Structure/Function Studies of DNA Replication Initiation

描述（由申请人提供）： 项目摘要：我的研究计划的一个长期目标是了解细胞DNA复制启动的分子机制。起始是细胞增殖的决定性承诺;不适当的复制起始可导致基因拷贝数的变化、DNA损伤和遗传不稳定性。从生物医学的角度来看，启动是一个关键的途径，应该容易受到控制细菌感染和癌症的治疗干预;然而，我们对启动过程的理解是不够完整的，以推进这样的努力。本申请集中于细菌中DNA复制的起始。虽然这一进程的基本框架已经存在了25年多，但其机械原理仍然非常神秘。通过采用创新的结构方法，新的生化检测和分析技术的组合，我们将回答基本问题，涉及起始蛋白如何合作，以打开一个复制起点和存款环形解旋酶的DNA泡沫。我们将确定分子的细节：1）如何DnaA引发剂熔化DNA和新生的单链DNA随后是否调节DnaA活性，2）是否六聚体DnaB解旋酶自动调节其DNA解旋活性，和3）如何DnaC解旋酶加载器，连同DnaA和DnaG引发酶，系统地合作，在牧羊的解旋酶到DNA上。拟议的研究的结果将是一个逐步的结构和功能的图片涉及的主要步骤，在转换成双向复制叉的双链体染色体区域。这些调查结果反过来将：1）为DNA复制领域和ATP依赖性机器和开关的广泛作用定义新的原理，2）建立新的测定和模型，以推进靶向起始系统的药物发现工作。

DESCRIPTION (provided by applicant): Project Summary: A long-term goal of my research program is to understand the molecular mechanisms underlying the initiation of cellular DNA replication. Initiation is a defining commitment to cell proliferation; inappropriate onset of replication can lead to changes in gene copy number, DNA damage, and genetic instabilities. From a biomedical perspective, initiation is a keystone pathway that should be susceptible to therapeutic intervention for controlling bacterial infections and cancers; however, our understanding of initiation processes is insufficiently complete to advance such efforts. The present application focuses on the initiation of DNA replication in bacteria. Although a basic framework for this process has been in place for more than 25 years, its mechanistic principles have remained highly enigmatic. By employing an innovative mix of structural methods, new biochemical assays, and analytic technologies, we will answer fundamental questions involving how initiation proteins collaborate to open a bubble in a replication origin and deposit ring-shaped helicases onto the DNA. We will determine in molecular detail: 1) how the DnaA initiator melts DNA and whether nascent single DNA strands subsequently regulate DnaA activity, 2) whether the hexameric DnaB helicase autoregulates its DNA unwinding activity, and 3) how the DnaC helicase loader, together with DnaA and the DnaG primase, systematically cooperate in shepherding the helicase onto DNA. The outcome of the proposed studies will be a stepwise structural and functional picture of the major steps involved in converting a duplex chromosomal region into a bidirectional replication fork. These findings in turn will: 1) define new principles for both the field of DNA replication and the broadr action of ATP-dependent machines and switches, and 2) establish new assays and models for advancing drug-discovery efforts that target initiation systems.