MOLECULAR INTERACTION IN T4 DNA REPLICATION COMPLEX

T4 DNA 复制复合体中的分子相互作用

• 批准号: 3276659
• 负责人: PETER H VON HIPPEL
• 金额: $ 15.39万
• 依托单位: UNIVERSITY OF OREGON
• 依托单位国家: 美国
• 财政年份: 1981
• 资助国家: 美国
• 起止时间: 1981-04-01 至 1995-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3276659
• 关键词: DNA binding protein; DNA directed DNA polymerase; DNA primase; DNA replication; atomic absorption spectrometry; bacteriophage T4; crosslink; electron microscopy; enzyme activity; enzyme complex; fluorescence spectrometry; helicase; laboratory rabbit; molecular biology; monoclonal antibody; nucleic acid structure; protein structure function; thermodynamics; tissue /cell culture; virus DNA; virus genetics; virus protein

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.

继续进行的物理生化研究的下一阶段 蛋白质和核酸的结构和功能相互作用 噬菌体T4 DNA复制系统的组件在 这项提议。以艾伯茨和诺萨尔及其联合的结果为基础 工作人员，他们已经定义了这个体外系统，以及我们自己 在前期工作中，我们将研究各个官能团的作用机制 蛋白质的子集，以及这些子集是如何组装成整合的 DNA复制复合体。这些亚群包括中心DNA聚合酶 (基因43蛋白)，聚合酶辅助蛋白复合体(基因44/62 和45种蛋白质)，以及解旋酶-底物酶复合体(基因41和61 蛋白质)，每个蛋白质也与T4单链DNA相互作用 结合蛋白(基因32)。在下一个批准期内，我们将使用 许多酶和生物物理方法来“探测”结构， 辅助蛋白复合体的组装和功能，解旋酶- 底物酶复合体，五蛋白全酶复合体(聚合酶加 辅助蛋白)，以及整合的七种蛋白系统(全酶 加上解旋酶-底物酶)。使用的方法包括紫外线激光蛋白质-DNA 交联剂、快速淬灭动力学和低温电子显微镜 与通常的物理、生化和酶技术一样。这个 聚合酶的加工性(以及保真度)将被用作 对不同组件的功能集成的度量 系统(既在DNA合成中“向前”工作，也在“向后”工作 核酸外切酶“编辑”的不断增长的引物链)。我们将继续 发展一个分子和动力学模型的各个步骤 基因的进行性单核苷酸加(或切除)循环 聚合酶，以确定这些步骤中的哪些是由 辅助蛋白复合体，并询问配对是如何发挥功能的 全酶复合体在结构上、机械上和通孔上都是集成的 解旋酶-底物酶复合体。我们希望这些研究将有助于 进一步从分子水平上了解各种蛋白质--蛋白质 以及控制这种DNA功能的蛋白质-核酸相互作用 伸长复合体，以及对复合体如何可能的进一步洞察 在各种生理状态下调节的。