Part of the Methods in Molecular Biology™ book series (MIMB, volume 113)
Physical Monitoring of HO-Induced Homologous Recombination
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).
KeywordsAlkaline Buffer Bromocresol Green Strand Invasion Physical Monitoring Meiotic DSBs
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© Humana Press Inc. 1999