Skip to main content
NCBI home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1992 Jun 1;117(5):921–934. doi: 10.1083/jcb.117.5.921

Sister chromatid separation in frog egg extracts requires DNA topoisomerase II activity during anaphase

PMCID: PMC2289485  PMID: 1315785

Abstract

We have produced metaphase spindles and induced them to enter anaphase in vitro. Sperm nuclei were added to frog egg extracts, allowed to replicate their DNA, and driven into metaphase by the addition of cytoplasm containing active maturation promoting factor (MPF) and cytostatic factor (CSF), an activity that stabilizes MPF. Addition of calcium induces the inactivation of MPF, sister chromatid separation and anaphase chromosome movement. DNA topoisomerase II inhibitors prevent chromosome segregation at anaphase, demonstrating that the chromatids are catenated at metaphase and that decatenation occurs at the start of anaphase. Topoisomerase II activity towards exogenous substrates does not increase at the metaphase to anaphase transition, showing that chromosome separation at anaphase is not triggered by a bulk activation of topoisomerase II.

Full Text

The Full Text of this article is available as a PDF (4.2 MB).

Selected References

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

  1. Alexander S. P., Rieder C. L. Chromosome motion during attachment to the vertebrate spindle: initial saltatory-like behavior of chromosomes and quantitative analysis of force production by nascent kinetochore fibers. J Cell Biol. 1991 May;113(4):805–815. doi: 10.1083/jcb.113.4.805. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Cande W. Z., McDonald K. L. In vitro reactivation of anaphase spindle elongation using isolated diatom spindles. Nature. 1985 Jul 11;316(6024):168–170. doi: 10.1038/316168a0. [DOI] [PubMed] [Google Scholar]
  3. Chen G. L., Yang L., Rowe T. C., Halligan B. D., Tewey K. M., Liu L. F. Nonintercalative antitumor drugs interfere with the breakage-reunion reaction of mammalian DNA topoisomerase II. J Biol Chem. 1984 Nov 10;259(21):13560–13566. [PubMed] [Google Scholar]
  4. Cooke C. A., Heck M. M., Earnshaw W. C. The inner centromere protein (INCENP) antigens: movement from inner centromere to midbody during mitosis. J Cell Biol. 1987 Nov;105(5):2053–2067. doi: 10.1083/jcb.105.5.2053. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Dawson D. S., Murray A. W., Szostak J. W. An alternative pathway for meiotic chromosome segregation in yeast. Science. 1986 Nov 7;234(4777):713–717. doi: 10.1126/science.3535068. [DOI] [PubMed] [Google Scholar]
  6. DiNardo S., Voelkel K., Sternglanz R. 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 U S A. 1984 May;81(9):2616–2620. doi: 10.1073/pnas.81.9.2616. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Downes C. S., Mullinger A. M., Johnson R. T. Inhibitors of DNA topoisomerase II prevent chromatid separation in mammalian cells but do not prevent exit from mitosis. Proc Natl Acad Sci U S A. 1991 Oct 15;88(20):8895–8899. doi: 10.1073/pnas.88.20.8895. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Earnshaw W. C., Cooke C. A. Analysis of the distribution of the INCENPs throughout mitosis reveals the existence of a pathway of structural changes in the chromosomes during metaphase and early events in cleavage furrow formation. J Cell Sci. 1991 Apr;98(Pt 4):443–461. doi: 10.1242/jcs.98.4.443. [DOI] [PubMed] [Google Scholar]
  9. 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]
  10. Evans L., Mitchison T., Kirschner M. Influence of the centrosome on the structure of nucleated microtubules. J Cell Biol. 1985 Apr;100(4):1185–1191. doi: 10.1083/jcb.100.4.1185. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. GRELL R. F. A new hypothesis on the nature and sequence of meiotic events in the female of Drosophila melanogaster. Proc Natl Acad Sci U S A. 1962 Feb;48:165–172. doi: 10.1073/pnas.48.2.165. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Hepler P. K. Calcium restriction prolongs metaphase in dividing Tradescantia stamen hair cells. J Cell Biol. 1985 May;100(5):1363–1368. doi: 10.1083/jcb.100.5.1363. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Hiramoto Y., Nakano Y. Micromanipulation studies of the mitotic apparatus in sand dollar eggs. Cell Motil Cytoskeleton. 1988;10(1-2):172–184. doi: 10.1002/cm.970100122. [DOI] [PubMed] [Google Scholar]
  14. Holm C., Goto T., Wang J. C., Botstein D. DNA topoisomerase II is required at the time of mitosis in yeast. Cell. 1985 Jun;41(2):553–563. doi: 10.1016/s0092-8674(85)80028-3. [DOI] [PubMed] [Google Scholar]
  15. Holm C., Stearns T., Botstein D. DNA topoisomerase II must act at mitosis to prevent nondisjunction and chromosome breakage. Mol Cell Biol. 1989 Jan;9(1):159–168. doi: 10.1128/mcb.9.1.159. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Hsieh T., Brutlag D. ATP-dependent DNA topoisonmerase from D. melanogaster reversibly catenates duplex DNA rings. Cell. 1980 Aug;21(1):115–125. doi: 10.1016/0092-8674(80)90119-1. [DOI] [PubMed] [Google Scholar]
  17. Hunt T., Luca F. C., Ruderman J. V. The requirements for protein synthesis and degradation, and the control of destruction of cyclins A and B in the meiotic and mitotic cell cycles of the clam embryo. J Cell Biol. 1992 Feb;116(3):707–724. doi: 10.1083/jcb.116.3.707. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Hyman A. A., White J. G. Determination of cell division axes in the early embryogenesis of Caenorhabditis elegans. J Cell Biol. 1987 Nov;105(5):2123–2135. doi: 10.1083/jcb.105.5.2123. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Ikegami S., Taguchi T., Ohashi M., Oguro M., Nagano H., Mano Y. Aphidicolin prevents mitotic cell division by interfering with the activity of DNA polymerase-alpha. Nature. 1978 Oct 5;275(5679):458–460. doi: 10.1038/275458a0. [DOI] [PubMed] [Google Scholar]
  20. Karsenti E., Newport J., Hubble R., Kirschner M. Interconversion of metaphase and interphase microtubule arrays, as studied by the injection of centrosomes and nuclei into Xenopus eggs. J Cell Biol. 1984 May;98(5):1730–1745. doi: 10.1083/jcb.98.5.1730. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Kline D. Calcium-dependent events at fertilization of the frog egg: injection of a calcium buffer blocks ion channel opening, exocytosis, and formation of pronuclei. Dev Biol. 1988 Apr;126(2):346–361. doi: 10.1016/0012-1606(88)90145-5. [DOI] [PubMed] [Google Scholar]
  22. Koshland D., Hartwell L. H. The structure of sister minichromosome DNA before anaphase in Saccharomyces cerevisiae. Science. 1987 Dec 18;238(4834):1713–1716. doi: 10.1126/science.3317838. [DOI] [PubMed] [Google Scholar]
  23. Lehner C. F., O'Farrell P. H. Expression and function of Drosophila cyclin A during embryonic cell cycle progression. Cell. 1989 Mar 24;56(6):957–968. doi: 10.1016/0092-8674(89)90629-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Lohka M. J., Maller J. L. Induction of nuclear envelope breakdown, chromosome condensation, and spindle formation in cell-free extracts. J Cell Biol. 1985 Aug;101(2):518–523. doi: 10.1083/jcb.101.2.518. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Lorca T., Galas S., Fesquet D., Devault A., Cavadore J. C., Dorée M. Degradation of the proto-oncogene product p39mos is not necessary for cyclin proteolysis and exit from meiotic metaphase: requirement for a Ca(2+)-calmodulin dependent event. EMBO J. 1991 Aug;10(8):2087–2093. doi: 10.1002/j.1460-2075.1991.tb07741.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. MOLE-BAJER J. Cine-micrographic analysis of C-mitosis in endosperm. Chromosoma. 1958;9(4):332–358. doi: 10.1007/BF02568085. [DOI] [PubMed] [Google Scholar]
  27. Marini J. C., Miller K. G., Englund P. T. Decatenation of kinetoplast DNA by topoisomerases. J Biol Chem. 1980 Jun 10;255(11):4976–4979. [PubMed] [Google Scholar]
  28. Masui Y., Markert C. L. Cytoplasmic control of nuclear behavior during meiotic maturation of frog oocytes. J Exp Zool. 1971 Jun;177(2):129–145. doi: 10.1002/jez.1401770202. [DOI] [PubMed] [Google Scholar]
  29. McNeill P. A., Berns M. W. Chromosome behavior after laser microirradiation of a single kinetochore in mitotic PtK2 cells. J Cell Biol. 1981 Mar;88(3):543–553. doi: 10.1083/jcb.88.3.543. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Meyerhof P. G., Masui Y. Ca and Mg control of cytostatic factors from Rana pipiens oocytes which cause metaphase and cleavage arrest. Dev Biol. 1977 Dec;61(2):214–229. doi: 10.1016/0012-1606(77)90293-7. [DOI] [PubMed] [Google Scholar]
  31. Murray A. W., Kirschner M. W. Cyclin synthesis drives the early embryonic cell cycle. Nature. 1989 May 25;339(6222):275–280. doi: 10.1038/339275a0. [DOI] [PubMed] [Google Scholar]
  32. Murray A. W., Kirschner M. W. Dominoes and clocks: the union of two views of the cell cycle. Science. 1989 Nov 3;246(4930):614–621. doi: 10.1126/science.2683077. [DOI] [PubMed] [Google Scholar]
  33. Murray A. W., Solomon M. J., Kirschner M. W. The role of cyclin synthesis and degradation in the control of maturation promoting factor activity. Nature. 1989 May 25;339(6222):280–286. doi: 10.1038/339280a0. [DOI] [PubMed] [Google Scholar]
  34. Murray A. W., Szostak J. W. Chromosome segregation in mitosis and meiosis. Annu Rev Cell Biol. 1985;1:289–315. doi: 10.1146/annurev.cb.01.110185.001445. [DOI] [PubMed] [Google Scholar]
  35. Nicklas R. B. The forces that move chromosomes in mitosis. Annu Rev Biophys Biophys Chem. 1988;17:431–449. doi: 10.1146/annurev.bb.17.060188.002243. [DOI] [PubMed] [Google Scholar]
  36. Nurse P. Universal control mechanism regulating onset of M-phase. Nature. 1990 Apr 5;344(6266):503–508. doi: 10.1038/344503a0. [DOI] [PubMed] [Google Scholar]
  37. Osheroff N., Shelton E. R., Brutlag D. L. DNA topoisomerase II from Drosophila melanogaster. Relaxation of supercoiled DNA. J Biol Chem. 1983 Aug 10;258(15):9536–9543. [PubMed] [Google Scholar]
  38. Poenie M., Alderton J., Steinhardt R., Tsien R. Calcium rises abruptly and briefly throughout the cell at the onset of anaphase. Science. 1986 Aug 22;233(4766):886–889. doi: 10.1126/science.3755550. [DOI] [PubMed] [Google Scholar]
  39. Rieder C. L., Alexander S. P. Kinetochores are transported poleward along a single astral microtubule during chromosome attachment to the spindle in newt lung cells. J Cell Biol. 1990 Jan;110(1):81–95. doi: 10.1083/jcb.110.1.81. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Sagata N., Watanabe N., Vande Woude G. F., Ikawa Y. The c-mos proto-oncogene product is a cytostatic factor responsible for meiotic arrest in vertebrate eggs. Nature. 1989 Nov 30;342(6249):512–518. doi: 10.1038/342512a0. [DOI] [PubMed] [Google Scholar]
  41. Sawin K. E., Mitchison T. J. Mitotic spindle assembly by two different pathways in vitro. J Cell Biol. 1991 Mar;112(5):925–940. doi: 10.1083/jcb.112.5.925. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Schollmeyer J. E. Calpain II involvement in mitosis. Science. 1988 May 13;240(4854):911–913. doi: 10.1126/science.2834825. [DOI] [PubMed] [Google Scholar]
  43. Sluder G. Role of spindle microtubules in the control of cell cycle timing. J Cell Biol. 1979 Mar;80(3):674–691. doi: 10.1083/jcb.80.3.674. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Sundin O., Varshavsky A. Arrest of segregation leads to accumulation of highly intertwined catenated dimers: dissection of the final stages of SV40 DNA replication. Cell. 1981 Sep;25(3):659–669. doi: 10.1016/0092-8674(81)90173-2. [DOI] [PubMed] [Google Scholar]
  45. Sundin O., Varshavsky A. Terminal stages of SV40 DNA replication proceed via multiply intertwined catenated dimers. Cell. 1980 Aug;21(1):103–114. doi: 10.1016/0092-8674(80)90118-x. [DOI] [PubMed] [Google Scholar]
  46. Tombes R. M., Borisy G. G. Intracellular free calcium and mitosis in mammalian cells: anaphase onset is calcium modulated, but is not triggered by a brief transient. J Cell Biol. 1989 Aug;109(2):627–636. doi: 10.1083/jcb.109.2.627. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. 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]
  48. Watanabe N., Hunt T., Ikawa Y., Sagata N. Independent inactivation of MPF and cytostatic factor (Mos) upon fertilization of Xenopus eggs. Nature. 1991 Jul 18;352(6332):247–248. doi: 10.1038/352247a0. [DOI] [PubMed] [Google Scholar]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press

RESOURCES