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. 1993 Sep 15;90(18):8407–8411. doi: 10.1073/pnas.90.18.8407

DNA topoisomerase II alpha is the major chromosome protein recognized by the mitotic phosphoprotein antibody MPM-2.

S Taagepera 1, P N Rao 1, F H Drake 1, G J Gorbsky 1
PMCID: PMC47365  PMID: 7690961

Abstract

We have determined that the major mitotic phosphoprotein in chromosomes recognized by the antiphosphoprotein antibody MPM-2 is the 170-kDa isoform of topoisomerase II (topo II), the isoform predominant in proliferating cells. As a prerequisite to making this discovery, it was necessary to develop protocols to protect chromosomal proteins from dephosphorylation during cell extraction and chromosome isolation procedures. Immunofluorescence analysis of the large chromosomes prepared from Indian Muntjac cells revealed colocalization of MPM-2 and anti-topo II antibodies to the chromosomal centromeres and to the axial regions of the chromosomal arms. For biochemical fractionation studies, large quantities of chromosomes from the P388D1 mouse lymphocyte cell line were isolated and treated to remove DNA and histone proteins. Immunoblot and immunoprecipitation experiments with this chromosome scaffold fraction identified the major MPM-2-reactive phosphoprotein to be DNA topo II. Using a panel of anti-peptide antibodies specific to the isoforms of topo II, we determined that the major phosphoprotein recognized by MPM-2 is the 170-kDa isoform of topo II, topo II alpha. The 180-kDa isoform, topo II beta, present in the isolated chromosomes in much smaller quantities, is also recognized by MPM-2. The mitotic phosphorylation of the topo II proteins may be critical for proper chromosome condensation and segregation.

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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., 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]
  4. Adolphs K. W., Cheng S. M., Paulson J. R., Laemmli U. K. Isolation of a protein scaffold from mitotic HeLa cell chromosomes. Proc Natl Acad Sci U S A. 1977 Nov;74(11):4937–4941. doi: 10.1073/pnas.74.11.4937. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bae Y. S., Kawasaki I., Ikeda H., Liu L. F. Illegitimate recombination mediated by calf thymus DNA topoisomerase II in vitro. Proc Natl Acad Sci U S A. 1988 Apr;85(7):2076–2080. doi: 10.1073/pnas.85.7.2076. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Boy de la Tour E., Laemmli U. K. The metaphase scaffold is helically folded: sister chromatids have predominantly opposite helical handedness. Cell. 1988 Dec 23;55(6):937–944. doi: 10.1016/0092-8674(88)90239-5. [DOI] [PubMed] [Google Scholar]
  7. 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]
  8. Chung T. D., Drake F. H., Tan K. B., Per S. R., Crooke S. T., Mirabelli C. K. Characterization and immunological identification of cDNA clones encoding two human DNA topoisomerase II isozymes. Proc Natl Acad Sci U S A. 1989 Dec;86(23):9431–9435. doi: 10.1073/pnas.86.23.9431. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Cockerill P. N., Garrard W. T. Chromosomal loop anchorage of the kappa immunoglobulin gene occurs next to the enhancer in a region containing topoisomerase II sites. Cell. 1986 Jan 31;44(2):273–282. doi: 10.1016/0092-8674(86)90761-0. [DOI] [PubMed] [Google Scholar]
  10. D'Arpa P., Machlin P. S., Ratrie H., 3rd, Rothfield N. F., Cleveland D. W., Earnshaw W. C. cDNA cloning of human DNA topoisomerase I: catalytic activity of a 67.7-kDa carboxyl-terminal fragment. Proc Natl Acad Sci U S A. 1988 Apr;85(8):2543–2547. doi: 10.1073/pnas.85.8.2543. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Davis F. M., Tsao T. Y., Fowler S. K., Rao P. N. Monoclonal antibodies to mitotic cells. Proc Natl Acad Sci U S A. 1983 May;80(10):2926–2930. doi: 10.1073/pnas.80.10.2926. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Drake F. H., Hofmann G. A., Bartus H. F., Mattern M. R., Crooke S. T., Mirabelli C. K. Biochemical and pharmacological properties of p170 and p180 forms of topoisomerase II. Biochemistry. 1989 Oct 3;28(20):8154–8160. doi: 10.1021/bi00446a029. [DOI] [PubMed] [Google Scholar]
  13. Drake F. H., Zimmerman J. P., McCabe F. L., Bartus H. F., Per S. R., Sullivan D. M., Ross W. E., Mattern M. R., Johnson R. K., Crooke S. T. Purification of topoisomerase II from amsacrine-resistant P388 leukemia cells. Evidence for two forms of the enzyme. J Biol Chem. 1987 Dec 5;262(34):16739–16747. [PubMed] [Google Scholar]
  14. 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]
  15. Earnshaw W. C., Laemmli U. K. Architecture of metaphase chromosomes and chromosome scaffolds. J Cell Biol. 1983 Jan;96(1):84–93. doi: 10.1083/jcb.96.1.84. [DOI] [PMC free article] [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. Heck M. M., Hittelman W. N., Earnshaw W. C. In vivo phosphorylation of the 170-kDa form of eukaryotic DNA topoisomerase II. Cell cycle analysis. J Biol Chem. 1989 Sep 15;264(26):15161–15164. [PubMed] [Google Scholar]
  18. Hirano T., Mitchison T. J. Cell cycle control of higher-order chromatin assembly around naked DNA in vitro. J Cell Biol. 1991 Dec;115(6):1479–1489. doi: 10.1083/jcb.115.6.1479. [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. Hyman A. A., Mitchison T. J. Two different microtubule-based motor activities with opposite polarities in kinetochores. Nature. 1991 May 16;351(6323):206–211. doi: 10.1038/351206a0. [DOI] [PubMed] [Google Scholar]
  22. Kroll D. J., Rowe T. C. Phosphorylation of DNA topoisomerase II in a human tumor cell line. J Biol Chem. 1991 Apr 25;266(12):7957–7961. [PubMed] [Google Scholar]
  23. 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]
  24. Mirkovitch J., Gasser S. M., Laemmli U. K. Scaffold attachment of DNA loops in metaphase chromosomes. J Mol Biol. 1988 Mar 5;200(1):101–109. doi: 10.1016/0022-2836(88)90336-1. [DOI] [PubMed] [Google Scholar]
  25. Nelson W. G., Liu L. F., Coffey D. S. Newly replicated DNA is associated with DNA topoisomerase II in cultured rat prostatic adenocarcinoma cells. Nature. 1986 Jul 10;322(6075):187–189. doi: 10.1038/322187a0. [DOI] [PubMed] [Google Scholar]
  26. Ohnuki Y. Structure of chromosomes. I. Morphological studies of the spiral structure of human somatic chromosomes. Chromosoma. 1968;25(4):402–428. doi: 10.1007/BF02327721. [DOI] [PubMed] [Google Scholar]
  27. Paulson J. R. Sulfhydryl reagents prevent dephosphorylation and proteolysis of histones in isolated HeLa metaphase chromosomes. Eur J Biochem. 1980 Oct;111(1):189–197. doi: 10.1111/j.1432-1033.1980.tb06092.x. [DOI] [PubMed] [Google Scholar]
  28. Rottmann M., Schröder H. C., Gramzow M., Renneisen K., Kurelec B., Dorn A., Friese U., Müller W. E. Specific phosphorylation of proteins in pore complex-laminae from the sponge Geodia cydonium by the homologous aggregation factor and phorbol ester. Role of protein kinase C in the phosphorylation of DNA topoisomerase II. EMBO J. 1987 Dec 20;6(13):3939–3944. doi: 10.1002/j.1460-2075.1987.tb02735.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. 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]
  30. 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]
  31. Schröder H. C., Trölltsch D., Friese U., Bachmann M., Müller W. E. Mature mRNA is selectively released from the nuclear matrix by an ATP/dATP-dependent mechanism sensitive to topoisomerase inhibitors. J Biol Chem. 1987 Jun 25;262(18):8917–8925. [PubMed] [Google Scholar]
  32. 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]
  33. Takano H., Kohno K., Ono M., Uchida Y., Kuwano M. Increased phosphorylation of DNA topoisomerase II in etoposide-resistant mutants of human cancer KB cells. Cancer Res. 1991 Aug 1;51(15):3951–3957. [PubMed] [Google Scholar]
  34. Tan K. B., Mattern M. R., Boyce R. A., Schein P. S. Elevated DNA topoisomerase II activity in nitrogen mustard-resistant human cells. Proc Natl Acad Sci U S A. 1987 Nov;84(21):7668–7671. doi: 10.1073/pnas.84.21.7668. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. 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]
  36. Uemura T., Tanagida M. Mitotic spindle pulls but fails to separate chromosomes in type II DNA topoisomerase mutants: uncoordinated mitosis. EMBO J. 1986 May;5(5):1003–1010. doi: 10.1002/j.1460-2075.1986.tb04315.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Vandre D. D., Davis F. M., Rao P. N., Borisy G. G. Phosphoproteins are components of mitotic microtubule organizing centers. Proc Natl Acad Sci U S A. 1984 Jul;81(14):4439–4443. doi: 10.1073/pnas.81.14.4439. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Woessner R. D., Chung T. D., Hofmann G. A., Mattern M. R., Mirabelli C. K., Drake F. H., Johnson R. K. Differences between normal and ras-transformed NIH-3T3 cells in expression of the 170kD and 180kD forms of topoisomerase II. Cancer Res. 1990 May 15;50(10):2901–2908. [PubMed] [Google Scholar]
  39. Woessner R. D., Mattern M. R., Mirabelli C. K., Johnson R. K., Drake F. H. Proliferation- and cell cycle-dependent differences in expression of the 170 kilodalton and 180 kilodalton forms of topoisomerase II in NIH-3T3 cells. Cell Growth Differ. 1991 Apr;2(4):209–214. [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. Yang L., Wold M. S., Li J. J., Kelly T. J., Liu L. F. Roles of DNA topoisomerases in simian virus 40 DNA replication in vitro. Proc Natl Acad Sci U S A. 1987 Feb;84(4):950–954. doi: 10.1073/pnas.84.4.950. [DOI] [PMC free article] [PubMed] [Google Scholar]

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