EUKARYOTIC TOPOISOMERASE I

将采用综合生物化学和遗传学方法， 研究其机制、特异性、细胞内靶向和 人I型拓扑异构酶（topo I）的多种功能。 与 可获得毫克量的纯拓扑异构酶I和高滴度 针对蛋白质的亲和纯化的抗体， 可以研究生物学上重要的问题。 具体目标是 (i)来生产足够数量的不同形式的纯拓扑异构酶I 具有和不具有结合的双链体寡核苷酸用于晶体学 研究，（ii）使用含有取代的拓扑异构酶I的突变形式， 苯丙氨酸的活性位点酪氨酸（Y723F突变体），以研究如何 DNA序列和DNA拓扑结构影响蛋白质与DNA的结合， (iii)使用连接介导的聚合酶链反应，以避免 喜树碱在鉴定与肿瘤相关的拓扑异构酶I断裂位点中的用途 （iv）确定人细胞中的转录和DNA复制， 过量表达野生型和Y723F突变型topo I在人细胞中的表达，和（v）分离编码蛋白质的cDNA， 与拓扑异构酶I特异性相互作用。 生物化学和晶体学 研究将阐明拓扑异构酶I催化机制的细节 并提供了一个理解的基础， 抗癌药物喜树碱的作用。 这些研究还将 阐明了顺序要求之间的关系 酶与DNA的结合具有拓扑I的序列要求- 加入变性剂后介导的DNA断裂。 的 过表达研究结合寻找相互作用的 蛋白质的合作伙伴应该提供重要的见解， 拓扑异构酶I与细胞中的其他蛋白质结合， 是DNA复制和转录所必需的。 研究涉及 拓扑异构酶I参与非法重组可能揭示了一种作用， 对于癌细胞遗传不稳定性中的酶，特别是 结肠癌细胞表现出升高的Topo I水平。

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.