FUNCTION OF SGS1, A HOMOLOG OF BLM AND WRN

该提案的具体目的是研究Sgs 1的功能。 该基因最初是作为top3的缓慢生长抑制因子分离的。 酿酒酵母中的突变体，并发现与 急诊coli RecQ解旋酶。 具有Sgs 1突变的细胞表现出超- 重复序列之间的重组，显示增加 染色体不分离，孢子形成差，为纯合 二倍体 最近，导致两种人类疾病的基因， Bloom和Werner综合征被克隆， 与Sgs 1同源。 因此，对酵母中Sgs 1的研究可以提供 深入了解这个多基因家族成员的功能， 可能为这两种癌症的病因学提供重要线索。 综合征 具体方法为：PI将调查 Sgs 1，拓扑异构酶， 检查点基因和其它酵母基因，包括解旋酶。 他将 开发一种新的等位基因替代技术，以帮助研究 这些互动。 (2)他将调查 sgs 1及其人类对应物的交叉互补研究， 酵母和哺乳动物细胞。 具体来说，他将在这些基因之间交换结构域，以定义 功能所需的单位。 此外，他还将确定 在酵母中发生的相同物理相互作用可以在哺乳动物中发生。 细胞 此外，还将测试对各种抑制剂的敏感性， 人类同源物的特征。 (3)调查员将净化 Sgs 1及其相互作用的组件， 定义其生化功能。 此外， 天然序列和引入的质粒都将通过 改变Sgs 1及其相互作用组分的基因剂量。 （四） 他将确定影响超重组的参数 Sgs 1缺陷导致的重复序列之间的差异。 具体来说，他将检查来自两个地点的序列， Sgs 1缺失下的超重组--rDNA阵列和 SUP 4区域。 PI还将调查以下因素之间的关系： 复制叉暂停和超重组。

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.