MOLECULAR INTERACTION IN T4 DNA REPLICATION COMPLEX

在这份提案中，我们概述了对该结构的详细的物理化学研究 以及各种蛋白质和核酸成分的相互作用 噬菌体T4 DNA复制复合体。以艾伯茨的结果为基础 以及诺萨尔和他们的同事，他们也定义了这个体外系统 就像我们以前的研究一样，我们将使用化学和光化学 确定结构和拓扑关系的交联实验 以及定位蛋白质与蛋白质和蛋白质与核酸的接触。的研究 不同组件之间的结合平衡，以及这些组件之间的依赖 盐浓度、pH和其他环境变量的结合常数 将进行计算以确定其性质和自由能贡献 不同类型官能团的络合形成及稳定性 互动。DNA合成和复制分叉移动的速度以及 不同部分催化合成的加工性和保真度 我们将对复制系统进行检查，以尝试了解 分子基础，每种蛋白质在偶联(领先和滞后)中所起的作用 链)DNA延伸阶段的整合复制过程，以及 蛋白质-蛋白质和蛋白质-核酸相互作用机制参与 整个系统的部分反应。

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.