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Meiosis pp 253-266 | Cite as

Analysis of Chromatin Structure at Meiotic DSB Sites in Yeasts

  • Kouji Hirota
  • Tomoyuki Fukuda
  • Takatomi Yamada
  • Kunihiro Ohta
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 557)

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.

Key words

Meiotic recombination recombination hotspot chromatin structure nucleosome linker DNA micrococcal nuclease (MNase) yeast indirect end labeling 

Notes

Acknowledgments

We thank Dr. Hajime Murakami for the original image of Fig. 16.1 .

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Copyright information

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

Authors and Affiliations

  • Kouji Hirota
    • 1
  • Tomoyuki Fukuda
    • 1
  • Takatomi Yamada
    • 1
  • Kunihiro Ohta
    • 1
  1. 1.Department of Life Sciences, Graduate School of Arts and SciencesThe University of Tokyo, and Shibata Distinguished Senior Scientist Laboratory, RIKEN Discovery Research InstituteJapan

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