Abstract
DNA topoisomerase I and II catalyze topological changes of DNA by transient breakage of the nucleic acid backbone. Topoisomerase I transiently cleaves one DNA strand and allows the passage of single-stranded DNA through the generated nick, whereas topoisomerase II introduces a transient double-stranded break and permits passage of another double helix through the gap (1–4). Owing to the physiological importance of the topoisomerase-mediated cleavage reactions, they have been intensively studied by use of an in vitro assay involving treatment with a strong protein denaturant (5,6). On this treatment, the cleavage-religation cycle of the enzyme is interrupted, and abortive topoisomerase-DNA complexes are formed, where the enzyme is covalently linked to the cleaved DNA. Topoisomerase I is covalently linked to the newly generated 3′-end, whereas a subunit of topoisomerase II is covalently linked to each of the generated 5′-ends. Several studies have utilized the detergent method to define the sites of interaction between DNA and topoisomerase I and II in vitro as well as in vivo (7,85). One of the major problems in these studies has been ascribed to the involvement of SDS in trapping cleavage complexes, as the detergent denatures the enzyme and thus prevents a subsequent study of the topoisomerase-mediated religation reaction. To overcome this problem, different attempts have been performed to separate the cleavage and religation half-reactions. Thus, circular or linear single-stranded DNA has been used as substrate for both topoisomerase I and II. The generated cleavage complexes can catalyze recircularization or ligation to double-stranded DNA containing either a 3′-overhang, a 5′-overhang, or a blunt end (9–12). However, the cleavage complexes generated with single-stranded DNA are heterogenous in DNA sequence and structure, making these systems inadequate for detailed studies of the ligation reaction.
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References
Vosberg H.-P. (1985) DNA topoisomerases: enzymes that control DNA conformation. Curr. Top. Microbiol. Immunol. 114, 19–102.
Osheroff N. (1989) Biochemical basis for the interactions of type I and type II topoisomerase with DNA. Pharmacol. Ther. 41, 223–241.
Gupta M. and Pommier Y. (1995) Eukaryotic DNA topoisomerase I. Biochim. Biophys. Acta 1262, 1–14.
Watt P. M. and Hickson I. D. (1994) Structure and function of type II DNA topoisomerases. Biochem. J. 303, 681-95.
Edwards K. A., Halligan B. D., Davis J. L., Nivera N. L., and Liu L. F. (1982) Recognition sites of eukaryotic DNA topoisomerase I: DNA nucleotide sequencing analysis of topoisomerase I cleavage sites on SV40 DNA. Nucleic Acids Res. 10, 2565–2576.
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, 15,365–15,370.
Bonven B. J., Gocke E., and Westergaard O. (1985) A high affinity topoisomerase I binding sequence is clustered at DNAase I hypersensitive sites in Tetrahymena R-chromatin. Cell 41, 541–551.
Yang L., Rowe T. C., Nelson E. M., and Liu L. F. (1985) In vivo mapping of DNA topoisomerase II-specific cleavage sites on SV40 chromatin. Cell 41, 127–132.
Been M. D. and Champoux J. J. (1981) DNA breakage and closure by rat liver type I topoisomerase: separation of the half-reactions by using a single-stranded substrate. Proc. Natl. Acad. Sci. USA 78, 2883–2887.
Halligan B. D., Davis J. L., Edwards K. A., and Liu L. F. (1982) Intra-and intermolecular strand transfer by HeLa DNA topoisomerase I. J. Biol. Chem. 257, 3995–4000.
Gale K. C. and Osheroff N. (1990) Uncoupling the DNA cleavage and religation activities of topoisomerase II with a single-stranded nucleic acid substrate: evidence for an active enzyme-cleaved DNA intermediate. Biochemistry 29, 9538–9545.
Gale K. C. and Osheroff N. (1992) Intrinsic intermolecular DNA-ligation activity of eucaryotic topoisomerase II. Potential roles in recombination. J. Biol. Chem. 267, 12,090–12,097.
Christiansen K., Bonven B. J., and Westergaard O. (1987) Mapping of sequence-specific chromatin proteins by a novel method: topoisomerase I on Tetrahymena ribosomal chromatin. J. Mol. Biol. 193, 517–525.
Andersen A. H., Gocke E., Bonven B., Westergaard O. (1985) Topoisomerse I has a strong binding preference for a conserved hexadecameric sequence in the promoter region of the rRNA gene from Tetrahymena pyriformis. Nucleic Acids Res. 13, 1543–1557.
Ness P. J., Koller T., and Thoma F. (1988) Topoisomerase I cleavage sites identified and mapped in the chromatin of Dictyostelium ribosomal RNA genes. J. Mol. Biol. 200, 127–1397.
Christiansen K., Svejstrup A. B. D., Andersen A. H., and Westergaard O. (1993) Eukaryotic topoisomerase I-mediated cleavage requires bipartite DNA interaction. Cleavage of DNA substrates containing strand interruptions implicates a role for topoisomerase I in illegitimate recombination. J. Biol. Chem. 268, 9690–9701.
Christiansen K. and Westergaard O. (1994) Characterization of intra-and inter-molecular DNA ligation mediated by eukaryotic topoisomerase I. Role of bipartite DNA interaction in the ligation process. J. Biol. Chem. 269,721–729.
Svejstrup J. Q., Christiansen K., Gromova I. I., Andersen A. H., and Westergaard O. (1991) New technique for uncoupling the cleavage and religation reactions of eukaryotic topoisomerase I. The mode of action of camptothecin at a specific recognition site. J. Mol. Biol. 222, 669–678.
Svejstrup J. Q., Christiansen K., Andersen A. H., Lund K., and Westergaard O. (1990) Minimal DNA duplex requirements for topoisomerase I-mediated cleavage in vitro. J. Biol. Chem. 265, 12,529–12,535.
Christiansen K. and Westergaard O. (1993) Involvement of eukaryotic topoisomerase I in illigitimate recombination: Generation of deletions and insertions, in DNA Repair Mechanisms, proceedings of the 35th Alfred Benzon Symposium (Bohr W. A., Wassermann K., and Krämer K. H., eds.), Munksgaard, Copenhagen, pp. 361–371.
Kjeldsen E., Mollerup S., Thomsen B., Bonven B. J., Bolund L., and Westergaard O. (1988) Sequence-dependent effect of camptothecin on human topoisomerase I DNA cleavage. J. Mol. Biol. 202, 333–342.
Jaxel C., Capranico G., Kerrigan D., Kohn K. W., and Pommier Y. (1991) Effect of local DNA sequence on topoisomerase I cleavage in the presence or absence of camptothecin. J. Biol. Chem. 266, 20,418–20,423.
Christiansen K. and Westergaard O. (1996) The effect of camptothecin on topoisomerase I catalysis, in: The Camptothecins from Discovery to the Patient (Pantasiz P., Giovanella D. C., and Rothenberg M. L., eds.), Annals of the New York Academy of Sciences, pp. 50–59.
Sander M. and Hsieh T. (1983) Double strand DNA cleavage by type II DNA topoisomerase from Drosophila melanogaster. J. Biol. Chem. 258, 8421–8428.
Lund K., Andersen A. H., Christiansen K., Svejstrup J. Q., and Westergaard O. (1990) Minimal DNA requirement for topoisomerase II-mediated cleavage in vitro. J. Biol. Chem. 265, 13,856–13,863.
Thomsen B., Bendixen C., Lund K., Andersen A. H., Sørensen B. S., and Westergaard O. (1990) Characterization of the interaction between topoisomerase II and DNA by transcriptional footprinting. J. Mol. Biol. 215, 237–244.
Andersen A. H., Sørensen B. S., Christiansen K., Svejstrup J. Q., Lund K., and Westergaard O. (1991) Studies of the topoisomerase II-mediated cleavage and religation reactions by use of a suicidal double-stranded DNA substrate. J. Biol. Chem. 266, 9203–9210.
Schmidt V. K., Sorensen B. S., Sorensen H. V, Alsner J. and Westergaard O. (1994) Intramolecular and intermolecular DNA ligation mediated by topoisomerase II. J. Mol. Biol. 241, 18–25.
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Andersen, A.H., Christiansen, K., Westergaard, O. (2001). Uncoupling of Topoisomerase-Mediated DNA Cleavage and Religation. In: Osheroff, N., Bjornsti, MA. (eds) DNA Topoisomerase Protocols. Methods in Molecular Biology™, vol 95. Humana Press. https://doi.org/10.1385/1-59259-057-8:101
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DOI: https://doi.org/10.1385/1-59259-057-8:101
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