DYNAMICS OF PROTEIN-DNA INTERACTIONS IN DNA REPLICATION

描述：这项提议的长期目标是更好地定义 DNA复制中蛋白质-DNA和蛋白质-蛋白质的动态相互作用。 将详细检查的系统是滑块的加载 多亚基夹持加载器对DNA的夹持(加工系数) 很复杂。调查人员建议从两个不同的角度来检查这个系统 微生物、大肠杆菌和T4。在具体目标1中，调查员将检查 DNA(DNA引物-模板，RNA)的结构变化如何 底漆-模板、钝端等)影响滑动夹具的载荷 (测试版)由夹具加载器复合体(伽马复合体)提供。一种新的荧光材料 基于各向异性的结合分析将用于鉴定蛋白质-蛋白质和 蛋白质与DNA的相互作用。各种不同的子复合体，可以是 从Pol III中分离出来的物质将被检查，包括Th tau复合体。ATP 将确定相关步骤。在具体目标2中，预稳定状态 将使用动力学方法来定义装货过程中的限速过程 以及事件在加载中的顺序。贝塔和伽马之间的相互作用 将与蛋白质-DNA相互作用一起被研究。需要采取的步骤 将确定三磷酸腺苷结合或水解。预稳定状态 将使用引物延伸分析来区分多产和 非生产性的β-DNA复合体。在具体目标3中，类似的实验 将使用滑动夹子蛋白(Gp45)和夹子加载器执行 噬菌体T4的复合体(gp44/62)，以定义机制上的相似性 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.