REGULATION OF PAPILLOMAVIRUS DNA REPLICATION

描述(改编自申请者摘要)：长期目标 这项建议是为了了解牛的发病机制和调控 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VERT的病原体 常见的性传播疾病，这种病毒已经强烈 与某些人类癌症的原因有关。因此，对这些问题的研究 病毒将使试剂的开发能够防止 这些病毒在体内的复制和随后的传播 这个项目的具体目标有五个方面。首先， BPV复制源中的基本结构域将是 BPV Ori将受到定点突变 突变的Ori DNA分子然后用三个 分析：ORI解离反应依赖于BPV EL蛋白和DNA 体外复制和体内DNA复制。第二，分子 ORI与BPVE1和E2蛋白之间的相互作用将是 由蛋白质-DNA的酶和化学探针法确定 第三，与ORI结合的E1的多聚体结构将是 用扫描电子显微镜进行了表征。这个 牛细小病毒E_2蛋白和三磷酸腺苷对E_1结合的寡聚体状态的影响 将对Ori进行检查。第四，将使用双混合系统来 从小鼠cdna文库中鉴定能与之相互作用的蛋白质 在体内表达的是E1蛋白。这些cdna分子的序列将是 下定决心。第五，细胞周期调控的相互作用 BPVE1和E2蛋白与ORI的关系将被研究。金额的多少 蛋白质与ORI的结合及ORI内结构的变化 将在非同步化细胞中进行化学和酶探测 在淘洗出的细胞中。淋巴提取液对小鼠的作用 细胞对E1和E2蛋白与ORI的结合以及对 还将研究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.