Human adenoviruses cause respiratory infections in children and are oncogenic viruses that cause tumors in rodents. The focus of this proposal is on the initiation of adenovirus (Ad) DNA replication as a model for the assembly of replication complexes at the nuclear matrix and to study the structure and function of the adenovirus DNA polymerase (Ad Pol), a member of the pol alpha family of DNA polymerases. An important advantage for these studies is the well characterized in vitro system for replication of the Ad genome: three virus proteins (Ad Pol, the precursor to the terminal protein (pTP), and the DNA binding protein (DBP) and three cellular proteins (NFI, NFII, and NFIII) are sufficient to replicate the Ad genome in vitro. Three specific areas for study are proposed: 1) Binding of pTP to the nuclear matrix. The applicant's hypothesis is that pTP mediates the assembly of Ad replication complexes at the nuclear matrix, by binding to one or more matrix proteins. The applicant will map domains within pTP that bind to nuclear matrix. As a long term goal, the applicant will identify the matrix protein(s) to which pTP (and perhaps other replication proteins) binds. These studies will be important for understanding the role of nuclear matrix in promoting cellular DNA replication and transcription. 2) Binding of Ad Pol to pTP and to NFI. The applicant's hypothesis is that Ad Pol is the essential intermediary between pTP bound on the nuclear matrix and an NFI/Ad DNA origin complex. Domains within these three proteins that mediate complex formation will be mapped. 3) Ad Pol as a model DNA polymerase. Ad Pol shares 5 out of 6 regions of homology with other pol alpha DNA polymerases. The applicant will expand our current mutagenesis studies from region I to other conserved regions, as well as to temperature-sensitive mutants of Ad Pol. Together with the studies in (2), the applicant desires to understand how eukaryotic DNA polymerases are functionally organized: what domains encode DNA synthesis activities (DNA binding, nucleotide binding) or are responsible for interactions with other regulatory proteins. The applicant has a unique collection of Ad Pol and pTP mutants and of polyclonal and monoclonal antibody reagents with which to study the steps in the assembly of initiation complexes and subsequent elongation reactions. The applicant has also developed a recombinant vaccinia virus system with which mutant Ad Pol and pTP proteins can be rapidly screened by transient expression from plasmid DNA; those mutants with unusual phenotypes can then be recombined into vaccinia for expression of higher levels of protein for purification and for further biochemical studies.
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