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. 1990 Dec;9(13):4555–4562. doi: 10.1002/j.1460-2075.1990.tb07908.x

Eukaryotic topoisomerases recognize nucleic acid topology by preferentially interacting with DNA crossovers.

E L Zechiedrich 1, N Osheroff 1
PMCID: PMC552253  PMID: 2176156

Abstract

Eukaryotic topoisomerases recognize DNA topology and preferentially react with positively or negatively supercoiled molecules over relaxed substrates. To elucidate the mechanism of this recognition, we examined the interaction of topoisomerases with DNA by electron microscopy. Under all conditions employed, approximately 90% of the bound type I or II enzyme was observed at points of helix--helix juxtaposition on negatively supercoiled plasmids which contained as few as four crossovers. Recognition was independent of torsional stress, as enzyme molecules were also found at crossovers on linear DNA. Since juxtaposed helices are more prevalent in supercoiled compared with relaxed nucleic acids, we propose that eukaryotic topoisomerases I and II recognize underwound or overwound substrates by interacting preferentially with DNA crossovers. This may represent a general mechanism for the recognition of DNA topology by proteins.

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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. Andersen A. H., Christiansen K., Zechiedrich E. L., Jensen P. S., Osheroff N., Westergaard O. Strand specificity of the topoisomerase II mediated double-stranded DNA cleavage reaction. Biochemistry. 1989 Jul 25;28(15):6237–6244. doi: 10.1021/bi00441a015. [DOI] [PubMed] [Google Scholar]
  3. 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]
  4. Baker T. A., Sekimizu K., Funnell B. E., Kornberg A. Extensive unwinding of the plasmid template during staged enzymatic initiation of DNA replication from the origin of the Escherichia coli chromosome. Cell. 1986 Apr 11;45(1):53–64. doi: 10.1016/0092-8674(86)90537-4. [DOI] [PubMed] [Google Scholar]
  5. Benedetti P., Baldi M. I., Mattoccia E., Tocchini-Valentini G. P. Purification and characterization of Xenopus laevis topoisomerase II. EMBO J. 1983;2(8):1303–1308. doi: 10.1002/j.1460-2075.1983.tb01585.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. 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]
  7. Bonven B. J., Gocke E., Westergaard O. A high affinity topoisomerase I binding sequence is clustered at DNAase I hypersensitive sites in Tetrahymena R-chromatin. Cell. 1985 Jun;41(2):541–551. doi: 10.1016/s0092-8674(85)80027-1. [DOI] [PubMed] [Google Scholar]
  8. Brill S. J., DiNardo S., Voelkel-Meiman K., Sternglanz R. Need for DNA topoisomerase activity as a swivel for DNA replication for transcription of ribosomal RNA. 1987 Mar 26-Apr 1Nature. 326(6111):414–416. doi: 10.1038/326414a0. [DOI] [PubMed] [Google Scholar]
  9. Busk H., Thomsen B., Bonven B. J., Kjeldsen E., Nielsen O. F., Westergaard O. Preferential relaxation of supercoiled DNA containing a hexadecameric recognition sequence for topoisomerase I. Nature. 1987 Jun 18;327(6123):638–640. doi: 10.1038/327638a0. [DOI] [PubMed] [Google Scholar]
  10. Camilloni G., Di Martino E., Caserta M., di Mauro E. Eukaryotic DNA topoisomerase I reaction is topology dependent. Nucleic Acids Res. 1988 Jul 25;16(14B):7071–7085. doi: 10.1093/nar/16.14.7071. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Camilloni G., Di Martino E., Di Mauro E., Caserta M. Regulation of the function of eukaryotic DNA topoisomerase I: topological conditions for inactivity. Proc Natl Acad Sci U S A. 1989 May;86(9):3080–3084. doi: 10.1073/pnas.86.9.3080. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Champoux J. J., Dulbecco R. An activity from mammalian cells that untwists superhelical DNA--a possible swivel for DNA replication (polyoma-ethidium bromide-mouse-embryo cells-dye binding assay). Proc Natl Acad Sci U S A. 1972 Jan;69(1):143–146. doi: 10.1073/pnas.69.1.143. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. 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]
  14. 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]
  15. Dillehay L. E., Jacobson-Kram D., Williams J. R. DNA topoisomerases and models of sister-chromatid exchange. Mutat Res. 1989 Nov;215(1):15–23. doi: 10.1016/0027-5107(89)90213-3. [DOI] [PubMed] [Google Scholar]
  16. 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]
  17. 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]
  18. Fuller R. S., Kaguni J. M., Kornberg A. Enzymatic replication of the origin of the Escherichia coli chromosome. Proc Natl Acad Sci U S A. 1981 Dec;78(12):7370–7374. doi: 10.1073/pnas.78.12.7370. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Garg L. C., DiAngelo S., Jacob S. T. Role of DNA topoisomerase I in the transcription of supercoiled rRNA gene. Proc Natl Acad Sci U S A. 1987 May;84(10):3185–3188. doi: 10.1073/pnas.84.10.3185. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Gasser S. M., Laemmli U. K. The organisation of chromatin loops: characterization of a scaffold attachment site. EMBO J. 1986 Mar;5(3):511–518. doi: 10.1002/j.1460-2075.1986.tb04240.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. 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]
  22. Gilmour D. S., Pflugfelder G., Wang J. C., Lis J. T. Topoisomerase I interacts with transcribed regions in Drosophila cells. Cell. 1986 Feb 14;44(3):401–407. doi: 10.1016/0092-8674(86)90461-7. [DOI] [PubMed] [Google Scholar]
  23. Goto T., Wang J. C. Cloning of yeast TOP1, the gene encoding DNA topoisomerase I, and construction of mutants defective in both DNA topoisomerase I and DNA topoisomerase II. Proc Natl Acad Sci U S A. 1985 Nov;82(21):7178–7182. doi: 10.1073/pnas.82.21.7178. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Goto T., Wang J. C. Yeast DNA topoisomerase II is encoded by a single-copy, essential gene. Cell. 1984 Apr;36(4):1073–1080. doi: 10.1016/0092-8674(84)90057-6. [DOI] [PubMed] [Google Scholar]
  25. Goto T., Wang J. C. Yeast DNA topoisomerase II. An ATP-dependent type II topoisomerase that catalyzes the catenation, decatenation, unknotting, and relaxation of double-stranded DNA rings. J Biol Chem. 1982 May 25;257(10):5866–5872. [PubMed] [Google Scholar]
  26. Griffith J. D., Christiansen G. Electron microscope visualization of chromatin and other DNA-protein complexes. Annu Rev Biophys Bioeng. 1978;7:19–35. doi: 10.1146/annurev.bb.07.060178.000315. [DOI] [PubMed] [Google Scholar]
  27. Harland R. M., Weintraub H., McKnight S. L. Transcription of DNA injected into Xenopus oocytes is influenced by template topology. Nature. 1983 Mar 3;302(5903):38–43. doi: 10.1038/302038a0. [DOI] [PubMed] [Google Scholar]
  28. Higgins N. P., Cozzarelli N. R. The binding of gyrase to DNA: analysis by retention by nitrocellulose filters. Nucleic Acids Res. 1982 Nov 11;10(21):6833–6847. doi: 10.1093/nar/10.21.6833. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. 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]
  30. 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]
  31. Kelly T. J. SV40 DNA replication. J Biol Chem. 1988 Dec 5;263(34):17889–17892. [PubMed] [Google Scholar]
  32. Kirkegaard K., Wang J. C. Bacterial DNA topoisomerase I can relax positively supercoiled DNA containing a single-stranded loop. J Mol Biol. 1985 Oct 5;185(3):625–637. doi: 10.1016/0022-2836(85)90075-0. [DOI] [PubMed] [Google Scholar]
  33. 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]
  34. Moore C. L., Klevan L., Wang J. C., Griffith J. D. Gyrase . DNA complexes visualized as looped structures by electron microscopy. J Biol Chem. 1983 Apr 10;258(7):4612–4617. [PubMed] [Google Scholar]
  35. 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]
  36. Muller M. T. Quantitation of eukaryotic topoisomerase I reactivity with DNA. Preferential cleavage of supercoiled DNA. Biochim Biophys Acta. 1985 Mar 20;824(3):263–267. doi: 10.1016/0167-4781(85)90057-0. [DOI] [PubMed] [Google Scholar]
  37. Osheroff N. Biochemical basis for the interactions of type I and type II topoisomerases with DNA. Pharmacol Ther. 1989;41(1-2):223–241. doi: 10.1016/0163-7258(89)90108-3. [DOI] [PubMed] [Google Scholar]
  38. Osheroff N. Eukaryotic topoisomerase II. Characterization of enzyme turnover. J Biol Chem. 1986 Jul 25;261(21):9944–9950. [PubMed] [Google Scholar]
  39. Osheroff N. Role of the divalent cation in topoisomerase II mediated reactions. Biochemistry. 1987 Oct 6;26(20):6402–6406. doi: 10.1021/bi00394a015. [DOI] [PubMed] [Google Scholar]
  40. 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]
  41. Osheroff N., Zechiedrich E. L. Calcium-promoted DNA cleavage by eukaryotic topoisomerase II: trapping the covalent enzyme-DNA complex in an active form. Biochemistry. 1987 Jul 14;26(14):4303–4309. doi: 10.1021/bi00388a018. [DOI] [PubMed] [Google Scholar]
  42. Register J. C., 3rd, Christiansen G., Griffith J. Electron microscopic visualization of the RecA protein-mediated pairing and branch migration phases of DNA strand exchange. J Biol Chem. 1987 Sep 15;262(26):12812–12820. [PubMed] [Google Scholar]
  43. 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]
  44. 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]
  45. Sander M., Hsieh T., Udvardy A., Schedl P. Sequence dependence of Drosophila topoisomerase II in plasmid relaxation and DNA binding. J Mol Biol. 1987 Mar 20;194(2):219–229. doi: 10.1016/0022-2836(87)90370-6. [DOI] [PubMed] [Google Scholar]
  46. Schomburg U., Grosse F. Purification and characterization of DNA topoisomerase II from calf thymus associated with polypeptides of 175 and 150 kDa. Eur J Biochem. 1986 Nov 3;160(3):451–457. doi: 10.1111/j.1432-1033.1986.tb10061.x. [DOI] [PubMed] [Google Scholar]
  47. 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]
  48. Singleton C. K., Wells R. D. The facile generation of covalently closed, circular DNAs with defined negative superhelical densities. Anal Biochem. 1982 May 15;122(2):253–257. doi: 10.1016/0003-2697(82)90277-9. [DOI] [PubMed] [Google Scholar]
  49. Snapka R. M. Topoisomerase inhibitors can selectively interfere with different stages of simian virus 40 DNA replication. Mol Cell Biol. 1986 Dec;6(12):4221–4227. doi: 10.1128/mcb.6.12.4221. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Spengler S. J., Stasiak A., Cozzarelli N. R. The stereostructure of knots and catenanes produced by phage lambda integrative recombination: implications for mechanism and DNA structure. Cell. 1985 Aug;42(1):325–334. doi: 10.1016/s0092-8674(85)80128-8. [DOI] [PubMed] [Google Scholar]
  51. Sperry A. O., Blasquez V. C., Garrard W. T. Dysfunction of chromosomal loop attachment sites: illegitimate recombination linked to matrix association regions and topoisomerase II. Proc Natl Acad Sci U S A. 1989 Jul;86(14):5497–5501. doi: 10.1073/pnas.86.14.5497. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Sugino A., Cozzarelli N. R. The intrinsic ATPase of DNA gyrase. J Biol Chem. 1980 Jul 10;255(13):6299–6306. [PubMed] [Google Scholar]
  53. 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]
  54. Tabuchi H., Hirose S. DNA supercoiling facilitates formation of the transcription initiation complex on the fibroin gene promoter. J Biol Chem. 1988 Oct 25;263(30):15282–15287. [PubMed] [Google Scholar]
  55. Thrash C., Bankier A. T., Barrell B. G., Sternglanz R. Cloning, characterization, and sequence of the yeast DNA topoisomerase I gene. Proc Natl Acad Sci U S A. 1985 Jul;82(13):4374–4378. doi: 10.1073/pnas.82.13.4374. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. 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]
  57. 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]
  58. 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]
  59. 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]
  60. Wang J. C. DNA topoisomerases. Annu Rev Biochem. 1985;54:665–697. doi: 10.1146/annurev.bi.54.070185.003313. [DOI] [PubMed] [Google Scholar]
  61. 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]
  62. Wasserman S. A., Cozzarelli N. R. Biochemical topology: applications to DNA recombination and replication. Science. 1986 May 23;232(4753):951–960. doi: 10.1126/science.3010458. [DOI] [PubMed] [Google Scholar]
  63. Weaver D. T., Fields-Berry S. C., DePamphilis M. L. The termination region for SV40 DNA replication directs the mode of separation for the two sibling molecules. Cell. 1985 Jun;41(2):565–575. doi: 10.1016/s0092-8674(85)80029-5. [DOI] [PubMed] [Google Scholar]
  64. Wells R. D. Unusual DNA structures. J Biol Chem. 1988 Jan 25;263(3):1095–1098. [PubMed] [Google Scholar]
  65. 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]
  66. Zechiedrich E. L., Christiansen K., Andersen A. H., Westergaard O., Osheroff N. Double-stranded DNA cleavage/religation reaction of eukaryotic topoisomerase II: evidence for a nicked DNA intermediate. Biochemistry. 1989 Jul 25;28(15):6229–6236. doi: 10.1021/bi00441a014. [DOI] [PubMed] [Google Scholar]
  67. von Hippel P. H., Berg O. G. Facilitated target location in biological systems. J Biol Chem. 1989 Jan 15;264(2):675–678. [PubMed] [Google Scholar]

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