FUNCTIONAL RELATEDNESS OF VIRAL DNA POLYMERASES

病毒 DNA 聚合酶的功能相关性

• 批准号: 3088574
• 负责人: WILLIAM R BEBRIN
• 金额: $ 6.19万
• 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/3088574
• 关键词: Alphaherpesvirinae; Betaherpesvirinae; DNA directed DNA polymerase; Epstein Barr virus; Escherichia coli; binding proteins; 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。 如本提案所述，对这些病毒的分析可能 导致改善理解并可能治疗疾病 由这些常见病原体引起的，可以呈现给牙医 或医师。 对分子感兴趣的牙科科学家 颅面发育的各个方面，疱疹病毒已知 打扰开发并为相对简单的系统 探测真核基因表达和DNA的方面 复制。 拟议的实验将重点放在 DNA复制，这对于开发和 生长。 CMV，EBV的DNA聚合酶的功能相关性 VZV和HSV-1 DNA聚合酶的VACINIA病毒将是 检查。 带有这些非HSV1 DNA聚合酶的质粒 在HSV启动子的控制下，基因（POL）将进行测试 因为它们可以补充HSV DNA聚合酶功能 使用瞬态和稳定的细胞系测定。 嵌合 将使用体外和 体内方法并测试了功能。 质粒 在控制下的基因（HSV Wildtype，突变体和嵌合体） 也将构建强大的大肠杆菌启动子。 这些基因会 在大肠杆菌中生产过多，纯化和表征 生化。 该特征将包括动力学分析 和使用适当底物和底物的结合研究 类似物和主要HSV结合蛋白与 刺激聚合酶活性。