title: Quantitative Genome-Wide Measurements of Meiotic DNA Double-Strand Breaks and Protein Binding in S. pombe.
abstract: The fission yeast Schizosaccharomyces pombe is especially well suited for studying meiosis in molecular detail. Experiments with S. pombe strains that undergo a nearly synchronous meiosis-at variable temperatures-have elucidated the mechanisms of meiotic progression and the proteins that are involved. For example, studies focused on the initiation of meiotic recombination by programmed DNA double-strand breaks (DSBs) have proven exceptionally informative. In meiosis, some regions of DNA have more frequent DSBs than the surrounding regions. These DSB hotspots can be visualized by Southern blot hybridization of restriction fragments ranging from kilobases (kb) to megabases (Mb) in size. More recently, the benefits of genome-wide analysis to map the distribution and frequency of meiotic DSBs have been attained, with resolution down to the nucleotide level. Infrequent, non-hotspot DSBs previously not detectable have been observed, creating a better understanding of how recombination is regulated. Additional genome-wide analyses have shown proteins that bind specifically to DSB hotspots, providing insight into how the DSB initiation complex functions. We describe here detailed methods for achieving these results.
authors.affiliation: Fred Hutchinson Cancer Research Center, Seattle, WA, USA.
authors.forename: Randy W
authors.initials: RW
authors.lastname: Hyppa
authors.affiliation: Fred Hutchinson Cancer Research Center, Seattle, WA, USA.|Department of Microbiology and Immunology, University of California at San Francisco, San Francisco, CA, USA.
authors.forename: Kyle R
authors.initials: KR
authors.lastname: Fowler
authors.affiliation: Fred Hutchinson Cancer Research Center, Seattle, WA, USA. gsmith@fredhutch.org.
authors.forename: Gerald R
authors.initials: GR
authors.lastname: Smith
mesh_terms: D047369:Chromatin Immunoprecipitation / Q000379:methods; D053903:DNA Breaks, Double-Stranded; D016681:Genome, Fungal; D055106:Genome-Wide Association Study; D017403:In Situ Hybridization / Q000379:methods; D008540:Meiosis; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D012568:Schizosaccharomyces / Q000235:genetics*; D029702:Schizosaccharomyces pombe Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D029702:Schizosaccharomyces pombe Proteins
keywords: Chromatin immunoprecipitation (ChIP); DNA double-strand breaks (DSBs); DNA microarray hybridization; Fission yeast; Massive parallel sequencing; Meiotic induction; Pulsed-field gel electrophoresis (PFGE); Rec12 (Spo11); Schizosaccharomyces pombe