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: 3276663
• 负责人: PETER H VON HIPPEL
• 金额: $ 16万
• 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 1991-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3276663
• 关键词: DNA binding protein; adenosine triphosphate; atomic absorption spectrometry; bacteriophage T4; binding proteins; chemical group; crosslink; fluorescence spectrometry; guanosine triphosphate; molecular biology; nucleic acid structure; photochemistry; protein structure; radiation genetics; thermodynamics; tissue /cell culture; virus DNA; virus genetics; virus protein; virus replication

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.

在此提案中，我们概述了持续物理的下一个阶段 对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成分 系统。 建立在艾伯特和诺萨尔及其同事的结果的基础上 谁已经定义了这种体外系统，以及我们自己早些时候定义的 研究，我们将继续专注于逐步的“建筑” 复杂以进一步了解每个组件的功能作用 在集成系统中。 为此，我们计划继续使用新的 开发了紫外线激光交联方法来确定接触（和 亲和力）在系统的各种蛋白质与DNA之间 底漆连接，以及由DNA链移位 推进不对称（仅限链）DNA中的复制叉 合成。 我们还将继续研究组件和分子 使用酶促的复制系统功能子集的活动 和物理化学“探针”。 这些子集包括DNA聚合酶 （基因43蛋白）本身，聚合酶辅助螺旋酶复合物（基因 44/62 + 45个蛋白质）和滞后链培根酶 - 甲基酸酶复合物（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。 我们还将继续研究 聚合酶作为功能整合的度量的加工性 系统的各个组件（在DNA中都“向前”工作 合成和“向后”作为增长底漆的外切酶“编辑器” 链），并计划使用这种方法开始研究 DNA合成的保真度。 我们希望这些研究将有所帮助 至少对各种集合的部分分子理解 蛋白质蛋白质和蛋白质核酸相互作用 该DNA伸长复合物的功能以及这些相互作用的工作方式 共同形成一个耦合系统，进行领先和滞后链 DNA合成以生理速率和生理保真度。