INITIATION OF SV40 DNA REPLICATION AND ITS REGULATION

对真核生物中DNA复制的启动知之甚少 生物体或这些事件是如何被调控的。关于儿童心理健康问题的研究 DNA复制及其调控非常重要，因为这些过程 被认为在不同的生物事件中发挥核心作用，例如 控制发展和癌症。 猿猴病毒40(SV40)体外复制系统是一种优秀的 真核生物复制起始研究的模型。敏感 已开发出可用于研究SV40启动的检测方法 DNA合成之前的事件和复制事件秒之后的事件 在DNA合成启动之后。同样重要的是，现在有可能 检查这些事件的规则；一个从理论上可以 发生在启蒙的任何阶段。 本方案中的实验旨在建立该机制 在体外启动SV40 DNA合成。它们还被设计成 进一步鉴定启动SV40的酶的要求 DNA复制。最后，本方案中的实验是预期的 更全面地描述启动SV40的规则的各个方面 DNA的体外合成。 在DNA合成之前的启动事件将使用 先前描述的解卷分析。脉冲和脉冲追逐反应 将被用来产生新生DNA，包括形成的引物DNA 在DNA合成开始后几秒钟。这些DNA将被用来 建立DNA合成的启动机制。类似的研究 将定义启动DNA合成的酶需求。 对dNTP依赖于预引发复合体的洞察 SV40原点的附近将通过确定大小和 形式为UR的展开区域的位置。派生出的知识 SV40 DNA复制启动的研究 非许可提取物应提供对启动 染色体DNA复制。

Little is known about initiation of DNA replication in eukaryotic organisms or how these events are regulated. Studies of initiation of DNA replication and its regulation are important because these processes are believed to play central roles in diverse biological events, such as control of development and cancer. The Simian Virus 40 (SV40) in vitro replication system is an excellent model for studies of initiation of eukaryotic replication. Sensitive assays have been developed that can be used to study SV40 initiation events prior to DNA synthesis and to follow replication events seconds after DNA synthesis initiates. Equally important, it is now possible to examine the regulation of these events; a process that can theoretically occur at any stage of initiation. The experiments in this proposal are designed to establish the mechanism of initiation of SV40 DNA synthesis in vitro. They are also designed to further characterize the enzymatic requirements for initiation of SV40 DNA replication. Finally, the experiments in this proposal are intended to more fully describe aspects of the regulation of initiation of SV40 DNA synthesis in vitro. Initiation events prior to DNA synthesis will be followed using previously described unwinding assays. Pulse and pulse-chase reactions will be used to generate nascent DNA, including primer-DNA, formed seconds after the start of DNA synthesis. These DNAs will be used to establish the mechanism of initiation of DNA synthesis. Similar studies will define the enzymatic requirements for initiation of DNA synthesis. Insights into the dNTP dependent of the preinitiation complex in the vicinity of the SV40 origin will be obtained by determining the size and position of the unwound region in the form UR. The knowledge derived from studies of initiation of SV40 DNA replication in permissive and nonpermissive extracts should provide insights into initiation of chromosomal DNA replication.