Wang Chia-Lin, Landry Joseph, Sternglanz Rolf
Department of Biochemistry and Cell Biology, Stony Brook University, Stony Brook, New York 11794-5215, USA.
Genetics. 2008 Dec;180(4):1955-62. doi: 10.1534/genetics.108.094516. Epub 2008 Oct 9.
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.
一项针对酿酒酵母温度敏感型沉默突变体的筛选鉴定出了一株在SIR2基因中存在点突变的菌株。该突变将Ser276变为Cys。这个氨基酸位于Sir2蛋白家族高度保守的NAD(+)结合口袋中。携带该突变的两种交配型单倍体菌株在37℃时交配严重缺陷，但在25℃时正常。对来自HMR位点的RNA的测量表明，将突变体从低温转移到高温后，沉默迅速丧失，但在转回25℃后重新建立沉默需要超过8小时。rDNA位点的沉默也对温度敏感。另一方面，端粒沉默在两个温度下都完全有缺陷。通过三种不同的测定方法比较了重组野生型和突变型Sir2蛋白的酶活性。在37℃和25℃时，突变体的脱乙酰酶活性均低于野生型蛋白。有趣的是，该突变体的NAD(+)-烟酰胺交换活性比野生型高得多，Sir2同源物Hst2中该蛋白相同区域的突变也是如此。因此，该蛋白NAD(+)结合口袋这一区域的突变能够将NAD(+)裂解为烟酰胺，但在反应的后续脱乙酰化步骤中存在缺陷。
