FUNCTION OF SGS1, A HOMOLOG OF BLM AND WRN

这项建议的具体目的是研究SGS1的职能。 该基因最初是作为top3的慢生长抑制基因被分离出来的。 酿酒酵母中发现的与其同源的突变体 大肠杆菌RecQ解旋酶。带有SGS1突变的细胞表现出高度的 重复序列之间的重组，显示增加 染色体不分离和产孢量低为纯合子 二倍体。最近，导致人类两种疾病的基因， Bloom和Werner综合征被克隆并发现是 与SGS1同源。因此，对酵母中SGS1的研究可能会提供 对这个多基因家族成员功能的洞察和 可能为这两种癌症的病因学提供重要线索 综合症。具体的方法是：PI将调查这两个 SGS1、拓扑异构酶、 检查点基因和包括解旋酶在内的其他酵母基因。他会的 开发一种新的等位基因替换技术来辅助研究 这些互动。(2)他将调查两国之间的关系 SGS1及其人类同行的交叉互补研究 酵母和哺乳动物细胞都有。 具体地说，他将在这些基因之间交换结构域，以定义 运行所必需的单元。此外，他将决定是否 在酵母中发生的相同的物理相互作用也可以在哺乳动物中发生 细胞。此外，将测试对各种抑制剂的敏感性，以 描述了人类同源物的特征。(3)调查员将净化 SG1和它与之交互的组件 定义它们的生化功能(S)。此外，DNA拓扑学 自然序列和引入的质粒都将由 改变SGS1及其相互作用组分的基因剂量。(4) 他将确定影响超重组的参数 在SGS1缺失导致的重复序列之间。 具体地说，他将检查来自两个地点的序列 缺少SGS1的超重组--rDNA阵列和 Sup4区域。公安局还将调查两国之间的关系 复制分叉暂停和超重组。

The specific aim of this proposal is to study the function of Sgs1. This gene was first isolated as a slow growth suppressor of top3 mutants in Saccharomyces cerevisiae and found to be homologous to the E. coli RecQ helicase. Cells with Sgs1 mutations exhibit hyper- recombination between repeated sequences, show increased chromosome non-disjunction and sporulate poorly as homozygous diploids. Recently, the genes responsible for two human diseases, Bloom and Werner syndromes were cloned and found to be homologous with Sgs1. Thus, the study of Sgs1 in yeast may provide insights into the function of the members of this multigene family and may yield important clues to the etiology of cancer in these two syndromes. The specific approaches are: the PI will investigate both the physical and genetic interactions between Sgs1, topoisomerases, checkpoint genes and other yeast genes including helicases. He will develop a novel allele replacement technique to aid in the study of these interactions. (2) He will investigate the relationship between Sgs1 and its human counterparts by cross-complementation studies in both yeast and mammalian cells. Specifically, he will swap domains among these genes to define the units necessary for function. In addition, he will determine if the same physical interactions that occur in yeast can occur in mammalian cells. Furthermore, sensitivity to various inhibitors will be tested to characterized the human homologs. (3) The investigator will purify both Sgs1 and the components with which it interacts in order to define their biochemical function(s). In addition, DNA topology of both native sequences and introduced plasmids will be investigated by varying the gene dosage of Sgs1 and its interacting components. (4) He will determine the parameters that affect hyper-recombination between repeated sequences resulting from a Sgs1 deficiency. Specifically, he will examine sequences from two locations that exhibit hyper-recombination in the absence of Sgs1 -- the rDNA array and the SUP4 region. The PI will also investigate the relationship between replication fork pausing and hyper-recombination.