Mechanisms of Transcription-Coupled DNA Supercoiling

转录偶联 DNA 超螺旋机制

基本信息

批准号：
6766530
负责人：
Fenfei Leng
金额：
$ 18.06万
依托单位：
FLORIDA INTERNATIONAL UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2004
资助国家：
美国
起止时间：
2004-04-01 至 2008-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/6766530
关键词：
DNA DNA binding protein DNA replication DNA topoisomerases Escherichia coli circular DNA gel mobility shift assay gene mutation genetic transcription high performance liquid chromatography mathematical model nucleic acid chemical synthesis nucleic acid structure plasmids protein purification protein structure function western blottings

项目摘要

Many genetic diseases and cancers are caused by inappropriate transcription of certain genes, which can be affected by DNA topology. DNA topoisomerases that regulate the cellular DNA topology in vivo are the target of many anti-cancer drugs. It has been recognized that transcription and DNA supercoiling are mutually linked. Therefore, a detailed mechanistic study of transcription-coupled DNA supercoiling and its biological functions is needed. The long-term goal of the proposed research is to understand how transcription affects DNA topology, chromosome structure, and the coupled DNA transactions. Specific aims are: (1) In vitro biochemical studies to directly test the "twin-supercoiled-domain" model of transcription. Different aspects of transcription-coupled DNA supercoiling, especially the stimulation by sequence-specific DNA-binding proteins, will be explored. Results are expected to provide a basis to propose a detailed mechanism to explain transcription-coupled DNA supercoiling. (2) Studies of transcription-coupled DNA supercoiling in E. coil cells. A two plasmid system will be designed to study transcription-coupled DNA supercoiling in vivo. These studies will mirror the in vitro studies for supporting the proposed mechanism. (3). Design and synthesis of novel DNA templates to study DNA supercoiling and transcription-coupled DNA supercoiling. In the proposed studies, two types of novel DNA templates will be designed and synthesized. The first type of DNA templates are circular DNA molecules with one or two interstrand covalent cross-links. The second type of DNA templates are linear supercoiled DNA molecules. These proposed novel supercoiled DNA templates will provide new tools to study DNA supercoiling and transcription-coupled DNA supercoiling. Results of these combined studies are anticipated to significantly enhance our understanding of the mechanism of transcription-coupled DNA supercoiling and its roles in other DNA transactions such as DNA replication. Models will be proposed to explain transcription-coupled DNA supercoiling both in vitro and in vivo.

许多遗传疾病和癌症是由某些基因的不适当转录引起的，这可能会受到 DNA 拓扑的影响。 DNA拓扑异构酶在体内调节细胞DNA拓扑结构，是许多抗癌药物的靶标。人们已经认识到转录和DNA超螺旋是相互关联的。因此，需要对转录耦合DNA超螺旋及其生物学功能进行详细的机制研究。该研究的长期目标是了解转录如何影响 DNA 拓扑、染色体结构和耦合的 DNA 事务。具体目标是：（1）体外生化研究，直接测试转录的“双超螺旋结构域”模型。将探索转录偶联 DNA 超螺旋的不同方面，特别是序列特异性 DNA 结合蛋白的刺激。预计结果将为提出解释转录偶联 DNA 超螺旋的详细机制提供基础。 (2) 大肠杆菌细胞中转录耦合 DNA 超螺旋的研究。两个质粒系统将是旨在研究体内转录耦合 DNA 超螺旋。这些研究将反映支持所提出机制的体外研究。（3）。设计和合成新型 DNA 模板以研究 DNA 超螺旋和转录耦合 DNA 超螺旋。在拟议的研究中，将设计和合成两种类型的新型 DNA 模板。第一种类型的 DNA 模板是具有一个或两个链间共价交联的环状 DNA 分子。第二种类型的 DNA 模板是线性超螺旋 DNA 分子。这些提出的新型超螺旋 DNA 模板将为研究 DNA 超螺旋和转录耦合 DNA 超螺旋提供新工具。这些综合研究的结果预计将显着增强我们对转录耦合 DNA 超螺旋机制及其在 DNA 复制等其他 DNA 事务中的作用的理解。将提出模型来解释体外和体内转录耦合 DNA 超螺旋。