Mechanistic Studies on DNA Packaging by Bacteriophage Lambda

噬菌体 Lambda 包装 DNA 的机理研究

• 批准号: 9018767
• 负责人: Carlos Catalano
• 金额: $ 19万
• 依托单位: University of Colorado at Boulder
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-02-15 至 1993-02-15
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9018767&HistoricalAwards=false
• 关键词: Mechanistic; Studies; DNA; Packaging; Bacteriophage

The objective of this proposal is to examine at the molecular level the mechanism of assembly of viral precursors into an infectious virus particle. A common mechanism for DNA packaging by the phage viruses has been proposed and consists of 1) binding of an enzyme called terminase to concatemeric (immature) viral DNA, 2) binding of this complex to a pre- formed capsid, 3) unidirectional of DNA into the capsid in an ATP-dependent reaction and4) nicking of the duplex and strand separation such that a single (mature) genome is packaged. Phage terminase is now obtainable in high yields and represents an ideal system in which to study viral DNA packaging. The enzyme possesses DNA-dependent ATPase, DNA translocase, restriction endonuclease and DNA helicase activities all manifest within a heterodimeric protein structure. Thermodynamic binding constants for ATP and DNA will be determined using equilibrium dialysis, gel-retardation and DNase footprinting techniques. The interactions between enzyme and ATP as well as enzyme and viral DNA will be examined using photoaffinity labeling techniques. Covalently modified residues will be identified by proteolytic digestion of the protein, isolation of the labeled peptide(s) and N-terminal amino sequencing analysis. A complete kinetic analysis of the endonuclease and helicase activities will be performed and a minimal kinetic mechanism defined using steady state and pre- steady state kinetic techniques. The experiments outlined in this proposal will allow a detailed characterization of phage terminase and serve as a basis for comparative studies with terminases from other phages Examination of these enzymes may reveal evolutionary relationships not only between terminases, but also other functionally related enzymes such as ATPases, restriction enzymes and helicases. Moreover, DNA packaging, the procaryotic version of chromosomal condensation, may yield insight into the basis mechanisms and energetics of cellular chromosome manipulation.

本文的目的是在分子水平上研究病毒前体组装成感染性病毒颗粒的机制。人们提出了噬菌体病毒包装DNA的一种常见机制，包括：1)将一种被称为终止酶的酶与未成熟的病毒DNA结合；2)将这种复合物与预先形成的衣壳结合；3) DNA在atp依赖反应中单向进入衣壳，4)双链和链分离的切口，这样一个单一的（成熟的）基因组被包装。噬菌体终止酶现在可以高产地获得，是研究病毒DNA包装的理想系统。该酶具有DNA依赖的atp酶、DNA转位酶、限制性内切酶和DNA解旋酶活性，这些活性都在异二聚体蛋白结构中表现出来。ATP和DNA的热力学结合常数将使用平衡透析，凝胶阻滞和DNA酶足迹技术来确定。酶和ATP以及酶和病毒DNA之间的相互作用将使用光亲和标记技术进行检测。共价修饰残基将通过蛋白质水解酶切、分离标记肽段和n端氨基测序分析来鉴定。我们将对核酸内切酶和解旋酶的活性进行完整的动力学分析，并利用稳态和准稳态动力学技术确定最小的动力学机制。本提案中概述的实验将允许对噬菌体末端酶进行详细的表征，并作为与其他噬菌体末端酶进行比较研究的基础。这些酶的检查可能揭示不仅是末端酶之间的进化关系，而且还有其他功能相关的酶，如atp酶，限制性内切酶和解旋酶。此外，DNA包装，染色体凝聚的原核版本，可能产生洞察基本机制和细胞染色体操作的能量学。