INITIATION OF SV40 DNA REPLICATION AND ITS REGULATION

SV40 DNA复制的启动及其调控

• 批准号: 2685124
• 负责人: PETER Augustus BULLOCK
• 金额: $ 24.76万
• 依托单位: TUFTS UNIVERSITY BOSTON
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-01-01 至 2001-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2685124
• 关键词: DNA replication; DNA replication origin; HeLa cells; disease /disorder model; nucleic acid sequence; plasmids; protein purification; simian virus 40; tissue /cell culture; virus genetics; virus protein; virus replication

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.

关于真核生物中DNA复制的起始知之甚少 生物或这些事件是如何被调节的。启动研究 DNA复制及其调控非常重要，因为这些过程 被认为在各种生物事件中发挥着重要作用， 控制发育和癌症。 猿猴病毒40（SV 40）体外复制系统是一种极好的 用于研究真核生物复制起始的模型。敏感 已经开发了可用于研究SV 40启动的检测方法 在DNA合成之前的事件和复制事件之后的秒数 在DNA合成开始后。同样重要的是，现在可以 研究这些事件的规则;一个理论上可以 发生在任何初始阶段。 本方案中的实验旨在建立该机制 启动SV 40 DNA合成。也是为了 进一步表征启动SV 40的酶要求 DNA复制。最后，本建议中的实验旨在 为了更全面地描述SV 40启动的调节方面， 体外DNA合成。 DNA合成前的起始事件将使用 先前描述的解旋测定。脉冲和脉冲追踪反应 将用于生成新生DNA，包括形成的引物DNA 在DNA合成开始后几秒钟。这些DNA将用于 建立DNA合成起始的机制。类似研究 将定义启动DNA合成的酶促要求。 深入了解dNTP依赖的preinitiation复合物在 SV 40原点附近将通过确定大小获得， 以UR形式的展开区域的位置。得到的知识 从研究SV 40 DNA复制的启动在允许和 非允许性摘录应该提供对启动的见解 染色体DNA复制