Abstract
One of the major features of meiosis is a high frequency of homologous recombination that not only confers genetic diversity to a successive generation but also ensures proper segregation of chromosomes. Meiotic recombination is initiated by DNA double-strand breaks that require many proteins including the catalytic core, Spo11. In this regard, like transcription and repair, etc., recombination is hindered by a compacted chromatin structure because trans-acting factors cannot easily access the DNA. Such inhibitory effects must be alleviated prior to recombination initiation. Indeed, a number of groups showed that chromatin around recombination hotspots is less condensed, by using nucleases as a probe to assess local DNA accessibility. Here we describe a method to analyze chromatin structure of a recombination hotspot in the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe. This method, combining micrococcal nuclease (MNase) digestion of chromatin DNA and subsequent Southern blotting, is expected to provide information as to chromatin context around a hotspot. Moreover, by virtue of MNase preferentially targeting linker DNA, positions of several nucleosomes surrounding a hotspot can also be determined. Our protocol is a very powerful way to analyze several-kb regions of interest and can be applied to other purposes.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsReferences
Wolffe, A. (1997) in: Chromatin: Structure and function, 3rd edn, Academic Press, San Diego, USA.
Nightingale, K.P., O’Neill, L.P. and Turner, B.M. (2006) Histone modifications: signalling receptors and potential elements of a heritable epigenetic code. Curr. Opin. Genet. Dev. 16, 125–36.
Eberharter, A. and Becker, P.B. (2004) ATP-dependent nucleosome remodelling: factors and functions. J. Cell Sci. 117, 3707–11.
Felsenfeld, G., Boyes, J., Chung, J., Clark, D. and Studitsky, V. (1996) Chromatin structure and gene expression. Proc. Natl. Acad. Sci. U. S. A. 93, 9384–8.
Keeney, S. and Neale, M.J. (2006) Initiation of meiotic recombination by formation of DNA double-strand breaks: mechanism and regulation. Biochem. Soc. Trans. 34, 523–5.
Wu, T.C. and Lichten, M. (1994) Meiosis-induced double-strand break sites determined by yeast chromatin structure. Science 263, 515–8.
Ohta, K., Shibata, T. and Nicolas, A. (1994) Changes in chromatin structure at recombination initiation sites during yeast meiosis. EMBO J. 13, 5754–63.
Mizuno, K., Emura, Y., Baur, M., Kohli, J., Ohta, K. and Shibata, T. (1997) The meiotic recombination hot spot created by the single-base substitution ade6-M26 results in remodeling of chromatin structure in fission yeast. Genes Dev. 11, 876–86.
Moreno, S., Klar, A. and Nurse, P. (1991) Molecular genetic analysis of fission yeast Schizosaccharomyces pombe. Methods Enzymol. 194, 795–823.
Isshiki, T., Mochizuki, N., Maeda, T. and Yamamoto, M. (1992) Characterization of a fission yeast gene, gpa2, that encodes a G alpha subunit involved in the monitoring of nutrition. Genes Dev. 6, 2455–62.
Hirota, K., Steiner, W.W., Shibata, T. and Ohta, K. (2007) Multiple modes of chromatin configuration at natural meiotic recombination hotspots in fission yeast. Eukaryot. Cell 6, 2072–80.
Hirota, K., Hasemi, T., Yamada, T., Mizuno, K.I., Hoffman, C.S., Shibata, T. and Ohta, K. (2004) Fission yeast global repressors regulate the specificity of chromatin alteration in response to distinct environmental stresses. Nucleic Acids Res. 32, 855–62.
Hirota, K., Hoffman, C.S. and Ohta, K. (2006) Reciprocal nuclear shuttling of two antagonizing Zn finger proteins modulates Tup family corepressor function to repress chromatin remodeling. Eukaryot Cell. 5, 1980–9.
Hirota, K., Hoffman, C.S., Shibata, T. and Ohta, K. (2003) Fission yeast tup1-like repressors repress chromatin remodeling at the fbp1(+) promoter and the ade6-M26 recombination hotspot. Genetics 165, 505–15.
Acknowledgments
We thank Dr. Hajime Murakami for the original image of Fig. 16.1 .
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2009 Humana Press, a part of Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Hirota, K., Fukuda, T., Yamada, T., Ohta, K. (2009). Analysis of Chromatin Structure at Meiotic DSB Sites in Yeasts. In: Keeney, S. (eds) Meiosis. Methods in Molecular Biology, vol 557. Humana Press. https://doi.org/10.1007/978-1-59745-527-5_16
Download citation
DOI: https://doi.org/10.1007/978-1-59745-527-5_16
Published:
Publisher Name: Humana Press
Print ISBN: 978-1-934115-66-4
Online ISBN: 978-1-59745-527-5
eBook Packages: Springer Protocols