Biochemical Characterization of Bacteriophage Lambda Terminase

本研究的目的是在分子水平上研究病毒前体组装成感染性病毒颗粒的机制。病毒组装的最后步骤之一是将病毒基因组包装成一层被称为衣壳或头的保护性蛋白质外壳。类似的DNA包装机制已被提出用于所有的双链DNA噬菌体，并可能适用于哺乳动物病毒，如腺病毒和疱疹病毒。终止酶是所有这些病毒共有的酶，它负责将单个基因组从串联前体中包装出来。噬菌体lambda多年来被广泛研究，是研究病毒DNA包装的理想系统。噬菌体末端酶具有位点特异性内切酶和三磷酸腺苷酶催化活性，它们协同作用影响DNA包装。P.I.先前利用明确的生化分析系统表征了这种酶的内切酶和三磷酸腺苷酶活性。然而，这些实验是在没有病毒前体的情况下进行的，因此检查了包装过程的开始。这里描述的实验扩展了这项工作，重点是包装的终止。利用物理和动力学技术来剖析蛋白质和DNA在终止过程中的作用。利用高能ATP类似物解决了包装过程的能量学问题。噬菌体末端酶结合ATP和DNA作为底物用于包装反应。光亲和标记实验旨在定位蛋白质一级序列内的核苷酸结合位点。对酶的结合和/或催化至关重要的氨基酸也通过修饰蛋白的蛋白水解消化和肽图谱技术进行了检测。最后，实验将最终导致这种DNA包装酶的小亚基的晶体结构进行了描述。来自噬菌体lambda的终止酶与所有双链DNA病毒的终止酶具有功能上的相似性，虽然机制细节不同，但从这些实验中获得的数据可用于模拟所有这些病毒的DNA包装。了解多种催化活性如何协同作用以影响病毒组装，可能有助于深入了解多功能、多蛋白酶复合物操纵DNA的一般机制。因此，从这些实验中获得的信息超出了对病毒组装的研究，并可能深入了解多蛋白酶复合物的基本特性。本实验室对病毒是如何由受感染细胞内合成的蛋白质和DNA前体组装而成感兴趣。研究中心是噬菌体lambda，一种感染大肠杆菌的DNA病毒。这种病毒利用一种被称为终止酶的酶将其染色体包装到一种被称为前衣壳或前体的保护性蛋白质外壳中。这种酶，连同其他来自病毒和宿主的蛋白质，组装到病毒DNA上，并利用ATP水解产生的能量将染色体“塞”进衣壳内的密闭空间。该实验室正在研究噬菌体末端酶的生化和生物物理特性，作为几种细菌病毒和哺乳动物病毒（如疱疹病毒）包装DNA的模型。他们对包装病毒DNA所需的每种蛋白质的作用以及这些蛋白质如何聚集在一起以准确有效地组装感染性病毒感兴趣。＊＊＊

9419087 Catalano The objective of this research is to examine, at the molecular level, the mechanism of assembly of viral precursors into an infectious virus particle. One of the final steps in viral assembly is the packaging of the viral genome into a protective protein coat known as the capsid, or head. Similar mechanisms for DNA packaging have been proposed for all of the doublestranded DNA bacteriophages and likely apply to mammalian viruses such as adenovirus and herpesvirus. Terminases are enzymes common to all of these viruses and are responsible for packaging of a single genome from a concatameric precursor. Bacteriophage lambda has been extensively studied over the years and represents an ideal system in which to study viral DNA packaging. Phage lambda terminase possesses site-specific endonuclease and ATPase catalytic activities which work in concert to effect DNA packaging. The P.I. has previously characterized the endonuclease and ATPase activities of this enzyme utilizing well defined biochemical assay systems. These experiments were performed in the absence of viral proheads, however, and thus examined the initiation of the packaging process. The experiments described here extend this work and focus on the termination of packaging . Physical and kinetic techniques are utilized in order to dissect the role of protein and DNA in the termination process. The energetics of the packaging process are addressed with the use of high-energy ATP analogs. Phage lambda terminase binds both ATP and DNA as substrates utilized in the packaging reaction. Photo-affinity labeling experiments are designed to locate the nucleotide binding sites within the primary sequence of the protein. Amino acids critical to binding and/or catalysis by the enzyme are also examined by proteolytic digestion of the modified protein and peptide mapping techniques. Finally, experiments which will ultimately lead to a crystal structure for the small subunit of this DNA packaging enzyme are described. The terminase enzyme from bacteriophage lambda bears functional similarities to terminases from all of the double-stranded DNA viruses and, while the mechanistic details differ, the data derived from these experiments may be used to model DNA packaging by all of these viruses. An understanding of how the multiple catalytic activities work in concert to effect viral assembly may yield insight into the general mechanisms of DNA manipulation by multifunction, multiprotein enzyme complexes. The information derived from these experiments thus extends beyond a study of viral assembly and may yield insight into the basic properties of multiprotein enzyme complexes. %%% This laboratory is interested in how a virus is assembled from protein and DNA precursors synthesized within the infected cell. The research centers on bacteriophage lambda,a DNA virus that infects E. coli. This virus utilizes an enzyme known as terminase to package its chromosome into a protective protein coat known as the pre-capsid, or prohead. This enzyme, along with other proteins of both viral and host origin, assemble onto viral DNA and use the energy derived from ATP hydrolysis to "stuff" the chromosome into the confined space found within the capsid. This laboratory is investigating the biochemical and biophysical properties of phage lambda terminase as a model for DNA packaging by several bacterial viruses as well as mammalian viruses such as herpesvirus. They are interested in the role of each of the proteins required to package viral DNA and how these proteins come together to accurately and efficiently assemble an infectious virus. ***