Biochemical Characterization of Phage Lambda Assembly

感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。终止酶是许多双链DNA病毒所共有的，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“包装”到衣壳中，这是病毒组装的关键步骤。该项目以λ噬菌体的末端酶为中心，并试图定义参与DNA包装的核蛋白中间体的生化、生物物理和结构特征。三种方法旨在提供深入了解病毒组装的机制进行了检查。(I)包装途径最吸引人和最不了解的方面之一是从复合物I（一种极其稳定的包装前体）到移动的包装复合物的转变。生物物理，动力学和结构实验是用来提供这种转变的机械基础。(II)目前描述包装途径的模型是基于遗传和粗的体外包装研究。使用生物化学研究，将在分子水平上定义末端酶的作用，并在包装途径的每一步中辅助包装蛋白。(III)遗传学研究表明，一个结构域组织的gpA，大的末端酶亚基。生物化学和生物物理实验将严格定义gpA中的功能结构域。这三种实验方法代表了离散的研究方向，它们在试图提供λ末端酶的详细描述及其在病毒组装中的作用时相互交织在一起。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，如DNA复制，转录和位点特异性重组之间存在机制相似性。因此，了解这种包装装置的结构和催化性能具有广泛的影响，在我们的理解DNA操作的大型多蛋白酶复合物在生物学中。

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.