MOLECULAR INTERACTION IN T4 DNA REPLICATION COMPLEX

在此资助申请中，我们描述了我们的计划，继续我们的体外研究的分子机制，相互作用和调节的组成部分的DNA复制复合物的噬菌体T4。 T4系统已经作为高等生物体中复制研究的范例，因为在其七种蛋白质组分中，它包括模板指导的复制DNA聚合酶（gp 43）、单链DNA结合蛋白（gp 32）、用于控制聚合酶在复制叉处的持续合成能力的三聚体滑动夹（gp 45）、ATP驱动的夹加载子组件（gp 44/62）、以及含有在复制叉之前打开DNA的六聚体T4复制解旋酶（gp 41）和滞后链合成的RNA引发所需的引发酶（gp 61）的引发体。在下一个报告期间，我们的具体目标是：（i）完成对涉及ATP驱动的gp 45夹加载到引物-模板DNA上并随后加载到gp 43聚合酶上的详细反应循环的阐明，以及确定在复制循环的各个阶段如何控制加载的滑动夹的稳定性;（ii）继续研究T4解旋酶（gp 41）能够打开双链DNA并在移动的复制叉之前暴露模板序列的机制（这项研究对其他六聚体解旋酶的功能也有普遍意义）;（iii）了解原小体子组件内的分子活动和相互作用如何与前导链和滞后链DNA合成协调;和（iv）阐明同步和控制进行性聚合酶，引发酶，和解旋酶。 就其对生物医学研究的长期意义而言，这些研究将作为类似DNA复制系统如何在高等生物中工作的分子模型，以及这些系统的控制如何在癌症和其他不适当的DNA复制和修复疾病中出错。

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.