FUNCTIONAL RELATEDNESS OF VIRAL DNA POLYMERASES

病毒 DNA 聚合酶的功能相关性

• 批准号: 3088576
• 负责人: WILLIAM R BEBRIN
• 金额: $ 5.54万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-07-01 至 1993-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3088576
• 关键词: Alphaherpesvirinae; DNA directed DNA polymerase; DNA replication; Epstein Barr virus; Escherichia coli; binding proteins; cytomegalovirus; enzyme mechanism; eukaryote; face; gene expression; herpes simplex virus 1; human subject; molecular biology; plasmids; skull; vaccinia virus; varicella zoster virus; virus DNA; virus genetics

Research will be in the area of herpesvirus molecular biology. This system allows sophisticated genetic and molecular biological analysis. Analysis of these viruses, as stated in this proposal, may lead to improved understanding and possibly treatment of diseases caused by these common pathogens that can present to a dentist or physician. To a dental scientist interested in the molecular aspects of craniofacial development, herpesviruses are known to perturb development and provide a relatively simple system for probing aspects of eukaryotic gene expression and DNA replication. The proposed experiments will focus on aspects of DNA replication, a process that is important for development and growth. The functional relatedness of the DNA polymerases of CMV, EBV, VZV, and Vaccinia virus to the HSV-1 DNA polymerase will be examined. Plasmids bearing these non-HSV1 DNA polymerase genes (pol), under the control of an HSV promoter, will be tested for their ability to complement HSV DNA polymerase function using both transient and stable cell line assays. Chimeric nonHSV/HSV pol genes will be constructed using both in vitro and in vivo methods and tested for function. Plasmids with the pol genes (HSV wildtype, mutants, and chimeras) under the control of a strong E.coli promoter will also be constructed. The genes will be overproduced in E.coli, purified, and characterized biochemically. This characterization will include kinetic analysis and binding studies using appropriate substrates and substrate analogs and the ability of the major HSV binding protein to stimulate polymerase activities.

研究将集中在疱疹病毒分子生物学领域。 该系统允许复杂的遗传和分子生​​物学 分析。 如本提案中所述，对这些病毒的分析可能会 提高对疾病的认识并可能治疗疾病 由这些常见病原体引起，可以向牙医提出 或医生。 对于对分子感兴趣的牙科科学家 众所周知，疱疹病毒在颅面发育方面 干扰开发并提供一个相对简单的系统 真核基因表达和 DNA 的探索 复制。 拟议的实验将重点关注以下方面 DNA复制，一个对于发育和发育非常重要的过程 生长。 CMV、EBV、DNA聚合酶的功能相关性 VZV 和牛痘病毒的 HSV-1 DNA 聚合酶将 检查了。 带有这些非 HSV1 DNA 聚合酶的质粒 将测试 HSV 启动子控制下的基因 (pol) 因其能够补充 HSV DNA 聚合酶功能 使用瞬时和稳定细胞系测定。 嵌合体 nonHSV/HSV pol 基因将使用体外和 体内方法并进行功能测试。 带有 pol 的质粒 基因（HSV野生型、突变体和嵌合体）受以下控制 还将构建强大肠杆菌启动子。 基因会 在大肠杆菌中过量生产、纯化和表征 生化上。 该表征将包括动力学分析 使用适当的底物和底物进行结合研究 类似物和主要 HSV 结合蛋白的能力 刺激聚合酶活性。