engleski

Inactivation of the SGS1 and EXO1 genes sinergistically stimulates plasmid integration in yeast

Different procedures used for targeted genetic manipulations are based on homologous recombination between chromosomal and exogenous DNA. A double-strand break (DSB) present on the plasmid molecule stimulates and directs plasmid integration to the homologous sequence in the yeast genome and we wondered whether this process could be further enhanced in selected DNA repair mutants. In order to compare the results obtained in different yeast strains, the efficiency of transformation with an linear integrative plasmid was compared to that obtained with a circular replicative plasmid. With respect to the wild type, the relative efficiency of transformation was increased in the sgs1 and exo1 mutants and decreased in the rad1 and srs2 mutants. Inactivation of the SGS1 or EXO1 gene stimulated plasmid integration 4- to 5-fold, while 15-fold increase was observed in the double sgs1 exo1 mutant. This result indicates that the two proteins participate in different cellular processes that limit plasmid integration in the wild type yeast. Southern blot analysis of 20 transformants obtained in the double mutant confirmed that they occured by homologous integration to the target sequence. Homologues to both EXO1 and SGS1 genes have been found in other organisms and we suggest that their inactivation may also lead to enhanced gene targeting.

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

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