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 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 >8 hours to reestablish 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.
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Evidence ID | Analyze ID | Gene/Complex | Systematic Name/Complex Accession | Qualifier | Gene Ontology Term ID | Gene Ontology Term | Aspect | Annotation Extension | Evidence | Method | Source | Assigned On | Reference |
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Evidence ID | Analyze ID | Gene | Gene Systematic Name | Phenotype | Experiment Type | Experiment Type Category | Mutant Information | Strain Background | Chemical | Details | Reference |
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Evidence ID | Analyze ID | Gene | Gene Systematic Name | Disease Ontology Term | Disease Ontology Term ID | Qualifier | Evidence | Method | Source | Assigned On | Reference |
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Evidence ID | Analyze ID | Regulator | Regulator Systematic Name | Target | Target Systematic Name | Direction | Regulation of | Happens During | Regulator Type | Direction | Regulation Of | Happens During | Method | Evidence | Strain Background | Reference |
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Site | Modification | Modifier | Source | Reference |
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Evidence ID | Analyze ID | Interactor | Interactor Systematic Name | Interactor | Interactor Systematic Name | Allele | Assay | Annotation | Action | Phenotype | SGA score | P-value | Source | Reference | Note |
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Evidence ID | Analyze ID | Interactor | Interactor Systematic Name | Interactor | Interactor Systematic Name | Assay | Annotation | Action | Modification | Source | Reference | Note |
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Complement ID | Locus ID | Gene | Species | Gene ID | Strain background | Direction | Details | Source | Reference |
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Evidence ID | Analyze ID | Dataset | Description | Keywords | Number of Conditions | Reference |
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