Reference: Wang CL, et al. (2008) A yeast sir2 mutant temperature sensitive for silencing. Genetics 180(4):1955-62

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Abstract


A screen for S. 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 degrees but normal at 25 degrees. 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 more than 8 hours to re-establish silencing after a shift back to 25 degrees. 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 degrees and 25 degrees. 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.

Reference Type
Journal Article
Authors
Wang CL, Landry J, Sternglanz R
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