MOLECULAR INTERACTION IN T4 DNA REPLICATION COMPLEX

在本提案中，我们概述了持续体检的下一阶段 生物化学研究的结构和功能的相互作用， 噬菌体T4 DNA复制的蛋白质和核酸成分 系统 基于Alberts和Nossal及其同事的研究结果， 他们已经定义了这个体外系统，以及我们自己的早期系统， 我们将继续集中精力，逐步“建立” 复杂，以进一步了解每个组件的功能作用 在综合系统中。 为此，我们计划继续使用我们的新 开发了紫外激光交联方法，以确定接触（和 系统的各种蛋白质和系统的DNA之间的亲和力）。 引物-模板连接处，以及由引物-模板连接处置换的DNA链。 不对称（仅前导链）DNA中的前进复制叉 合成. 我们还将继续研究组装和分子 复制系统的功能子集的活性，使用酶 和物理化学“探针”。 这些子集包括DNA聚合酶 (gene 43蛋白）本身，聚合酶附件-解旋酶复合物（基因 44/62 + 45蛋白）和滞后链引物酶-解旋酶复合物（基因 41和61蛋白），它们中的每一个也与T4单链 DNA结合（基因32）蛋白。 我们亦会继续研究 聚合酶的持续合成能力作为功能整合的量度 系统的各个组成部分（在DNA中“向前”工作 合成和“反向”作为外切核酸酶“编辑器”的生长引物 股），并计划使用这一办法开始研究 DNA合成的保真度。 我们希望这些研究将有助于 提供了至少部分的分子理解的各种集合， 蛋白质-蛋白质和蛋白质-核酸的相互作用， 这种DNA延伸复合物的功能，以及这些相互作用如何起作用， 一起形成一个耦合系统， 以生理速率和生理稳定性进行DNA合成。

In this proposal we outline the next stages of a continuing physical biochemical study of the structural and functional interactions of the protein and nucleic acid components of the bacteriophage T4 DNA replication system. 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 continue to focus on a step-wise "building-up" of the complex to gain further insight into the functional role of each component in the integrated system. To this end we plan to continue to use our newly developed UV laser cross-linking methodology to determine contacts (and affinities) between the various proteins of the system and the DNA of the primer-template junction, as well as the DNA strand displaced by the advancing replication fork in asymmetric (leading-strand-only) DNA synthesis. We will also continue to study the assembly and molecular activities of functional subsets of the replication system, using enzymatic and physical chemical "probes". These subsets include the DNA polymerase (gene 43 protein) itself, the polymerase accessory-helicase complex (genes 44/62 + 45 proteins) and the lagging strand primase-helicase complex (genes 41 and 61 protein), each of which also interacts with the T4 single-strand DNA binding (gene 32) protein. We will also continue to study the processivity of the polymerase as a measure of the functional integration of the various components of the system (working both "forward" in DNA synthesis and "backward" as an exonuclease "editor" of the growing primer strand), and plan to use this approach to begin to study the issue of the fidelity of DNA synthesis as well. We hope that these studies will help to provide at least a partial molecular understanding of the various sets of protein-protein and protein-nucleic acid interactions that control the function of this DNA elongation complex, and of how these interactions work together to form a coupled system that conduct leading- and lagging-strand DNA synthesis at physiological rates and with physiological fidelities.