Physical Monitoring of HO-Induced Homologous Recombination

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


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).


Alkaline Buffer Bromocresol Green Strand Invasion Physical Monitoring Meiotic DSBs 
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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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