Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 1987 Jan 1;241(1):111–119. doi: 10.1042/bj2410111

Mechanism of inhibition of mammalian DNA topoisomerase I by heparin.

K Ishii, S Futaki, H Uchiyama, K Nagasawa, T Andoh
PMCID: PMC1147532  PMID: 3032152

Abstract

We have previously shown that heparin is a potent inhibitor of a mammalian DNA topoisomerase I. We have now investigated the mechanism of its inhibition. This was carried out first by scrutinizing the structural features of heparin molecules responsible for the inhibition. Commercial heparin preparation was fractionated by antithrombin III-Sepharose into non-adsorbed, low-affinity and high-affinity fractions, of which only the high-affinity fraction of heparin is known to contain a specific oligosaccharide sequence responsible for the binding to antithrombin III. These fractions all exhibited essentially similar inhibitory activities. Furthermore, when chemically sulphated to an extent comparable with or higher than heparin, otherwise inactive glycosaminoglycans such as heparan sulphate, chondroitin 4-sulphate, dermatan sulphate and neutral polysaccharides such as dextran and amylose were converted into potent inhibitors. Sulphated dermatan sulphate, one of the model compounds, was further shown to bind competitively to the same sites on the enzyme as heparin. These observations strongly suggested that topoisomerase inhibition by heparin is attributable primarily, if not entirely, to the highly sulphated polyanionic nature of the molecules. In a second series of experiments we examined whether heparin inhibits only one or both of the topoisomerase reactions, i.e. nicking and re-joining. It was demonstrated that both reactions were inhibited by heparin, but the nicking reaction was more severely affected than was the re-joining reaction.

Full text

PDF
111

Images in this article

114Fig. 1.
on p.114
114Fig. 2.
on p.114
116Fig. 3.
on p.116
117Fig. 4.
on p.117
117Fig. 5.
on p.117

Selected References

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

  1. Azizkhan R. G., Azizkhan J. C., Zetter B. R., Folkman J. Mast cell heparin stimulates migration of capillary endothelial cells in vitro. J Exp Med. 1980 Oct 1;152(4):931–944. doi: 10.1084/jem.152.4.931. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Been M. D., Champoux J. J. DNA breakage and closure by rat liver type 1 topoisomerase: separation of the half-reactions by using a single-stranded DNA substrate. Proc Natl Acad Sci U S A. 1981 May;78(5):2883–2887. doi: 10.1073/pnas.78.5.2883. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bengtsson G., Olivecrona T. The hepatic heparin releasable lipase binds to high density lipoproteins. FEBS Lett. 1980 Oct 6;119(2):290–292. doi: 10.1016/0014-5793(80)80274-2. [DOI] [PubMed] [Google Scholar]
  4. Björk I., Lindahl U. Mechanism of the anticoagulant action of heparin. Mol Cell Biochem. 1982 Oct 29;48(3):161–182. doi: 10.1007/BF00421226. [DOI] [PubMed] [Google Scholar]
  5. Castellot J. J., Jr, Addonizio M. L., Rosenberg R., Karnovsky M. J. Cultured endothelial cells produce a heparinlike inhibitor of smooth muscle cell growth. J Cell Biol. 1981 Aug;90(2):372–379. doi: 10.1083/jcb.90.2.372. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Cozzarelli N. R. DNA gyrase and the supercoiling of DNA. Science. 1980 Feb 29;207(4434):953–960. doi: 10.1126/science.6243420. [DOI] [PubMed] [Google Scholar]
  7. DODGSON K. S., PRICE R. G. A note on the determination of the ester sulphate content of sulphated polysaccharides. Biochem J. 1962 Jul;84:106–110. doi: 10.1042/bj0840106. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. DiCioccio R. A., Srivastava B. I. Inhibition of deoxynucleotide-polymerizing enzyme activities of human cells and of simian sarcoma virus by heparin. Cancer Res. 1978 Aug;38(8):2401–2407. [PubMed] [Google Scholar]
  9. DiNardo S., Voelkel K. A., Sternglanz R., Reynolds A. E., Wright A. Escherichia coli DNA topoisomerase I mutants have compensatory mutations in DNA gyrase genes. Cell. 1982 Nov;31(1):43–51. doi: 10.1016/0092-8674(82)90403-2. [DOI] [PubMed] [Google Scholar]
  10. Fairfield F. R., Bauer W. R., Simpson M. V. Studies on mitochondrial type I topoisomerase and on its function. Biochim Biophys Acta. 1985 Jan 29;824(1):45–57. doi: 10.1016/0167-4781(85)90028-4. [DOI] [PubMed] [Google Scholar]
  11. Fleischmann G., Pflugfelder G., Steiner E. K., Javaherian K., Howard G. C., Wang J. C., Elgin S. C. Drosophila DNA topoisomerase I is associated with transcriptionally active regions of the genome. Proc Natl Acad Sci U S A. 1984 Nov;81(22):6958–6962. doi: 10.1073/pnas.81.22.6958. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Funahashi M., Matsumoto I., Seno N. Preparation of three types of heparin-sepharose and their binding activities to thrombin and antithrombin III. Anal Biochem. 1982 Nov 1;126(2):414–421. doi: 10.1016/0003-2697(82)90537-1. [DOI] [PubMed] [Google Scholar]
  13. Gellert M. DNA topoisomerases. Annu Rev Biochem. 1981;50:879–910. doi: 10.1146/annurev.bi.50.070181.004311. [DOI] [PubMed] [Google Scholar]
  14. Germond J. E., Hirt B., Oudet P., Gross-Bellark M., Chambon P. Folding of the DNA double helix in chromatin-like structures from simian virus 40. Proc Natl Acad Sci U S A. 1975 May;72(5):1843–1847. doi: 10.1073/pnas.72.5.1843. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Gocke E., Bonven B. J., Westergaard O. A site and strand specific nuclease activity with analogies to topoisomerase I frames the rRNA gene of Tetrahymena. Nucleic Acids Res. 1983 Nov 25;11(22):7661–7678. doi: 10.1093/nar/11.22.7661. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Hsiang Y. H., Hertzberg R., Hecht S., Liu L. F. Camptothecin induces protein-linked DNA breaks via mammalian DNA topoisomerase I. J Biol Chem. 1985 Nov 25;260(27):14873–14878. [PubMed] [Google Scholar]
  17. Ishii K., Hasegawa T., Fujisawa K., Andoh T. Rapid purification and characterization of DNA topoisomerase I from cultured mouse mammary carcinoma FM3A cells. J Biol Chem. 1983 Oct 25;258(20):12728–12732. [PubMed] [Google Scholar]
  18. Ishii K., Katase A., Andoh T., Seno N. Inhibition of topoisomerase I by heparin. Biochem Biophys Res Commun. 1982 Jan 29;104(2):541–547. doi: 10.1016/0006-291x(82)90671-4. [DOI] [PubMed] [Google Scholar]
  19. Jacques L. B. Heparin: an old drug with a new paradigm. Science. 1979 Nov 2;206(4418):528–533. doi: 10.1126/science.386509. [DOI] [PubMed] [Google Scholar]
  20. Javaherian K., Liu L. F. Association of eukaryotic DNA topoisomerase I with nucleosomes and chromosomal proteins. Nucleic Acids Res. 1983 Jan 25;11(2):461–472. doi: 10.1093/nar/11.2.461. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Lam L. H., Silbert J. E., Rosenberg R. D. The separation of active and inactive forms of heparin. Biochem Biophys Res Commun. 1976 Mar 22;69(2):570–577. doi: 10.1016/0006-291x(76)90558-1. [DOI] [PubMed] [Google Scholar]
  22. Laurent T. C., Tengblad A., Thunberg L., Hök M., Lindahl U. The molecular-weight-dependence of the anti-coagulant activity of heparin. Biochem J. 1978 Nov 1;175(2):691–701. doi: 10.1042/bj1750691. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Liu L. F. DNA topoisomerases--enzymes that catalyse the breaking and rejoining of DNA. CRC Crit Rev Biochem. 1983;15(1):1–24. doi: 10.3109/10409238309102799. [DOI] [PubMed] [Google Scholar]
  24. Muller M. T., Pfund W. P., Mehta V. B., Trask D. K. Eukaryotic type I topoisomerase is enriched in the nucleolus and catalytically active on ribosomal DNA. EMBO J. 1985 May;4(5):1237–1243. doi: 10.1002/j.1460-2075.1985.tb03766.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Nakano N. Establishment of cell lines in vitro from a mammary ascites tumor of mouse and biological properties of the established lines in a serum containing medium. Tohoku J Exp Med. 1966 Jan 25;88(1):69–84. doi: 10.1620/tjem.88.69. [DOI] [PubMed] [Google Scholar]
  26. Nimrod A., Lindner H. R. Heparin facilitates the induction of LH receptors by FSH in granulosa cells cultured in serum-enriched medium. FEBS Lett. 1980 Sep 22;119(1):155–157. doi: 10.1016/0014-5793(80)81019-2. [DOI] [PubMed] [Google Scholar]
  27. Ogamo A., Nagai A., Nagasawa K. Binding of heparin fractions and other polysulfated polysaccharides to plasma fibronectin: effects of molecular size and degree of sulfation of polysaccharides. Biochim Biophys Acta. 1985 Jul 26;841(1):30–41. [PubMed] [Google Scholar]
  28. Ogamo A., Uchiyama H., Nagasawa K. Separation of heparin into fractions with different anticoagulant activity by hydrophobic interaction chromatography. Biochim Biophys Acta. 1980 Dec 16;626(2):477–485. doi: 10.1016/0005-2795(80)90144-0. [DOI] [PubMed] [Google Scholar]
  29. Olivecrona T., Bengtsson G., Marklund S. E., Lindahl U., Hök M. Heparin-lipoprotein lipase interactions. Fed Proc. 1977 Jan;36(1):60–65. [PubMed] [Google Scholar]
  30. Pfeffer S. R., Stahl S. J., Chamberlin M. J. Binding of Escherichia coli RNA polymerase to T7 DNA. Displacement of holoenzyme from promoter complexes by heparin. J Biol Chem. 1977 Aug 10;252(15):5403–5407. [PubMed] [Google Scholar]
  31. Pruss G. J., Manes S. H., Drlica K. Escherichia coli DNA topoisomerase I mutants: increased supercoiling is corrected by mutations near gyrase genes. Cell. 1982 Nov;31(1):35–42. doi: 10.1016/0092-8674(82)90402-0. [DOI] [PubMed] [Google Scholar]
  32. Rosenberg R. D. Chemistry of the hemostatic mechanism and its relationship to the action of heparin. Fed Proc. 1977 Jan;36(1):10–18. [PubMed] [Google Scholar]
  33. Sakakibara Y., Tomizawa J. I. Replication of colicin E1 plasmid DNA in cell extracts. Proc Natl Acad Sci U S A. 1974 Mar;71(3):802–806. doi: 10.1073/pnas.71.3.802. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Shing Y., Folkman J., Sullivan R., Butterfield C., Murray J., Klagsbrun M. Heparin affinity: purification of a tumor-derived capillary endothelial cell growth factor. Science. 1984 Mar 23;223(4642):1296–1299. doi: 10.1126/science.6199844. [DOI] [PubMed] [Google Scholar]
  35. Slobin L. I. The inhibition of elongation factor 1 activity by heparin. Biochem Biophys Res Commun. 1976 Dec 6;73(3):539–547. doi: 10.1016/0006-291x(76)90844-5. [DOI] [PubMed] [Google Scholar]
  36. Stathakis N. E., Mosesson M. W. Interactions among heparin, cold-insoluble globulin, and fibrinogen in formation of the heparin-precipitable fraction of plasma. J Clin Invest. 1977 Oct;60(4):855–865. doi: 10.1172/JCI108840. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Sternglanz R., DiNardo S., Voelkel K. A., Nishimura Y., Hirota Y., Becherer K., Zumstein L., Wang J. C. Mutations in the gene coding for Escherichia coli DNA topoisomerase I affect transcription and transposition. Proc Natl Acad Sci U S A. 1981 May;78(5):2747–2751. doi: 10.1073/pnas.78.5.2747. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Thornton S. C., Mueller S. N., Levine E. M. Human endothelial cells: use of heparin in cloning and long-term serial cultivation. Science. 1983 Nov 11;222(4624):623–625. doi: 10.1126/science.6635659. [DOI] [PubMed] [Google Scholar]
  39. Thrash C., Voelkel K., DiNardo S., Sternglanz R. Identification of Saccharomyces cerevisiae mutants deficient in DNA topoisomerase I activity. J Biol Chem. 1984 Feb 10;259(3):1375–1377. [PubMed] [Google Scholar]
  40. Uemura T., Yanagida M. Isolation of type I and II DNA topoisomerase mutants from fission yeast: single and double mutants show different phenotypes in cell growth and chromatin organization. EMBO J. 1984 Aug;3(8):1737–1744. doi: 10.1002/j.1460-2075.1984.tb02040.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Waldman A. A., Goldstein J. Inhibition by heparin of globin messenger rbinucleic acid translation in a mammalian cell-free system. Biochemistry. 1973 Jul 3;12(14):2706–2711. doi: 10.1021/bi00738a025. [DOI] [PubMed] [Google Scholar]
  42. Walter G., Zillig W., Palm P., Fuchs E. Initiation of DNA-dependent RNA synthesis and the effect of heparin on RNA polymerase. Eur J Biochem. 1967 Dec;3(2):194–201. doi: 10.1111/j.1432-1033.1967.tb19515.x. [DOI] [PubMed] [Google Scholar]
  43. Wang J. C. DNA topoisomerases. Annu Rev Biochem. 1985;54:665–697. doi: 10.1146/annurev.bi.54.070185.003313. [DOI] [PubMed] [Google Scholar]
  44. Wang J. C. Interaction between DNA and an Escherichia coli protein omega. J Mol Biol. 1971 Feb 14;55(3):523–533. doi: 10.1016/0022-2836(71)90334-2. [DOI] [PubMed] [Google Scholar]
  45. Weisbrod S. T. Properties of active nucleosomes as revealed by HMG 14 and 17 chromatography. Nucleic Acids Res. 1982 Mar 25;10(6):2017–2042. doi: 10.1093/nar/10.6.2017. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. van de Water L., 3rd, Schroeder S., Crenshaw E. B., 3rd, Hynes R. O. Phagocytosis of gelatin-latex particles by a murine macrophage line is dependent on fibronectin and heparin. J Cell Biol. 1981 Jul;90(1):32–39. doi: 10.1083/jcb.90.1.32. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES