INTERACTION OF DNA TOPOISOMERASES WITH CHROMATIN

目的是了解拓扑异构酶在 染色质结构和功能。 鸟类系统已经发展成为一个 模型来确定拓扑异构酶I和II的催化位点， DNA序列水平。 专门开发的技术 这一目标，并以此为目标。 这些研究取决于 证明内源性拓扑异构酶形成瞬时共价 与DNA（染色质中）的复合物，可以被捕获并纯化 从游离蛋白质和游离DNA。 纯化的复合物中的DNA将被 使用当前的分子生物学工具进行表征。 具体地说， 通过与克隆基因（发育或管家基因）杂交 与拓扑异构酶偶联的不同DNA序列的富集试验 将被执行。 催化作用位点的高分辨率绘图 然后进行拓扑异构酶I和II的检测， 染色质中具有拓扑异构酶切割位点的二级结构。 的 作图实验需要产生单特异性抗体 对拓扑异构酶。 这些免疫试剂也将用于 在细胞学水平上定位拓扑异构酶分布， 除了免疫电镜外，还用光学显微镜进行免疫荧光 用蛋白A-胶体金进行显微镜检查。 第二个目标是调查收集类型II 拓扑异构酶已经在一个步骤中通过亲和分离， 新生霉素-琼脂糖凝胶层析。 一个实验性的方案已经 旨在从亲和纯化的蛋白质中寻找真核促旋酶， 活动 此外，将针对亲和力制备抗体。 用于免疫选择DNA片段的纯化的活性， 内源性拓扑异构酶II。 DNA片段将被识别， 以与克隆基因杂交为特征。 这项工作的主要意义是增进我们对 染色质结构改变的结构基础， 在分化过程中基因的时间表达。 提案涉及 使用一个非常好的特点发展系统，并借鉴 在过去十年中积累的关于染色质结构、DNA 结构和基因开关在发展过程中明确界定，易于处理， 呼叫血统。 此外，我们还结合了广泛的知识， 该系统采用了本实验室开发的强大技术， 我们研究DNA结合蛋白本身以及DNA结合 这些蛋白质在染色质中的序列。

The objective is to gain an understanding of the role of topoisomerases in chromatin structure and function. The avian system has been developed as a model to identify the catalytic sites of topoisomerases I and II at the level of DNA sequence. Technologies have been developed specifically for this purpose and with this goal in mind. These studies hinge on the demonstration that endogenous topoisomerases form a transient covalent complex with DNA (in chromatin) which can be trapped, and purified away from free protein and free DNA. The DNA in the purified complexes will be characterized using the current tools of molecular biology. Specifically, by hybridization with cloned genes (developmental or housekeeping genes) tests for enrichment of different DNA sequences coupled to topoisomerases will be carried out. High resolution mapping of catalytic sites of action of topo I and II will then be carried out to correllate alterations in DNA secondary structure with topoisomerase cleavage sites in chromatin. The mapping experiments require the production of monospecific antibodies against topoisomerases. These immunologic reagents will also be used to localize topoisomerase distribution at the cytological level using immunofluorescence with the light microscope in addition to immunoelectron microscopy with protein A-colloidal gold. A second objective is to investigate the collection of type II topoisomerases that have been isolated in a single step by affinity chromatography over novobiocin-Sepharose. An experimental scheme has been devised to search for a eukaryotic gyrase among the affinity purified activities. In addition, antibodies will be prepared against the affinity purified activities for use in immuno-selecting DNA fragments containing endogenous topoisomerase II. The DNA fragments will be identified and characterized by hybridization to cloned genes. The primary significance of the work is to advance our knowledge of the structural basis for alterations in chromatin structure which attend the temporal expression of genes during differentiation. The proposal involves the use of a very well characterized developmental system and draws on knowledge accumulated during the past decade on chromatin structure, DNA structure and gene switching during development in well defined, tractable call lineages. In addition, we are combining the extensive knowledge on this system with powerful technologies developed in this lab which allow us to study the DNA binding proteins themselves as well as the DNA binding sequence of these proteins in chromatin.