REGULATION OF PAPILLOMAVIRUS DNA REPLICATION

乳头状病毒 DNA 复制的调控

• 批准号: 2330858
• 负责人: JAMES A. BOROWIEC
• 金额: $ 21.42万
• 依托单位: NEW YORK UNIVERSITY SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-04-01 至 1999-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2330858
• 关键词: DNA binding protein; DNA replication; DNA replication origin; animal genetic material tag; bovine papillomavirus; cell growth regulation; complementary DNA; electroporation; genetic library; host organism interaction; nucleic acid sequence; oncogenic virus; polymerase chain reaction; scanning transmission electron microscopy; site directed mutagenesis; virus DNA; virus protein; virus related neoplasm /cancer; virus replication; yeasts

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.

描述（改编自申请人的摘要）：长期目标 该提案旨在了解牛的机制和调节 乳头瘤病毒 (BPV) DNA 复制。 研究这种病毒很重要 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严重的病原体 常见的性传播疾病，这种病毒已被强烈 被认为是某些人类癌症的原因。 因此，研究这些 病毒将允许开发试剂来阻止 这些病毒的复制和随后的传播 人类。该项目的具体目标有五个。 首先， BPV 复制起点内的重要结构域将是 BPV ori 将进行定点诱变 然后使用三种方法测试突变 ori DNA 分子的活性 测定：ori解旋反应依赖于BPV El蛋白、DNA 体外复制和体内DNA复制。 二、分子 ori 与 BPV E1 和 E2 蛋白之间的相互作用将是 通过蛋白质-DNA 的酶促和化学探测来确定 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进行表征。 这 BPV E2 蛋白和 ATP 对 E1 结合寡聚状态的影响 到ori将被检查。 第四，双混合动力系统将用于 从小鼠 cDNA 文库中鉴定可与 体内E1蛋白。 cDNA分子的序列为 决定。 五、细胞周期的相互作用的调控 将研究带有 ori 的 BPV E1 和 E2 蛋白。 金额 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 和淘洗细胞中。 小鼠淘洗提取物的作用 细胞对 E1 和 E2 蛋白与 ori 的结合以及 还将检查 ori 结构变化的诱导。