Biochemical Characterization of Bacteriophage Lambda Terminase

9419087 Catalano本研究的目的是在分子水平上检查病毒前体组装成感染性病毒颗粒的机制。病毒组装的最后一步是将病毒基因组包装到一个保护性的蛋白质外壳中，称为衣壳或头部。对于所有的双链DNA噬菌体都提出了类似的DNA包装机制，并且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，负责从多联体前体包装单个基因组。噬菌体λ已经被广泛研究了多年，并代表了一个理想的系统，在其中研究病毒DNA包装。 噬菌体λ末端酶具有位点特异性核酸内切酶和ATP酶催化活性，其协同作用以影响DNA包装。私家侦探先前已经利用明确的生物化学测定系统表征了这种酶的内切核酸酶和ATP酶活性。然而，这些实验是在不存在病毒前体的情况下进行的，因此检查了包装过程的起始。这里描述的实验扩展了这项工作，并专注于包装的终止。 利用物理和动力学技术，以剖析蛋白质和DNA在终止过程中的作用。能量的包装过程中解决了使用高能量ATP类似物。 噬菌体λ末端酶结合ATP和DNA作为包装反应中使用的底物。设计光亲和标记实验以定位蛋白质的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的蛋白质的蛋白水解消化和肽图谱技术来检查。最后，实验将最终导致这种DNA包装酶的小亚基的晶体结构的描述。来自λ噬菌体的末端酶与来自所有双链DNA病毒的末端酶具有功能相似性，尽管机理细节不同，但来自这些实验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，可能会深入了解多功能、多蛋白酶复合物对DNA操作的一般机制。因此，从这些实验中获得的信息超出了病毒组装的研究，并可能深入了解多蛋白酶复合物的基本性质。 该实验室对病毒如何从受感染细胞内合成的蛋白质和DNA前体组装而成感兴趣。 这项研究的中心是λ噬菌体，一种感染大肠杆菌的DNA病毒。杆菌这种病毒利用一种称为末端酶的酶将其染色体包装成一种保护性蛋白质外壳，称为前衣壳或前头。这种酶，沿着病毒和宿主来源的其他蛋白质，组装到病毒DNA上，并利用ATP水解产生的能量将染色体“塞进”衣壳内的有限空间。该实验室正在研究噬菌体λ末端酶作为几种细菌病毒以及哺乳动物病毒（如疱疹病毒）DNA包装模型的生物化学和生物物理特性。他们感兴趣的是包装病毒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准确有效地组装感染性病毒。 ***

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. ***