Analysis of DNA Topology in Yeast Chromatin

  • Randall H. Morse
Part of the Methods in Molecular Biology book series (MIMB, volume 523)


Topology of closed circular DNA is affected by its packaging into nucleosomes and potentially by alteration of nucleosome structure. Changes in topology that reflect alterations in chromatin structure can be measured and quantified using closed circular plasmids from living yeast. Here we describe detailed protocols for measuring DNA topology in yeast chromatin.

Key words

Chromatin yeast nucleosomes topology 



I thank Dr. Len Lutter for providing Fig. 7.2 .


  1. 1.
    Keller, W., and Wendel, I. (1974) Stepwise relaxation of supercoiled SV40 DNA. Cold Spring Harbor Symp. Quant. Biol., 39, 199–208.Google Scholar
  2. 2.
    Germond, J.E., Hirt, B., Oudet, P., Gross-Bellard, M., and Chambon, P. (1975) Folding of the DNA double helix in chromatin-like structures from simian virus 40. Proc. Natl. Acad. Sci. U.S.A., 72, 1843–1847.PubMedCrossRefGoogle Scholar
  3. 3.
    Simpson, R.T., Thoma, F., and Brubaker, J.M. (1985) Chromatin reconstituted from tandemly repeated cloned DNA fragments and core histones: a model system for study of higher order structure. Cell, 42, 799–808.PubMedCrossRefGoogle Scholar
  4. 4.
    Norton, V.G., Imai, B.S., Yau, P., and Bradbury, E.M. (1989) Histone acetylation reduces nucleosome core particle linking number change. Cell, 57, 449–457.PubMedCrossRefGoogle Scholar
  5. 5.
    Keller, W., Muller, U., Eicken, I., Wendel, I., and Zentgraf, H. (1978) Biochemical and ultrastructural analysis of SV40 chromatin. Cold Spring Harbor Symp. Quant. Biol., 42, 227–243.PubMedGoogle Scholar
  6. 6.
    Pederson, D.S., Venkatesan, M., Thoma, F., and Simpson, R.T. (1986) Isolation of an episomal yeast gene and replication origin as chromatin. Proc. Natl. Acad. Sci. U.S.A., 83, 7206–7210.PubMedCrossRefGoogle Scholar
  7. 7.
    Morse, R.H., Pederson, D.S., Dean, A., and Simpson, R.T. (1987) Yeast nucleosomes allow thermal untwisting of DNA. Nucl. Acids. Res., 15, 10311–10330.PubMedCrossRefGoogle Scholar
  8. 8.
    White, J.H., Cozzarelli, N.R., and Bauer, W.R. (1988) Helical repeat and linking number of surface wrapped DNA. Science, 241, 323–327.PubMedCrossRefGoogle Scholar
  9. 9.
    Hayes, J.J., Tullius, T.D., and Wolffe, A.P. (1990) The structure of DNA in a nucleosome. Proc. Natl. Acad. U.S.A., 87, 7405–7409.CrossRefGoogle Scholar
  10. 10.
    Freeman, L.T., and Garrard, W.T. (1992) DNA supercoiling in chromatin structure and gene expression. Crit. Rev. Euk. Exp., 2, 165–209.Google Scholar
  11. 11.
    Lenfant, F., Mann, R.K., Thomsen, B., Ling, X., and Grunstein, M. (1996) All four core histone N-termini contain sequences required for the repression of basal transcription in yeast. EMBO J., 15, 3974–3985.PubMedGoogle Scholar
  12. 12.
    Smith, M.M., Yang, P., Santisteban, M.S., Boone, P.W., Goldstein, A.T., and Megee, P.C. (1996) A novel histone H4 mutant defective in nuclear division and mitotic chromosome transmission. Mol. Cell. Biol., 16, 1017–1026.PubMedGoogle Scholar
  13. 13.
    Wechsler, M.A., Kladde, M.P., Alfieri, J.A., and Peterson, C.L. (1997) Effects of Sin-versions of histone H4 on yeast chromatin structure and function. EMBO J., 16, 2086–2095.CrossRefGoogle Scholar
  14. 14.
    Kim, U.J., Han, M., Kayne, P., and Grunstein, M. (1988) Effects of histone H4 depletion on the cell cycle and transcription of Saccharomyces cerevisiae. EMBO J., 7, 2211–2219.PubMedGoogle Scholar
  15. 15.
    Norton, V.G., Marvin, K.W., Yau, P., and Bradbury, E.M. (1990) Nucleosome linking number change controlled by acetylation of histones H3 and H4. J. Biol. Chem., 265, 19848–19852.PubMedGoogle Scholar
  16. 16.
    Morse, R.H., and Cantor, C.R. (1986) Effect of trypsinization and histone H5 addition on DNA twist and topology in reconstituted minichromosomes. Nucl. Acids Res., 14, 3293–3310.PubMedCrossRefGoogle Scholar
  17. 17.
    Thomsen, B., Bendixen, C., and Westegaard, O. (1991) Histone hyperacetylation is accompanied by changes in DNA topology in vivo. Eur. J. Biochem., 201, 107–111.PubMedCrossRefGoogle Scholar
  18. 18.
    Lutter, L.C., Judis, L., and Paretti, R.F. (1992) The effects of histone acetylation on chromatin topology in vivo. Mol. Cell. Biol., 12, 5004–5014.PubMedGoogle Scholar
  19. 19.
    Stafford, G.A., and Morse, R.H. (1997) Chromatin remodeling by transcriptional activation domains in a yeast episome. J. Biol. Chem., 272, 11526–11534.PubMedCrossRefGoogle Scholar
  20. 20.
    Wong, J., Shi, Y.-B., and Wolffe, A.P. (1997) Determinants of chromatin disruption and transcriptional regulation instigated by the thyroid hormone receptor: hormone-regulated chromatin disruption is not sufficient for transcriptional activation. EMBO J., 16, 3158–3171.PubMedCrossRefGoogle Scholar
  21. 21.
    Boeger, H., Griesenbeck, J., Strattan, J.S., and Kornberg, R.D. (2003) Nucleosomes unfold completely at a transcriptionally active promoter. Mol. Cell, 11, 1587–1598.PubMedCrossRefGoogle Scholar
  22. 22.
    Korber, P., Luckenbach, T., Blaschke, D., and Horz, W. (2004) Evidence for histone eviction in trans upon induction of the yeast PHO5 promoter. Mol. Cell. Biol., 24, 10965–10974.PubMedCrossRefGoogle Scholar
  23. 23.
    Depew, R.E., and Wang, J.C. (1975) Conformational fluctuations of DNA helix. Proc. Natl. Acad. Sci. U.S.A., 72, 4275–4280.PubMedCrossRefGoogle Scholar
  24. 24.
    Pulleyblank, D.E., Shure, M., Tang, D., Vinograd, J., and Vosberg, H.-S. (1975) Action of nicking-closing enzyme on supercoiled and nonsupercoiled closed circular DNA: formation of a Boltzmann distribution of topological isomers. Proc. Natl. Acad. Sci. U.S.A., 72, 4280–4284.PubMedCrossRefGoogle Scholar
  25. 25.
    Morse, R.H., and Cantor, C.R. (1985) Nucleosome core particles suppress the thermal untwisting of core DNA and adjacent linker DNA. Proc. Natl. Acad. Sci. U.S.A., 82, 4653–4657.PubMedCrossRefGoogle Scholar
  26. 26.
    Ambrose, C., McLaughlin, R., and Bina, M. (1987) The flexibility and topology of simian virus 40 DNA in minichromosomes. Nucl. Acids Res., 15, 3703–3721.PubMedCrossRefGoogle Scholar
  27. 27.
    Lutter, L.C. (1989) Thermal unwinding of simian virus 40 transcription complex DNA. Proc. Natl. Acad. Sci. U.S.A., 86, 8712–8716.PubMedCrossRefGoogle Scholar
  28. 28.
    Saavedra, R.A., and Huberman, J.A. (1986) Both DNA topoisomerases I and II relax 2 m plasmid DNA in living yeast cells. Cell, 45, 65–70.PubMedCrossRefGoogle Scholar
  29. 29.
    White, J.H., Gallo, R., and Bauer, W.R. (1989) Dependence of the linking deficiency of supercoiled minichromosomes upon nucleosome distortion. Nucl. Acids Res., 17, 5827–5835.PubMedCrossRefGoogle Scholar
  30. 30.
    White, J.H., Gallo, R., and Bauer, W.R. (1989) Effect of nucleosome distortion on the linking deficiency in relaxed minichromosomes. J. Mol. Biol., 207, 193–199.PubMedCrossRefGoogle Scholar
  31. 31.
    Bauer, W.R., Hayes, J.J., White, J.H., and Wolffe, A.P. (1994) Nucleosome structural changes due to acetylation. J. Mol. Biol., 236, 685–690.PubMedCrossRefGoogle Scholar
  32. 32.
    Pederson, D.S., and Morse, R.H. (1990) Effect of transcription of yeast chromatin on DNA topology in vivo. EMBO J., 9, 1873–1881.PubMedGoogle Scholar
  33. 33.
    Bauer, W., and Vinograd, J. (1970) Interaction of closed circular DNA with intercalative dyes. II. The free energy of superhelix formation in SV40 DNA. J. Mol. Biol., 47, 419–435.PubMedCrossRefGoogle Scholar
  34. 34.
    Hsieh, T.-S., and Wang, J.C. (1975) Thermodynamic properties of superhelical DNAs. Biochemistry, 14, 527–535.PubMedCrossRefGoogle Scholar
  35. 35.
    Bates, A.D., and Maxwell, A. (1993) DNA Topology. IRL Press at Oxford University Press, Oxford, UK.Google Scholar
  36. 36.
    Morse, R.H. (1991) Topoisomer heterogeneity of plasmid chromatin in living cells. J. Mol. Biol., 222, 133–137.PubMedCrossRefGoogle Scholar
  37. 37.
    Sambrook, J., Fritsch, E.F., and Maniatis, T. (1989) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.Google Scholar
  38. 38.
    Church, G., and Gilbert, W. (1984) Genomic sequencing. Proc. Natl. Acad. Sci. USA, 81, 1991–1995.PubMedCrossRefGoogle Scholar
  39. 39.
    Drabik, C.E., Nicita, C.A., and Lutter, L.C. (1997) Measurement of the linking number change in transcribing chromatin. J. Mol. Biol., 267, 794–806.PubMedCrossRefGoogle Scholar
  40. 40.
    Lee, C.-H., Mizusawa, H., and Kakefuda, T. (1981) Unwinding of double-stranded DNA helix by dehydration. Proc. Natl. Acad. Sci. U.S.A., 78, 2838–2842.PubMedCrossRefGoogle Scholar
  41. 41.
    Givens, R.M., Saavedra, R.A., and Huberman, J.A. (1996) Topological complexity of SV40 minichromosomes. J. Mol. Biol., 257, 53–65.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press, a part of Springer Science+Business Media, LLC 2009

Authors and Affiliations

  • Randall H. Morse
    • 1
  1. 1.NY State Department of Health and SUNY School of Public HealthWadsworth CenterAlbanyUSA

Personalised recommendations