In vivo biochemistry: Physical monitoring of recombination induced by site-specific endonucleases

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Abstract

The recombinational repair of chromosomal double-strand breaks (DSBs) is of critical importance to all organisms, who devote considerable genetic resources to ensuring such repair is accomplished. In Saccharomyces cerevisiae, DSB-mediated recombination can be initiated synchronously by the conditional expression of two site-specific endonucleases, HO or I-Scel. DNA undergoing recombination can then be extracted at intervals and analyzed. Recombination initiated by meiotic-specific DSBs can be followed in a similar fashion. This type of ‘in vivo biochemistry’ has been used to describe several discrete steps in two different homologous recombination pathways: gene conversion and single-strand annealing. The roles of specific proteins during recombination can be established by examining DNA in strains deleted for the corresponding gene. These same approaches are now becoming available for the study of recombination in both higher plants and animals. Physical monitoring can also be used to analyze nonhomologous recombination events, whose mechanisms appear to be conserved from yeast to mammals.

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