SpringerProtocols: Abstract: 9. In Situ Assay for Analyzing the Chromatin Binding of Proteins in Fission Yeast

9. In Situ Assay for Analyzing the Chromatin Binding of Proteins in Fission Yeast

By: Stephen E. Kearsey, Lydia Brimage, Mandana Namdar, Emma Ralph, Xiaowen Yang

Abstract

Full Text

Download PDF (285K)

An in situ technique for studying the chromatin binding of proteins in single fission yeast cells (Schizosaccharomyces pombe) is described. Cells are permeabilized by enzymatic digestion and extracted with a detergent-containing buffer. This procedure removes soluble proteins, but proteins that are bound to insoluble cell structures such as chromatin are retained, and overall cell morphology is maintained. Extraction of proteins is monitored by fluorescence microscopy, either using fluorescently tagged proteins or by indirect immunofluorescence. This method allows the chromatin association of proteins to be correlated with other cell cycle events without the need for cell synchronization.

Book Title: Cell Cycle Control: Mechanisms and Protocols

Series: Methods in Molecular Biology | Volume: 296 | Pub. Date: Dec-01-2004 | Page Range: 181-188 | DOI: 10.1385/1-59259-857-9:181

Subject: Cell Biology

Key Words: DNA replication - chromatin - cell cycle - Schizosaccharomyces pombe fission yeast - GFP

References

Download References

1.	Gregan, J., Lindner, K., Brimage, L., et al. (2003) Fission yeast Cdc23/Mcm10 functions after pre-replicative complex formation to promote Cdc45 chromatin binding. Mol. Biol. Cell 14, 3876–3887.

2.	Donovan, S., Drury, L., and Diffley, J. (1997) Cdc6p-dependent loading of Mcm proteins onto pre-replicative chromatin in budding yeast. Proc. Natl. Acad. Sci. USA 94, 5611–5616.

3.	Tanaka, T., Knapp, D., and Nasmyth, K. (1997) Loading of an Mcm protein onto DNA replication origins is regulated by Cdc6p and CDKs. Cell 90, 649–660.

4.	Todorov, I. T., Attaran, A., and Kearsey, S. E. (1995) BM28, a human member of the MCM2-3-5 family, is displaced from chromatin during DNA replication. J. Cell Biol. 129, 1433–1446.

5.	Klein, F., Laroche, T., Cardenas, M. E., Hofmann, J. F., Schweizer, D., and Gasser, S. M. (1992) Localization of RAP1 and topoisomerase II in nuclei and meiotic chromosomes of yeast.J. Cell Biol. 117, 935–948.

6.	Kearsey, S. E., Montgomery, S., Labib, K., and Lindner, K. (2000) Chromatin binding of the fission yeast replication factor mcm4 occurs during anaphase and requires ORC and cdc18. EMBOJ. 19, 1681–1690.

7.	Lindner, K., Gregan, J., Montgomery, S., and Kearsey, S. (2002) Essential role of MCM proteins in pre-meiotic DNA replication. Mol. Biol. Cell 13, 435–444.

8.	Gomez, E. B., Catlett, M. G., and Forsburg, S. L. (2002) Different phenotypes in vivo are associated with ATPase motif mutations in Schizosaccharomyces pombe minichromosome maintenance proteins. Genetics 160, 1305–1318.

9.	Wolkow, T. and Enoch, T. (2003) Fission yeast Rad26 responds to DNA damage independently of Rad3. BMC Genet. 4, 6.

10.	Williams, D. R. and McIntosh, J. R. (2002) mcl1+, the Schizosaccharomyces pombe homologue of CTF4, is important for chromosome replication, cohesion, and segregation. Eukaryot. Cell 1, 758–773.

11.	Tomonaga, T., Nagao, K., Kawasaki, Y., et al. (2000) Characterization of fission yeast cohesin: essential anaphase proteolysis of Rad21 phosphorylated in the S phase. Genes Dev. 14, 2757–2770.

12.	Moreno, S., Klar, A., and Nurse, P. (1991) Molecular genetic analysis of the fission yeast Schizosaccharomyces pombe. Methods Enzymol. 194, 795–823.

13.	Wu, L. C., Fisher, P. A., and Broach, J. R. (1987) A yeast plasmid partitioning protein is a karyoskeletal component. J. Biol. Chem. 262, 883–891.

14.	Bahler, J., Wu, J. Q., Longtine, M. S., et al. (1998) Heterologous modules for efficient and versatile PCR-based gene targeting in Schizosaccharomyces pombe. Yeast 14, 943–951.

Comments (Loading...)

Post comment/View all comments