Abstract
We have successfully expressed enzymatically active plant topoisomerase II in Escherichia coli for the first time, which has enabled its biochemical characterization. Using a PCR-based strategy, we obtained a full-length cDNA and the corresponding genomic clone of tobacco topoisomerase II. The genomic clone has 18 exons interrupted by 17 introns. Most of the 5′ and 3′ splice junctions follow the typical canonical consensus dinucleotide sequence GU-AG present in other plant introns. The position of introns and phasing with respect to primary amino acid sequence in tobacco TopII and Arabidopsis TopII are highly conserved, suggesting that the two genes are evolved from the common ancestral type II topoisomerase gene. The cDNA encodes a polypeptide of 1482 amino acids. The primary amino acid sequence shows a striking sequence similarity, preserving all the structural domains that are conserved among eukaryotic type II topoisomerases in an identical spatial order. We have expressed the full-length polypeptide in E. coli and purified the recombinant protein to homogeneity. The full-length polypeptide relaxed supercoiled DNA and decatenated the catenated DNA in a Mg2+- and ATP-dependent manner, and this activity was inhibited by 4′-(9-acridinylamino)-3′-methoxymethanesulfonanilide (m-AMSA). The immunofluorescence and confocal microscopic studies, with antibodies developed against the N-terminal region of tobacco recombinant topoisomerase II, established the nuclear localization of topoisomerase II in tobacco BY2 cells. The regulated expression of tobacco topoisomerase II gene under the GAL1 promoter functionally complemented a temperature-sensitive TopII ts yeast mutant.
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References
Adachi, N., Miyaike, M., Kato, S., Kanamaru, R., Koyama, H. and Kikuchi, A. 1997b. Cellular distribution of mammalian DNA topoisomerase II is determined by its catalytically dispensable C-terminal domain. Nucl. Acids Res. 25: 3135-3142.
Austin, C.A. and Marsh, K.L. 1998. Eukaryotic DNA topoisomerase II beta. Bioessays 20: 215-226.
Berger, J.M., Gamblin, S.J., Harrison, S.C. and Wang, J.C. 1996. Structure and mechanism of DNA topoisomerase II. Nature 379: 225-232.
Brown, J.W. 1986. A catalogue of splice junction and putative branch point sequences from plant introns. Nucl. Acids Res. 14: 9549-9559.
Burden, D.A. and Osheroff, N. 1998. Mechanism of action of eukaryotic topoisomerase II and drugs targeted to the enzyme. Biochim. Biophys. Acta 1400: 139-154.
Burge, C. and Karlin, S. 1997. Prediction of complete gene structures in human genomic DNA. J. Mol. Biol. 268: 78-94.
Carballo, M., Gine, R., Santos, M. and Puigdomènech, P. 1991. Characterization of topoisomerase I and II activities in nuclear extracts during callogenesis in immature embryos of Zea mays. Plant Mol. Biol. 16: 59-70.
Carbonera, D., Rovati, L., Guano, F. and Balestrazzi, A. 1995. Puri-fication and properties of DNA topoisomerase II from Daucus carota cells. J. Exp. Bot. 46: 347-354.
Caron, P.R. and Wang, J.C. 1994. Appendix. II: Alignment of primary sequences of DNA topoisomerases. Adv. Pharmacol. 29B: 271-297.
Caron, P.R., Watt, P. and Wang, J.C. 1994. The C-terminal domain of Saccharomyces cerevisiae DNA topoisomerase II. Mol. Cell. Biol. 14: 3197-3207.
Champoux J.J 1990. Mechanistic aspects of type I topoisomerases. In: N.R. Cozzarelli and J.C. Wang (Eds.) DNA Topology and its Biological Effects, Cold Spring Harbor Laboratory Press, Plainview, NY, pp. 217-242.
Champoux, J.J. 2001. DNA topoisomerases: structure, function, and mechanism. Annu. Rev. Biochem. 70: 369-413.
Chiatante, D., Claut V. and Bryant, J.A. 1993. Nuclear DNA topoisomerases in Pisum sativum L. J. Exp. Bot. 44: 1045-1051.
Cowell, I.G., Willmore, E., Chalton, D., Marsh, K.L., Jazrawi, E., Fisher, L.M. and Austin, C.A. 1998. Nuclear distribution of human DNA topoisomerase IIbeta: a nuclear targeting signal resides in the 116-residue C-terminal tail. Exp. Cell. Res. 243: 232-240.
Crenshaw, D.G. and Hsieh, T. 1993. Function of the hydrophilic carboxyl terminus of type II DNA topoisomerase from Drosophila melanogaster. II. In vivo studies. J. Biol. Chem. 268: 21335-21343.
DiNardo, S., Voelkel, K. and Sternglanz, R. 1984. DNA topoisomerase II mutant of Saccharomyces cerevisiae: topoisomerase II is required for segregation of daughter molecules at the termination of DNA replication. Proc. Natl. Acad. Sci. USA 81: 2616-2620.
Dingwall, C. and Laskey, R.A. 1991. Nuclear targeting sequences: a consensus? Trends Biochem. Sci. 16: 478-481.
Falquet, L., Pagni, M., Bucher, P., Hulo, N., Sigrist, C.J., Hofmann, K. and Bairoch, A. 2002. The PROSITE database, its status in 2002. Nucl. Acids Res. 30: 235-238.
Fukata, H. and Fukasawa, H. 1982. Isolation and partial characterization of two distinct DNA topoisomerases from cauliflower inflorescence. J. Biochem. (Tokyo) 91: 1337-1342.
Fukata, H., Ohgami, K.L. and Fukasawa, H. 1986. Isolation and characterization of DNA topoisomerase II from cauliflower inflorescences. Plant. Mol. Biol. 6: 137-144.
Giaever, G., Lynn, R., Goto, T. and Wang, J.C. 1986. The complete nucleotide sequence of the structural gene TOP2 of yeast DNA topoisomerase II. J. Biol. Chem. 261: 12448-12454.
Goto, T. and Wang, J.C. 1984. Yeast DNA topoisomerase II is encoded by a single-copy, essential gene. Cell 36: 1073-1080.
Grelon, M., Vezon, D., Gendrot, G. and Pelletier, G. 2001. AtSPO11-1 is necessary for efficient meiotic recombination in plants. EMBO J. 20: 589-600.
Hartung, F. and Puchta, H. 2000. Molecular characterisation of two paralogous SPO11 homologues in Arabidopsis thaliana. Nucl. Acids Res. 28: 1548-1554.
Hartung, F. and Puchta, H. 2001. Molecular characterization of homologues of both subunits A (SPO11) and B of the archaebacterial topoisomerase 6 in plants. Gene 271: 81-86.
Hartung, F., Angelis, K.J., Meister, A., Schubert, I., Melzer, M. and Puchta, H. 2002. An archaebacterial topoisomerase homolog not present in other eukaryotes is indispensable for cell proliferation of plants. Curr. Biol. 12: 1787-1791.
Holm, C., Stearns, T. and Botstein, D. 1989. DNA topoisomerase II must act at mitosis to prevent non-disjunction and chromosome breakage. Mol. Cell. Biol. 9: 159-168.
Hsieh T.S. 1990. Mechanistic aspects of type II topoisomerases. In: N.R. Cozzarelli and J.C.Wang (Eds.) DNA Topology and its Biological Effects, Cold Spring Harbor Laboratory Press, Plainview, NY, pp. 243-263.
Lam, E. and Chua, N.H. 1987. Chloroplast DNA gyrase and in vitro regulation of transcription by template topology and novobiocin. Plant Mol. Biol. 8: 415-424.
Lang, A.J., Mirski, S.E., Cummings, H.J., Yu, Q., Gerlach, J.H. and Cole, S.P. 1998. Structural organization of the human TOP2A and TOP2B genes. Gene 221: 255-266.
Lynn, R., Giaever, G., Swanberg, S.L. and Wang, J.C. 1986. Tandem regions of yeast DNA topoisomerase II share homology with different subunits of bacterial gyrase. Science 233: 647-649.
Marrison, J. L. and Leech, R.M. 1992. Co-immunolocalization of topoisomerase II and chloroplast DNA in developing, dividing and mature wheat chloroplasts. Plant J. 2: 783-790.
Petruti-Mot, A.S. and Earnshaw, W.C. 2000. Two differentially spliced forms of topoisomerase II? and ? mRNAs are conserved between birds and humans. Gene 258: 183-192.
Pringle, J.R., Adams, A.E., Drubin, D.G. and Haarer, B.K. 1991. Immunofluorescence methods for yeast. Meth. Enzymol. 194: 565-602.
Proust, J., Houlne, G., Schantz, M.L., Shen, W.H. and Schantz, R. 1999. Regulation of biosynthesis and cellular localization of Sp32 annexins in tobacco BY2 cells. Plant Mol. Biol. 39: 361-372.
Pyke, K. A., Marrison, J. and Leech, R.M. 1989. Evidence for a type II topoisomerase in wheat chloroplasts. FEBS Lett. 242: 305-308.
Reddy, M.K., Nair, S. and Tewari, K.K. 1998. Cloning, expression and characterization of a gene which encodes a topoisomerase I with positive supercoiling activity in pea. Plant Mol. Biol. 37: 773-784.
Reddy, M.K., Nair, S., Tewari, K.K., Mudgil, Y., Yadav, B.S. and Sopory, S.K. 1999. Cloning and characterization of a cDNA encoding topoisomerase II in pea and analysis of its expression in relation to cell proliferation. Plant Mol. Biol. 41: 125-137.
Rudenko, G.N. 1992. Plant DNA-topoisomerase type-II: isolation, characterization and properties. Molekulyarnaya Biol. 25: 1125-1135.
Sugimoto-Shirasu, K., Stacey, N.J., Corsar, J., Roberts, K. and McCann, M.C. 2002. DNA topoisomerase VI is essential for endoreduplication in Arabidopsis. Curr. Biol. 12: 1782-1786.
Trigueros, S. and Roca, J. 2002. A GyrB-GyrA fusion protein expressed in yeast cells is able to remove DNA supercoils but cannot substitute eukaryotic topoisomerase II. Genes Cells 7: 249-257.
Wang, J.C. 1996. DNA topoisomerases. Annu. Rev. Biochem. 65: 635-692.
Wang, J.C. 2002. Cellular roles of DNA topoisomerases: a molecular perspective. Natl. Rev. Mol. Cell. Biol. 3: 430-440.
Wyckoff, E., Natalie, D., Nolan, J.M., Lee, M. and Hsieh, T. 1989. Structure of the Drosophila DNA topoisomerase II gene. Nucleotide sequence and homology among topoisomerases II. J. Mol. Biol. 205: 1-13.
Xie, S., and Lam, E. 1994. Abundance of nuclear DNA topoisomerase II is correlated with proliferation in Arabidopsis thaliana. Nucl. Acids Res. 22: 5729-5736.
Yin, Y., Cheong, H., Friedrichsen, D., Zhao, Y., Hu, J., Mora-Garcia, S. and Chory, J. 2002. A crucial role for the putative Arabidopsis topoisomerase VI in plant growth and development. Proc. Natl. Acad. Sci. USA 99: 10191-10196.
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Singh, B., Mudgil, Y., Sopory, S. et al. Molecular characterization of a nuclear topoisomerase II from Nicotiana tabacum that functionally complements a temperature-sensitive topoisomerase II yeast mutant. Plant Mol Biol 52, 1063–1076 (2003). https://doi.org/10.1023/A:1025427700337
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DOI: https://doi.org/10.1023/A:1025427700337