INITIATION OF BACTERIOPHAGE DNA REPLICATION IN VITRO

噬菌体 DNA 体外复制的启动

• 批准号: 3280913
• 负责人: ROGER MCMACKEN
• 金额: $ 35.27万
• 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 1995-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3280913
• 关键词: DNA binding protein; DNA directed RNA polymerase; DNA footprinting; DNA replication; DNA replication origin; Escherichia coli; bacteriophage lambda; calorimetry; conformation; crystallization; dimer; electron microscopy; gel electrophoresis; genetic promoter element; genetic transcription; immunoelectron microscopy; intermolecular interaction; molecular dynamics; molecular site; nucleic acid sequence; nucleic acid structure; nucleoproteins; nucleosomes; plasmids; polymerase chain reaction; protein engineering; protein structure function; site directed mutagenesis; virus DNA; virus genetics; virus protein

We propose to continue our studies of the initiation of bacterlophage lambda DNA replication in a system that is reconstituted with highly purified lambda0 and E. coli proteins. Our long range goal is to achieve a detailed mechanistic understanding of the biochemical events that occur in the initiation, propagation and regulation of lambda DNA replication. We will perform mechanistic studies of a ten protein system that specifically initiates DNA replication at a lambda replication origin (ori lambda) present on a superconed plasmid template. We will attempt to establish bidirectional replication in this system. We will conduct high-resolution supercoil footprinting analysis of the various nucleoprotein prepriming structures that are formed at ori lambda prior to the initiation of localized DNA unwinding, priming and DNA chain synthesis. The effect of template superhelicity on the efficiency of initiation of lambda replication will be determined. We will examine how E. coli HU protein, a strong inhibitor of lambda DNA replication, blocks the initiation process. The molecular mechanisms involved in the transcriptional activation of lambda DNA replication will be thoroughly explored. We will mutagenize the A/T-rich region of ori lambda and select for origin defective mutants to determine which nucleotide residues play a critical role in the initiation of chromosomal DNA replication. We will continue our studies of the propagation of lambda replication forks on rolling-cycle DNA templates. In particular, we will investigate how highly processive replication forks deal with stable nucleoprotein structures that they encounter during their rapid movement along the chromosome. We will use immunoelectron microscopy to determine which replication proteins are directly associated with the replication fork on a rollingcycle template. Much of our efforts during the next project period will be devoted to studies of the lambda O initiator protein. These studies will include (a) a determination of the binding constant for interaction of O with its recognition site; (b) quantitative analysis of the individual-site binding isotherms for O binding to ori lambda, using footprinting and isothermal titration calorimetry; (c) formation and analysis of cocrystals of O protein with its DNA binding site; (d) isolation and characterization of DNA-binding mutants in O protein; (e) isolation and characterization of mutants in the O protein recognition site; and (f) purification and characterization of the carboxy-terminal domain of O protein. Biochemical studies of lambda DNA replication have provided and will undoubtedly continue to provlde important insights into the biological mechanisms used in the initiation and regulation of chromosomal DNA replication. This knowledge, furthermore, provides important guidelines for studies of these processes in more complex eukaryotic systems.

我们建议继续研究噬菌体的开始 LAMBDA DNA复制在与高度重构的系统中 纯化的lambda0和大肠杆菌蛋白。 我们的远程目标是 对生化事件有详细的机械理解 这发生在lambda DNA的启动，传播和调节中 复制。 我们将对十个蛋白质系统进行机械研究 专门在lambda复制来源启动DNA复制 （ori lambda）存在于超融合的质粒模板上。 我们将尝试 在此系统中建立双向复制。 我们将进行 高分辨率的超级围足迹分析各种 核蛋白蛋白培养结构，在ori lambda之前形成 启动局部DNA，启动和DNA链 合成。 模板超智能对效率的影响 将确定lambda复制的启动。 我们将检查 大肠杆菌Hu蛋白如何，强烈的lambda DNA复制抑制剂， 阻止启动过程。 涉及的分子机制 Lambda DNA复制的转录激活将彻底进行 探索。 我们将诱变Ori Lambda的A/T丰富区域， 选择原点缺陷突变体以确定哪种核苷酸 残留物在染色体DNA的启动中起关键作用 复制。 我们将继续研究Lambda的传播 滚动周期DNA模板上的复制叉。 特别是我们 将调查高度处理的复制叉如何处理 它们在快速时遇到的稳定核蛋白结构 沿着染色体运动。 我们将使用免疫电子显微镜 确定哪种复制蛋白与 滚动模板上的复制叉。 我们在下一个项目期间的大部分努力将致力于 Lambda O引发剂蛋白的研究。 这些研究将包括 （a）确定O与O相互作用的结合常数 其认可地点； （b）对个体位置的定量分析 使用足迹和 等温滴定量热法； （c）形成和分析 O蛋白与其DNA结合位点的共晶； （d）隔离和 O蛋白中DNA结合突变体的表征； （e）隔离和 O蛋白识别位点突变体的表征； （f） O的纯化和表征O 蛋白质。 Lambda DNA复制的生化研究已提供，并且将 毫无疑问，继续为生物学的重要见解提供重要的见解 染色体DNA的启动和调节中使用的机制 复制。 此外，这些知识提供了重要的指南 用于对更复杂的真核系统中这些过程的研究。