Understanding the biochemistry of DNA replication and the associated regulation of cell cycle in eukaryotes is difficult at a molecular level due to the complexity of these genomes. As an alternative, small extrachromosomal genomes, such as yeast ARS plasmids and certain DNA viruses, have been explored as model systems for cellular DNA replication and its regulation. Papillomaviruses are an attractive model system because replication of the viral genome is coordinately regulated with the host cell cycle during stable infection, which implies that the viral genome is utilizing normal cellular control mechanisms. The long-range goal of this work will be the identification and characterization of all the cellular and viral proteins required for authentic, regulated papillomavirus replication. As a first step in this process, the immediate goal will be to define the mechanism(s) of action of viral proteins known to be required for replication in vivo. This proposal will focus specifically on the two viral proteins required for replication of bovine papillomavirus (BPV) in vivo, the E1 and E2 proteins. Recent work has shown that the E1 protein binds to BPV DNA in the region that functions as an origin of replication in vivo (Wilson and Ludes-Meyer, manuscript submitted), and that this E1 binding site is adjacent to a binding site for E2 protein (V. Wilson, unpublished observations). Both the E1 and E2 proteins will be expressed in a bacterial system and purified for assays of function in vitro.
Specific aims will be to 1) define the location, organization, and critical nucleotides of the E1 binding site in vitro, 2) determine the effect of E1 binding on BPV DNA structure, 3) establish whether or not origin binding is required for the replication activity of E1 protein, and 4) examine the physical and functional relationship between the E1 and E2 proteins bound at the origin region. These studies should provide the initial information about critical protein-DNA interactions at a functional origin of replication in BPV.
