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. 1994 Sep 2;126(6):1331–1340. doi: 10.1083/jcb.126.6.1331

An RNase-sensitive particle containing Drosophila melanogaster DNA topoisomerase II

PMCID: PMC2290960  PMID: 8089168

Abstract

Most DNA topoisomerase II (topo II) in cell-free extracts of 0-2-h old Drosophila embryos appears to be nonnuclear and remains in the supernatant after low-speed centrifugation (10,000 g). Virtually all of this apparently soluble topo II is particulate with a sedimentation coefficient of 67 S. Similar topo II-containing particles were detected in Drosophila Kc tissue culture cells, 16-19-h old embryos and extracts of progesterone-matured oocytes from Xenopus. Drosophila topo II- containing particles were insensitive to EDTA, Triton X-100 and DNase I, but could be disrupted by incubation with 0.3 M NaCl or RNase A. After either disruptive treatment, topo II sedimented at 9 S. topo II- containing particles were also sensitive to micrococcal nuclease. Results of chemical cross-linking corroborated those obtained by centrifugation. Immunoblot analyses demonstrated that topo II- containing particles lacked significant amounts of lamin, nuclear pore complex protein gp210, proliferating cell nuclear antigen, RNA polymerase II subunits, histones, coilin, and nucleolin. Northern blot analyses demonstrated that topo II-containing particles lacked U RNA. Thus, current data support the notion that nonnuclear Drosophila topo II-containing particles are composed largely of topo II and an unknown RNA molecule(s).

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Selected References

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

  1. 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]
  2. 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]
  3. Allis C. D., Waring G. L., Mahowald A. P. Mass isolation of pole cells from Drosophila melanogaster. Dev Biol. 1977 Apr;56(2):372–381. doi: 10.1016/0012-1606(77)90277-9. [DOI] [PubMed] [Google Scholar]
  4. Andrade L. E., Tan E. M., Chan E. K. Immunocytochemical analysis of the coiled body in the cell cycle and during cell proliferation. Proc Natl Acad Sci U S A. 1993 Mar 1;90(5):1947–1951. doi: 10.1073/pnas.90.5.1947. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Berrios M., Osheroff N., Fisher P. A. In situ localization of DNA topoisomerase II, a major polypeptide component of the Drosophila nuclear matrix fraction. Proc Natl Acad Sci U S A. 1985 Jun;82(12):4142–4146. doi: 10.1073/pnas.82.12.4142. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bogenhagen D. F. Proteolytic footprinting of transcription factor TFIIIA reveals different tightly binding sites for 5S RNA and 5S DNA. Mol Cell Biol. 1993 Sep;13(9):5149–5158. doi: 10.1128/mcb.13.9.5149. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Buchenau P., Saumweber H., Arndt-Jovin D. J. Consequences of topoisomerase II inhibition in early embryogenesis of Drosophila revealed by in vivo confocal laser scanning microscopy. J Cell Sci. 1993 Apr;104(Pt 4):1175–1185. doi: 10.1242/jcs.104.4.1175. [DOI] [PubMed] [Google Scholar]
  8. Clemens K. R., Wolf V., McBryant S. J., Zhang P., Liao X., Wright P. E., Gottesfeld J. M. Molecular basis for specific recognition of both RNA and DNA by a zinc finger protein. Science. 1993 Apr 23;260(5107):530–533. doi: 10.1126/science.8475383. [DOI] [PubMed] [Google Scholar]
  9. 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]
  10. 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]
  11. 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]
  12. Filson A. J., Lewis A., Blobel G., Fisher P. A. Monoclonal antibodies prepared against the major Drosophila nuclear Matrix-pore complex-lamina glycoprotein bind specifically to the nuclear envelope in situ. J Biol Chem. 1985 Mar 10;260(5):3164–3172. [PubMed] [Google Scholar]
  13. Fisher P. A. Chromosomes and chromatin structure: the extrachromosomal karyoskeleton. Curr Opin Cell Biol. 1989 Jun;1(3):447–453. doi: 10.1016/0955-0674(89)90004-5. [DOI] [PubMed] [Google Scholar]
  14. Fisher P. A., Lin L., McConnell M., Greenleaf A., Lee J. M., Smith D. E. Heat shock-induced appearance of RNA polymerase II in karyoskeletal protein-enriched (nuclear "matrix") fractions correlates with transcriptional shutdown in Drosophila melanogaster. J Biol Chem. 1989 Feb 25;264(6):3464–3469. [PubMed] [Google Scholar]
  15. Gasser S. M., Laemmli U. K. Cohabitation of scaffold binding regions with upstream/enhancer elements of three developmentally regulated genes of D. melanogaster. Cell. 1986 Aug 15;46(4):521–530. doi: 10.1016/0092-8674(86)90877-9. [DOI] [PubMed] [Google Scholar]
  16. 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]
  17. Greber U. F., Senior A., Gerace L. A major glycoprotein of the nuclear pore complex is a membrane-spanning polypeptide with a large lumenal domain and a small cytoplasmic tail. EMBO J. 1990 May;9(5):1495–1502. doi: 10.1002/j.1460-2075.1990.tb08267.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Heine M. A., Rankin M. L., DiMario P. J. The Gly/Arg-rich (GAR) domain of Xenopus nucleolin facilitates in vitro nucleic acid binding and in vivo nucleolar localization. Mol Biol Cell. 1993 Nov;4(11):1189–1204. doi: 10.1091/mbc.4.11.1189. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. 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]
  20. 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]
  21. Honda B. M., Roeder R. G. Association of a 5S gene transcription factor with 5S RNA and altered levels of the factor during cell differentiation. Cell. 1980 Nov;22(1 Pt 1):119–126. doi: 10.1016/0092-8674(80)90160-9. [DOI] [PubMed] [Google Scholar]
  22. Howard M. T., Lee M. P., Hsieh T. S., Griffith J. D. Drosophila topoisomerase II-DNA interactions are affected by DNA structure. J Mol Biol. 1991 Jan 5;217(1):53–62. doi: 10.1016/0022-2836(91)90610-i. [DOI] [PubMed] [Google Scholar]
  23. Izaurralde E., Mirkovitch J., Laemmli U. K. Interaction of DNA with nuclear scaffolds in vitro. J Mol Biol. 1988 Mar 5;200(1):111–125. doi: 10.1016/0022-2836(88)90337-3. [DOI] [PubMed] [Google Scholar]
  24. Jazwinski S. M., Edelman G. M. Evidence for participation of a multiprotein complex in yeast DNA replication in vitro. J Biol Chem. 1984 Jun 10;259(11):6852–6857. [PubMed] [Google Scholar]
  25. Jenkins J. R., Ayton P., Jones T., Davies S. L., Simmons D. L., Harris A. L., Sheer D., Hickson I. D. Isolation of cDNA clones encoding the beta isozyme of human DNA topoisomerase II and localisation of the gene to chromosome 3p24. Nucleic Acids Res. 1992 Nov 11;20(21):5587–5592. doi: 10.1093/nar/20.21.5587. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Klein F., Laroche T., Cardenas M. E., Hofmann J. F., Schweizer D., Gasser S. M. Localization of RAP1 and topoisomerase II in nuclei and meiotic chromosomes of yeast. J Cell Biol. 1992 Jun;117(5):935–948. doi: 10.1083/jcb.117.5.935. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. 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]
  28. Lee M. P., Sander M., Hsieh T. Nuclease protection by Drosophila DNA topoisomerase II. Enzyme/DNA contacts at the strong topoisomerase II cleavage sites. J Biol Chem. 1989 Dec 25;264(36):21779–21787. [PubMed] [Google Scholar]
  29. Liu L. F., Liu C. C., Alberts B. M. Type II DNA topoisomerases: enzymes that can unknot a topologically knotted DNA molecule via a reversible double-strand break. Cell. 1980 Mar;19(3):697–707. doi: 10.1016/s0092-8674(80)80046-8. [DOI] [PubMed] [Google Scholar]
  30. Luke M., Bogenhagen D. F. Quantitation of type II topoisomerase in oocytes and eggs of Xenopus laevis. Dev Biol. 1989 Dec;136(2):459–468. doi: 10.1016/0012-1606(89)90271-6. [DOI] [PubMed] [Google Scholar]
  31. 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]
  32. 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]
  33. Mirkovitch J., Gasser S. M., Laemmli U. K. Relation of chromosome structure and gene expression. Philos Trans R Soc Lond B Biol Sci. 1987 Dec 15;317(1187):563–574. doi: 10.1098/rstb.1987.0081. [DOI] [PubMed] [Google Scholar]
  34. Ng L., Prelich G., Anderson C. W., Stillman B., Fisher P. A. Drosophila proliferating cell nuclear antigen. Structural and functional homology with its mammalian counterpart. J Biol Chem. 1990 Jul 15;265(20):11948–11954. [PubMed] [Google Scholar]
  35. Noguchi H., Prem veer Reddy G., Pardee A. B. Rapid incorporation of label from ribonucleoside disphosphates into DNA by a cell-free high molecular weight fraction from animal cell nuclei. Cell. 1983 Feb;32(2):443–451. doi: 10.1016/0092-8674(83)90464-6. [DOI] [PubMed] [Google Scholar]
  36. Nolan J. M., Lee M. P., Wyckoff E., Hsieh T. S. Isolation and characterization of the gene encoding Drosophila DNA topoisomerase II. Proc Natl Acad Sci U S A. 1986 Jun;83(11):3664–3668. doi: 10.1073/pnas.83.11.3664. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Pelham H. R., Brown D. D. A specific transcription factor that can bind either the 5S RNA gene or 5S RNA. Proc Natl Acad Sci U S A. 1980 Jul;77(7):4170–4174. doi: 10.1073/pnas.77.7.4170. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. 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]
  39. Rose D., Thomas W., Holm C. Segregation of recombined chromosomes in meiosis I requires DNA topoisomerase II. Cell. 1990 Mar 23;60(6):1009–1017. doi: 10.1016/0092-8674(90)90349-j. [DOI] [PubMed] [Google Scholar]
  40. STUDIER F. W. SEDIMENTATION STUDIES OF THE SIZE AND SHAPE OF DNA. J Mol Biol. 1965 Feb;11:373–390. doi: 10.1016/s0022-2836(65)80064-x. [DOI] [PubMed] [Google Scholar]
  41. Sander M., Hsieh T. Double strand DNA cleavage by type II DNA topoisomerase from Drosophila melanogaster. J Biol Chem. 1983 Jul 10;258(13):8421–8428. [PubMed] [Google Scholar]
  42. Shamu C. E., Murray A. W. Sister chromatid separation in frog egg extracts requires DNA topoisomerase II activity during anaphase. J Cell Biol. 1992 Jun;117(5):921–934. doi: 10.1083/jcb.117.5.921. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Shelton E. R., Osheroff N., Brutlag D. L. DNA topoisomerase II from Drosophila melanogaster. Purification and physical characterization. J Biol Chem. 1983 Aug 10;258(15):9530–9535. [PubMed] [Google Scholar]
  44. Smith D. E., Fisher P. A. Interconversion of Drosophila nuclear lamin isoforms during oogenesis, early embryogenesis, and upon entry of cultured cells into mitosis. J Cell Biol. 1989 Feb;108(2):255–265. doi: 10.1083/jcb.108.2.255. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Spikes D., Bingham P. M. Analysis of spliceosome assembly and the structure of a regulated intron in Drosophila in vitro splicing extracts. Nucleic Acids Res. 1992 Nov 11;20(21):5719–5727. doi: 10.1093/nar/20.21.5719. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. 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]
  47. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. 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]
  49. Wang J. C. DNA topoisomerases. Annu Rev Biochem. 1985;54:665–697. doi: 10.1146/annurev.bi.54.070185.003313. [DOI] [PubMed] [Google Scholar]
  50. Weeks J. R., Coulter D. E., Greenleaf A. L. Immunological studies of RNA polymerase II using antibodies to subunits of Drosophila and wheat germ enzyme. J Biol Chem. 1982 May 25;257(10):5884–5892. [PubMed] [Google Scholar]
  51. Whalen A. M., McConnell M., Fisher P. A. Developmental regulation of Drosophila DNA topoisomerase II. J Cell Biol. 1991 Jan;112(2):203–213. doi: 10.1083/jcb.112.2.203. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. 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]
  53. Wozniak R. W., Bartnik E., Blobel G. Primary structure analysis of an integral membrane glycoprotein of the nuclear pore. J Cell Biol. 1989 Jun;108(6):2083–2092. doi: 10.1083/jcb.108.6.2083. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. 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]

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