MOLECULAR INTERACTION IN T4 DNA REPLICATION COMPLEX

在这个建议中，我们概述了详细的物理化学研究的结构 以及蛋白质的各种蛋白质和核酸组分之间的相互作用。 噬菌体T4 DNA复制复合物。 基于Alberts的结果 以及Nossal和他们的同事，他们也定义了这种体外系统， 正如我们自己早期的研究一样，我们将使用化学和光化学方法， 交联实验以确定结构和拓扑关系 并定位蛋白质-蛋白质和蛋白质-核酸接触。 研究 各种成分之间的结合平衡，以及这些成分之间的依赖性 盐浓度、pH值和其他环境变量的结合常数 将进行，以确定性质和自由能的贡献， 复合物的形成和各种类型官能团的稳定性 交互. DNA合成和复制叉运动的速率，以及 通过各种部分催化合成的持续合成能力和保真度， 复制系统将被检查，以试图理解，在一个 分子基础上，每个蛋白质在耦合（领先和落后）中发挥的作用 链）DNA延伸阶段的整合复制过程中，和 蛋白质-蛋白质和蛋白质-核酸相互作用机制参与了 整个系统的局部反应。

In this proposal we outline a detailed physico-chemical study of the structure and interactions of the various protein and nucleic acid components of the bacteriophage T4 DNA replication complex. Building on the results of Alberts and Nossal and their coworkers, who have defined this in vitro system, as well as on our own earlier studies, we will use chemical and photochemical cross-linking experiments to determine structural and topological relationships and to localize protein-protein and protein-nucleic acid contacts. Studies of binding equilibria between various components, and the dependence of these binding constants on salt concentration, pH and other environmental variables will be carried out to determine the nature and the free energy contributions to complex formation and stability of various types of functional group interactions. Rates of DNA synthesis and replication fork movement, as well as the processivity and fidelity of the synthesis catalyzed by various partial replication systems will be examined in order to attempt to understand, on a molecular basis, the role each protein plays in the coupled (leading and lagging strand) DNA elongation phase of the integrated replication process, and the protein-protein and protein-nucleic acid interaction mechanisms involved in the partial reactions of the overall system.