Mechanistic Studies on DNA Packaging by Bacteriophage Lambda

这项建议的目的是在分子水平上研究病毒前体组装成具有感染性的病毒颗粒的机制。已经提出了一种由噬菌体病毒包装DNA的常见机制，该机制包括1)称为终止酶的酶与串联(未成熟)病毒DNA的结合，2)该复合体与预先形成的衣壳的结合，3)DNA在依赖于ATP的反应中单向进入衣壳；4)切割双链和链分离，从而包装单个(成熟的)基因组。噬菌体终止酶现在可以高产量获得，是研究病毒DNA包装的理想系统。该酶具有DNA依赖的ATPase、DNA转位酶、限制性内切酶和DNA解旋酶活性，均存在于异源二聚体蛋白结构中。ATP和DNA的热力学结合常数将使用平衡透析、凝胶延迟和DNA酶足迹技术来确定。将使用光亲和标记技术来检测酶与ATP以及酶与病毒DNA之间的相互作用。共价修饰残基将通过蛋白水解酶、标记肽(S)的分离和N-末端氨基酸序列分析来鉴定。将对核酸内切酶和解旋酶活性进行完整的动力学分析，并使用稳态和稳态前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终止酶进行详细的表征，并作为与其他噬菌体终止酶进行比较研究的基础。对这些酶的检查不仅可以揭示终止酶之间的进化关系，而且还可以揭示其他功能相关的酶，如ATPase、限制性内切酶和解旋酶。此外，DNA包装，即染色体凝聚的原核版本，可能会深入了解细胞染色体操纵的基本机制和能量学。

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.