MOLECULAR INTERACTION IN T4 DNA REPLICATION COMPLEX

在这份拨款申请中，我们描述了我们继续在体外研究噬菌体T4 DNA复制复合体的分子机制、相互作用和调控的计划。T4系统已成为高等生物复制研究的范例，因为在其七个蛋白质成分中，它包括模板导向复制DNA聚合酶（gp43），单链DNA结合蛋白（gp32），用于控制复制叉处聚合酶的三聚体滑动夹（gp45）， atp驱动的夹加载亚组件（gp44/62），以及包含在复制叉前打开DNA的六聚体T4复制解旋酶（gp41）的引物体和后链合成所需的RNA引物酶（gp61）。在下一个报告期内，我们的具体目标是：(i)完成atp驱动的gp45夹加载到引物模板DNA上并随后加载到gp43聚合酶上的详细反应周期的阐明，以及确定在复制周期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（gp41）能够在移动复制叉之前打开双链DNA并暴露模板序列的机制（该研究也对其他六聚解旋酶的功能具有一般意义）；（iii）了解原体亚组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；（iv）阐明同步和控制复制叉内的过程聚合酶、引物酶和解旋酶功能的分子偶联机制。就其对生物医学研究的长远意义而言，这些研究将作为分子模型，研究类似的DNA复制系统如何在高等生物中工作，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.