ENZYMATIC MECHANISM OF HERPES VIRUS DNA REPLICATION

这项研究计划的目的是了解分子细节 单纯疱疹病毒1型复制的机制 病毒DNA被启动并持续存在。我们建议在 用纯化的酶进行体外实验，我们将了解其结构和机制 详细检查，有序的，半保守的复制 单纯疱疹病毒1型基因组。我们期望这些研究能为我们提供一个 洞察真核生物基因组复制的机制 是启动的。它们还应提供有关 复制一类具有很大公共性的动物病毒 健康的重要性。调查将沿着 以下几行： A.纯化的HSV-1编码复制酶的分析 1.来源结合蛋白(UL9蛋白) A.原产地特定解旋酶活性 B.UL9蛋白与其他HSV-1复制酶的相互作用 C.LTL9蛋白的磷酸化 2.DNA聚合酶-UL42蛋白复合体 A.DNA聚合酶的三维结构 B.DNA聚合酶反应的机制 DNA聚合酶的C.5‘-GT；3’外切酶(RNase H)活性 D.UL42蛋白的磷酸化 3.DNA解旋酶-底物酶 4.单链DNA结合蛋白-ICP8 B.依赖于起源(Ori-S)的DNA复制的重建 1.起源结合蛋白在启动中的作用 2.奥里-S依赖的DNA重组的具体方法 复制 A.启动的转录激活 B.对宿主因素的要求 C.机械论研究 3.单纯疱疹病毒L DNA复制的调控

The aim of this research program is to understand in molecular detail the mechanism by which the replication of Herpes Simplex-1 (HSV-1) virus DNA is initiated and sustained. We propose to reconstitute in vitro with purified enzymes whose structure and mechanism we will examine in detail, the orderly, semiconservative replication of the HSV-1 genome. We expect that these studies should provide us with an insight into the mechanism by which replication of a eukaryotic genome is initiated. They should also yield information about the replication of a class of animal viruses that are of great public health importance. The investigation will be organized along the following lines: A. Analysis of purified HSV-1 encoded replication enzymes 1. Origin binding protein (UL9 protein) a. Origin specific helicase activity b. Interaction of UL9 protein with other HSV-1 replication enzymes c. Phosphorylation of LTL9 protein 2. DNA polymerase - UL42 protein complex a. Three dimensional structure of DNA polymerase b. Mechanism of DNA polymerase reaction c. 5' -> 3' exonuclease (RNase H) activity of DNA polymerase d. Phosphorylation of UL42 protein 3. DNA helicase - primase 4. Single-strand DNA binding protein - ICP8 B. Reconstitution of Origin (Ori-s) dependent DNA replication 1. Role of origin binding protein in initiation 2. Specific approaches to reconstitution of Ori-s dependent DNA replication a. Transcriptional activation of initiation b. Requirement for host factors c. Mechanistic studies 3. Regulation of HSV-L DNA replication