DYNAMICS OF PROTEIN-DNA INTERACTIONS IN DNA REPLICATION

说明：本提案的长期目标是更好地定义 DNA复制中的动态蛋白质-DNA和蛋白质-蛋白质相互作用。 将被详细检查的系统是滑动的加载 通过多亚基夹钳加载器将夹钳（持续合成因子）夹在DNA上 复杂. 研究者建议从2个不同的角度检查该系统 organisms，E. coli和T4. 在具体目标1中，研究人员将检查 如何改变DNA的结构（DNA引物模板，RNA 引物-模板、平端等）影响滑动夹的加载 （beta）由夹钳加载器复合体（gamma复合体）。 一种新的荧光 基于各向异性的结合测定将用于鉴定蛋白质-蛋白质， 蛋白质-DNA相互作用 各种不同的子复合体， 将检查从pol III分离的，包括tau复合物。 ATP 将确定相关步骤。 在特定目标2中，预稳态 动力学方法将被用来定义速率限制过程中加载 以及加载中事件的顺序。 beta和gamma之间的相互作用 将被检查，沿着与蛋白质-DNA相互作用。 需要的步骤 将鉴定ATP结合或水解。 准稳态 引物延伸分析将用于区分生产性和 非生产性β-DNA复合物。 在具体目标3中，类似的实验 将使用滑动夹蛋白（gp 45）和夹加载器进行 复合物（gp 44/62），以确定机制的相似性 以及两个系统之间的差异。

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.