Mechanisms of Action of Pre-Replicative Complexes

预复制复合物的作用机制

• 批准号: 7082206
• 负责人: ROGER MCMACKEN
• 金额: $ 29.94万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-07-01 至 2009-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7082206
• 关键词: DNA replication; DNA replication origin; Escherichia coli; X ray crystallography; bacteriophage lambda; genetic transcription; helicase; intermolecular interaction; nucleoproteins; protein binding; virus assembly; virus protein; virus replication

DESCRIPTION (provided by applicant): The long range goal of the proposed studies is to define the fundamental molecular mechanisms that underpin initiation and regulation of bacteriophage ? DNA replication in a model in vitro system composed of 20 or more highly purified ? and E. coli replication proteins. The proposed studies are expected to provide insights that will guide studies of the replication of chromosomes of more complex organisms, including all eubacteria and many human DNA tumor viruses. The ? O and P replication initiators promote the assembly of a pre-replicative complex (pre-RC) at the phage replication origin (ori?) that contains the 2 initiators and the E. coli DnaB replicative helicase. A critical stage in the initiation pathway is the conversion of this functionally inert nucleoprotein complex into an "activated pre-RC" upon negative supercoiling of the DNA template. We will use a variety of biochemical approaches, including the use of photoaffinity protein-DNA crosslinking or mutant initiator proteins, to define the nature of the protein-DNA interactions in this key replication intermediate. We will seek to confirm our hypothesis that cryptic single-stranded-DNA (ssDNA) binding activities associated with the ?, O and P initiator proteins are responsible for stabilizing a conformational variant of the DNA duplex at ori?, to activate the DNA for loading of DnaB helicase. Related approaches will be used to precisely define the pathway by which a ring-shaped DnaB hexamer is transferred onto the ? chromosome at the Arich region of ori?, during nucleoprotein remodeling catalyzed by E. coli molecular chaperones. We will pursue crystallographic studies to determine the three-dimensional (3D) structure of the DNA-binding domain of O, bound to its recognition site. A related aim is to elucidate the structure of the ? pre-RC assembled on forked ori? DNA templates or on origin DNA that contains small bubbles or mismatches. Biochemical and genetic studies will be performed to define the ssDNA-binding motifs in ? P and the C-terminal domain of O. We will determine the molecular mechanism by which transcription of the DNA template in cis activates ? DNA replication.

描述（由申请人提供）：拟议的研究的远距离目标是定义基于噬菌体开始和调节的基本分子机制？ DNA在体外系统模型中复制，由20个或更高纯化组成？和大肠杆菌复制蛋白。拟议的研究预计将提供见解，以指导研究更复杂生物体的染色体的复制研究，包​​括所有Eubacteria和许多人类DNA肿瘤病毒。这 ？ O和P复制启动者在包含2个启动器和大肠杆菌DNAB复制酶的噬菌体复制起源（ORI？）处促进了复制前复合物（PRE-RC）的组装。起始途径中的关键阶段是该功能惰性核蛋白复合物转化为DNA模板负面涂层的“激活的PER-RC”。我们将使用多种生化方法，包括使用光接触蛋白-DNA交联或突变引发剂蛋白来定义此键复制中间体中蛋白-DNA相互作用的性质。我们将寻求确认我们的假设，即与？，O和P引发剂蛋白相关的隐性单链DNA（SSDNA）结合活性负责稳定ORI的DNA双链体的构象变体，以激活DNA的DNA，以载入DNAB旋转酶的载荷。相关方法将用于精确定义环形DNAB六角形的途径？在大肠杆菌分子伴侣催化的核蛋白重塑期间，Ori？Arich区域的染色体。我们将进行晶体学研究，以确定与其识别位点结合的O的DNA结合域的三维（3D）结构。相关的目的是阐明？ RC预先组装在分叉的ORI上？ DNA模板或包含小气泡或不匹配的原点DNA。将进行生化和遗传研究以定义ssDNA结合基序中的基因序。 P和O的C末端结构域。我们将确定CIS中DNA模板转录的分子机制？ DNA复制。