Biochemical Characterization of Phage Lambda Assembly

传染性病毒颗粒的组装需要许多蛋白质的协调作用，以及与病毒DNA的复杂相互作用。终端酶是许多双链(DS)DNA病毒的常见酶，包括痘病毒、疱疹病毒群和大多数双链DNA噬菌体。终止酶将病毒DNA“打包”到衣壳中，这是病毒组装的关键一步。该项目以噬菌体lambda的终止酶为中心，试图确定参与DNA包装的核蛋白中间体的生化、生物物理和结构特征。研究了三种旨在提供对病毒组装的机械性洞察的方法。(I)包装途径中最令人着迷但最不为人所知的方面之一是从极其稳定的包装前体复合体I向移动式包装复合体的过渡。生物物理、动力学和结构实验被用来为这一转变提供机械支撑。(Ii)目前描述包装途径的模型是基于遗传和粗略的体外包装研究。生物化学研究将在分子水平上确定终止酶和线虫包装蛋白在包装途径的每一步中的作用。(Iii)遗传学研究表明，GPA是一种大的末端酶亚单位，有一个结构域组织。生化和生物物理实验将严格定义GPA中的功能域。这三种实验方法代表了相互交织的不同研究方向，试图提供对lambda终止酶及其在病毒组装中的作用的详细描述。这项工作将为其他dsDNA病毒的基因组包装提供一个范例。此外，基因组包装和复杂的细胞过程之间存在机械上的相似之处，如DNA复制、转录和定点重组。因此，了解这种包装装置的结构和催化性能对于我们理解生物学中大型多蛋白酶复合体对DNA的操纵具有广泛的意义。

The assembly of an infectious virus particle requires the coordinated action of a number of proteins, and complex interactions with viral DNA. Terminase enzymes are common to many double-stranded (ds) DNA viruses, including poxvirus, the herpesvirus groups, and most dsDNA bacteriophage. Terminases "package" viral DNA into the capsid, a critical step in virus assembly. This project centers on the terminase enzyme from bacteriophage lambda and attempts to define the biochemical, biophysical, and structural characteristics of nucleoprotein intermediates involved in DNA packaging. Three approaches designed to provide mechanistic insight into virus assembly are examined. (I) One of the most fascinating and least understood aspects of the packaging pathway is the transition from complex I, an extremely stable packaging precursor, to a mobile packaging complex. Biophysical, kinetic, and structural experiments are used to provide a mechanistic underpinning for this transition. (II) Current models describing the packaging pathway are based on genetic and crude in vitro packaging studies. Biochemical studies are used that will define, at a molecular level, the role of terminase, and inancillaryls packaging proteins at each step of the packaging pathway. (III) Genetic studies suggest a domain organization for gpA, the large terminase subunit. Biochemical and biophysical experiments will rigorously define functional domains in gpA. These three experimental approaches represent discrete research directions that are intertwined in their attempt to provide a detailed description of lambda terminase, and its role in virus assembly. This work will provide a paradigm for genome packaging on other dsDNA viruses. Moreover, mechanistic similarities exist between genome packaging and complex cellular processes such as DNA replication, transcription, and site-specific recombination. Thus, an understanding of the structural and catalytic properties of this packaging apparatus has broad implications in our understanding of DNA manipulation by large multiprotein enzyme complexes in biology.