GENOMIC STABILITY AND AGING IN YEAST

酵母的基因组稳定性和老化

• 批准号: 6214603
• 负责人: STEVEN J. BRILL
• 金额: $ 2.63万
• 依托单位: RUTGERS, THE STATE UNIV OF N.J.
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-05-01 至 2004-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6214603
• 关键词: DNA repair; Werner's syndrome; alleles; cell senescence; enzyme activity; fungal genetics; gene complementation; genome; helicase; lethal genes; mutant; phenotype; protein localization; yeast two hybrid system

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.

Werner's综合征（WS）是一种遗传疾病，其特征是早衰的特征。 负责WS（WRN）的基因类似于酵母的SGS1基因，并且像WS一样，SGS1突变体显示早龄和基因组不稳定性。 WRN和SGS1均编码属于DNA解旋酶的RECQ家族的160 kDa蛋白。 由于WRN和SGS1可能在相同的遗传途径中起作用，因此搜索与SGS1相互作用的突变应发现控制细胞衰老和基因组稳定性的基因。 提出了基因实验来识别和表征与SGS1直接和间接相互作用的基因，并且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合成致死突变筛选，以鉴定导致细胞需要SGS1的突变。 负责这些突变（SLX基因）的基因可能在与SGS1平行的遗传途径中起作用。一些SLX基因被克隆并发现用保守的人类同源物编码新型蛋白质。 SLX基因的完整集将被克隆，所有SLX突变体将被特征在于对酵母老化和基因组稳定性的影响。 将确定SLX蛋白的表达模式，细胞定位和酶活性。 这些结果将通过对人类细胞中SLX1基因的类似研究来证实。 为了鉴定直接与SGS1相互作用的基因，我们分离了一组新型的SGS1条件性致命等位基因。 这些等位基因的外部抑制剂有望直接在SGS1途径中起作用。 抑制菌株将被隔离，并将受影响的基因克隆。 通过搜索显示SGS1特异性表型的突变，并通过SLX突变体进行合成致死筛选，可以识别该途径中的其他基因。 为了确定SLX和SGS1蛋白的生化功能，它们将以其天然形式从酵母中纯化。 这些纯化的蛋白质将用于参与DNA代谢的酶活性。 与SLX和SGS1蛋白共纯化的稳定鉴定的蛋白质将接受氨基酸测序，分子克隆和反向遗传学，以鉴定其在控制细胞衰老和基因组稳定性方面的功能。