INTERACTION OF DNA TOPOISOMERASES WITH CHROMATIN

DNA 拓扑异构酶与染色质的相互作用

• 批准号: 3279797
• 负责人: MARK T MULLER
• 金额: $ 15.96万
• 依托单位: OHIO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1983
• 资助国家: 美国
• 起止时间: 1983-01-01 至 1988-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3279797
• 关键词: DNA; DNA gyrase; DNA topoisomerases; affinity chromatography; biomechanics; cell differentiation; chromatin; developmental genetics; enzyme linked immunosorbent assay; eukaryote; gene expression; genetic library; genetic mapping; genetic models; genetic transcription; immunoelectron microscopy; immunoelectrophoresis; immunofluorescence technique; molecular cloning; monoclonal antibody; neoplastic transformation; novobiocin; nucleic acid sequence; nucleosomes; radiotracer

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.

目的是了解拓扑异构酶在 染色质结构和功能。 禽类系统已被开发为 模型来识别拓扑异构酶 I 和 II 的催化位点 DNA 序列的水平。 技术是专门为 这个目的并牢记这个目标。 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）形成的复合物，可以被捕获并纯化掉 来自游离蛋白质和游离DNA。 纯化后的复合物中的 DNA 将是 使用当前的分子生物学工具进行表征。 具体来说， 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 序列的富集测试 将进行。 催化作用位点的高分辨率绘图 然后将进行拓扑 I 和 II 的分析以关联 DNA 的变化 染色质中具有拓扑异构酶切割位点的二级结构。 这 作图实验需要产生单特异性抗体 对抗拓扑异构酶。 这些免疫试剂也将用于 使用以下方法在细胞学水平上定位拓扑异构酶分布 除免疫电镜外，还使用光学显微镜进行免疫荧光 蛋白 A-胶体金显微镜检查。 第二个目标是调查 II 类的收集 通过亲和力一步分离的拓扑异构酶 新生霉素-琼脂糖层析。 一个实验方案已经 设计用于在亲和纯化的亲和力中寻找真核旋转酶 活动。 此外，将针对亲和力制备抗体 用于免疫选择 DNA 片段的纯化活性，其中含有 内源性拓扑异构酶 II。 DNA 片段将被识别并 其特征是与克隆基因杂交。 这项工作的主要意义是增进我们对 参与染色质结构改变的结构基础 分化过程中基因的时间表达。 该提案涉及 使用一个非常明确的发展系统并借鉴 过去十年积累的关于染色质结构、DNA 的知识 发育过程中的结构和基因转换是明确的、易于处理的 呼唤血统。 此外，我们还结合了广泛的知识 该系统采用本实验室开发的强大技术，允许 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。