Meiosis pp 117-142

Part of the Methods in Molecular Biology book series (MIMB, volume 557)

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

  • Hajime Murakami
  • Valérie Borde
  • Alain Nicolas
  • Scott Keeney


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 


  1. 1.
    Petes, T. D. (2001) Meiotic recombination hot spots and cold spots. Nat. Rev. Genet. 2, 360–369.PubMedCrossRefGoogle Scholar
  2. 2.
    Baudat, F. and Nicolas, A. (1997) Clustering of meiotic double-strand breaks on yeast chromosome III. Proc. Natl. Acad. Sci. U. S. A. 94, 5213–5218.PubMedCrossRefGoogle Scholar
  3. 3.
    Xu, F. and Petes, T. D. (1996) Fine-structure mapping of meiosis-specific double-strand DNA breaks at a recombination hotspot associated with an insertion of telomeric sequences upstream of the HIS4 locus in yeast. Genetics 143, 1115–1125.PubMedGoogle Scholar
  4. 4.
    Xu, L. and Kleckner, N. (1995) Sequence non-specific double-strand breaks and interhomolog interactions prior to double-strand break formation at a meiotic recombination hot spot in yeast. EMBO J. 14, 5115–5128.PubMedGoogle Scholar
  5. 5.
    Liu, J., Wu, T. C., and Lichten, M. (1995) The location and structure of double-strand DNA breaks induced during yeast meiosis: evidence for a covalently linked DNA-protein intermediate. EMBO J. 14, 4599–4608.PubMedGoogle Scholar
  6. 6.
    de Massy, B., Rocco, V., and Nicolas, A. (1995) The nucleotide mapping of DNA double-strand breaks at the CYS3 initiation site of meiotic recombination in Saccharomyces cerevisiae. EMBO J. 14, 4589–4598.PubMedGoogle Scholar
  7. 7.
    Vedel, M. and Nicolas, A. (1999) CYS3, a hotspot of meiotic recombination in Saccharomyces cerevisiae. Effects of heterozygosity and mismatch repair functions on gene conversion and recombination intermediates. Genetics 151, 1245–1259.PubMedGoogle Scholar
  8. 8.
    Borde, V., Goldman, A. S., and Lichten, M. (2000) Direct coupling between meiotic DNA replication and recombination initiation. Science 290, 806–809.PubMedCrossRefGoogle Scholar
  9. 9.
    Neale, M. J., Pan, J., and Keeney, S. (2005) Endonucleolytic processing of covalent protein-linked DNA double-strand breaks. Nature 436, 1053–1057.PubMedCrossRefGoogle Scholar
  10. 10.
    Keeney, S. and Kleckner, N. (1995) Covalent protein-DNA complexes at the 5′ strand termini of meiosis-specific double-strand breaks in yeast. Proc. Natl. Acad. Sci. U. S. A. 92, 11274–11278.PubMedCrossRefGoogle Scholar
  11. 11.
    Blitzblau, H. G., Bell, G. W., Rodriguez, J., Bell, S. P., and Hochwagen, A. (2007) Mapping of meiotic single-stranded DNA reveals double-strand-break hotspots near centromeres and telomeres. Curr. Biol. 17, 2003–2012PubMedCrossRefGoogle Scholar
  12. 12.
    Buhler, C., Borde, V., and Lichten, M. (2007) Mapping meiotic single-strand DNA reveals a new landscape of DNA double-strand breaks in Saccharomyces cerevisiae. PLoS Biol. 5, e324.PubMedCrossRefGoogle Scholar
  13. 13.
    Goyon, C. and Lichten, M. (1993) Timing of molecular events in meiosis in Saccharomyces cerevisiae: stable heteroduplex DNA is formed late in meiotic prophase. Mol. Cell. Biol. 13, 373–382.PubMedGoogle Scholar
  14. 14.
    Borde, V., Wu, T. C., and Lichten, M. (1999) Use of a recombination reporter insert to define meiotic recombination domains on chromosome III of Saccharomyces cerevisiae. Mol. Cell. Biol. 19, 4832–4842.PubMedGoogle Scholar
  15. 15.
    Buhler, C., Lebbink, J. H., Bocs, C., Ladenstein, R., and Forterre, P. (2001) DNA topoisomerase VI generates ATP-dependent double-strand breaks with two-nucleotide overhangs. J. Biol. Chem. 276, 37215–37222.PubMedCrossRefGoogle Scholar

Copyright information

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

Authors and Affiliations

  • Hajime Murakami
    • 1
  • Valérie Borde
    • 2
  • Alain Nicolas
    • 3
  • Scott Keeney
    • 4
  1. 1.Molecular Biology ProgramMemorial Sloan-Kettering Cancer CenterNew YorkUSA
  2. 2.Institut Curie, Centre de Recherche, UMR7147-CNRS, UniversitéPierre et Marie CurieFrance
  3. 3.Institut Curie, Centre de Recherche, UMR7147-CNRS, UniversitéPierre et Marie CurieFrance
  4. 4.Howard Hughes Medical Institute and Molecular Biology ProgramMemorial Sloan-Kettering Cancer CenterNew YorkUSA

Personalised recommendations