Catalano 9728550 1. Technical Packaging of DNA within an empty pre-formed capsid proceeds through an ordered progression of nucleoprotein intermediates requires terminase, an enzyme common to all of the double-stranded DNA bacteriophages. The enzyme subunits, gpNu1 and gpA, assemble onto a multi-element DNA binding site generating a nucleoprotein complex, excising a single genome from the concatamer and inserting it into the capsid. This study examines, at the molecular level, the biochemical and biophysical properties of the lambda phage 1 gpNu1 protein which are responsible for the cooperative assembly and unusual stability of the nucleoprotein intermediates involved. The aims are to understand the roles of gpNu1 self-association interactions and gpNu1 gpA protein protein interactions in the assembly and stability of the nucleoprotein complexes critical to the assembly. The experiments are designed to provide a detailed understanding of the thermodynamic linkages between gpNu1 self-association and cooperative binding to DNA, and to the subsequent assembly of gpA and a catalytically competent genome packaging complex. This study also seeks to define functional domains within the gpNu1 polypeptide which are responsible for the observed oligomerization of gpNu1, for gpNu1 gpA interactions, and for cooperative binding to DNA. Since the terminase enzyme is common to many double-stranded DNA viruses, findings from this study may be generally applicable to the understanding of other viral assembly and packing mechanisms. 2. Non-Technical A variety of biological processes require the assembly of multiple proteins onto specific sites within the chromosomes of cells and/or viruses. Many of these complexes initially assemble as stable intermediates which are subsequently transformed into highly mobile biological machines. Elaborate nucleoprotein (DNA and protein) complexes are responsible for critical biological processes including the replication of chromosomes, the synthesis of messenger RNA, and the assembly of infectious viruses. This study is to elucidate the process of viral assembly within the cell and the complex process of viral chromosome packaging, specifically in the involvement of the terminase enzyme, the multi-protein machines which insert the viral chromosome within a protective protein shell in bacteriophage lambda, a virus which infects the bacteria E. coli. This enzyme, which is common to many double-stranded DNA viruses, is an integral part of an ordered series of nucleoprotein complexes which are critical to viral assembly. Experiments are carried out to examine the biochemical and biophysical properties of gpNu1, a phage l packaging protein, which are responsible for the assembly and unusual stability of the nucleoprotein intermediates involved in viral chromosome packaging. Importantly, the data derived from these studies will additionally yield insight into the basic principles driving the assembly of stable nucleoprotein complexes involved in a variety of biological processes critical to both viral and cellular replication.
