Originally published as Genetics Published Articles Ahead of Print on October 9, 2008.

Genetics, Vol. 180, 1955-1962, December 2008, Copyright © 2008
doi:10.1534/genetics.108.094516

A Yeast Sir2 Mutant Temperature Sensitive for Silencing

Chia-Lin Wang, Joseph Landry1 and Rolf Sternglanz2

Department of Biochemistry and Cell Biology, Stony Brook University, Stony Brook, New York 11794-5215

2 Corresponding author: Department of Biochemistry and Cell Biology, Stony Brook University, Stony Brook, NY 11794-5215.
E-mail: rolf{at}life.bio.sunysb.edu

A screen for Saccharomyces cerevisiae temperature-sensitive silencing mutants identified a strain with a point mutation in the SIR2 gene. The mutation changed Ser276 to Cys. This amino acid is in the highly conserved NAD+ binding pocket of the Sir2 family of proteins. Haploid strains of either mating type carrying the mutation were severely defective at mating at 37° but normal at 25°. Measurements of RNA from the HMR locus demonstrated that silencing was lost rapidly upon shifting the mutant from the low to the high temperature, but it took >8 hours to reestablish silencing after a shift back to 25°. Silencing at the rDNA locus was also temperature sensitive. On the other hand, telomeric silencing was totally defective at both temperatures. Enzymatic activity of the recombinant wild-type and mutant Sir2 protein was compared by three different assays. The mutant exhibited less deacetylase activity than the wild-type protein at both 37° and 25°. Interestingly, the mutant had much more NAD+–nicotinamide exchange activity than wild type, as did a mutation in the same region of the protein in the Sir2 homolog, Hst2. Thus, mutations in this region of the NAD+ binding pocket of the protein are able to carry out cleavage of NAD+ to nicotinamide but are defective at the subsequent deacetylation step of the reaction.