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.
Tsalik
E L
EL
Department of Pharmacology, UMDNJ-Robert Wood Johnson Medical School, Piscataway, NJ 08854, USA.
Gartenberg
M R
MR
D015139:Blotting, Southern; D002262:Carboxy-Lyases / Q000235:genetics / Q000378:metabolism; D003470:Culture Media; D004249:DNA Damage; D004254:DNA Nucleotidyltransferases / Q000235:genetics / Q000378:metabolism; D005690:Galactose / Q000378:metabolism; D005947:Glucose / Q000378:metabolism; D010957:Plasmids / Q000235:genetics*; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development
D003470:Culture Media; D004254:DNA Nucleotidyltransferases; C045119:FLP recombinase; D002262:Carboxy-Lyases; C022378:phosphoribosylaminoimidazole carboxylase; D005947:Glucose; D005690:Galactose