DYNAMICS OF PROTEIN-DNA INTERACTIONS IN DNA REPLICATION

DNA 复制中蛋白质-DNA 相互作用的动力学

• 批准号: 6768071
• 负责人: Linda B Bloom
• 金额: $ 5.98万
• 依托单位: UNIVERSITY OF FLORIDA
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-07-01 至 2004-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6768071
• 关键词: DNA directed DNA polymerase; DNA replication; adenosine triphosphate; bacteriophage T4; chemical kinetics; enzyme activity; fluorescence microscopy; intermolecular interaction; microorganism culture; molecular dynamics; protein protein interaction; virus DNA; virus genetics

DESCRIPTION: The long-term goal of this proposal is to better define dynamic protein-DNA and protein-protein interactions in DNA replication. The system that will be examined in detail is the loading of the sliding clamp (processivity factor) onto DNA by the multi-subunit clamp loader complex. The investigator proposes to examine this system from 2 different organisms, E. coli and T4. In Specific Aim 1, the investigator will examine how varying the structure of the DNA (DNA primer-template, RNA primer-template, blunt end, etc.) affects loading of the sliding clamp (beta) by the clamp loader complex (gamma complex). A novel fluorescence anisotropy-based binding assay will be used to identify protein-protein and protein-DNA interactions. A variety of different sub-complexes that can be isolated from pol III will be examined, including th tau complex. ATP dependent steps will be identified. In Specific Aim 2, pre-steady-state kinetic approaches will be used to define rate-limiting processes in loading and the order of events in loading. Interactions between beta and gamma will be examined, along with protein-DNA interactions. Steps that require either ATP binding or hydrolysis will be identified. Pre-steady-state primer extension assays will be used to differentiate between productive and non-productive beta-DNA complexes. In Specific Aim 3, similar experiments will be performed using the sliding clamp protein (gp45) and clamp loader complex (gp44/62) from phage T4 in order to define mechanistic similarities and differences between the two systems.

描述：该提议的长期目标是更好地定义 DNA复制中的动态蛋白-DNA和蛋白质蛋白相互作用。 将要详细检查的系统是滑动的加载 夹具（加工性因子）通过多支亚基夹具到DNA上 复杂的。 研究人员建议从2个不同的 生物，大肠杆菌和T4。 在特定目标1中，调查人员将检查 DNA的结构如何变化（DNA引物 - 板，RNA 底漆，钝端等）影响滑动夹的负载 （beta）由夹具加载器复合物（伽马络合物）。 一种新型的荧光 基于各向异性的结合测定法将用于鉴定蛋白质蛋白质和 蛋白-DNA相互作用。 可以是各种不同的子复合物 将检查从Pol III中分离出来，包括TAU复合物。 ATP 将确定因步骤。 在特定的目标2中，预态状态 动力学方法将用于定义加载速率的过程 以及加载事件的顺序。 Beta和Gamma之间的相互作用 将研究以及蛋白-DNA相互作用。 需要的步骤 将确定AT​​P结合或水解。 稳态的状态 底漆扩展测定将用于区分生产力和 非生产性β-DNA复合物。 在特定的目标3中，类似的实验 将使用滑动夹蛋白（GP45）和夹具装载机进行 从噬菌体T4的复合物（GP44/62）以定义机械相似性 和两个系统之间的差异。