Abstract
Topoisomerase II is an essential enzyme that is required for a number of critical nuclear processes. All of the catalytic functions of topoisomerase II require the enzyme to generate a transient double-stranded break in the backbone of the double helix. To maintain genomic integrity during the cleavage event, topoisomerase II forms covalent bonds between active site tyrosyl residues and the newly generated 5′-DNA termini. In addition to the critical cellular functions of the type II enzyme, several important anticancer drugs kill cells by increasing levels of covalent topoisomerase II–DNA cleavage complexes. Due to the physiological importance of topoisomerase II and its role in cancer chemotherapy, several methods have been developed to monitor the in vitro DNA cleavage activity of the type II enzyme. The plasmid-based system described in this chapter quantifies enzyme-mediated double-stranded DNA cleavage by monitoring the conversion of covalently closed supercoiled DNA to linear molecules. The assay is simple, straightforward, and does not require the use of radiolabeled substrates.
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Wang, J.C. (1996) DNA Topoisomerases. Annu Rev Biochem 65, 635–692.
Fortune, J.M. and Osheroff, N. (2000) Topoisomerase II as a target for anticancer drugs: When enzymes stop being nice. Prog Nucleic Acid Res Mol Biol 64, 221–253.
Champoux, J.J. (2001) DNA topoisomerases: Structure, function, and mechanism. Annu Rev Biochem 70, 369–413.
Wang, J.C. (2002) Cellular roles of DNA topoisomerases: A molecular perspective. Nat Rev Mol Cell Biol 3, 430–440.
McClendon, A.K. and Osheroff, N. (2007) DNA topoisomerase II, genotoxicity, and cancer. Mutat Res 623, 83–97.
Baguley, B.C. and Ferguson, L.R. (1998) Mutagenic properties of topoisomerase-targeted drugs. Biochim Biophys Acta 1400, 213–222.
Kaufmann, S.H. (1998) Cell death induced by topoisomerase-targeted drugs: More questions than answers. Biochim Biophys Acta 1400, 195–211.
Walker, J.V. and Nitiss, J.L. (2002) DNA topoisomerase II as a target for cancer chemotherapy. Cancer Invest 20, 570–589.
Nelson, E.M., Tewey, K.M. and Liu, L.F. (1984) Mechanism of antitumor drug action: Poisoning of mammalian DNA topoisomerase II on DNA by 4′-(9-acridinylamino)-methanesulfon-m-anisidide. Proc Natl Acad Sci USA 81, 1361–1365.
Sander, M. and Hsieh, T. (1983) Double strand DNA cleavage by type II DNA topoisomerase from Drosophila melanogaster. J Biol Chem 258, 8421–8428.
Liu, L.F., Rowe, T.C., Yang, L., Tewey, K.M. and Chen, G.L. (1983) Cleavage of DNA by mammalian DNA topoisomerase II. J Biol Chem 258, 15365–15370.
Osheroff, N. and Zechiedrich, E.L. (1987) Calcium-promoted DNA cleavage by eukaryotic topoisomerase II: Trapping the covalent enzyme-DNA complex in an active form. Biochemistry 26, 4303–4309.
Worland, S.T. and Wang, J.C. (1989) Inducible overexpression, purification, and active site mapping of DNA topoisomerase II from the yeast Saccharomyces cerevisiae. J Biol Chem 264, 4412–4416.
Kingma, P.S., Greider, C.A. and Osheroff, N. (1997) Spontaneous DNA lesions poison human topoisomerase IIα and stimulate cleavage proximal to leukemic 11q23 chromosomal breakpoints. Biochemistry 36, 5934–5939.
Hu, T., Chang, S. and Hsieh, T. (1998) Identifying Lys359 as a critical residue for the ATP-dependent reactions of Drosophila DNA topoisomerase II. J Biol Chem 273, 9586–9592.
Lavrukhin, O.V., Fortune, J.M., Wood, T.G., Burbank, D.E., Van Etten, J.L., Osheroff, N. and Lloyd, R.S. (2000) Topoisomerase II from Chlorella virus PBCV-1. Characterization of the smallest known type II topoisomerase. J Biol Chem 275, 6915–6921.
Baldwin, E.L., Byl, J.A. and Osheroff, N. (2004) Cobalt enhances DNA cleavage mediated by human topoisomerase IIα in vitro and in cultured cells. Biochemistry 43, 728–735.
Osheroff, N. (1987) Role of the divalent cation in topoisomerase II mediated reactions. Biochemistry 26, 6402–6406.
Osheroff, N., Shelton, E.R. and Brutlag, D.L. (1983) DNA topoisomerase II from Drosophila melanogaster. Relaxation of supercoiled DNA. J Biol Chem 258, 9536–9543.
Zechiedrich, E.L. and Osheroff, N. (1990) Eukaryotic topoisomerases recognize nucleic acid topology by preferentially interacting with DNA crossovers. EMBO J 9, 4555–4562.
McClendon, A.K. and Osheroff, N. (2006) The geometry of DNA supercoils modulates topoisomerase-mediated DNA cleavage and enzyme response to anticancer drugs. Biochemistry 45, 3040–3050.
Osheroff, N. (1986) Eukaryotic topoisomerase II. Characterization of enzyme turnover. J Biol Chem 261, 9944–9950.
Corbett, A.H., Zechiedrich, E.L., Lloyd, R.S. and Osheroff, N. (1991) Inhibition of eukaryotic topoisomerase II by ultraviolet-induced cyclobutane pyrimidine dimers. J Biol Chem 266, 19666–19671.
Froelich-Ammon, S.J., Burden, D.A., Patchan, M.W., Elsea, S.H., Thompson, R.B. and Osheroff, N. (1995) Increased drug affinity as the mechanistic basis for drug hypersensitivity of a mutant type II topoisomerase. J Biol Chem 270, 28018–28021.
Pommier, Y. and Marchand, C. (2005) Interfacial inhibitors of protein-nucleic acid interactions. Curr Med Chem Anticancer Agents 5, 421–429.
Lindsey, R.H., Jr., Bromberg, K.D., Felix, C.A. and Osheroff, N. (2004) 1,4-Benzoquinone is a topoisomerase II poison. Biochemistry 43, 7563–7574.
Bandele, O.J. and Osheroff, N. (2008) (–)-Epigallocatechin gallate, a major constituent of green tea, poisons human type II topoisomerases. Chem Res Toxicol 21, 936–943.
Acknowledgments
This protocol was developed in part under the auspices of Grant GM33944 from the National Institutes of Health. O.J.B. was a trainee under grant 5 T32 CA09582 from the National Institutes of Health and was supported in part by Ruth L. Kirschstein National Research Service Award Predoctoral Fellowship F31 GM78744 from the National Institutes of Health. We are grateful to Amanda C. Gentry for critical reading of the manuscript.
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Bandele, O.J., Osheroff, N. (2009). Cleavage of Plasmid DNA by Eukaryotic Topoisomerase II. In: Clarke, D. (eds) DNA Topoisomerases. Methods in Molecular Biology™, vol 582. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60761-340-4_4
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DOI: https://doi.org/10.1007/978-1-60761-340-4_4
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