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Inactivation of the SGS1 and EXO1 genes synergistically stimulates the efficiencies of yeast transformation

In the contrast to many other eukaryotic organisms integration of exogenous DNA in the yeast Saccharomyces cerevisiae genome is almost always due to homologous recombination. Therefore plasmids and linear DNA fragments are routinely used to introduce any desired genetic modification in the yeast genome. A double-strand break (DSB) present on the plasmid molecule stimulates and directs plasmid integration to the homologous sequence, while linear DNA fragments participate in replacement of genomic allele by the allele present on the transforming fragment. In this work we investigated whether these processes can be further enhanced in DNA repair mutants. In order to compare the results obtained in different yeast strains, the efficiency of plasmid integration and gene replacement was normalised to the efficiency of transformation with a circular replicative plasmid. The inactivation of several genes had opposite effect on plasmid integration and gene replacement suggesting that the transforming DNA was subjected to the different recombination mechanisms. However, the inactivation of SGS1 and EXO1 synergistically enhanced yeast transformation 15- to 20-fold in both assays. Homologues to both SGS1and EXO1 genes have been found in other organisms and we suggest that their transient inactivation may facilitate gene manipulation.

gene targeting; SGS1; EXO1; RAD1; SRS2; plasmid integration

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