CONTROL OF PHASE SPECIFIC INITIATION EVENTS AT BPV ORI

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Papillomaviruses are important human pathogens which have been linked to a number of cancers. The study of papillomaviruses is valuable as a model for cellular replication because of their non-lytic nature and the availability of an in vitro replication system. Bovine papillomavirus type 1 (BPV) maintains its DNA as an extrachromosomal plasmid within the infected mammalian cell and is dependent upon host replication machinery, in addition to the viral E1 and E2 initiation protein complex, for S-phase specific replication. The cell-cycle- specific replication of BPV DNA and its use of mammalian replication proteins can be exploited to identify cellular factors which regulate the initiation of the BPV DNA in vivo. Maintenance of a high copy number of extrachromosomal BPV DNA allows the use of enzymatic and chemical probing of the BPV ori within the living cells. The DNA will be analyzed by primer extension to detect variations in origin occupancy and structure which result from host factor interaction. This in situ probing will be performed in populations of cells enriched for distinct phases of the cell cycle (i.e., G1, S, and G2). Footprinting of BPV ori in living cells will be compared to footprint patterns obtained from in vitro probing in the presence of purified E1 and E2 proteins and cell extracts derived from synchronized cultures in the G1, S, or G2 phase. The comparison of the in vivo and in vitro footprinting results will provide a unique look at a subpopulation of host replication proteins involved in the cell cycle specific initiation of DNA replication.