Abstract
The mechanisms of biological chromatin assembly and their regulation have been studied intensively using cellular extracts, particularly those from the embryonic cells of various metazoans. Here we describe how to prepare and use a crude chromatographic fraction from budding yeast, which also supports biological chromatin assembly. In this system, nucleosomes are assembled by a replication-independent mechanism into physiologically spaced arrays that significantly protect underlying DNA from restriction endonuclease digestion. The formation of correctly spaced nucleosome arrays absolutely requires ATP and exogenous core histones of yeast or Drosophila. We have explored how cell cycle and DNA damage signals affect assembly activity in this system.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Mello, J. A. and Almouzni, G. (2001) The ins and outs of nucleosome assembly. Curr. Opin. Genet. Dev. 11, 136–141.
Haushalter, K. A. and Kadonaga, J. T. (2003) Chromatin assembly by DNA-translocating motors. Nat. Rev. Mol. Cell. Biol. 4, 613–620.
Ahmad, K. and Henikoff, S. (2002) The histone variant H3.3 marks active chromatin by replication-independent nucleosome assembly. Mol. Cell 9, 1191–1200.
Ray-Gallet, D., Quivy, J. P., Scamps, C., Martini, E. M., Lipinski, M., and Almouzni, G. (2002) HIRA is critical for a nucleosome assembly pathway independent of DNA synthesis. Mol. Cell 9, 1091–1100.
Wolffe, A. (1998) Chromatin Structure and Function, 3rd ed., Academic Press, San Diego, CA.
Schultz, M. C. (1999) Chromatin assembly in yeast cell-free extracts. Methods 17, 161–172.
Robinson, K. M. and Schultz, M. C. (2003) Replication-independent assembly of nucleosome arrays in a novel yeast chromatin reconstitution system involves antisilencing factor Asf1p and chromodomain protein Chd1p. Mol. Cell. Biol. 23, 7937-7946.
van Holde, K. E. (1988) Chromatin. Springer-Verlag, New York, NY.
Pilon, J., Terrell, A., and Laybourn, P. J. (1997) Yeast chromatin reconstitution system using purified yeast core histones and yeast nucleosome assembly protein-1. Protein Expr. Purif. 10, 132–140.
Dunn, B. and Wobbe, C. R. (1997) Preparation of protein extracts from yeast, in Current Protocols in Molecular Biology (Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., and Struhl, K., eds.), John Wiley & Sons Inc., Hoboken, NJ, pp. 13.13.1–13.13.9.
Pazin, M. J., Hermann, J. W., and Kadonaga, J. T. (1998) Promoter structure and transcriptional activation with chromatin templates assembled in vitro. A single Gal4-VP16 dimer binds to chromatin or to DNA with comparable affinity. J. Biol. Chem. 273, 34653–34660.
Jones, E. W. (1991) Tackling the protease problem in Saccharomyces cerevisiae. Methods Enzymol. 194, 428–453.
Robinson, K. M. and Schultz, M. C. (2005) Gal4-VP16 directs ATP-independent chromatin reorganization in a yeast chromatin assembly system. Biochemistry 44, 4551–4561.
Kamakaka, R. T., Bulger, M., and Kadonaga, J. T. (1993) Potentiation of RNA polymerase II transcription by Gal4-VP16 during but not after DNA replication and chromatin assembly. Genes Dev. 7, 1779–1795.
Stuart, D. and Wittenberg, C. (1998) CLB5 and CLB6 are required for premeiotic DNA replication and activation of the meiotic S/M checkpoint. Genes Dev. 12, 2698–2710.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2006 Humana Press Inc., Totowa, NJ
About this protocol
Cite this protocol
Robinson, K.M., Schultz, M.C. (2006). Chromatin Assembly in a Crude Fraction From Yeast Cells. In: Xiao, W. (eds) Yeast Protocol. Methods in Molecular Biology, vol 313. Humana Press, Totowa, NJ. https://doi.org/10.1385/1-59259-958-3:209
Download citation
DOI: https://doi.org/10.1385/1-59259-958-3:209
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-58829-437-1
Online ISBN: 978-1-59259-958-5
eBook Packages: Springer Protocols