Mechanistic Studies on a Viral DNA Packaging Machine

我们的长期目标是在分子水平上了解病毒组装。DNA包装是许多双链DNA病毒组装的关键步骤，包括痘病毒、腺病毒、疱疹病毒和许多噬菌体。终止酶是这些病毒共有的，其功能是将病毒DNA包装到衣壳中。对于所有这些病毒，已经提出了基因组包装的共同机制。 基因组包装的过程是病毒所独有的，因此是抗病毒治疗的理想靶点;然而，缺乏机制细节阻碍了药物设计的合理方法。 λ噬菌体是研究DNA包装的理想系统。λ末端酶是一系列有序包装中间体的核心组成部分，是包装马达的组成部分。 这个项目的目标是定义一个连贯的物理和动力学模型的病毒DNA包装马达的组装。任何复杂生物过程的机理模型都需要描述所涉及的大分子复合物的物理性质，对过程所需的催化活性进行动力学分析，以及清楚描述催化、结构和功能之间联系的机制。不幸的是，实际上对λ中包装中间体的物理性质一无所知。此外，催化活性和功能之间的机械联系仍然难以捉摸，部分原因是缺乏结构信息。该项目旨在（i）表征负责病毒DNA上包装马达特异性组装的相互作用，（ii）定义功能性包装马达的组装状态，（iii）表征关键包装中间体，（iv）表征定义的酶种类的活性，以提供结构和功能之间的直接联系。 白蚁结合结构，生物物理和动力学的方法将定义关键方面的核心，我们了解的机制和酶学的DNA包装。

Our long-range goal is to understand virus assembly at the molecular level. DNA packaging is a critical step in the assembly of many double-stranded DNA viruses, including poxvirus, adenovirus, herpesviruses, and many bacteriophages. Terminase enzymes are common to these viruses and function to package viral DNA into the capsid. Common mechanisms for genome packaging have been proposed for these all viruses. The process of genome packaging is unique to viruses, and thus represents an ideal target for antiviral therapy; however, the lack of mechanistic detail precludes a rational approach to drug design. Bacteriophage lambda presents an ideal system to study DNA packaging. Lambda terminase is a central component of an ordered series of packaging intermediates, and is an integral part of the packaging motor. The goal of this project is to define a coherent physical and kinetic model for the assembly of a viral DNA packaging motor. A mechanistic model for any complex biological process requires a description of the physical nature of the macromolecular complexes involved, a kinetic dissection of the catalytic activities required for the process, and a mechanism that clearly describes the linkage between catalysis, structure, and function. Unfortunately, virtually nothing is known about the physical properties of the packaging intermediates in lambda. Moreover, a mechanistic link between catalytic activity and function remains elusive, due in part to the dearth of structural information. This project seeks to (i) characterize the interactions responsible for specific assembly of the packaging motor on viral DNA, (ii) define the assembly state of the functional packaging motor, to (iii) characterize critical packaging intermediates, and (iv) characterize the activity of defined enzyme species to provide a direct link between stucture and function of termiase. A combined structural, biophysical, and kinetic approach will define critical aspects central to our understanding of the mechanism and enzymology of DNA packaging.