Mechanistic Studies of Replication Initiation in Prokaryotes

原核生物复制起始的机制研究

基本信息

批准号：
10189628
负责人：
JAMES M BERGER
金额：
$ 44.49万
依托单位：
JOHNS HOPKINS UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2005
资助国家：
美国
起止时间：
2005-05-01 至 2022-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10189628
关键词：
ATP phosphohydrolase ATPase Domain Accounting Address Affect Anti-Bacterial Agents Award Bacteria Bacterial Infections Binding Biochemical Biochemistry Biological Assay Biological Models Bypass C-terminal Cell Proliferation Cell Survival Cells Closure by clamp Complex Coupled DNA DNA Damage DNA Primase DNA biosynthesis DNA replication fork DNA-Directed RNA Polymerase Data Defect Deposition DnaB helicase Elements Escherichia coli Eukaryota Gene Dosage Genetic Goals Homo Investigation Lead Malignant Neoplasms Mediating Methods Molecular Motor N-terminal Nucleoproteins Organism Outcome Pathway interactions Peptide Initiation Factors Positioning Attribute Process Prokaryotic Cells Proteins Reagent Regulation Replication Initiation Replication Origin Replication-Associated Process Research Single-Stranded DNA Stretching Structure Substrate Interaction System Technology Therapeutic Intervention Time Work drug discovery ds-DNA helicase innovation melting metaplastic cell transformation molecular modeling operation prevent recruit single molecule structural biology tau Proteins translocase

项目摘要

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.

我们研究的长期目标是了解潜在的分子机制细胞DNA复制的起始。启动是对细胞的明确承诺增殖;不适当的复制起始可能导致遗传不稳定、DNA 损伤和基因拷贝数的变化。从生物医学的角度来看，启动是一个关键途径，应该容易受到治疗干预的控制细菌感染和癌症；然而，对引发的分子水平理解推动此类努力的因素和活动还不够完善。本申请的重点是使用大肠杆菌启动细菌中的 DNA 复制。大肠杆菌作为模型系统。尽管这个过程的基本框架已经到位 25 年多来，其机械原理一直非常神秘。经过采用结构方法、新生化分析的创新组合，分析技术，我们将回答涉及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环形解旋酶到 DNA 上。我们将确定分子细节：1) DnaA 如何在加载 DnaB 解旋酶之前处理复制起点 oriC，以及如何 DnaA 的 ATP 酶活性控制该活性，2) 复制解旋酶装载机如何， DnaC，协调 ATP 周转和 ssDNA 结合，以有效地将 DnaB 加载到 DNA 上并促进解旋酶介导的 DNA 解旋，以及 3) DnaB 的伴侣蛋白如何协调从解旋酶招募到解旋酶加载的过渡，以及它们如何调节不同的 DnaB 易位活动。拟议研究的结果将是转换复式公寓所涉及的主要步骤的结构和功能图染色体区域形成双向复制叉。这些发现反过来将：1) 为 DNA 复制领域和 ATP 更广泛的作用定义新原理相关机器和开关，以及 2) 建立新的试剂和测定方法推进针对引发系统的药物发现工作。