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: 6097375
• 负责人: PETER H VON HIPPEL
• 金额: $ 28.82万
• 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 2004-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6097375
• 关键词: DNA binding protein; DNA directed DNA polymerase; DNA primase; DNA replication; DNA replication origin; adenosinetriphosphatase; bacteriophage T4; enzyme complex; enzyme mechanism; genetic models; helicase; immunologic assay /test; intermolecular interaction; laboratory mouse; laboratory rabbit; protein structure function; thermodynamics; virus DNA; virus genetics; virus protein; virus replication

In this grant application we describe our plans to continue our in vitro studies on the molecular mechanisms, interactions, and regulation of the components of the DNA replication complex of bacteriophage T4. The T4 system has served as a paradigm for replication studies in higher organisms because, within its seven protein components, it includes a template-directed replication DNA polymerase (gp43), a single-stranded DNA binding protein (gp32), a trimeric sliding clamp (gp45) for controlling the processivity of the polymerases at the replication fork, an ATP-driven clamp-loading subassembly (gp44/62), and a primosome that contains the hexameric T4 replication helicase (gp41) that opens the DNA ahead of the replication fork and the primase (gp61) required for the RNA priming of lagging strand synthesis. During the next reporting period our Specific Aims are: (i) to complete the elucidation of the detailed reaction cycle involved in the ATP-driven loading of the gp45 clamp onto the primer- template DNA and subsequently onto the gp43 polymerase, as well as to determine how the stability of the loaded sliding clamp is controlled at various stages of the replication cycle; (ii) to continue our studies of the mechanisms whereby the T4 helicase (gp41) is able to open double-stranded DNA and expose the template sequences ahead of the moving replication fork (this study has general implications for the function of other hexameric helicases as well); (iii) to understand how the molecular activities and interactions within the primosome subassembly are coordinated with leading- and lagging-strand DNA synthesis; and (iv) to elucidate the molecular coupling mechanisms that synchronize and control the functions of the processive polymerases, primase, and helicase within the replication fork. In terms of their longer range significance for biomedical research, these studies will serve as molecular models for how the analogous DNA replication systems work in higher organisms and how the control of these systems may go awry in cancer and other diseases of inappropriate DNA replication and repair.

在此赠款应用中，我们描述了我们的计划，以继续我们对噬菌体T4的DNA复制复合物的分子机制，相互作用和调控的体外研究。 T4系统已成为较高生物体复制研究的范式，因为在其七个蛋白质成分中，它包括一个模板定向的复制DNA聚合酶（GP43），一种单链DNA结合蛋白（GP32）（GP32），一种三聚合物夹（GP45），用于控制Polimistation and promimymerase and promimymymersed inp inp inp inp inp inp artper inp inp artper inp inp inp inp inp art inp inp ark inp ark inp ark ark ark inp ark ark ark ark ark ark ark ark ark ark ark ark ark ark in夹具加载子组件（GP44/62）和一个含有六聚体T4复制解旋酶（GP41）的原始体，该六型六角叉和初次叉（GP61）在滞后链链合成所需的RNA启动所需的DNA之前打开DNA。在下一个报告期内，我们的具体目的是：（i）完成对ATP驱动的GP45夹在引物DNA上以及随后在GP43聚合酶上的详细反应周期的阐明，以确定如何在各种阶段的稳定下控制载荷滑动板的稳定性； （ii）继续我们对T4解旋酶（GP41）能够打开双链DNA并在移动复制叉之前暴露模板序列的机制的研究（这项研究对其他甲式化剂解放液的功能也具有一般影响）； （iii）了解原始体子组装中的分子活性和相互作用如何与前链和链链DNA合成协调； （iv）阐明了复制叉中同步和控制过程聚合酶，原始酶和解旋酶的功能的分子耦合机制。 就其对生物医学研究的较长范围而言，这些研究将作为分子模型，用于类似的DNA复制系统如何在较高的生物体中起作用，以及这些系统的控制如何在癌症和其他不适当DNA复制和修复的疾病中产生问题。