Advertisement

Characterization of Meiotic Recombination Initiation Sites Using Pulsed-Field Gel Electrophoresis

  • Sarah Farmer
  • Wing-Kit Leung
  • Hideo TsubouchiEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 745)

Abstract

High levels of homologous recombination are induced during meiosis. This meiotic recombination is initiated by programmed formation of DNA double-strand breaks (DSBs) by a conserved meiosis-specific protein, Spo11. Meiotic DSBs are not formed at random along chromosomes but are formed in clusters known as recombination hot spots. To understand the regulation of this initiation step of meiotic recombination, determining the timing and location of meiotic DSBs is essential. In this chapter, we describe a method to detect genome-wide meiotic DSBs by using a combination of pulsed-field gel electrophoresis and Southern blotting.

Key words

Budding yeast chromosomes double-strand breaks meiosis pulsed-field gel electrophoresis recombination recombination hot spot Spo11 

Notes

Acknowledgments

We would like to thank Prof. Alan Lehmann for critical reading of the manuscript. This work was supported by a Marie Curie Cancer Care transitional program grant.

References

  1. 1.
    Gerton, J.L. and Hawley, R.S. (2005) Homologous chromosome interactions in meiosis: diversity amidst conservation. Nat Rev Genet 6, 477–487.PubMedCrossRefGoogle Scholar
  2. 2.
    Roeder, G.S. (1997) Meiotic chromosomes: it takes two to tango. Genes Dev 11, 2600–2621.PubMedCrossRefGoogle Scholar
  3. 3.
    Petes, T.D. (2001) Meiotic recombination hot spots and cold spots. Nat Rev Genet 2, 360–369.PubMedCrossRefGoogle Scholar
  4. 4.
    Sun, H., Treco, D., and Szostak, J.W. (1991) Extensive 3-overhanging, single-stranded DNA associated with the meiosis-specific double-strand breaks at the ARG4 recombination initiation site. Cell 64, 1155–1161.PubMedCrossRefGoogle Scholar
  5. 5.
    Bergerat, A., de Massy, B., Gadelle, D., Varoutas, P.C., Nicolas, A., and Forterre, P. (1997) An atypical topoisomerase II from Archaea with implications for meiotic recombination. Nature 386, 414–417.PubMedCrossRefGoogle Scholar
  6. 6.
    Keeney, S., Giroux, C.N., and Kleckner, N. (1997) Meiosis-specific DNA double-strand breaks are catalyzed by Spo11, a member of a widely conserved protein family. Cell 88, 375–384.PubMedCrossRefGoogle Scholar
  7. 7.
    Sehorn, M.G. and Sung, P. (2004) Meiotic recombination: an affair of two recombinases. Cell Cycle 3, 1375–1377.PubMedCrossRefGoogle Scholar
  8. 8.
    Masson, J.Y. and West, S.C. (2001) The Rad51 and Dmc1 recombinases: a non-identical twin relationship. Trends Biochem Sci 26, 131–136.PubMedCrossRefGoogle Scholar
  9. 9.
    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
  10. 10.
    Blitzblau, H.G., Bell, G.W., Rodriguez, J., Bell, S.P., and Hochwagen, A. (2007) Mapping of meiotic single-stranded DNA reveals double-stranded-break hotspots near centromeres and telomeres. Curr Biol 17, 2003–2012.PubMedCrossRefGoogle Scholar
  11. 11.
    Game, J.C. (1992) Pulsed-field gel analysis of the pattern of DNA double-strand breaks in the Saccharomyces genome during meiosis. Dev Genet 13, 485–497.PubMedCrossRefGoogle Scholar
  12. 12.
    Zenvirth, D., Arbel, T., Sherman, A., Goldway, M., Klein, S., and Simchen, G. (1992) Multiple sites for double-strand breaks in whole meiotic chromosomes of Saccharomyces cerevisiae. EMBO J 11, 3441–3447.PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  1. 1.MRC Genome Damage and Stability Centre, University of SussexSussexUK
  2. 2.MRC Genome Damage and Stability Centre, University of SussexBrightonUK

Personalised recommendations