SpringerProtocols: Abstract: End-Labeling and Analysis of Spo11-Oligonucleotide Complexes in Saccharomyces cerevisiae

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End-Labeling and Analysis of Spo11-Oligonucleotide Complexes in Saccharomyces cerevisiae

By: Matthew J. Neale², Scott Keeney¹

Abstract

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During meiosis Spo11 catalyzes the formation of DNA double-strand breaks, becoming covalently attached to the 5′ ends on both sides of the break during this process. Spo11 is removed from the DSB by single-stranded endonucleolytic cleavage flanking the DSB, liberating a short-lived species consisting of Spo11 protein covalently linked to a short oligonucleotide. The method presented here details how to detect these Spo11-oligo complexes in extracts made from meiotic yeast cells.

Affiliation(s): (1) MRC Genome Damage and Stability Centre, University of Sussex, Falmer, UK
(2) Howard Hughes Medical Institute and Molecular Biology Program, Memorial Sloan-Kettering Cancer Center, New York, NY, USA

Book Title: Meiosis: Volume 1, Molecular and Genetic Methods

Series: Methods in Molecular Biology | Volume: 557 | Pub. Date: Aug-01-2009 | Page Range: 183-195 | DOI: 10.1007/978-1-59745-527-5_12

Subject: Cell Biology

Key Words: Spo11 - meiosis - DNA double-strand break - end-labeling - terminal transferase

References

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1.	Keeney, S. (2001) Mechanism and control of meiotic recombination initiation. Curr. Top. Dev. Biol. 52, 1–53.

2.	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.

3.	Diaz, R. L., Alcid, A. D., Berger, J. M., and Keeney, S. (2002) Identification of residues in yeast Spo11p critical for meiotic DNA double-strand break formation. Mol. Cell. Biol. 22, 1106–1115.

4.	Maleki, S., Neale, M. J., Arora, C., Henderson, K. A., and Keeney, S. (2007) Interactions between Mei4, Rec114, and other proteins required for meiotic DNA double-strand break formation in Saccharomyces cerevisiae. Chromosoma 116, 471–486.

5.	Arora, C., Kee, K., Maleki, S., and Keeney, S. (2004) Antiviral protein Ski8 is a direct partner of Spo11 in meiotic DNA break formation, independent of its cytoplasmic role in RNA metabolism. Mol. Cell 13, 549–559.

6.	Li, J., Hooker, G. W., and Roeder, G. S. (2006) Saccharomyces cerevisiae Mer2, Mei4 and Rec114 form a complex required for meiotic double-strand break formation. Genetics 173, 1969–1981.

7.	Neale, M. J. and Keeney, S. (2006) Clarifying the mechanics of DNA strand exchange in meiotic recombination. Nature 442, 153–158.

8.	Neale, M. J., Pan, J., and Keeney, S. (2005) Endonucleolytic processing of covalent protein-linked DNA double-strand breaks. Nature 436, 1053–1057.

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