Analysis of chromatin structure at meiotic DSB sites in yeasts.
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 ofchromatin 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.
Hirota
Kouji
K
Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo, Tokyo, Japan.
Fukuda
Tomoyuki
T
Yamada
Takatomi
T
Ohta
Kunihiro
K
D018929:Cell Culture Techniques / Q000379:methods; D002843:Chromatin / Q000737:chemistry*; D053903:DNA Breaks, Double-Stranded; D008540:Meiosis / Q000235:genetics*; D008954:Models, Biological; D009707:Nucleosomes / Q000737:chemistry; D011995:Recombination, Genetic / Q000502:physiology; D012045:Regulatory Sequences, Nucleic Acid / Q000502:physiology; D015003:Yeasts / Q000737:chemistry / Q000235:genetics* / Q000254:growth & development
D002843:Chromatin; D009707:Nucleosomes