MOLECULAR INTERACTION IN T4 DNA REPLICATION COMPLEX

在这项拨款申请中，我们描述了我们继续对T4噬菌体DNA复制复合体的分子机制、相互作用和调控进行体外研究的计划。T4系统已经成为高等生物复制研究的范例，因为在它的七个蛋白质组分中，它包括一个模板导向的复制DNA聚合酶(Gp43)，一个单链DNA结合蛋白(Gp32)，一个用于控制复制叉处聚合酶加工能力的三聚体滑动夹(Gp45)，一个由ATP驱动的钳制加载组件(gp44/62)，以及一个含有六角体T4复制解旋酶(Gp41)的初体，它打开复制叉前的DNA，以及启动滞后和合成的RNA所需的引物酶(Gp61)。在下一个报告期间，我们的具体目标是：(I)完成在ATP驱动下将gp45夹子加载到引物-模板DNA上并随后加载到gp43聚合酶中所涉及的详细反应周期的阐明，以及确定加载的滑动夹子在复制周期的不同阶段如何控制稳定性；(Ii)继续我们对T4解旋酶(Gp41)能够打开双链DNA并在复制叉子移动之前暴露模板序列的机制的研究(这项研究也对其他六角螺旋酶的功能具有普遍意义)；(Iii)了解原始体组件内的分子活性和相互作用如何与先导和滞后链DNA合成相协调；以及(Iv)阐明同步和控制复制叉内的前向聚合酶、启动酶和解旋酶功能的分子耦合机制。就其对生物医学研究的更长期意义而言，这些研究将作为分子模型，研究类似的DNA复制系统在高等生物体中如何工作，以及在癌症和其他不适当的DNA复制和修复疾病中，这些系统的控制可能会出现问题。

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.