BIOLOGICAL FUNCTIONS OF TOPOISOMERASE I IN S CEREVISIAE

该项目的目标是确定以下关键功能 酿酒酵母拓扑异构酶I(Topo I)的分子遗传学研究 与TOP 1结合后不能存活的突变分析 突变。拓扑异构酶I被认为是重要的释放 在DNA复制和转录过程中遇到的扭转应力。 然而，top 1基因零突变的酵母突变株是可行的。 对排名前1位的突变体的生存能力的一种解释是 提供重叠功能的蛋白质。识别编码基因 重叠的功能，我们已经分离出了不可行的突变体，除非 Topo I正在表达(Topo I-Required的TPR突变体)。TPR 基因定义了至少五个互补基团。我们还发现， TOP 1单个突变体在核糖体DNA中存在以前未被发现的缺陷 复制。 I.TPR的分子遗传学和生化分析(TOPO I-要求) 基因。 A.TPR1(编码与TOP 1同源的蛋白质的基因 地区) 我们将检测TOP1tpr1(Ts)突变体的DNA和RNA合成。 以确定这一功能是什么。Tpr1蛋白将是 进行免疫定位，以确定它是否像Top 1一样位于核仁中。 Tpr1是否编码拓扑异构酶，将在 纯化的蛋白质。 B.TPR2(RNA聚合酶II亚单位RPB4) TOP1可以在功能上取代RNA聚合酶II亚单位的事实 这表明类似的生化活性可能是由 亚单位。转录诱导模板DNA和mRNA的超螺旋启动 Tpr2突变体的位点效率，以及拓扑异构酶和DNA解旋酶的测定 对Tpr2蛋白的研究将为类似的研究提供证据 功能。 C.TPR3、4和5(功能目前未知) TPR3、TPR4和TPR5的DNA序列将确定它们之间的同源性 和其他基因。将检测Top1、tpr3、4和5(Ts)双突变体 对于生理缺陷(DNA、RNA合成和细胞周期停滞)，以及 检查核仁结构是否有缺陷。 二、TOP1中rDNA复制缺陷的分析及TPR检测 突变者 我们将研究rdna顺反子中顺式作用突变对 Topo I在rDNA复制过程中的作用 哪些点突变和缺失可以很容易地引入 数组。迷你顺反管将与Fangman 2D结合使用 复制凝胶和电子显微镜检查rDNA复制 Top1和TPR突变体中的中间体。

The goal of this project is to identify the critical functions of topoisomerase I (topo I) in S. cerevisiae through a molecular-genetic analysis of mutations that are inviable in combination with top 1 mutations. Topoisomerase I is presumed to be important to relieve torsional stress encountered during DNA replication and transcription. However, yeast mutants with a null mutation in the TOP 1 gene are viable. One explanation for the viability of top 1 mutants is that there are proteins providing overlapping functions. To identify genes encoding overlapping functions, we have isolated mutants that are inviable unless topo I is being expressed (tpr mutants for topo I-requiring). The TPR genes define at least five complementation groups. We have also found that top 1 single mutants have a previously unrecognized defect in ribosomal DNA replication. I. Molecular-genetic and biochemical analysis of TPR (topo I-requiring) genes. A. TPR1 (a gene encoding a protein homologous to TOP 1 over two short regions) We will examine DNA and RNA synthesis in top1 tpr1 (ts) mutants made in vitro to define what that function is. The Tpr1 protein will be immunolocalized to see if it is in the nucleolus like Top 1. To determine whether Tpr1 encodes a topoisomerase, assays will be performed on the purified protein. B. TPR2 (RNA polymerase II subunit RPB4) The fact that TOP1 can functionally replace an RNA polymerase II subunit suggests that a similar biochemical activity may be provided by that subunit. Transcription induced supercoiling of template DNA and mRNA start site efficiency in tpr2 mutants, and topoisomerase and DNA helicase assays on the Tpr2 protein will be performed to provide evidence for similar functions. C. TPR3, 4 and 5 (function currently unknown) The DNA sequences of TPR3, 4 and 5 will determine homologies between these and other genes. top1 tpr3, 4 and 5 (ts) double mutants will be examined for physiological defects (DNA, RNA synthesis, and cell-cycle arrest), and the examined for defects in nucleolar structure. II. Analysis of the rDNA replication defect in top1 and examination of tpr mutants We will examine the effect of cis-acting mutations in an rDNA cistron on topo I function during rDNA replication by constructing a mini-cistron with which point mutations and deletions can be readily introduced into the array. The mini-cistron will be used in combination with Fangman 2D replication gels and electron microscopy to examine rDNA replication intermediates in top1 and tpr mutants.