REGULATION OF PAPILLOMAVIRUS DNA REPLICATION

描述（改编自申请人的摘要）： 这一建议是为了了解牛的机制和调节， 乳头瘤病毒（BPV）DNA复制。 对这种病毒的研究很重要 有两个原因。首先，BPV系统可以提供 人类染色体DNA复制的机制和调控。 第二、 密切相关的人乳头瘤病毒是非常 常见的性传播疾病，这种病毒已经强烈 被认为是某些人类癌症的病因。 因此，研究这些 病毒将允许开发试剂，防止 复制和随后的传播这些病毒在 该项目的具体目标有五个方面。 一是 BPV复制起点内的重要结构域将是 将BPV ori进行定点诱变， 然后用三种不同的方法测试突变的ori DNA分子的活性， 测定：依赖于BPV El蛋白、DNA的ori-解旋反应 体外复制和体内DNA复制。 第二，分子 ori和BPV E1和E2蛋白之间的相互作用将是 通过蛋白质-DNA的酶和化学探测确定 第三，与ori结合的E1的多聚体结构将是 使用扫描透射电子显微镜表征。 的 BPV E2蛋白和ATP对E1结合寡聚状态的影响 将对ORI进行检查。 第四，双混合动力系统将用于 从小鼠cDNA文库中鉴定可以与 体内E1蛋白。 cDNA分子的序列将是 测定 第五，细胞周期调控的相互作用， 将研究带有ori的BPV E1和E2蛋白。 的量 蛋白质与ori的结合以及ori内结构变化的存在 将在非同步细胞中进行化学和酶促探测 和淘析的细胞中。 淘洗小鼠提取物的作用 细胞对E1和E2蛋白与ori的结合以及对 还将检查ori结构变化的诱导。

DESCRIPTION (Adapted from Applicant's Abstract): The long term goals of this proposal are to understand the mechanism and regulation of bovine papillomavirus (BPV) DNA replication. Study of this virus is important for two reasons.First, the BPV system can provide information of the mechanism and regulation of human chromosomal DNA replication. Second, the closely related human papillomavirus is the causal agent of very common sexually transmitted diseases, and this virus has been strongly implicated as a cause of certain human cancers. Thus, study of these viruses will allow the development of reagents that prevent the replication and the subsequent propagation of these viruses in humans.The specific aims of this project are five-fold. First, the essential domains within the BPV origin of replication will be characterized.The BPV ori will be subjected to site-directed mutagenesis and the mutant ori DNA molecules then tested for activity using three assays: the ori-unwinding reaction dependent on the BPV El protein, DNA replication in vitro, and DNA replication in vivo. Second, the molecular interactions between ori and the BPV E1 and E2 proteins will be determined by enzymatic and chemical probing of the protein-DNA complexes.Third, the multimeric structure of E1 bound to ori will be characterized using scanning transmission electron microscopy. The effect of the BPV E2 protein and ATP on the oligomeric state of E1 bound to ori will be examined. Fourth the two- hybrid system will be used to identify proteins from a mouse cDNA library that can interact with the E1 protein in vivo. The sequences of the cDNA molecules will be determined. Fifth, the cell-cycle regulation of the interaction of the BPV E1 and E2 proteins with ori will be investigated. The amount of protein binding to ori and the presence of structural changes within ori will be probed chemically and enzymatically in non-synchronized cells and in elutriated cells. The effect of extracts of elutriated mouse cells on the binding of the E1 and E2 proteins to ori and on the induction of ori structural changes will also be examined.