Proteins encoded by the small DNA viruses change the growth regulatory networks of the cell for the advantage of the viral growth cycle. The oncogenic virus Bovine papilloma virus-1 (BPV-1) establishes itself as a stable plasmid in latently transformed fibroblasts, and as such provides an ideal system to uncover mechanisms that are of general importance for DNA replication control. This proposal focuses upon the cellular protein p53, its interactions with another cellular encoded protein, RPA, and the roles that the viral encoded E6 protein plays in deregulating the p53 checkpoint, which monitors DNA damage and unscheduled DNA replication. The investigator also proposes to continue his efforts to study the roles of the E1 and E2 family of viral proteins in directly and indirectly controlling plasmid copy number.
The specific aims are as follows. 1. Does the single strand DNA binding protein RPA block p53 transactivation activity in vivo? Does DNA damage lead to breakdown of a specific p53:RPA complex in vivo which then initiates a pathway to cell cycle arrest? Can one map a specific domain in the large sub-unit of RPA that mediates the formation of this complex? 2. Ectopic expression of p53 in CHO cells blocks BPV-1 transient replication, and this interference is counteracted by the BPV-1 E6 protein. Specific experiments are outlined to address the mechanism underlying these phenomena. 3. The viral protein E2C represses DNA replication in vitro and in vivo, and is critical for copy number control. The investigator has shown the heterodimers (E2:E2C) can function as activators in vitro and he wishes to ask if heterodimers can function as activators in vivo. Dr. Botchan wishes to probe further with his cell-free replication system the mechanisms of repression of replication. 4. The investigator has mapped by direct protein sequencing and by mass spectrometry three new phosphorylation sites on the E2 protein which turn over rapidly and accumulate upon treatment of cells with okadaic acid. The investigator wants to explore the roles of these phosphates in the assembly and disassembly of the preinitiation complex, and to further characterize viral mutants having substitutions at these residues. 5. The investigator proposes to examine the structure of the E1:ori DNA complex and the E1:E2:DNA ternary complex by the application of scanning force microscopy (SFM). This method should readily tell us how many E1 monomers are bound in the preinitiation complex, and may provide important details as to how the conformation of E1 or DNA might change as a result of assembly with E2. SFM is a new microscopic imaging technique which potentially can provide considerable detail with respect to protein shape in nucleoprotein complexes, because it doesn't rely on shadowing for visualization, and has a high resolving power. The investigator also would like to extend his electron microscope studies on unwound DNA created by E1, by employing immuno-electron microscopic techniques. Such methods should tell if E2 can track with the moving helicase. 6. The investigator has developed a yeast assay which measures the assembly of E1 and E2 on the BPV-1 origin DNA within the cell. The investigator proposes to characterize by a genetic approach the interaction surfaces of these two proteins with this system. Further, Dr. Botchan wishes to select for mutant alleles of these proteins which are resistant to compounds which are found to block their interaction in vitro and block in vitro replication.
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