate the topoisomerase-covalent complex biology, including the effect of specific topoisomerase inhibitors. Finally, the same assay can be more generally applied to study covalent complexes of other enzymes with DNA.

Original languageEnglish
Title of host publicationHelicase Enzymes Part A
EditorsMichael A. Trakselis
PublisherAcademic Press Inc.
Pages369-381
Number of pages13
ISBN (Print)9780323914765
DOIs
StatePublished - Jan 2022

Publication series

NameMethods in Enzymology
Volume672
ISSN (Print)0076-6879
ISSN (Electronic)1557-7988

Keywords

  • DNA topoisomerase
  • DNA topoisomerase–covalent complex
  • DNA-protein crosslink
  • Protein–DNA interaction
  • RADAR
  • Slot blot

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