Biochemical dissection of the replication process for oncogenic DNA viruses has provided insight into cellular mechanisms of DNA replication. We propose to continue our studies on the DNA replication of papovaviruses and to initiate new studies which use the virus BPV-1 as a tool to study chromosomal DNA replication more directly.
Our aims : 1. To exploit a new cell system within which the viral E1 and E2 proteins are under the control of conditional promoters which can be regulated by tetracycline. This system can be used to follow BPV-1 plasmid levels (amplification and loss) in stably transformed cells and, most importantly, we believe that this system will allow us to genetically map the cis acting determinants required for replication at chromosomal loci. Withdrawal of E1 and E2 from plasmids previously replicating in the cell under their control should allow for the selection of inserted DNA fragments, which contain sequences supporting autonomous DNA replication. Our initial targets will be the origins of replication found at the hamster DHFR and the CAD gene origins of replication. 2. To clone a functional cDNA for the Drosophila melanogaster homologue of the yeast protein origin recognition protein-2 (ORC-2). We will raise antibodies to the protein expressed in E. coli and create tagged expression vectors to produce this gene product in insect cell culture and in whole flies. The immediate goal will be to ask if drosophila ORC-2 exists in a complex with other proteins as it does in yeast cells, and does it bind site specifically to DNA fragments known to contain replication origins. By characterizing such proteins we hope to generate reagents which will be useful in similar probes for mammalian systems. In particular we wish to explore, ultimately, the notion that cis acting sequences or ori's (aim #1) bind ORC-like complexes. 3. Neutralizing monoclonal antibodies specifically directed to the cellular polymerase epsilon abolish in vitro cell free replication of both BPV-1 and SV-40 as directed by E1 and E2 or T antigen, respectively. We wish to explore the role of the epsilon polymerase in lagging strand DNA synthesis in fractionated extracts with viral and model templates. We also want to know if epsilon interacts with the viral helicases or associates with other cellular factors which allow it to coordinate synthesis with the delta polymerase. 4. The BPV-1 E1 interacts with DNA polymerase alpha: primase (pol alpha), and this interaction is important for primosome initiation at the viral ori. Both the p180 and p70 subunits of pol alpha bind to E1, though the p70 interactions are stronger. We want to map the protein domains on p70 and E1 which are responsible for this binding, and to use this information in a yeast two hybrid screen to search for proteins which might bind to p70 in a manner analogous to E1. 5. We have isolated and characterized over 30 amino acid substitution mutants in the activation domain of the enhancer protein E2. Many of these mutants are temperature sensitive and some allow for separation of replication and transcription activity. We propose to exploit these mutants for structural and functional studies of the protein both in vivo and in vitro. An important aim is to obtain a protease resistant domain of E2 for NMR studies and to ask if some of our mutants are temperature sensitive folding mutants.
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