Abstract
During meiosis, programmed double strand breaks give rise to crossover and non-crossover recombination products. Meiotic recombination products are formed via several branched intermediates, including single end invasions and double Holliday junctions. Two-dimensional gel electrophoresis provides a sensitive and specific approach for detecting branched recombination intermediates, determining their genetic requirements, and enriching intermediates for further analysis. Here, we describe analysis of branched recombination intermediates in the yeast Saccharomyces cerevisiae by two-dimensional gel electrophoresis. We also provide an introduction to meiotic time-course procedures, stabilization of branched DNA molecules by interstrand crosslinking, extraction of genomic DNA from meiotic cultures, and quantitative analysis of two-dimensional gel blots.
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References
Neale, M.J., and Keeney, S. (2006) Clarifying the mechanics of DNA strand exchange in meiotic recombination. Nature 442, 153–158.
Hunter, N., and Kleckner, N. (2001) The single-end invasion: an asymmetric intermediate at the double-strand break to double-Holliday junction transition of meiotic recombination. Cell 106, 59–70.
Schwacha, A., and Kleckner, N. (1995) Identification of double Holliday junctions as intermediates in meiotic recombination. Cell 83, 783–791.
Allers, T., and Lichten, M. (2001) Differential timing and control of noncrossover and crossover recombination during meiosis. Cell 106, 47–57.
Börner, G.V., Kleckner, N., and Hunter, N. (2004) Crossover/noncrossover differentiation, synaptonemal complex formation, and regulatory surveillance at the leptotene/zygotene transition of meiosis. Cell 117, 29–45
Schwacha, A., and Kleckner, N. (1994) Identification of joint molecules that form frequently between homologs but rarely between sister chromatids during yeast meiosis. Cell 76, 51–63.
Oh, S.D., Lao, J.P., Hwang, P.Y., Taylor, A.F., Smith, G.R., and Hunter, N. (2007) BLM ortholog, Sgs1, prevents aberrant crossing-over by suppressing formation of multichromatid joint molecules. Cell 130, 259–272.
Börner, G.V., Barot, A., and Kleckner, N. (2008) Yeast Pch2 promotes domainal axis organization, timely recombination progression, and arrest of defective recombinosomes during meiosis. Proc Natl Acad Sci USA 105, 3327–3332.
Lao, J.P., Oh, S.D., Shinohara, M., Shinohara, A., and Hunter, N. (2008) Rad52 promotes postinvasion steps of meiotic double-strand-break repair. Mol Cell 29, 517–524.
Oh, S.D., Jessop, L., Lao, J.P., Allers, T., Lichten, M., and Hunter, N. (2009) Stabilization and electrophoretic analysis of meiotic recombination intermediates in Saccharomyces cerevisiae. Methods Mol Biol 557, 209–234.
Hochwagen, A., Wrobel, G., Cartron, M., Demougin, P., Niederhauser-Wiederkehr, C., Boselli, M.G., et al. (2005) Novel response to microtubule perturbation in meiosis. Mol Cell Biol 25, 4767–4781.
Bell, L., and Byers, B. (1979) Occurrence of crossed strand-exchange forms in yeast DNA during meiosis. Proc Natl Acad Sci USA 76, 3445–3449.
Goldstein, A.L., and McCusker, J.H. (1999) Three new dominant drug resistance cassettes for gene disruption in Saccharomyces cerevisiae. Yeast 14, 1541–1553.
Acknowledgments
Work in the Börner lab is supported by NIH NIGMS grant GM080715.
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Ahuja, J.S., Börner, G.V. (2011). Analysis of Meiotic Recombination Intermediates by Two-Dimensional Gel Electrophoresis. In: Tsubouchi, H. (eds) DNA Recombination. Methods in Molecular Biology, vol 745. Humana Press. https://doi.org/10.1007/978-1-61779-129-1_7
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DOI: https://doi.org/10.1007/978-1-61779-129-1_7
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