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Physical Monitoring of HO-Induced Homologous Recombination

  • Allyson Holmes
  • James E. Haber
Part of the Methods in Molecular Biology™ book series (MIMB, volume 113)

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).
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).

Keywords

Alkaline Buffer Bromocresol Green Strand Invasion Physical Monitoring Meiotic DSBs 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

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Copyright information

© Humana Press Inc. 1999

Authors and Affiliations

  • Allyson Holmes
    • 1
  • James E. Haber
    • 1
  1. 1.Rosenstiel Center and Department of BiologyBrandeis UniversityWaltham

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