Regulated expression of endonuclease EcoRI in Saccharomyces cerevisiae: nuclear entry and biological consequences

In an investigation to determine how proteins are localized within the nucleus of a cell, we demonstrate that the restriction endonuclease EcoRI is able to enter and function within the nucleus of Saccharomyces cerevisiae when this prokaryotic protein is synthesized in vivo. The EcoRI endonuclease was produced in yeast under the transcriptional control of a regulated yeast promoter by ligating a DNA fragment containing only coding sequences for the endonuclease to the promoter element of the yeast GAL1 gene (the structural gene for galactokinase, EC 2.7.1.6). Yeast cells harboring a plasmid containing this promoter-gene fusion are able to grow under conditions that repress transcription from the GAL1 promoter. However, under inducing conditions, these yeast cells are unable to grow. Moreover, rad52 mutants, which are deficient in the repair of double-strand breaks, are more sensitive to the presence of the promoter-gene fusion plasmid than are wild-type cells. We demonstrate that the EcoRI endonuclease activity is present in lysates prepared from yeast transformants grown under conditions that induce transcription of GAL1, but this activity is not detectable in cells grown under conditions that repress transcription from the promoter. Furthermore, analysis of yeast chromosomal DNA shows that the endonuclease enters the yeast nucleus and cleaves DNA specifically at EcoRI recognition sites.