Mechanistic Studies on a DNA Packaging Machine

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DESCRIPTION (provided by the applicant): The assembly of nucleoprotein structures that perform complex biological functions is a common theme in nature. Relevant examples include the nucleoprotein complexes involved in chromosomal replication, DNA transcription, and genome "packaging" in many double-stranded DNA (dsDNA) viruses. Terminase enzymes are common to a diverse group of dsDNA viruses, including the herpesvirus groups. These enzymes are an integral part of a series of nucleoprotein complexes critical to genome packaging. All of the known terminases possess ATPase and nuclease activities that play an important role in the packaging process. The principal investigator and his group are interested in the biochemical properties of the packaging machinery, and specifically the enzymology of the terminase enzymes. Studies described in this proposal seek to understand the molecular mechanism of genome packaging by lambda terminase. The enzyme possesses site-specific nuclease and ATPase catalytic activities that work in concert to package viral DNA. Viable models describing the packaging pathway have been developed. Many aspects of the packaging pathway remain obscure, however, and most lack mechanistic rigor. Studies are designed to provide a mechanistic basis for the delicate interplay between these catalytic activities, and their role in the assembly and stability of nucleoprotein packaging intermediates. Biochemical and enzyme kinetic studies are designed to directly probe the catalytic mechanism of the enzyme, and to define the role of catalysis in nucleoprotein complex assembly and function. While mechanistic details may differ, the data derived from these experiments may be used to model DNA packaging by other double-stranded DNA viruses, including the medically relevant adenovirus and herpesvirus groups. Furthermore, this enzyme shares mechanistic similarities to the complex nucleoprotein machines involved in DNA replication and transcription. An understanding of the catalytic properties of this packaging machine may yield insight into the general mechanisms of DNA manipulation by large multiprotein enzyme complexes.