GENOMIC STABILITY AND AGING IN YEAST

沃纳综合征(WS)是一种以早衰和染色体不稳定为特征的遗传性疾病。WS(WRN)的致病基因与酵母的SGS1基因相似，与WS一样，SGS1突变体也表现出早衰和基因组不稳定。WRN和SGS1都编码160 kDa的蛋白质，属于DNA解旋酶的RecQ家族。由于WRN和SGS1可能在相同的遗传途径中发挥作用，因此对与SGS1相互作用的突变的搜索应该会发现控制细胞衰老和基因组稳定性的基因。建议进行基因实验来鉴定和表征与SGS1直接和间接相互作用的基因，并将使用生化实验来确定编码基因产物的功能。已经进行了SGS1合成致命突变筛选，以确定导致细胞需要SGS1才能存活的突变。导致这些突变的基因(SLX基因)很可能作用于与SGS1平行的遗传途径。一些SLX基因已经被克隆，并被发现编码具有保守的人类同源物的新蛋白。全套SLX基因将被克隆，所有SLX突变体将被鉴定对酵母老化和基因组稳定性的影响。SLX蛋白的表达模式、细胞定位和酶活性将被确定。这些结果将在人类细胞中对SLX1基因的类似研究中得到证实。为了确定与SGS1直接相互作用的基因，我们分离了一组新的SGS1条件致死等位基因。这些等位基因的外源抑制因子有望直接作用于SGS1途径。抑制菌株将被分离，受影响的基因将被克隆。这一途径中的其他基因将通过搜索显示SGS1特定表型的突变和对SLX突变体进行合成致死筛选来识别。为了确定SLX和SGS1蛋白的生化功能，我们将从酵母中提纯它们的天然形式。这些纯化的蛋白质将被检测与DNA新陈代谢有关的酶活性。与SLX和SGS1蛋白共纯化的稳定结合的蛋白质将接受氨基酸测序、分子克隆和反向遗传学，以确定它们在控制细胞衰老和基因组稳定性方面的功能。

Werner's Syndrome (WS) is a genetic disease characterized by premature aging and chromosomal instability. The gene responsible for WS (WRN) is similar to the SGS1 gene of yeast and, like WS, SGS1 mutants display premature aging and genomic instability. Both WRN and SGS1 encode 160 kDa proteins belonging to the RecQ family of DNA helicases. Because WRN and SGS1 are likely to function in identical genetic pathways, a search for mutations that interact with SGS1 should uncover genes that control cellular aging and genome stability. Genetic experiments are proposed to identify and characterize genes that interact directly and indirectly with SGS1, and biochemical experiments will be used to determine the function of the encoded gene products. An SGS1 synthetic-lethal mutant screen has been conducted to identify mutations that cause the cell to require SGS1 for viability. The genes responsible for these mutations (SLX genes) are likely to act in genetic pathways that are parallel to SGS1. Some of the SLX genes have been cloned and found to encode novel proteins with conserved human homologs. The complete set of SLX genes will be cloned and all SLX mutants will be characterized for effects on yeast aging and genomic stability. The expression pattern, cellular localization, and enzymatic activity of the SLX proteins will be determined. These results will be confirmed by similar studies of the SLX1 gene in human cells. To identify genes that interact directly with SGS1, we have isolated a set of novel conditional-lethal alleles of SGS1. Extragenic suppressors of these alleles are expected to act directly in the SGS1 pathway. Suppressor strains will be isolated and affected genes will be cloned. Additional genes in this pathway will be identified by searching for mutations that show SGS1-specific phenotypes and by performing a synthetic-lethal screen with SLX mutants. To determine the biochemical function of the SLX and SGS1 proteins, they will be purified from yeast in their native form. These purified proteins will be assayed for enzymatic activities involved in DNA metabolism. Stably-assoicated proteins that co-purify with the SLX and SGS1 proteins will be subjected to amino acid sequencing, molecular cloning and reverse genetics to identify their function in controlling cellular aging and genomic stability.