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. ***
