Mechanisms of Transcription-Coupled DNA Supercoiling

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拓扑结构的影响。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点。已经认识到转录和DNA超螺旋是相互联系的。因此，需要对转录偶联DNA超螺旋的机制及其生物学功能进行详细的研究。该研究的长期目标是了解转录如何影响DNA拓扑结构，染色体结构和耦合的DNA交易。具体目标是：（1）体外生物化学研究，直接测试“双超螺旋结构域”转录模型。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，将进行探讨。研究结果有望为提出一个详细的机制来解释转录偶联DNA超螺旋提供基础。（二） 大肠杆菌转录偶联DNA超螺旋的研究螺旋细胞两个质粒系统将是 旨在研究体内转录偶联的DNA超螺旋。这些研究将反映支持所提出的机制的体外研究。（三）、设计和合成新的DNA模板来研究DNA超螺旋和转录偶联DNA超螺旋。在本研究中，我们将设计并合成两种新型的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。这些新的超螺旋DNA模板将为研究DNA超螺旋和转录偶联DNA超螺旋提供新的工具。这些综合研究的结果预计将显着提高我们对转录偶联DNA超螺旋的机制及其在其他DNA交易，如DNA复制中的作用的理解。将提出模型来解释体外和体内转录偶联的DNA超螺旋。

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.