CONTROL OF SIMIAN VIRUS 40 AND CELLULAR DNA REPLICATION

我们提出的研究的长期目标是在分子水平上阐明 详述在哺乳动物细胞中控制DNA复制的机制。 乳头病毒DNA在感染细胞和无细胞条件下的复制 反应已被证明是一个非常有用的模型系统。它有 帮助确定和表征了许多 复制所需的细胞蛋白质并导致了一种基本的 了解SV40 DNA复制的机制。具体目标 在这项新申请中提出的研究计划如下： 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 以测试磷酸化状态控制的想法 T抗原六聚体之间的协同作用 解开双链起源DNA。2)细胞周期特异性的作用 DNA聚合酶α-启动子和复制蛋白A的磷酸化 (RP-A)在调节其病毒DNA复制活性方面将 检查过了。3)聚合酶物种特异性的分子基础 将对SV40中的α-Primase和多瘤DNA复制进行研究。 纯化的重组鼠-人杂交聚合酶α-引物将被 测试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，并使用杂交酶进行研究 它们与T抗原和RP-A的物理和功能相互作用 体外培养。4)基因功能所需的顺式作用元件 将确定哺乳动物染色体中DNA复制的起源 使用竞争性的聚合酶链式反应方法。

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.