ENZYMATIC MECHANISMS OF DNA REPLICATION

DNA 复制的酶促机制

• 批准号: 2174470
• 负责人: Judith L CAMPBELL
• 金额: $ 32.5万
• 依托单位: CALIFORNIA INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 1986
• 资助国家: 美国
• 起止时间: 1986-09-01 至 1995-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2174470
• 关键词: DNA binding protein; DNA directed DNA polymerase; DNA primase; DNA repair; DNA replication; active sites; affinity chromatography; enzyme mechanism; enzyme reconstitution; enzyme structure; fungal genetics; gene complementation; helicase; immunoprecipitation; intermolecular interaction; isozymes; molecular cloning; molecular site; phosphorylation; plasmids; protein purification; protein sequence; protein structure function; replicon; spleen exonuclease; temperature sensitive mutant; transcription factor

A working model for our studies is that initiation of DNA replication at a yeast chromosomal origin of replication occurs in multiple stages: unwinding initiation of DNA synthesis by DNA polymerase alpha, and fork assembly with pol delta and/or pol epsilon. The proposal, itself, is divided into two parts, the first of which concerns the DNA polymerases. There are three essential DNA polymerases in yeast, pol alpha, pol delta and pol epsilon, and both pol alpha and pol delta are required for DNA replication. We must resolve one open question by demonstrating whether the essential function of pol epsilon is in replication or repair. The nature of our approach to polymerase studies will then change from past emphasis on reverse genetics on the catalytic core subunits to more biochemical approaches. We will focus increasingly on the role of pol (alpha, because of its apparently central role in coupling unwinding and fork assembly. The long term goal is to isolate holoenzymes of each replicative DNA polymerase and then to investigate how they interact with each other on a physical basis and how each one stimulates the other in the various model reactiolis catalyzed by DNA polymerases. There is no paradigm in prokaryotic systems for two, much less three, genetically distinct polymerases interacting during DNA replication, so very basic studies are warranted. The second part of the grant takes advantage of the fact that yeast is the only eukaryote for which chromosomal origins of replication have been well characterized and can be isolated and studied in detail. We will investigate the protein/DNA and protein/protein interactions at ARS elements, yeast origins of replication. ARS binding factor I, ABFI, which we have shown to be involved in plasmid replication, the SSB, RP-A, and a new helicase we have identified that also binds to ARS DNA will be studied. If other critical initiation proteins are discovered, we will also add these to our work. We propose that one or more of the DNA binding proteins directs DNA polymerase to the origin of replication, and it is this proposed interaction that forms the link between the two parts of the proposal. Ultimately, we would like to reconstitute an unwinding and initiation event at an ARS with purified proteins. Study of the proteins described in this grant will provide useful background.

我们研究的一个工作模型是，DNA复制的起始是在 酵母染色体复制起点发生在多个阶段： 解旋启动DNA聚合酶α和叉的DNA合成 具有pol delta和/或pol delta的组件。 该提案本身是 分为两部分，第一部分涉及DNA聚合酶。 酵母中有三种必需的DNA聚合酶，pol α，pol δ pol alpha和pol delta都是DNA所必需的 复制的 我们必须解决一个悬而未决的问题， 聚合酶的基本功能是复制或修复。 的 我们的聚合酶研究方法的性质将从过去的 强调反向遗传学对催化核心亚基的研究， 生物化学方法。 我们将越来越重视警察的作用， （α，因为它在偶联解旋和 叉组件。 长期目标是分离每种的全酶 然后研究它们如何相互作用 在物理基础上相互作用，以及每个人如何刺激 另一种是由DNA聚合酶催化的各种模型反应。 在原核系统中没有两个的范例，更不用说三个了， 基因不同的聚合酶在DNA复制过程中相互作用， 非常基础的研究是必要的。 赠款的第二部分利用了酵母是 唯一一种染色体复制起点 已经被很好地表征，并且可以被分离和详细研究。 我们 将在ARS研究蛋白质/DNA和蛋白质/蛋白质相互作用 酵母复制起点 ARS结合因子I，ABFI， 我们已经证明它与质粒复制有关，SSB， RP-A，以及我们发现的一种也与ARS DNA结合的新解旋酶 将被研究。 如果发现其他关键起始蛋白， 我们亦会把这些工作纳入我们的工作中。 我们建议一个或多个 DNA结合蛋白将DNA聚合酶引导到 复制，正是这种相互作用形成了联系， 提案的两个部分之间。 最后，我们想重建一个解开和开始 事件在ARS与纯化的蛋白质。 对所述蛋白质的研究 将提供有用的背景资料。