A RADAR method to measure DNA topoisomerase covalent complexes.
DNA topoisomerase
DNA topoisomerase–covalent complex
DNA-protein crosslink
Protein–DNA interaction
RADAR
Slot blot
Journal
Methods in enzymology
ISSN: 1557-7988
Titre abrégé: Methods Enzymol
Pays: United States
ID NLM: 0212271
Informations de publication
Date de publication:
2022
2022
Historique:
entrez:
7
8
2022
pubmed:
8
8
2022
medline:
10
8
2022
Statut:
ppublish
Résumé
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.
Identifiants
pubmed: 35934484
pii: S0076-6879(22)00127-6
doi: 10.1016/bs.mie.2022.03.036
pmc: PMC9722332
mid: NIHMS1852117
pii:
doi:
Substances chimiques
DNA
9007-49-2
DNA Topoisomerases, Type I
EC 5.99.1.2
Types de publication
Journal Article
Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, Non-P.H.S.
Langues
eng
Sous-ensembles de citation
IM
Pagination
369-381Subventions
Organisme : NCI NIH HHS
ID : R01 CA237263
Pays : United States
Organisme : NCI NIH HHS
ID : R01 CA248526
Pays : United States
Informations de copyright
Copyright © 2022 Elsevier Inc. All rights reserved.
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