A genetic screen identified suppressors of Sir2’s catalytic activity. (A) Schematic of the screen to identify mutations that restored silencing in the absence of Sir2 catalytic activity. A sir2N345A sas2Δ strain had the URA3 reporter integrated in place of the a1 ORF at HMR. Expression of the URA3 gene resulted in cells that grow on complete synthetic media lacking uracil but are sensitive to 5-FOA. Expression of the α-genes from HML and a-genes from MAT result in a pseudodiploid cell identity that cannot mate. This strain was mutagenized with EMS and mutants were picked that reestablished silencing as assayed by resistance to 5-FOA and the ability to mate. (B) The phenotypes of a SIR2 wild-type strain (JRY10676) exhibiting complete silencing, the parent strain (JRY9097), and the four mutants that were isolated from the suppressor screen. Fivefold serial dilutions assayed for growth phenotypes on the indicated media. Growth in the patches on the right indicates successful matings as either an a- or α-cell identity.