MOLECULAR INTERACTION IN T4 DNA REPLICATION COMPLEX

在这份提案中，我们概述了持续体检的下一个阶段 肌动蛋白的结构和功能相互作用的生化研究 噬菌体T4 DNA复制的蛋白质和核酸组成 系统。基于艾伯茨和诺萨尔及其同事的研究结果， 是谁定义了这个体外系统，也是我们之前定义的 研究，我们将继续专注于循序渐进地“建立” 复杂以进一步了解每个组件的功能角色 在集成系统中。为此，我们计划继续使用我们的新产品 开发了紫外光激光交联法以确定接触点(和 亲和力)在系统的各种蛋白质和DNA之间 引物-模板连接，以及DNA链被 在不对称(仅前导链)DNA中推进复制分叉 综合。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“探头”。这些亚群包括DNA聚合酶 (基因43蛋白)本身，聚合酶附件-解旋酶复合体(基因 44/6245个蛋白质)和滞后链启动子-解旋酶复合体(基因 41和61蛋白)，每个蛋白也与T4单链相互作用 DNA结合(基因32)蛋白。我们亦会继续研究 聚合酶的加工性作为功能整合的衡量标准 系统的不同组件(在DNA中工作时既“向前” 合成与“反向”作为核酸外切酶“编辑”的成长中的引子 Strand)，并计划使用这种方法来开始研究 DNA合成的保真度也很高。我们希望这些研究将有助于 提供至少部分分子上的理解，了解不同的 蛋白质-蛋白质和蛋白质-核酸相互作用控制 这种DNA延长复合体的功能，以及这些相互作用是如何工作的 一起形成一个耦合的系统，引导领先和滞后的链 以生理速率和生理保真度合成DNA。

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.