EUKARYOTIC TOPOISOMERASE I

将使用一种综合的生化和遗传方法来 研究其机制、特异性、细胞内靶向和 人类I型拓扑异构酶(Topo I)的多种功能。与 可获得毫克量的纯Topo I和高滴度 亲和纯化的针对该蛋白的抗体，一些 生物学上重要的问题可以被调查。具体目标是 (I)生产足够数量的不同形式的纯TOPO I 结晶学研究中使用和不使用结合双链寡核苷酸 研究，(Ii)使用含有取代的TOPO I的突变形式 研究苯丙氨酸对酪氨酸活性部位(Y723F突变体)的影响 DNA序列和DNA拓扑影响蛋白质与DNA的结合， (3)使用连接介导的聚合酶链式反应，以避免 喜树碱在识别与TOPO I相关的断裂位点中的应用 在人类细胞中的转录和DNA复制，(Iv)确定 过表达TOPO野生型和Y723F突变型的效果 I在人类细胞中，以及(V)分离编码蛋白质的cDNA 与TOPO的特异性相互作用I.生化和结晶学 研究将阐明Topo I的催化机理的细节。 以及提供对抑制的基础的理解 抗癌药物喜树碱的效果。这些研究还将 的顺序要求之间的关系 具有TOPO I-序列要求的酶与DNA的结合 加入变性剂后DNA的中介断裂。这个 过度表达研究与互动研究相结合 蛋白质的伙伴应该提供重要的见解，让我们了解 Topo I与细胞中的其他蛋白质结合以提供旋转 DNA复制和转录所必需的。哪些研究涉及 Topo I参与非法重组可能揭示了一个作用 对于癌细胞遗传不稳定性中的酶，特别是 结肠癌细胞的Topo I水平升高。

An integrated biochemical and genetic approach will be used to investigate the mechanism, specificity, intracellular targeting and multiple functions of the human type I topoisomerase (topo I). With the availability of milligram quantities of pure topo I and high titer affinity purified antibodies against the protein, a number of biologically important issues can be investigated. The specific aims are (i) to produce sufficient quantities of different forms of pure topo I with and without bound duplex oligonucleotides for crystallographic studies, (ii) to use a mutant form of topo I containing a substitution of phenylalanine for the active site tyrosine (Y723F mutant) to study how DNA sequence and DNA topology affect the binding of DNA by the protein, (iii) to use the ligation-mediated polymerase chain reaction to avoid the use of camptothecin in identifying topo I break sites associated with transcription and DNA replication in human cells, (iv) to determine the effects of overexpressing the wild type and Y723F mutant forms of topo I in human cells, and (v) to isolate cDNAs which encode proteins that specifically interact with topo I. The biochemical and crystallographic studies will elucidate the details of the catalytic mechanism of topo I as well as provide an understanding of the basis for the inhibitory effects of the anti-cancer drug, camptothecin. These studies will also clarify the relationship between the sequence requirements for the binding of the enzyme to DNA with the sequence requirements for topo I- mediated breakage of DNA after addition of denaturants. The overexpression studies in combination with the search for interacting partners of the protein should provide important insights into how the topo I associates with other proteins in the cell to provide the swivels required for DNA replication and transcription. Studies which address the involvement of topo I in illegitimate recombination may reveal a role for the enzyme in the genetic instability of cancer cells, especially colon carcinoma cells which exhibit elevated levels of topo I.