engleski

Increased frequency of site-specific DSBs by addition of nuclear localization signal to the yeast mitochondrial I-SceI endonuclease

The Saccharomyces cerevisiae mitochondrial endonuclease I-SceI has been widely used for the induction of site-specific DSBs in various organisms. Increased levels of homologous recombination in cells expressing the synthetic I-SceI gene from cytoplasmic ribosomes, was indirect proof that although of mitochondrial origin, the I-SceI protein can enter the nucleus. However, the protein sequence of this nuclease does not contain any known nuclear localization signal (nls), suggesting incidental endonuclease transport into nucleus compartment. We tested the proficiency of the I-SceI in DSB induction in yeast in relationship to the presence of the nls, by a two plasmid vectors system. The first was a 2micron episomal vector expressing the I-SceI endonuclease with or w. o. the nls from the large T antigen of simian virus 40, under the control of GAL1 promoter. The second was a target centromeric plasmid carrying the 18 bp I-SceI recognition DNA sequence. The expression level of I-SceI endonuclease, its cellular localization and the DSB formation were monitored in yeast cells periodically after galactose induction. The frequency of induced DSB on target plasmid differed significantly between two I-SceI clones. High percentage of target plasmid was linearized by I-SceI-nls endonuclease already 2 hours after induction. In the case of the I-SceI w. o. nls, DSBs were not detected even after prolonged galactose induction, regardless of the high amount of expressed protein. Obvious difference in DSB proficiency between two endonucleases was connected with their cellular localization.

yeast; recombination; DSB; I-SceI endonuclease

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