ENZYMATIC MECHANISM OF HERPES VIRUS DNA REPLICATION

这项研究计划的目的是了解分子的细节 单纯疱疹病毒复制的机制 1个基因组启动并维持。 我们目前的模式是， 线性病毒基因组环化后，θ型 复制起始于三个HSV-1起点中的一个或多个， 然后切换到滚动循环复制。 我们建议 用纯化的酶在体外重组， 机制，我们将详细研究，无论是θ和滚动圈 HSV-1 DNA复制的阶段。 我们预计，这些研究将 为我们提供了一个重要的类复制的洞察力， 人类病原体。 调查将沿着 以下线路： A. HSV-1编码的结构和机制 复制酶 1.起源结合蛋白（UL 9蛋白）a. 通过UL 9蛋白B解旋口。化学计量的测定 UL 9蛋白与Oris c. UL 9蛋白与 细胞DNA聚合酶α-引发酶d.三个决定- UL 9蛋白2的三维结构。DNA聚合酶-UL 42蛋白 复杂的;复杂的增强的预稳态动力学分析 UL 42蛋白B的持续合成能力。UL 42磷酸化的影响 蛋白质对持续合成能力的增强3.解旋酶-引发酶（Primosome）a. 启动子UL 8亚基在其解旋酶活性中的作用B.分析 解旋酶活性c.引发酶活性分析B.复溶 依赖奥里斯的DNA复制1.一般方法2.审查 具体条件3.转录激活C. HSV-1复合物对滚环DNA复制的进一步研究 编码的复制酶1.复合体2的组装。机制 从theta转换到滚环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.