ENZYMATIC MECHANISM OF HERPES VIRUS DNA REPLICATION

这项研究计划的目的是了解分子细节 单纯疱疹病毒类型的复制机制 1基因组被启动和维持。我们目前的模式是 随着线性病毒基因组的环化，theta类型 在三个HSV-1起始点中的一个或多个处启动复制， 然后切换到滚圆复制。我们建议 用纯化的酶进行体外重组，其结构和 我们将详细研究这个机制，包括theta和滚动圈 单纯疱疹病毒1型DNA复制的时相。我们预期这些研究将会 为我们提供对一个重要类的复制的洞察 人类病原体。调查将沿着 主要内容如下：A.HSV-1编码的结构和机制 复制酶。1.来源结合蛋白(UL9蛋白)a. UL9蛋白b对口腔的解离作用 UL9蛋白与Oris的结合C.UL9蛋白与Oris的相互作用 细胞DNA聚合酶α-底物酶D.三种底物的测定 UL9蛋白的空间结构2.DNA聚合酶-UL42蛋白 复合a.强化的稳态前动力学分析 UL42蛋白的加工性b.UL42蛋白磷酸化的影响 蛋白质对加工性的促进作用3.解旋酶-启动子酶(原粒)a. 原粒UL8亚基在其解旋酶活性分析中的作用 解旋酶活性C.引物酶活性的分析B.重组 Oris依赖的DNA复制1.一般方法2.检查 特定条件3.转录C的转录激活。 单纯疱疹病毒1型复合体对滚环DNA复制的进一步研究 编码复制酶1.复合体的组装2.作用机理 从theta切换到滚圈DNA复制。

The aim of this research program is to understand in molecular detail the mechanism by which the replication of the herpes simplex virus type 1 genome is initiated and sustained. Our current model is that following circularization of the linear viral genome, theta type replication is initiated at one or more of the three HSV-1 origins, followed by a switch to rolling circle replication. We propose to reconstitute in vitro with purified enzymes whose structure and mechanism we will examine in detail, both the theta and rolling circle phases of HSV-1 DNA replication. We anticipate that these studies will provide us with an insight into the replication of a significant class of human pathogens. The investigation will be organized along the following lines: A. Structure and mechanism of HSV-1 encoded replication enzymes. 1. Origin binding protein (UL9 protein) a. Unwinding of oris by UL9 protein b. Determination of stoichiometry of binding of UL9 protein to Oris c. Interaction of UL9 protein with cellular DNA polymerase alpha-primase d. Determination of three- dimensional structure of UL9 protein 2. DNA polymerase-UL42 protein complex a. Pre-steady state kinetic analysis of enhancement of processivity by UL42 protein b. Effect of phosphorylation of UL42 protein on processivity enhancement 3. Helicase-primase (Primosome) a. Role of UL8 subunit of primosome in its helicase activity b. Analysis of helicase activity c. Analysis of primase activity B. Reconstitution of Oris-dependent DNA replication 1. General approach 2. Examination of specific conditions 3. Transcriptional Activation of transcription C. Further studies of rolling circle DNA replication by a complex of HSV-1 encoded replication enzymes 1. Assembly of complex 2. Mechanism of switch from theta to rolling circle DNA replication.