INTERACTION OF DNA TOPOISOMERASES WITH CHROMATIN

目的是了解拓扑异构酶在细胞周期中的作用。 染色质的结构和功能。禽类系统是作为一种 确定拓扑异构酶I和II的催化位置的模型 DNA序列的水平。专门为以下目的开发的技术 这一目的，并牢记这一目标。这些研究取决于 内源性拓扑异构酶形成瞬时共价键的证明 与DNA的络合物(在染色质中)，可被捕获并提纯 来自游离蛋白质和游离DNA。纯化的复合体中的DNA将是 使用当前的分子生物学工具进行了表征。具体来说， 通过与克隆的基因(发育基因或看家基因)杂交 不同拓扑异构酶偶联DNA序列的富集性试验 将会被执行。催化作用部位的高分辨率测绘 然后将进行TOPO I和II的相关DNA改变 染色质中含有拓扑异构酶裂解位点的二级结构。这个 测绘实验需要产生单特异性抗体 抗拓扑异构酶。这些免疫试剂还将用于 拓扑异构酶在细胞学水平的定位 除免疫电子外，还用光学显微镜进行免疫荧光 用蛋白A胶体金作显微镜观察。 第二个目标是调查第二类动物的收集情况 一步亲和分离得到的拓扑异构酶 用新生物素琼脂糖层析法。一项试验性计划已经完成 目的是在纯化的亲和力中寻找真核细胞旋转酶 活动。此外，还将制备针对亲和力的抗体 用于免疫选择DNA片段的纯化活性 内源性拓扑异构酶II。DNA片段将被鉴定和 以与克隆基因杂交为特征的。 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 基因在分化过程中的时间表达。该提案涉及 使用一个非常有特点的发展系统，并借鉴 在过去十年中积累的关于染色质结构、DNA 发育过程中的结构和基因切换，定义良好，易于处理 称其为血统。此外，我们正在结合关于 该系统与本实验室开发的强大技术相结合，可以 美国将研究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.