Physical Monitoring of HO-Induced Homologous Recombination
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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, HML α 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 HML α and thus switch to MAT α, whereas MAT α cells use HMR a to switch to MAT a . This change of mating type can be scored genetically and molecularly, since Y a and Y α 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, HML α. Strand invasion and repair synthesis can be monitored using a unique set of primers (pB and pA), located distal to MAT , and within HML α. Final product formation can also be detected by PCR using MAT -proximal and Yα primers (pD and pC).
