Bovine papilloma virus-1 replicates in transformed cells in synchrony with the cell as a plasmid, and as such an independent replicon it provides an ideal system to study the mechanisms by which DNA replication is regulated. The main focus of this application is the relationship between transcription and DNA replication in the cell cycle. The products of the E1 ORF are critical for replication, as are the products of the E2 ORF. The E1 ORF encodes for a 68 kda protein (R) which has structural and functional homologies to the SV-40 large T antigen. The E1 ORF also produces a truncated gene product (M). This 16 kda protein is important for regulated DNA replication. The E2 ORF encodes for a 45 kda transcriptional enhancer protein and two truncated proteins which can repress transcription. We have recently shown that the 68 kda E1 protein and activator E2 protein form a stable complex. This complex allows E1 to recognize the BPV-1 origin of DNA replication. 1. We will determine when in the cell cycle the E1 products (M and R) are made and when they are phosphorylated. Mutants will be placed in E1 ORF to abolish activities including nuclear localization, ATPase domains, and phosphorylation sites. A putative target site for the cellular CDC-2 kinase will be studied. The consequences of these mutations for biochemical and in vivo plasmid maintenance will be explored. 2. In this phosphorylated form the E2 protein does not bind E1. We would like to mutate the amino acids which get phosphorylated in E2 and examine the in vivo effects of such mutations upon BPV-1 replication. We will attempt to isolate the kinase which phosphorylates E2 and ask if the phosphatase PP2A activates E2 for its interaction with E-1. Cycles of phosphorylation and dephosphorylation on one protein may thus be a mechanism to coordinate transcription and replication. 3. Analysis of the ATPase and helicase properties of the E1:E2 complex will be performed. Questions such as the stoichiometry (subunit interactions) between the proteins and precise DNA specificity of the complex will be asked. 4. We will use both site directed mutagenesis and yeast genetics to create mutations in the E2 protein to define its structure. We wish to know if separate domains of E2 are involved in replication and transcription. 5. We wish to ask if E1 and E2 expression are sufficient for plasmid amplification, and how M and the E2 repressors modulate this amplification. 6. We will initiate studies on the highly oncogenic human papilloma virus type 16.
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