Mechanistic Studies of Replication Initiation in Prokaryotes

我们研究的一个长期目标是了解潜在的分子机制 细胞DNA复制的启动。启蒙是对细胞的决定性承诺 增殖；不适当的复制可能导致遗传不稳定，DNA 损伤，以及基因拷贝数的变化。从生物医学的角度来看，启蒙是 一种易受治疗干预控制的Keystone通路 细菌感染与癌症；然而，从分子水平上理解启动 这些因素和活动不够完整，不足以推动这类努力。 本应用的重点是利用E。 Coli作为模型系统。尽管这一进程的基本框架已经到位 在超过25年的时间里，它的机械原理一直非常神秘。通过 采用创新的结构方法、新的生化分析和 分析技术，我们将回答有关如何启动的基本问题 蛋白质协作在复制起点打开气泡并沉积成环状 DNA上的解旋酶。我们将在分子细节上确定：1)DNAA是如何 在加载DNAB解旋酶之前处理复制起点ORIC，以及如何 DNAA的ATPase活性控制这种活性，2)复制的解旋酶加载器如何， DNAC，协调ATP周转和单链DNA结合，有效地将DNAB装载到DNA上 并促进解旋酶介导的DNA解离，以及3)DNAB的伴侣蛋白如何 协调从解旋酶补充到解旋酶加载的过渡，以及它们如何 调节不同的DNAB转位活动。建议的研究结果将会 是转换双工系统所涉及的主要步骤的结构和功能图 染色体区域变成一个双向复制叉子。这些发现反过来将：1) 为DNA复制领域和更广泛的ATP行动定义新的原则- 依赖的机器和开关，以及2)建立新的试剂和检测方法 推进针对启动系统的药物发现工作。

A long-term goal of our research 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 genetic instabilities, DNA damage, and changes in gene copy number. From a biomedical perspective, initiation is a keystone pathway that should be susceptible to therapeutic intervention for controlling bacterial infections and cancers; however, a molecular-level understanding of initiation factors and activities is insufficiently complete to advance such efforts. The present application focuses on the initiation of DNA replication in bacteria, using E. coli as a model system. 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 processes the replication origin, oriC, prior to loading of the DnaB helicase and how the ATPase activity of DnaA controls this activity, 2) how the replicative helicase loader, DnaC, coordinates ATP turnover and ssDNA binding to efficiently load DnaB onto DNA and promote helicase-mediated DNA unwinding, and 3) how partner proteins of DnaB coordinate the transition from helicase recruitment to helicase loading, and how they regulate different DnaB translocation activities. The outcome of the proposed studies will be a 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 broader action of ATP- dependent machines and switches, and 2) establish new reagents and assays for advancing drug-discovery efforts that target initiation systems.