The repair of chromosomal double-strand breaks (DSBs) inSaccharomyces cerevisiaeoccurs 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 ).MATswitching is a genetically programmed event in yeast haploid cells, initiated by the site-specific HO endonuclease (Fig. 1 ). This creates a DSB atMATthat can be repaired by homologous donor sequences,HMLα orHMRa, 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 timeMATacells useHMLα and thus switch toMATα, whereasMATα cells useHMRato switch toMATa. This change of mating type can be scored genetically and molecularly, sinceYaandYα 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 toMAT, and withinHMLα. Final product formation can also be detected by PCR usingMAT-proximal and Yα primers (pD and pC).
