FIGURE 1.

pol30 mutants are defective in silencing a regulatable HMR locus. A, a regulatable HMR locus. The HMR contained a modified E silencer, HMR-GalSS, which comprised four Gal4 binding sites in place of the ORC binding site plus a Rap1p and an Abf1p binding site and the a1 and a2 genes, but lacked the I silencer. Expression of the chimeric Gal4-Sir1p via the MET3-repressible promoter in these cells enabled Sir protein association at HMR. Upon induction of Flp1p from the GAL10 promoter, Flp1p binds to the FRT sites that flank HMR and excises HMR from chromosome III as a 2.6-kb double-stranded circular DNA molecule. B, experimental strategy. Cells grown in methionine lacked Gal4-Sir1p and expressed a1 mRNA. Cells were arrested in G₁ with α-factor. Flp1p expression was then induced with galactose to excise HMR from the chromosome in G₁. Next, expression of Flp1p was repressed, and Gal4-Sir1p was induced in G₁ in medium containing raffinose and lacking methionine. Finally, cells expressing Gal4-Sir1p were released from G₁ and allowed to progress through the cell cycle until G₂/M where they were rearrested with benomyl plus nocodazole.