Abstract
DNA topoisomerases break and rejoin DNA strands through a covalent pro-tein-DNA intermediate. The reaction chemistry involves nucleophilic attack by a tyrosine moiety of the enzyme on the phosphodiester backbone of DNA to form a phosphotyrosyl linkage to one (in the case of type I enzymes) or both (type II enzymes) of the DNA strands. Although the distribution of topoisomerase cleavage sites in DNA is nonrandom, the principles governing cleavage site choice are not fully understood. The question of how topoisomerases recognize their DNA cleavage sites is of definite interest, insofar as site specificity may have implications for topoisomerase action in vivo, and because the topoisomerase-DNA complex is the pharmacological target of many antimicrobial and anticancer drugs. An extensive battery of footprinting techniques has been applied to analyzing the topoisomerase-DNA interface in selected model systems, including deoxyribonuclease protection, base-specific modification protection, base-specific modification interference, phosphate modification interference, and site-specific photocrosslinking. This chapter focuses on the use of the electrophoretic mobility shift assay (EMSA) to study topoisomerase binding to DNA.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Garner M. M. and Revzin A. (1981) A gel electrophoresis method for quantifying the binding of proteins to specific DNA regions: application to components of the Escherichia coli lactose operon regulatory system. Nucleic Acids Res. 9, 3047–3060.
Fried M. and Crothers D. M. (1981) Equilibrium and kinetics of lac repressor-operator interactions by polyacrylamide gel electrophoresis. Nucleic Acids Res. 9, 6505–6525.
Senear D. F. and Brenowitz M. (1991) Determination of binding constants for cooperative site-specific protein-DNA interactions using the gel-mobility shift assay. J. Biol. Chem. 266, 13,661–13,671.
Morham S. G. and Shuman S. (1992) Covalent and noncovalent DNA binding by mutants of vaccinia DNA topoisomerase I. J. Biol. Chem. 267, 15,984–15,992.
Wittschieben J. and Shuman S. (1994) Mutational analysis of vaccinia virus DNA topoisomerase defines amino acid residues essential for covalent catalysis. J. Biol. Chem. 269, 29,978–29,983.
Sekiguchi J. and Shuman S. (1994) Requirements for noncovalent binding of vaccinia topoisomerase I to duplex DNA. Nucleic Acids Res. 22, 5360–5365.
Sekiguchi J., Seeman N. C., and Shuman S. (1996) Resolution of Holliday junctions by eukaryotic DNA topoisomerase I. Proc. Natl Acad. Sci. USA 93, 785–789.
Maxwell A. and Gellert M. (1984) The DNA dependence of the ATPase activity of DNA gyrase. J. Biol. Chem. 259, 14,472–14,480.
Yang Y. and Ames G. F. (1988) DNA gyrase binds to a family of prokaryotic repetitive extragenic palindromic sequences. Proc. Natl. Acad. Sci. USA 85, 8850–8854.
Zhang H. L. and DiGate R. J. (1994) The carboxyl-terminal residues of Escheri-chia coli DNA topoisomerase III are involved in substrate binding. J. Biol. Chem. 269, 9052–9059.
Zhang H. L., Malpure S., and DiGate R. J. (1995) Escherichia coli DNA topoisomerase III is a site-specific DNA binding protein that binds asymmetrically to its cleavage site. J. Biol. Chem. 270, 23,700–23,705.
Shuman S. and Moss B. (1987) Identification of a vaccinia virus gene encoding a type I DNA topoisomerase. Proc. Natl. Acad. Sci. USA 84, 7478–7482.
Caron P. R. and Wang J. C. (1994) Alignment of primary sequences of DNA topoisomerases. Adv. Pharmocol. 29B, 271–297.
Shuman S. and Prescott J. (1990) Specific DNA cleavage and binding by vaccinia virus DNA topoisomerase I. J. Biol. Chem. 265, 17,826–17,836.
Shuman S., Kane E. M., and Morham S. G. (1989) Map** the active-site tyrosine of vaccinia virus DNA topoisomerase I. Proc. Natl. Acad. Sci. USA 86, 9793–9797.
Shuman S. (1991) Site-specific interaction of vaccinia virus topoisomerase I with duplex DNA. J. Biol. Chem. 266, 11,372–11,279.
Shuman S., Golder M., and Moss B. (1988) Characterization of vaccinia virus DNA topoisomerase I expressed in Escherichia coli. J. Biol. Chem. 263, 16,401–16,407.
McConaughy B. L., Young L. S., and Champoux J. J. (1981) The effect of salt on the binding of the eucaryotic DNA nicking-closing enzyme to DNA and chro-matin. Biochim. Biophys. Acta 655, 1–8.
Stivers J. T., Shuman S., and Mildvan A. S. (1994) Vaccinia DNA topoisomerase I: Single-turnover and steady-state kinetic analysis of the DNA strand cleavage and ligation reactions. Biochemistry 33, 327–339.
Sekiguchi J. and Shuman S. (1994) Stimulation of vaccinia DNA topoisomerase by nucleoside triphosphates. J. Biol. Chem. 269, 29,760–29,764.
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2001 Humana Press Inc.
About this protocol
Cite this protocol
Shuman, S. (2001). Analysis of Topoisomerase-DNA Interactions by Electrophoretic Mobility Shift Assay. In: Osheroff, N., Bjornsti, MA. (eds) DNA Topoisomerase Protocols. Methods in Molecular Biology™, vol 95. Humana Press. https://doi.org/10.1385/1-59259-057-8:65
Download citation
DOI: https://doi.org/10.1385/1-59259-057-8:65
Publisher Name: Humana Press
Print ISBN: 978-0-89603-512-6
Online ISBN: 978-1-59259-057-5
eBook Packages: Springer Protocols