MOLECULAR INTERACTION IN T4 DNA REPLICATION COMPLEX

下一个阶段的持续物理生化研究， 蛋白质和核酸的结构和功能相互作用 噬菌体T4 DNA复制系统的组成部分概述于 这个提议。 基于Alberts和Nossal及其合作者的结果， 工作者，他们定义了这个体外系统，以及我们自己 前期工作中，我们将研究每个功能的作用机制 蛋白质的子集，以及这些子集如何组装成整合的蛋白质。 DNA复制复合体。 这些子集包括中央DNA聚合酶 (gene 43蛋白），聚合酶辅助蛋白复合物（基因44/62 和45蛋白质）和解旋酶-引发酶复合物（基因41和61 蛋白质），其中每一个也与T4单链DNA相互作用 结合（基因32）蛋白。 在下一个授予期间，我们将使用 许多酶和生物物理方法来“探测”结构， 辅助蛋白质复合物、解旋酶- 引发酶复合物，五蛋白全酶复合物（聚合酶加 辅助蛋白）和整合的七蛋白系统（全酶 加解旋酶-引发酶）。 使用的方法包括UV激光蛋白质-DNA 交联，快速淬火动力学和低温电子显微镜，以及 如通常的物理生化和酶促技术。 的 聚合酶的持续合成能力（以及保真度）将被用作 的各个组成部分的功能整合的措施， 系统（在DNA合成中既“向前”工作，又“向后”工作， 生长引物链的外切核酸酶“编辑器”）。 我们将继续 发展的分子和动力学模型的各个步骤， 进行性单核苷酸添加（或切除）循环 聚合酶，以确定这些步骤中的哪一个是由 辅助蛋白复合物，并询问如何配对的功能， 全酶复合物在结构上、机械上和通过 解旋酶引发酶复合物。 我们希望这些研究将有助于 对各种蛋白质-蛋白质 以及控制DNA功能的蛋白质-核酸相互作用 延伸复合体，以及进一步了解如何复杂可能是 在各种生理状态下调节。

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 are outlined in this proposal. Building on the results of Alberts and Nossal and their co- workers, who have defined this in vitro system, as well as on our own earlier work, we will study the mechanism of action of each functional subset of proteins, and how these subsets are assembled into the integrated DNA replication complex. These subsets include the central DNA polymerase (gene 43 protein), the Polymerase accessory proteins complex (genes 44/62 and 45 proteins), and the helicase-primase complex (genes 41 and 61 proteins), each of which also interacts with the T4 single-stranded DNA binding (gene 32) protein. During the next granting period we will use a number of enzymatic and biophysical methods to "probe" the structure, assembly, and function of the accessory proteins complex, the helicase- primase complex, the five-protein holoenzyme complex (polymerase plus accessory proteins), and the integrated seven-protein system (holoenzyme plus helicase-primase). Methods to be used include UV laser protein-DNA crosslinking, rapid-quench kinetics, and cryoelectron microscopy, as well as the usual physical biochemical and enzymatic techniques. The processivity (as well as the fidelity) of the polymerase will be used 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). We will continue to develop a molecular and a kinetic model of the individual steps of the processive single-nucleotide addition (or excision) cycle of the polymerase, to determine which of these steps are regulated by the accessory proteins complex, and to ask how the functions of paired holoenzyme complexes are integrated structurally, mechanistically, and via the helicase-primase complex. We hope that these studies will contribute to a further molecular understanding of the various sets of protein-protein and protein-nucleic acid interactions that control the function of this DNA elongation complex, as well as further insights into how the complex may be regulated in various physiological states.