Photolyase

A Molecular Tool to Characterize Chromatin Structure in Yeast
  • Magdalena Livingstone-Zatchej
  • Bernhard Suter
  • Fritz Thoma
Part of the Methods in Molecular Biology™ book series (MIMB, volume 119)

Abstract

Folding of DNA into nucleosomes and higher order chromatin structures restricts its accessibility to proteins and drugs. Hence, the location of histone octamers on the DNA sequence (nucleosome positions) as well as structural and dynamic properties of nucleosomes may play important roles in gene regulation, replication and DNA repair. Conventional approaches to characterize chromatin structure include (partial) purification of chromatin and characterization of DNA accessibility to nucleases (micrococcal nuclease, DNaseI) and chemical cleavage reagents (hydroxyl radicals, methidium propyl-EDTA-iron, copper phenanthroline). The cleavage sites are monitored using low- and high-resolution footprinting protocols. These techniques, however, expose the problem that chromatin extraction procedures could alter chromatin composition and structure, including nucleosome positioning. To investigate chromatin structures in vivo, alternative approaches are applied, such as expression of prokaryotic methyltransferases in Saccharomyces cerevisiae, the genome of which contains no endogenous detectable methylation (1,2). The sites of methylation can be measured after DNA isolation using methylation-sensitive restriction enzymes. This approach, however, requires expression of a foreign gene, and the resolution is restricted because of the sequence specificity of the methyltransferases.

Keywords

Vortex Phenol EDTA Electrophoresis Boiling 

References

  1. 1.
    Singh, J. and Klar, A. J. S. (1992) Active genes in budding yeast display enhanced in vivo accessibility to foreign DNA methylases: a novel in vivo probe for chromatin structure of yeast. Genes Develop. 6, 186–196.PubMedCrossRefGoogle Scholar
  2. 2.
    Kladde, M. P. and Simpson, R. T. (1996) Chromatin structure mapping in vivo using methyltransferases. Meth. Enzymol. 274, 214–233.PubMedCrossRefGoogle Scholar
  3. 3.
    Friedberg, E. C., Walker, G. C., and Siede, W. (1995) DNA repair and mutagenesis. ASM Press, Washington, DC.Google Scholar
  4. 4.
    Sancar, A. (1996) No “End of History” for photolyases. Science 272, 48,49.PubMedCrossRefGoogle Scholar
  5. 5.
    Sancar, A. and Sancar, G. B. (1988) DNA repair enzymes. Ann. Rev. Biochem. 57, 29–67.PubMedCrossRefGoogle Scholar
  6. 6.
    Yasui, A., Eker, A. P. M., Yasuhira, S., Yajima, H., Kobayashi, T., Takao, M., and Oikawa, A. (1994) A new class of DNA photolyases present in various organisms including aplacental mammals. EMBO J 13, 6143–6151.PubMedGoogle Scholar
  7. 7.
    Suter, B., Livingstone-Zatchej, M., and Thoma, F. (1997) Chromatin structure modulates DNA repair by photolyase in vivo. EMBO J 16, 2150–2160.PubMedCrossRefGoogle Scholar
  8. 8.
    Livingstone-Zatchej, M., Meier, A., Suter, B., and Thoma, F. (1997) RNA-Polymerase II transcription inhibits DNA repair by photolyase in the transcribed strand of active yeast genes. Nucleic Acids Res. 25, 3795–3800.PubMedCrossRefGoogle Scholar
  9. 9.
    Gale, J. M., Nissen, K. A., and Smerdon, M. J. (1987) UV-induced formation of pyrimidine dimers in nucleosome core DNA is strongly modulated with a period of 10.3 bases. Proc. Natl. Acad. Sci. USA 84, 6644–6648.PubMedCrossRefGoogle Scholar
  10. 10.
    Pehrson, J. R. (1989) Thymine dimer formation as a probe of the path of DNA in and between nucleosome in intact chromatin. Proc. Natl. Acad. Sci. USA 86, 9149–9153.PubMedCrossRefGoogle Scholar
  11. 11.
    Schieferstein, U. and Thoma, F. (1996) Modulation of cyclobutane pyrimidine dimer formation in a positioned nucleosome containing polydA.dT tracts. Biochemistry 35, 7705–7714.PubMedCrossRefGoogle Scholar
  12. 12.
    Tornaletti, S. and Pfeifer, G. P. (1996) UV damage and repair mechanisms in mammalian cells. Bioessays 18, 221–228.PubMedCrossRefGoogle Scholar
  13. 13.
    Gordon, L. K. and Haseltine, W. A. (1980) Comparison of the cleavage of pyrimidine dimers by the bacteriophage T4 and Micrococcus luteus UV-specific endonucleases. J. Biol. Chem. 255, 12,047–12,050.PubMedGoogle Scholar
  14. 14.
    Smerdon, M. J. and Thoma, F. (1990) Site-specific DNA repair at the nucleosome level in a yeast minichromosome. Cell 61, 675–684.PubMedCrossRefGoogle Scholar
  15. 15.
    Maniatis, T., Fritsch, E., and Sambrook, J. (1982) Molecular cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press. Cold Spring Harbor, NY.Google Scholar
  16. 16.
    Sherman, F., Fink, G. R., and Hicks, J. B. (1986) Laboratory Course Manual for Methods in Yeast Genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.Google Scholar
  17. 17.
    Losa, R., Omari, S., and Thoma, F. (1990) Poly(dA)’poly(dT) rich sequences are not sufficient to exclude nucleosome formation in a constitutive yeast promoter. Nucleic Acids Res. 18, 3495–3502.PubMedCrossRefGoogle Scholar
  18. 18.
    Thoma, F., Bergman, L. W., and Simpson, R. T. (1984) Nuclease digestion of circular TRP1ARS1 chromatin reveals positioned nucleosomes separated by nuclease sensitive regions. J. Mol. Biol. 177, 715–733.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc. 1999

Authors and Affiliations

  • Magdalena Livingstone-Zatchej
    • 1
  • Bernhard Suter
    • 1
  • Fritz Thoma
    • 1
  1. 1.Institut für ZellbiologieEidgenossische Technische HochschuleZürichSwitzerland

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