Skip to main content
PMC home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1994 Oct;14(10):6962–6974. doi: 10.1128/mcb.14.10.6962

Topoisomerase II forms multimers in vitro: effects of metals, beta-glycerophosphate, and phosphorylation of its C-terminal domain.

Y S Vassetzky 1, Q Dang 1, P Benedetti 1, S M Gasser 1
PMCID: PMC359227  PMID: 7935413

Abstract

We present a novel assay for the study of protein-protein interactions involving DNA topoisomerase II. Under various conditions of incubation we observe that topoisomerase II forms complexes at least tetrameric in size, which can be sedimented by centrifugation through glycerol. The multimers are enzymatically active and can be visualized by electron microscopy. Dephosphorylation of topoisomerase II inhibits its multimerization, which can be restored at least partially by rephosphorylation of multiple sites within its 200 C-terminal amino acids by casein kinase II. Truncation of topoisomerase II just upstream of the major phosphoacceptor sites reduces its aggregation, rendering the truncated enzyme insensitive to either kinase treatments or phosphatase treatments. This is consistent with a model in which interactions involving the phosphorylated C-terminal domain of topoisomerase II aid either in chromosome segregation or in chromosome condensation.

Full text

PDF
6962

Images in this article

6964Image
on p.6964
6966Image
on p.6966
6967Image
on p.6967
6969Image
on p.6969
6970Image
on p.6970
6972Image
on p.6972
6972Image
on p.6972

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Ackerman P., Glover C. V., Osheroff N. Phosphorylation of DNA topoisomerase II by casein kinase II: modulation of eukaryotic topoisomerase II activity in vitro. Proc Natl Acad Sci U S A. 1985 May;82(10):3164–3168. doi: 10.1073/pnas.82.10.3164. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Ackerman P., Glover C. V., Osheroff N. Phosphorylation of DNA topoisomerase II in vivo and in total homogenates of Drosophila Kc cells. The role of casein kinase II. J Biol Chem. 1988 Sep 5;263(25):12653–12660. [PubMed] [Google Scholar]
  3. Adachi Y., Käs E., Laemmli U. K. Preferential, cooperative binding of DNA topoisomerase II to scaffold-associated regions. EMBO J. 1989 Dec 20;8(13):3997–4006. doi: 10.1002/j.1460-2075.1989.tb08582.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Adachi Y., Luke M., Laemmli U. K. Chromosome assembly in vitro: topoisomerase II is required for condensation. Cell. 1991 Jan 11;64(1):137–148. doi: 10.1016/0092-8674(91)90215-k. [DOI] [PubMed] [Google Scholar]
  5. Adolph K. W., Cheng S. M., Laemmli U. K. Role of nonhistone proteins in metaphase chromosome structure. Cell. 1977 Nov;12(3):805–816. doi: 10.1016/0092-8674(77)90279-3. [DOI] [PubMed] [Google Scholar]
  6. Cardenas M. E., Dang Q., Glover C. V., Gasser S. M. Casein kinase II phosphorylates the eukaryote-specific C-terminal domain of topoisomerase II in vivo. EMBO J. 1992 May;11(5):1785–1796. doi: 10.1002/j.1460-2075.1992.tb05230.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Cardenas M. E., Gasser S. M. Regulation of topoisomerase II by phosphorylation: a role for casein kinase II. J Cell Sci. 1993 Feb;104(Pt 2):219–225. doi: 10.1242/jcs.104.2.219. [DOI] [PubMed] [Google Scholar]
  8. Cardenas M. E., Walter R., Hanna D., Gasser S. M. Casein kinase II copurifies with yeast DNA topoisomerase II and re-activates the dephosphorylated enzyme. J Cell Sci. 1993 Feb;104(Pt 2):533–543. doi: 10.1242/jcs.104.2.533. [DOI] [PubMed] [Google Scholar]
  9. Caron P. R., Watt P., Wang J. C. The C-terminal domain of Saccharomyces cerevisiae DNA topoisomerase II. Mol Cell Biol. 1994 May;14(5):3197–3207. doi: 10.1128/mcb.14.5.3197. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Chung I. K., Muller M. T. Aggregates of oligo(dG) bind and inhibit topoisomerase II activity and induce formation of large networks. J Biol Chem. 1991 May 25;266(15):9508–9514. [PubMed] [Google Scholar]
  11. Crenshaw D. G., Hsieh T. Function of the hydrophilic carboxyl terminus of type II DNA topoisomerase from Drosophila melanogaster. I. In vitro studies. J Biol Chem. 1993 Oct 5;268(28):21328–21334. [PubMed] [Google Scholar]
  12. Earnshaw W. C., Halligan B., Cooke C. A., Heck M. M., Liu L. F. Topoisomerase II is a structural component of mitotic chromosome scaffolds. J Cell Biol. 1985 May;100(5):1706–1715. doi: 10.1083/jcb.100.5.1706. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Earnshaw W. C., Heck M. M. Localization of topoisomerase II in mitotic chromosomes. J Cell Biol. 1985 May;100(5):1716–1725. doi: 10.1083/jcb.100.5.1716. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Gasser S. M., Amati B. B., Cardenas M. E., Hofmann J. F. Studies on scaffold attachment sites and their relation to genome function. Int Rev Cytol. 1989;119:57–96. doi: 10.1016/s0074-7696(08)60649-x. [DOI] [PubMed] [Google Scholar]
  15. Gasser S. M., Laroche T., Falquet J., Boy de la Tour E., Laemmli U. K. Metaphase chromosome structure. Involvement of topoisomerase II. J Mol Biol. 1986 Apr 20;188(4):613–629. doi: 10.1016/s0022-2836(86)80010-9. [DOI] [PubMed] [Google Scholar]
  16. Hirano T., Hiraoka Y., Yanagida M. A temperature-sensitive mutation of the Schizosaccharomyces pombe gene nuc2+ that encodes a nuclear scaffold-like protein blocks spindle elongation in mitotic anaphase. J Cell Biol. 1988 Apr;106(4):1171–1183. doi: 10.1083/jcb.106.4.1171. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Hirano T., Mitchison T. J. Topoisomerase II does not play a scaffolding role in the organization of mitotic chromosomes assembled in Xenopus egg extracts. J Cell Biol. 1993 Feb;120(3):601–612. doi: 10.1083/jcb.120.3.601. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Hofmann J. F., Laroche T., Brand A. H., Gasser S. M. RAP-1 factor is necessary for DNA loop formation in vitro at the silent mating type locus HML. Cell. 1989 Jun 2;57(5):725–737. doi: 10.1016/0092-8674(89)90788-5. [DOI] [PubMed] [Google Scholar]
  19. Kaufmann S. H., Fields A. P., Shaper J. H. The nuclear matrix: current concepts and unanswered questions. Methods Achiev Exp Pathol. 1986;12:141–171. [PubMed] [Google Scholar]
  20. Kirchhausen T., Wang J. C., Harrison S. C. DNA gyrase and its complexes with DNA: direct observation by electron microscopy. Cell. 1985 Jul;41(3):933–943. doi: 10.1016/s0092-8674(85)80074-x. [DOI] [PubMed] [Google Scholar]
  21. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  22. Lewis C. D., Laemmli U. K. Higher order metaphase chromosome structure: evidence for metalloprotein interactions. Cell. 1982 May;29(1):171–181. doi: 10.1016/0092-8674(82)90101-5. [DOI] [PubMed] [Google Scholar]
  23. Lindsley J. E., Wang J. C. Proteolysis patterns of epitopically labeled yeast DNA topoisomerase II suggest an allosteric transition in the enzyme induced by ATP binding. Proc Natl Acad Sci U S A. 1991 Dec 1;88(23):10485–10489. doi: 10.1073/pnas.88.23.10485. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Littlewood T. D., Hancock D. C., Evan G. I. Characterization of a heat shock-induced insoluble complex in the nuclei of cells. J Cell Sci. 1987 Aug;88(Pt 1):65–72. doi: 10.1242/jcs.88.1.65. [DOI] [PubMed] [Google Scholar]
  25. McConnell M., Whalen A. M., Smith D. E., Fisher P. A. Heat shock-induced changes in the structural stability of proteinaceous karyoskeletal elements in vitro and morphological effects in situ. J Cell Biol. 1987 Sep;105(3):1087–1098. doi: 10.1083/jcb.105.3.1087. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Mirkovitch J., Mirault M. E., Laemmli U. K. Organization of the higher-order chromatin loop: specific DNA attachment sites on nuclear scaffold. Cell. 1984 Nov;39(1):223–232. doi: 10.1016/0092-8674(84)90208-3. [DOI] [PubMed] [Google Scholar]
  27. Osheroff N., Zechiedrich E. L., Gale K. C. Catalytic function of DNA topoisomerase II. Bioessays. 1991 Jun;13(6):269–273. doi: 10.1002/bies.950130603. [DOI] [PubMed] [Google Scholar]
  28. Paulson J. R., Laemmli U. K. The structure of histone-depleted metaphase chromosomes. Cell. 1977 Nov;12(3):817–828. doi: 10.1016/0092-8674(77)90280-x. [DOI] [PubMed] [Google Scholar]
  29. Roca J., Wang J. C. The capture of a DNA double helix by an ATP-dependent protein clamp: a key step in DNA transport by type II DNA topoisomerases. Cell. 1992 Nov 27;71(5):833–840. doi: 10.1016/0092-8674(92)90558-t. [DOI] [PubMed] [Google Scholar]
  30. Sahyoun N., Wolf M., Besterman J., Hsieh T., Sander M., LeVine H., 3rd, Chang K. J., Cuatrecasas P. Protein kinase C phosphorylates topoisomerase II: topoisomerase activation and its possible role in phorbol ester-induced differentiation of HL-60 cells. Proc Natl Acad Sci U S A. 1986 Mar;83(6):1603–1607. doi: 10.1073/pnas.83.6.1603. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Saijo M., Enomoto T., Hanaoka F., Ui M. Purification and characterization of type II DNA topoisomerase from mouse FM3A cells: phosphorylation of topoisomerase II and modification of its activity. Biochemistry. 1990 Jan 16;29(2):583–590. doi: 10.1021/bi00454a036. [DOI] [PubMed] [Google Scholar]
  32. Saijo M., Ui M., Enomoto T. Growth state and cell cycle dependent phosphorylation of DNA topoisomerase II in Swiss 3T3 cells. Biochemistry. 1992 Jan 21;31(2):359–363. doi: 10.1021/bi00117a007. [DOI] [PubMed] [Google Scholar]
  33. Scheller K. H., Abel T. H., Polanyi P. E., Wenk P. K., Fischer B. E., Sigel H. Metal ion/buffer interactions. Stability of binary and ternary complexes containing 2-[bis(2-hydroxyethyl)amino]-2(hydroxymethyl)-1,3-propanediol (Bistris) and adenosine 5'-triphosphate (ATP). Eur J Biochem. 1980 Jun;107(2):455–466. [PubMed] [Google Scholar]
  34. Shiozaki K., Yanagida M. Functional dissection of the phosphorylated termini of fission yeast DNA topoisomerase II. J Cell Biol. 1992 Dec;119(5):1023–1036. doi: 10.1083/jcb.119.5.1023. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Swedlow J. R., Sedat J. W., Agard D. A. Multiple chromosomal populations of topoisomerase II detected in vivo by time-lapse, three-dimensional wide-field microscopy. Cell. 1993 Apr 9;73(1):97–108. doi: 10.1016/0092-8674(93)90163-k. [DOI] [PubMed] [Google Scholar]
  36. Taagepera S., Rao P. N., Drake F. H., Gorbsky G. J. DNA topoisomerase II alpha is the major chromosome protein recognized by the mitotic phosphoprotein antibody MPM-2. Proc Natl Acad Sci U S A. 1993 Sep 15;90(18):8407–8411. doi: 10.1073/pnas.90.18.8407. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Uemura T., Ohkura H., Adachi Y., Morino K., Shiozaki K., Yanagida M. DNA topoisomerase II is required for condensation and separation of mitotic chromosomes in S. pombe. Cell. 1987 Sep 11;50(6):917–925. doi: 10.1016/0092-8674(87)90518-6. [DOI] [PubMed] [Google Scholar]
  38. Verdier J. M., Stalder R., Roberge M., Amati B., Sentenac A., Gasser S. M. Preparation and characterization of yeast nuclear extracts for efficient RNA polymerase B (II)-dependent transcription in vitro. Nucleic Acids Res. 1990 Dec 11;18(23):7033–7039. doi: 10.1093/nar/18.23.7033. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Wang J. C. DNA topoisomerases. Annu Rev Biochem. 1985;54:665–697. doi: 10.1146/annurev.bi.54.070185.003313. [DOI] [PubMed] [Google Scholar]
  40. Wood E. R., Earnshaw W. C. Mitotic chromatin condensation in vitro using somatic cell extracts and nuclei with variable levels of endogenous topoisomerase II. J Cell Biol. 1990 Dec;111(6 Pt 2):2839–2850. doi: 10.1083/jcb.111.6.2839. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Worland S. T., Wang J. C. Inducible overexpression, purification, and active site mapping of DNA topoisomerase II from the yeast Saccharomyces cerevisiae. J Biol Chem. 1989 Mar 15;264(8):4412–4416. [PubMed] [Google Scholar]
  42. Wyckoff E., Natalie D., Nolan J. M., Lee M., Hsieh T. Structure of the Drosophila DNA topoisomerase II gene. Nucleotide sequence and homology among topoisomerases II. J Mol Biol. 1989 Jan 5;205(1):1–13. doi: 10.1016/0022-2836(89)90361-6. [DOI] [PubMed] [Google Scholar]
  43. Zechiedrich E. L., Osheroff N. Eukaryotic topoisomerases recognize nucleic acid topology by preferentially interacting with DNA crossovers. EMBO J. 1990 Dec;9(13):4555–4562. doi: 10.1002/j.1460-2075.1990.tb07908.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Molecular and Cellular Biology are provided here courtesy of Taylor & Francis

RESOURCES