Vaccinia DNA Replication

痘苗 DNA 复制

• 批准号: 6887690
• 负责人: Paula Traktman
• 金额: $ 37.88万
• 依托单位: MEDICAL COLLEGE OF WISCONSIN
• 依托单位国家: 美国
• 财政年份: 1984
• 资助国家: 美国
• 起止时间: 1984-12-01 至 2009-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6887690
• 关键词: DNA binding protein; DNA directed DNA polymerase; DNA replication; DNA replication origin; N glycosidase; adenosinetriphosphatase; capsid; cytoplasm; enzyme activity; genetic regulatory element; genetically modified animals; laboratory mouse; laboratory rabbit; molecular genetics; protein kinase; protein protein interaction; telomere; vaccinia virus; virion; virus DNA; virus genetics; virus protein; virus replication

DESCRIPTION (provided by applicant): Few processes are as fundamental to biological systems as the faithful duplication of the genetic material. In this proposal, we present our plan for continued investigation of how vaccinia virus coordinates the duplication and encapsidation of its genome. Vaccinia, the prototypic poxvirus, replicates in the cytoplasm of infected cells and possesses a high degree of autonomy from the host. Among the approximately 200 viral genes are those encoding most, if not all, of the repertoire of proteins required for DNA replication. The experimental tractability of this system, amenable to genetic, biochemical, and cell biological dissection, has made vaccinia virus an intriguing model system. The need to gain a deeper understanding of viral replication has become even more compelling in light of a growing fear that life-threatening poxvirus infections might again threaten the human population through acts of bioterrorism. Our plans for the next five years of study are: AIM I: Understanding the mechanisms of genome replication and encapsidation. Our first goal for this aim is to gain an appreciation of the strategies used by the virus to ensure specific and efficient replication of the viral genome within the cytoplasm of infected cells. First, we will explore a new hypothesis that poxviral DNA replication occurs in localized cytoplasmic domains by virtue of protein interactions with intracellular membranes. Second, we will refine our understanding of which cis-acting elements within the terminal 200-bp of the genome are important for efficient DNA replication. Our second goal is to understand the mechanism whereby progeny genomes are encapsidated into nascent virions. We will test a new model which proposes that the interaction of the telomere-bound I6 protein with the virion-bound A32 protein, a putative ATPase, mediates genome packaging. AIM II: Understanding the enzymology of genome replication. Genetic and biochemical analyses have identified a repertoire of proteins involved in vaccinia DNA replication. Our goal for these studies is to understand the assembly of a replication fork complex that can duplicate the genome in a processive manner with high fidelity. The E9 DNA polymerase, A20 processivity factor, D4 uracil DNA glycosylase, D5 NTPase, I3 single-strand DNA binding protein, and B1 protein kinase will be the major topics of study.

描述（由申请人提供）：对于生物系统来说，很少有过程比遗传物质的忠实复制更重要。在这项提案中，我们提出了继续研究痘苗病毒如何协调其基因组的复制和衣壳化的计划。痘苗病毒是一种原型痘病毒，在受感染细胞的细胞质中复制，并具有相对于宿主的高度自主性。大约 200 个病毒基因中的基因编码了 DNA 复制所需的大部分（如果不是全部）蛋白质。该系统的实验易处理性，适合遗传、生化和细胞生物学解剖，使牛痘病毒成为一个有趣的模型系统。由于人们越来越担心危及生命的痘病毒感染可能会通过生物恐怖主义行为再次威胁人类，因此更深入地了解病毒复制的必要性变得更加迫切。我们未来五年的学习计划是： 目标 I：了解基因组复制和衣壳化的机制。我们的首要目标是了解病毒所使用的策略，以确保病毒基因组在受感染细胞的细胞质内进行特异性和有效的复制。首先，我们将探索一个新的假设，即痘病毒 DNA 复制通过蛋白质与细胞内膜的相互作用发生在局部细胞质结构域中。其次，我们将加深对基因组末端 200 bp 内哪些顺式作用元件对于有效 DNA 复制很重要的理解。我们的第二个目标是了解后代基因组被包裹成新生病毒粒子的机制。我们将测试一个新模型，该模型提出端粒结合的 I6 蛋白与病毒体结合的 A32 蛋白（一种假定的 ATP 酶）的相互作用介导基因组包装。 目标 II：了解基因组复制的酶学。遗传和生化分析已经鉴定出参与牛痘 DNA 复制的一系列蛋白质。我们这些研究的目标是了解复制叉复合体的组装，该复合体可以以高保真度的持续方式复制基因组。 E9 DNA聚合酶、A20持续因子、D4尿嘧啶DNA糖基化酶、D5 NTPase、I3单链DNA结合蛋白和B1蛋白激酶将是主要研究课题。