Skip to main content
SpringerLink
Log in

Analysis of DNA Topology in Yeast Chromatin

  • Protocol
  • First Online:
Chromatin Protocols

Part of the book series: Methods in Molecular Biology ((MIMB,volume 523))

Abstract

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.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 84.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 109.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 109.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  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. 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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  10. Freeman, L.T., and Garrard, W.T. (1992) DNA supercoiling in chromatin structure and gene expression. Crit. Rev. Euk. Exp., 2, 165–209.

    CAS  Google Scholar 

  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.

    PubMed  CAS  Google Scholar 

  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.

    PubMed  CAS  Google Scholar 

  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.

    Article  Google Scholar 

  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.

    PubMed  CAS  Google Scholar 

  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.

    PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  23. Depew, R.E., and Wang, J.C. (1975) Conformational fluctuations of DNA helix. Proc. Natl. Acad. Sci. U.S.A., 72, 4275–4280.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  27. Lutter, L.C. (1989) Thermal unwinding of simian virus 40 transcription complex DNA. Proc. Natl. Acad. Sci. U.S.A., 86, 8712–8716.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  34. Hsieh, T.-S., and Wang, J.C. (1975) Thermodynamic properties of superhelical DNAs. Biochemistry, 14, 527–535.

    Article  PubMed  CAS  Google Scholar 

  35. Bates, A.D., and Maxwell, A. (1993) DNA Topology. IRL Press at Oxford University Press, Oxford, UK.

    Google Scholar 

  36. Morse, R.H. (1991) Topoisomer heterogeneity of plasmid chromatin in living cells. J. Mol. Biol., 222, 133–137.

    Article  PubMed  CAS  Google Scholar 

  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. Church, G., and Gilbert, W. (1984) Genomic sequencing. Proc. Natl. Acad. Sci. USA, 81, 1991–1995.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  41. Givens, R.M., Saavedra, R.A., and Huberman, J.A. (1996) Topological complexity of SV40 minichromosomes. J. Mol. Biol., 257, 53–65.

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgment

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

Author information

Authors and Affiliations

  1. NY State Department of Health and SUNY School of Public Health, Wadsworth Center, Albany, NY, USA

    Randall H. Morse

Authors
  1. Randall H. Morse
    View author publications

    You can also search for this author in PubMed Google Scholar

Editor information

Editors and Affiliations

  1. Research Institute, H. Lee Moffitt Cancer Center &, Magnolia Drive 12902, Tampa, 33612, U.S.A.

    Srikumar P. Chellappan

Rights and permissions

Reprints and permissions

Copyright information

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

About this protocol

Cite this protocol

Morse, R.H. (2009). Analysis of DNA Topology in Yeast Chromatin. In: Chellappan, S. (eds) Chromatin Protocols. Methods in Molecular Biology, vol 523. Humana Press. https://doi.org/10.1007/978-1-59745-190-1_7

Download citation

  • DOI: https://doi.org/10.1007/978-1-59745-190-1_7

  • Published:

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-58829-873-7

  • Online ISBN: 978-1-59745-190-1

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation