engleski

Sgs1 protein prevents break-induced replication during gene targeting in yeast Saccharomyces cerevisiae

In contrast to higher eukaryotes, in yeast Saccharomyces cerevisiae DNA double strand breaks (DSB) on transforming DNA are almost always repaired by homologous recombination with homologous target in the genome. Therefore, transformation with non-replicative DNA fragments carrying selectable marker and containing ends homologous to the particular locus in the genome is widely used to precisely modify yeast genome. Transformation with cut plasmids almost exclusively results in targeted plasmid integration whereas illegitimate recombination is extremely rare. Additionally, illegitimate recombination is the only aberrant genetic event observed in plasmid integration assay so far. Contrary to plasmid integration, the spectrum of aberrant genetic events during gene-replacement is rather wider. Apart from illegitimate recombination, it can result in (i) integration of transforming DNA next to the homologous target and (ii) duplication of targeted chromosome yielding disomic transformants containing two copies of targeted chromosome, one containing transformed and another containing untransformed allele (heteroallelic transformants ; Svetec et al., 2007). In this study we showed that inactivation of EXO1 and SGS1 genes synergistically increased the efficiency of transformation in both, plasmid integration and gene replacement assay. However, almost all transformants obtained in gene replacement assay were heteroallelic transformants and this phenomenon was assigned to deletion of SGS1 gene. Further analysis showed that appearance of heteroallelic transformants is POL32 dependent strongly suggesting that duplication of targeted chromosome occurs by to break-induced replication (BIR). Additionally, this study is the first evidence that BIR also occurs in plasmid integration assay. In accordance with results obtained in gene replacement assay, the proportion of BIR incidence during plasmid integration strikingly increased in exo1 sgs1 background.

gene targeting; break-induced replication; Sgs1; yeast

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