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.

持续的物理生化研究的下一个阶段 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 这个建议。 建立在阿尔伯特和诺萨尔及其共同的结果的基础上 已经定义了这种体外系统的工人以及我们自己 较早的工作，我们将研究每个功能的作用机理 蛋白质的子集，以及如何将这些子集组装到集成的 DNA复制复合物。 这些子集包括中央DNA聚合酶 （基因43蛋白），聚合酶辅助蛋白复合物（基因44/62 和45种蛋白质）和解旋酶 - 蛋白酶复合物（基因41和61 蛋白质），每个蛋白也与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 “探测”结构的酶促和生物物理方法的数量， 辅助蛋白复合物的组装和功能，解旋酶 - 原始酶配合物，五蛋白全酶络合物（聚合酶Plus 辅助蛋白）和集成的七蛋白系统（Holoenzyme 加上解旋酶 - 蛋白酶）。 要使用的方法包括紫外线激光蛋白-DNA 还交联，快速淬火动力学和冷冻电子显微镜， 作为通常的物理生化和酶促技术。 这 聚合酶的加工性（以及保真度）将被用作 衡量各个组件的功能整合的度量 系统（DNA合成中的“向前”和“向后”起作用 外核酸酶的“编辑器”的生长底漆链）。 我们将继续 开发单个步骤的分子和动力学模型 过程中的单核苷酸添加（或切除）周期 聚合酶，确定这些步骤中的哪个是由 辅助蛋白复合物，并询问配对的功能如何 全酶络合物在结构，机械和通过 解旋酶 - 蛋白酶复合物。 我们希望这些研究能做出贡献 要进一步理解各种蛋白质蛋白 控制该DNA功能的蛋白质核酸相互作用 伸长复合物，以及进一步的见解 在各种生理状态下受到监管。