Mechanistic Studies on DNA Packaging by Bacteriophage Lambda

本提案的目的是在分子水平上检查 使病毒前体组装成 传染性病毒颗粒 DNA的共同机制 已经提出用噬菌体病毒包装， 1）一种称为末端酶的酶与多联体的结合 （未成熟）病毒DNA，2）该复合物与前- 形成的衣壳，3）单向的DNA进入衣壳在一个 ATP依赖性反应和4）双链体和链的切口 分离使得单个（成熟）基因组被包装。 噬菌体终止酶现在可以高产率获得，并代表 这是研究病毒DNA包装的理想系统。 的 酶具有DNA依赖性ATP酶、DNA转位酶、 限制性内切酶和DNA解旋酶活性全部 表现在异二聚体蛋白质结构中。 ATP和DNA的热力学结合常数为： 使用平衡透析、凝胶阻滞和 DNA酶足迹技术。 酶之间的相互作用 和ATP以及酶和病毒DNA将使用 光亲和标记技术。 共价修饰 残基将通过蛋白水解消化 蛋白质，标记肽的分离和N-末端 氨基酸序列分析。 一个完整的动力学分析， 将进行核酸内切酶和解旋酶活性， 最小动力学机制定义使用稳态和预 稳态动力学技术。 中概述的实验 该提议将允许噬菌体的详细表征 终止并作为比较研究的基础， 对这些酶的检测可以 不仅揭示了末端之间的进化关系， 还包括其它功能相关的酶如ATP酶， 限制酶和解旋酶。 此外，DNA包装， 染色体浓缩的原核版本，可能产生 深入了解细胞的基本机制和能量学 染色体操作

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.