BACTERIOPHAGE LAMBDA DNA REPLICATION IN VITRO

噬菌体 Lambda DNA 体外复制

• 批准号: 2838489
• 负责人: ROGER MCMACKEN
• 金额: $ 33.06万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-07-01 至 2000-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2838489
• 关键词: DNA replication; DNA replication origin; bacterial proteins; bacteriophage lambda; cell free system; conformation; nucleic acid structure; protein purification; protein structure function; radiotracer; stoichiometry; synthetic nucleic acid; transcription factor; virus genetics; virus protein; virus replication; DNA replication; DNA replication origin; bacterial proteins; bacteriophage lambda; cell free system; conformation; nucleic acid structure; protein purification; protein structure function; radiotracer; stoichiometry; synthetic nucleic acid; transcription factor; virus genetics; virus protein; virus replication

We propose to continue our studies of the replication of bacteriophage lambda DNA in an in vitro system that is reconstituted with 20 highly purified lambda and E. coli proteins. Our long range goal is to achieve a detailed mechanistic understanding of the biochemical events that occur during the initiation, propagation, termination, and regulation of lambda DNA replication. The viral O and P replication proteins promote the assembly of an ordered series of nucleoprotein structures at the lambda replication origin (ori- lambda) prior to priming and DNA chain elongation. Recent findings indicate that the first two of these structures, the O-some and the ori- lambda.O.P.DnaB complex, have the capacity to capture and hold alternate DNA conformations induced by a combination of negative DNA supercoiling and O protein binding to ori lambda. Moreover, the step leading to formation of these "pre-open" complexes appears to be a key point of regulation of lambda DNA replication. We will characterize this step further (a) by defining its dependence on superhelical tension; (b) by defining the amino acid residues of the lambda O and P proteins and E. coli DnaB helicase that interact with single stranded DNA and/or with the A/T-rich region of ori lambda; and (c) by analyzing how transcriptional events distant from orilambda facilitate the DNA melting step. Using radiolabeled proteins, we will define the stoichiometries of the O, P, and DnaB proteins in pre-initiation structures assemble at orilambda. The fate of radiolabeled O, P and DnaB following initiation will be monitored. We will continue our efforts to use x-ray crystallography to define the tertiary structure of the DNA-binding domain of the lambda O initiator. In related experiments, we will use a genetic approach to define the amino acid residues of the N-terminal domain of O involved in specific recognition of origin DNA sequences. We will also select mutant orilambda DNA replication and identify the molecular step or steps in the initiation pathway affected by individual mutations. Biochemical studies of lambda DNA replication will continue to provide important insights into the biological mechanisms used in the initiation and regulation of chromosomal DNA replication. This knowledge will help guide studies of these vital processes in more complex organisms.

我们建议继续研究噬菌体的复制 在体外系统中用20个高度重构的体外系统中的lambda DNA 纯化的lambda和大肠杆菌蛋白。我们的远距离目标是实现 对发生的生化事件的详细机械理解 在启动，传播，终止和调节期间 DNA复制。 病毒O和P复制蛋白促进了有序的组装 lambda复制起源的一系列核蛋白结构（ori- lambda）在启动和DNA链伸长之前。最新发现 表明这些结构中的前两个，O-Mos和Ori- lambda.o.p.dnab综合大楼，有能力捕获和保持替代 负DNA超螺旋的组合引起的DNA构象 O蛋白与Ori Lambda结合。而且，导致 这些“预开”配合物的形成似乎是 lambda DNA复制的调节。我们将表征这一步骤 进一步（a）定义其对超螺旋张力的依赖； （b）由 定义Lambda O和P蛋白和E的氨基酸残基。 与单链DNA相互作用的大肠杆菌DNAB解旋酶和/或与 Ori Lambda的A/T-T-Rich区域； （c）分析转录方式 远离Orilambda的事件有助于DNA熔化步骤。 使用放射标记的蛋白质，我们将定义O， P和DNAB蛋白在Orilambda聚集的生成结构中。 启动后的放射性标记的O，P和DNAB的命运将是 受监控。我们将继续努力使用X射线晶体学 定义Lambda O的DNA结合域的三级结构 发起人。在相关实验中，我们将使用一种遗传方法 定义与参与的N末端结构域的氨基酸残基 原点DNA序列的特定识别。我们还将选择突变体 Orilambda DNA复制并确定分子步骤或步骤 受单个突变影响的启动途径。 Lambda DNA复制的生化研究将继续提供 对开始中使用的生物学机制的重要见解 和调节染色体DNA复制。这些知识将有所帮助 指导对更复杂的生物中这些重要过程的研究。