Assembly of a large DNA virus includes a series of steps for packaging the viral genome. The viral DNA is first recognized for packaging by a packaging protein, then is processed and translocated into an empty protein shell (prohead). Translocation is the least understood stage of DNA packaging. The energy for translocation is known to be ATP hydrolysis, but the mechanism is unclear. In this proposal, experiments are presented for a molecular characterization of the translocation complex, using phage lambda as a model system. Genetic experiments will identify those parts of the translocation complex that are directly involved in the translocation process. Initially these genetic studies will focus on the large subunit of terminase, the viral protein involved in recognition, processing, and translocation. In parallel with the translocation studies, a series of structural studies will be carried out to identify ATP binding centers of terminase. The locations of globular domains of terminase will also be identified. The structural studies, coupled with the translocation studies will give much information about the role of terminase in translocation, and will be the first such detailed studies on the mechanism of translocation. Replication of lambda DNA produces end-to-end multimers of lambda chromosomes. The multimeric precursor DNA is processed by terminase, which introduces staggered nicks to produce the cohesive ends of virion DNA. Because shell mutants are defective for DNA processing, the DNA processing reaction is controlled somehow by the shell. Such shell control is also observed for the pathogenic herpes viruses. Tests of models for how the shell achieves this are presented. There are three adjacent sites used for recognition and processing; genetic experiments will examine in detail the interactions between packaging proteins and these sites. These studies are directed at understanding how packaging protein interactions with DNA account for the major reconfiguring of packaging proteins that occurs during DNA packaging.
Showing the most recent 10 out of 37 publications
