The objective of this proposal 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 double-stranded DNA bacteriophages and may also 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. We therefore propose to use phage lambda terminase as a model enzyme with which to study DNA packaging and virus assembly. The present project focuses on the catalytic activities of lambda terminase and their role in the packaging of viral DNA. This enzyme possesses a site-specific endonuclease activity, a DNA-stimulated ATPase activity and a DNA helicase activity, all of which work in concert to effect genome packaging. DNA packaging by lambda terminase initiates with the assembly of a stable multiprotein complex (termasome) onto the concatameric packaging substrate and site-specific nicking of the duplex. Prior to or immediately after strand separation by the enzyme, an empty prohead binds to the binary protein.DNA intermediate and the termasome releases from the assembly site. Translocation of terminase ensues, likely powered by the hydrolysis of ATP, and DNA is actively packaged into the viral prohead. The experiments described in this proposal systematically probe the protein-protein and protein-DNA interactions required to assemble a stable enzyme.DNA intermediate which nicks the DNA duplex, and the subsequent interactions required to disengage the complex so that packaging may ensue. The interplay between the three catalytic activities in the assembly-release processes as well as the role of ATP and ATP hydrolysis in these functions are examined in detail. Genetic studies have identified several mutant enzymes which are deficient in specific aspects of lambda assembly and these altered proteins will be utilized as tools to further probe the catalytic mechanisms of the packaging process. Both biophysical and kinetic techniques are utilized to examine in detail each of the steps involved in the initiation of genome packaging. While the mechanistic details differ, the data derived from these experiments may be used to model DNA packaging by all of the double- stranded DNA phages, and may include assembly in the eucaryotic adenovirus and herpesvirus groups. Mechanistic similarities to other DNA manipulating enzymes such as the recBCD nuclease/helicase of E. coli, the restriction endonucleases, particularly types IIS and III, and the assembly-release of an open-promoter complex during the initiation of transcription further suggest that an understanding of the catalytic properties of this packaging machine may yield insight into the general mechanisms of DNA manipulation by multiprotein enzyme complexes.
