John L Nitiss1, Kostantin Kiianitsa2, Yilun Sun3, Karin C Nitiss1,4, Nancy Maizels2. 1. Pharmaceutical Sciences Department, University of Illinois College of Pharmacy, Rockford, Illinois. 2. Departments of Immunology and Biochemistry, University of Washington, Seattle, Washington. 3. Developmental Therapeutics Branch and Laboratory of Molecular Pharmacology, Center for Cancer Research, NCI, NIH, Bethesda, Maryland. 4. Biomedical Sciences Department, University of Illinois College of Medicine, Rockford, Illinois.
Abstract
Topoisomerases are enzymes that play essential roles in DNA replication, transcription, chromosome segregation, and recombination. All cells have two major forms of DNA topoisomerases: type I enzymes, which make single-stranded cuts in DNA, and type II enzymes, which cut and decatenate double-stranded DNA. DNA topoisomerases are important targets of approved and experimental anti-cancer agents. Provided in this article are protocols to assess activities of topoisomerases and their inhibitors. Included are an assay for topoisomerase I activity based on relaxation of supercoiled DNA; an assay for topoisomerase II based on the decatenation of double-stranded DNA; and approaches for enriching and quantifying DNA-protein covalent complexes formed as obligatory intermediates in the reactions of type I and II topoisomerases with DNA; and assays for measuring DNA cleavage in vitro. Topoisomerases are not the only proteins that form covalent adducts with DNA in living cells, and the approaches described here are likely to find use in characterizing other protein-DNA adducts and exploring their utility as targets for therapy.
Topoisomerases are enzymes that play essential roles in DNA replication, transcription, chromosome segregation, and recombination. All cells have two major forms of DNA topoisomerases: type I enzymes, which make single-stranded cuts in DNA, and type II enzymes, which cut and decatenate double-stranded DNA. DNA topoisomerases are important targets of approved and experimental anti-cancer agents. Provided in this article are protocols to assess activities of topoisomerases and their inhibitors. Included are an assay for topoisomerase I activity based on relaxation of supercoiled DNA; an assay for topoisomerase II based on the decatenation of double-stranded DNA; and approaches for enriching and quantifying DNA-protein covalent complexes formed as obligatory intermediates in the reactions of type I and II topoisomerases with DNA; and assays for measuring DNA cleavage in vitro. Topoisomerases are not the only proteins that form covalent adducts with DNA in living cells, and the approaches described here are likely to find use in characterizing other protein-DNA adducts and exploring their utility as targets for therapy.
Authors: Curtis T Rueden; Johannes Schindelin; Mark C Hiner; Barry E DeZonia; Alison E Walter; Ellen T Arena; Kevin W Eliceiri Journal: BMC Bioinformatics Date: 2017-11-29 Impact factor: 3.169
Authors: Yilun Sun; Eroica Soans; Margarita Mishina; Elena Petricci; Yves Pommier; Karin C Nitiss; John L Nitiss Journal: Front Mol Biosci Date: 2022-09-23