CONTROL OF SIMIAN VIRUS 40 AND CELLULAR DNA REPLICATION

猿病毒 40 和细胞 DNA 复制的控制

• 批准号: 2192159
• 负责人: ELLEN Hope FANNING
• 金额: $ 24.43万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-08-01 至 1999-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2192159
• 关键词: DNA binding protein; DNA primase; DNA replication; chimeric proteins; genetic regulatory element; phosphorylation; polymerase chain reaction; recombinant proteins; simian virus 40; tissue /cell culture; virus antigen; virus infection mechanism

The long-term goal of our proposed research is to elucidate in molecular detail the mechanisms that control DNA replication in mammalian cells. Replication of papovaviral DNA in infected cells and in cell-free reactions has proven to be extremely useful as a model system. It has facilitated the identification and characterization of many of the cellular proteins required for replication and has led to a basic understanding of the mechanism of SV40 DNA replication. The specific aims of the research program proposed in this new application are as follows: 1) The phosphorylation state of the SV40 replication protein T antigen governs its ability to initiate viral DNA replication. Work will be performed to test the idea that the phosphorylation state controls cooperative interactions between T antigen hexamers that are required to unwind duplex origin DNA. 2) The role of cell cycle-specific phosphorylation of DNA polymerase alpha-primase and replication protein A (RP-A) in regulating their viral DNA replication activities will be examined. 3) The molecular basis for the species-specificity of polymerase alpha-primase in SV40 and polyoma DNA replication will be investigated. Purified recombinant mouse-human hybrid polymerase alpha-primases will be tested for their ability to replicate viral DNA in vitro. Based on these results, we will attempt to establish cell lines with altered species- specificity for viral DNA replication, and use the hybrid enzymes to study their physical and functional interactions with T antigen and RP-A in vitro. 4) Cis-acting elements that are required for function of a genetic original of DNA replication in mammalian chromosomes will be identified using a competitive PCR approach.

我们提出的研究的长期目标是阐明分子 详细说明控制哺乳动物细胞中DNA复制的机制。 在感染细胞和无细胞中复制木瓜病毒DNA 事实证明，反应作为模型系统非常有用。 它有 促进了许多许多人的识别和表征 复制所需的细胞蛋白，并导致了基本 了解SV40 DNA复制机制。 具体目标 在此新应用程序中提出的研究计划中如下： 1）SV40复制蛋白T抗原的磷酸化状态 控制其启动病毒DNA复制的能力。 工作将是 执行以测试磷酸化状态控制的想法 T抗原六聚体之间需要的合作互动 解开双链源DNA。 2）细胞周期特异性的作用 DNA聚合酶α-蛋白酶和复制蛋白A的磷酸化 （RP-A）在调节其病毒DNA复制活动时将是 检查。 3）聚合酶物种特异性的分子基础 将研究SV40中的α-蛋白酶和多瘤DNA复制。 纯化的重组小鼠 - 人类杂种聚合酶α-蛋白酶将是 测试了它们在体外复制病毒DNA的能力。 基于这些 结果，我们将尝试建立具有改变物种的细胞系 - 病毒DNA复制的特异性，并使用杂化酶进行研究 它们与T抗原和RP-A的物理和功能相互作用 体外。 4）遗传功能所需的顺式作用元素 将鉴定出哺乳动物染色体中DNA复制的原始复制 使用竞争性PCR方法。