CONTROL OF PHASE SPECIFIC INITIATION EVENTS AT BPV ORI

乳头瘤病毒是重要的人类病原体， to a number数of cancers癌症. 乳头状瘤病毒的研究是有价值的， 由于其非裂解性质， 体外复制系统的可用性。 牛 乳头瘤病毒1型（BPV）保持其DNA作为染色体外 在感染的哺乳动物细胞内的质粒，并依赖于宿主 复制机制，除了病毒E1和E2启动 蛋白复合物，用于S期特异性复制。 细胞周期 BPV DNA特异性复制及其在哺乳动物复制中的应用 蛋白质可以用来鉴定细胞因子， BPV DNA在体内的起始。 高拷贝的维护 染色体外BPV DNA的数量允许使用酶和 活细胞内BPV ori的化学探测。 dna将 通过引物延伸进行分析，以检测起源占据的变化 以及寄主因子相互作用的结果。 这种原位 探测将在富集不同的细胞群中进行， 细胞周期的阶段（即，G1、S和G2）。 BPV ori的足迹 将与从活细胞中获得的足迹模式进行比较， 在纯化的E1和E2蛋白和细胞存在下的体外探测 来自G1、S或G2期同步培养物的提取物。 体内和体外足迹结果的比较将 提供了一个独特的外观在一个亚群的主机复制蛋白质 参与DNA复制的细胞周期特异性起始。

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.