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Physical Monitoring of HO-Induced Homologous Recombination

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DNA Repair Protocols

Part of the book series: Methods in Molecular Biology™ ((MIMB,volume 113))

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Abstract

The repair of chromosomal double-strand breaks (DSBs) in Saccharomyces cerevisiae occurs most efficiently by homologous recombination. Homothallic mating-type (MAT) switching provides the most well-characterized system to study DSB repair by recombination in mitotic cells (1,2,3). MAT switching is a genetically programmed event in yeast haploid cells, initiated by the site-specific HO endonuclease (Fig. 1). This creates a DSB at MAT that can be repaired by homologous donor sequences, HMLα or HMR a, located near the ends of the same chromosome. These donor loci are maintained in a silent chromatin structure that prevents both their transcription and cleavage by HO, though they can still serve as donors in recombination. Most of the time MAT a cells use HMLα and thus switch to MATα, whereas MATα cells use HMR a to switch to MAT a. This change of mating type can be scored genetically and molecularly, since Y a and Yα sequences are different and have restriction endonuclease polymorphisms (Fig. 1).

Molecular model of mating type switching. A DSB is induced at the Y/Z junction by HO endonuclease. 5′–3′ Exonucleolytic degradation creates a 3′ single-stranded tail that invades the homologous silent donor sequence, HMLα. Strand invasion and repair synthesis can be monitored using a unique set of primers (pB and pA), located distal to MAT, and within HMLα. Final product formation can also be detected by PCR using MAT-proximal and Yα primers (pD and pC).

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Authors and Affiliations

  1. Rosenstiel Center and Department of Biology, Brandeis University, Waltham, MA

    Allyson Holmes & James E. Haber

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  1. Allyson Holmes
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  2. James E. Haber
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Editors and Affiliations

  1. University of Dundee, Dundee, UK

    Daryl S. Henderson

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© 1999 Humana Press Inc.

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Holmes, A., Haber, J.E. (1999). Physical Monitoring of HO-Induced Homologous Recombination. In: Henderson, D.S. (eds) DNA Repair Protocols. Methods in Molecular Biology™, vol 113. Humana Press. https://doi.org/10.1385/1-59259-675-4:403

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  • DOI: https://doi.org/10.1385/1-59259-675-4:403

  • Publisher Name: Humana Press

  • Print ISBN: 978-0-89603-802-8

  • Online ISBN: 978-1-59259-675-1

  • eBook Packages: Springer Protocols

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