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Meiosis

Volume 557 of the series Methods in Molecular Biology pp 117-142

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Gel Electrophoresis Assays for Analyzing DNA Double-Strand Breaks in Saccharomyces cerevisiae at Various Spatial Resolutions

  • Hajime MurakamiAffiliated withMolecular Biology Program, Memorial Sloan-Kettering Cancer Center
  • , Valérie BordeAffiliated withInstitut Curie, Centre de Recherche, UMR7147-CNRS, Université
  • , Alain NicolasAffiliated withInstitut Curie, Centre de Recherche, UMR7147-CNRS, Université
  • , Scott KeeneyAffiliated withHoward Hughes Medical Institute and Molecular Biology Program, Memorial Sloan-Kettering Cancer Center

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Abstract

Meiotic recombination is triggered by programmed DNA double-strand breaks (DSBs), which are catalyzed by Spo11 protein in a type II topoisomerase-like manner. Meiotic DSBs can be detected directly using physical assays (gel electrophoresis, Southern blotting, and indirect end-labeling) applied to samples of genomic DNA from sporulating cultures of budding and fission yeast. Such assays are extremely useful for quantifying and characterizing many aspects of the initiation of meiotic recombination, including the timing of DSB formation relative to other events, the distribution of DSBs across the genome, and the influence on DSB formation of mutations in recombination factors and other gene products. By varying the type of gel electrophoresis and other parameters, the spatial resolution of DSB analysis can range from single nucleotides up to whole yeast chromosomes.

Key words

yeast meiotic recombination Southern blotting pulsed-field gel electrophoresis