ENZYMATIC MECHANISM OF HERPES VIRUS DNA REPLICATION

这项研究计划的目的是了解分子的细节 单纯疱疹病毒1型（HSV-1）的复制机制 病毒DNA被启动和维持。 我们建议重组 体外纯化的酶，其结构和机制，我们将 仔细检查，有序的，保守的复制， HSV-1基因组。 我们希望这些研究能为我们提供一个 深入了解真核基因组复制的机制 开始了。 它们还应提供关于 一类动物病毒的复制， 健康的重要性。 调查将沿着 以下线路： A.纯化的HSV-1编码的复制酶的分析 1.来源结合蛋白（UL 9蛋白） a.起源特异性解旋酶活性 B. UL 9蛋白与其他HSV-1复制酶的相互作用 C. LTL 9蛋白的磷酸化 2. DNA聚合酶-UL 42蛋白复合物 a. DNA聚合酶的三维结构 B. DNA聚合酶反应的机理 C. DNA聚合酶的5' -> 3'核酸外切酶（RNase H）活性 D. UL 42蛋白的磷酸化 3. DNA解旋酶-引发酶 4.单链DNA结合蛋白-ICP 8 B。依赖于Origin（Ori-s）的DNA复制 1.起始结合蛋白在起始中的作用 2. Ori-s依赖性DNA重建的具体方法 复制 a.起始转录激活 B.东道国因素要求 C.机制研究 3. HSV-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