Curing Saccharomyces cerevisiae of the 2 micron plasmid by targeted DNA damage

Powerful mutagenic screens of yeast Saccharomyces cerevisiae have recently been developed which require strains that lack the endogenous 2 micron plasmid (Burns et al., 1994). Here, we describe a simple and reliable method for curing yeast of the highly stable genetic element. The approach employs heterologous expression of a 'step-arrest' mutant of the Flp recombinase. The mutant, Flp H305L (Parsons et al., 1988), forms long-lived covalent protein-DNA complexes exclusively at 2 micron-borne recombinase target sites. In vivo, the complexes serve as sites of targeted DNA damage. Using Southern hybridization and a colony color assay for plasmid loss, we show that expression of the mutant enzyme results in the effective elimination of the 2 micron from cells.