Abstract
The role of DNA topoisomerases in plant cell metabolism is currently under investigation in our laboratory. Using a purified type I topoisomerase from cultured tobacco, we have carried out a biochemical characterization of enzymatic behavior. The enzyme relaxes negatively supercoiled DNA in the presence of MgCl2, and to a lesser extent in the presence of KCl. Phosphorylation of the topoisomerase does not influence its activity and it is not stimulated by the presence of histones H1 or H5. The enzyme may act in either a processive or distributive manner depending on reaction conditions. The anti-tumor drug, camptothecin, induces significant breakage by the enzyme on purified DNA molecules unless destabilized by the addition of KCl. The tobacco topoisomerase I can catalyze the formation of stable nucleosomes on circular DNA templates, suggesting a role for the enzyme in chromatin assembly.
Similar content being viewed by others
References
Aronoff R, Champoux JJ: The effects of camptothecin on the reaction and the specificity of the wheat germ type I topoisomerase. J Biol Chem 264: 1010–1015 (1989).
Been MD, Burgess RR, Champoux JJ: DNA strand breakage by wheat germ type I topoisomerase. Biochim Biophys Acta 782: 304–312 (1984).
Brougham MJ, Rowe TC, Holloman WK: Topoisomerase from Ustilago Maydis forms a covalent complex with single-stranded DNA through a phosphodiester bond to tyrosine. Biochemistry 25: 7362–7368 (1986).
Champoux JJ, Dubbecco R: An activity from mammalian cells that untwists superhelical DNA: a possible swivel for DNA replication. Proc Natl Acad Sci USA 69: 143–146 (1972).
Champoux JJ: DNA Topoisomerases. Annu Rev Biochem 47: 449–479 (1978).
Charbonnera D, Cella R, Montescusco A, Ciarrocchi G: Isolation of a type I topoisomerase from carrot cells. J Exp Bot 39: 70–78 (1988).
Covey JM, Jaxel C, Kohn KW, Pommier Y: Proteinlinked DNA strand breaks induced in mammalian cells by camptothecin, an inhibitor of topoisomerase I. Cancer Res 49: 5016–5022 (1989).
Echeverria M, Martin MT, Ricard B, Litvack S: A DNA topoisomerase type I from wheat embryo mitochondria. Plant Mol Biol 6: 417–427 (1986).
Gilmour DS, Elgin SCR: Localization of specific topoisomerase I interactions within the transcribed region of active heat shock genes by using the inhibitor, camptothecin. Mol Cell Biol 7: 141–148 (1985).
Glikin G, Ruberti I, Worcel A: Chromatin assembly in Xenopus oocytes: in vitro studies. Cell 37: 33–41 (1984).
Heath-Pagliuso S, Cole AD, Kmiec EB: Purification and characterization of a type I topoisomerase from cultured tobacco cells. Plant Physiol 94: 599–606 (1990).
Hsiang Y-H, Hertzberg R, Hecht S, Liu LF: Camptothecin induces protein-linked DNA breaks via mammalian DNA topoisomerase I. J Biol Chem 260: 14873–14878 (1985).
Kaiserman HB, Ingebritsen TS, Benbow RM: Regulation of Xenopus laevis DNA topoisomerase I activity by phosphorylation in vitro. Biochemistry 27: 3216–3222 (1988).
Kmiec EB, Sekiguchi JM, Cole AD: Studies on the ATP requirements of in vitro chromatin assembly. Biochem Cell Biol 67: 443–454 (1989).
Laemmli UK: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680–685 (1975).
Liu LF, Miller KG: Two forms of type I DNA topoisomerases from the cell nuclei. Proc Natl Acad Sci USA 78: 3487–3491 (1981).
Maxwell A, Gellert M: Mechanistic aspects of DNA topoisomerases. Adv Prot Chem 38: 69–107 (1986).
Morse RL: Nucleosomes inhibit both transcriptional initiation and elongation by RNA polymerase III in vitro. EMBO J 8: 2343–2351 (1989).
Nielson BL, Tewari KK: Pea chloroplast topoisomerase I: purification, characterization and role in replication. Plant Mol Biol 11: 3–14 (1988).
Rowe TC, Rusche JR, Brougham MJ, Holloman WK: Purification and properties of a topoisomerase from Ustilago maydis. J Biol Chem 256: 10354–10361 (1981).
Sekiguchi JM, Kmiec EB: Studies on DNA topoisomerase activity during in vitro chromatin assembly. Mol Cell Biochem 83: 195–205 (1988).
Sekiguchi JM, Swank RA, Kmiec EB: Changes in DNA topology can modulate the expression of certain RNA polymerase III genes in vitro. Mol Cell Biochem 85: 123–133 (1989).
Singleton CK, Wells RD: The facile generation of covalently closed, circular DNAs with defined negative superhelical densities. Anal Biochem 122: 253–257 (1982).
Shaffer R, Traktman P: Vaccinia virus encapsidates a novel topoisomerase with the properties of a eukaryotic type I enzyme. J Biol Chem 262: 9309–9315 (1987).
Shuman S, Moss B: Identification of a vaccinia virus gene encoding a type I DNA topoisomerase. Proc Natl Acad Sci USA 84: 7478–7482 (1987).
Stevnsner T, Mortensen UH, Westergard O, Bonven BJ: Interactions between eukaryotic DNA topoisomerase I and a specific binding sequence. J Biol Chem 264: 10110–10113 (1989).
Wang JC: DNA topoisomerases. Ann Rev Biochem 54: 665–697 (1985).
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Cole, A.D., Heath-Pagliuso, S., Baich, A. et al. In vitro analysis of a type I DNA topoisomerase activity from cultured tobacco cells. Plant Mol Biol 19, 265–276 (1992). https://doi.org/10.1007/BF00027348
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00027348