ENZYMATIC MECHANISMS OF DNA REPLICATION

DNA 复制的酶促机制

• 批准号: 2518895
• 负责人: Judith L CAMPBELL
• 金额: $ 33.12万
• 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 1998-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2518895
• 关键词: DNA; DNA binding protein; DNA directed DNA polymerase; DNA primase; DNA replication; DNA replication origin; active sites; adenosinetriphosphatase; chemical kinetics; enzyme mechanism; enzyme reconstitution; enzyme structure; enzyme substrate; epitope mapping; fungal genetics; helicase; immunoaffinity chromatography; immunoprecipitation; intermolecular interaction; protein purification; protein sequence; protein structure function; replicon; site directed mutagenesis; temperature sensitive mutant

A working model for our studies is that assembly of a replication fork at a yeast chromosomal origin of replication occurs in multiple stages: recognition, unwinding, priming of DNA synthesis by DNA polymerase alpha - primase, and recruitment of pol delta and/or pol epsilon. In a complicated, multicomponent process such as DNA replication, even the most exhaustive characterization of the individual proteins cannot describe the dynamics of the actual physiological events. In order to do that, interaction between the components, no matter how many there are, must be studied. The experimental theme of this proposal is therefore to design both biochemical and genetic experiments to characterize the dynamic interactions between the replication proteins, especially the helicase that unwinds and the polymerases that form the primosome and elongation apparatus. Ultimately we would like to reconstitute an unwinding and initiation event at an origin with purified proteins. Study of the proteins described herein will provide essential background and materials for doing so. The proposal itself is divided into two parts, the first of which concerns unwinding. We have identified a new yeast mutant that affects a gene encoding a protein with DNA helicase activity. The specificity of the helicase suggests that it may be involved in the initiation of DNA replication and since such a helicase may form the linchpin of replication fork assembly, we will characterize the helicase and its interactions with the Origin Replication Complex, ORC, and the DNA polymerases. The second part of the proposal deals with further characterization of the three replication polymerases. The two least well characterized polymerases, pol delta and pol epsilon, will be emphasized. The goal is to isolate "holoenzymes" of each replicative DNA polymerase and then to investigate how they interact with each other physically and functionally. 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. Monoclonal affinity purification of pol e will be carried out. Furthermore, the gene will be used to establish the function of the C-terminal portion of the protein, which may be involved in protein/protein interactions. For studies of pol delta, we will take advantage of our ability to express the catalytic subunit free of other subunits in E. coli to purify the 55 kDa subunit and determine its function. We will continue using the gene to characterize pol delta's interaction with PCNA, the critical protein that tethers the polymerase to the DNA. We propose that a helicase directs the polymerases to the fork and this forms a link between the two parts of the proposal.

我们研究的一个工作模型是在以下位置组装复制叉： 酵母染色体复制起点发生在多个阶段： DNA 聚合酶 α 识别、解旋、引发 DNA 合成 - 引物酶，以及 pol δ 和/或 pol epsilon 的募集。在一个 复杂的多组分过程，例如 DNA 复制，甚至是最复杂的过程 单个蛋白质的详尽表征无法描述 实际生理事件的动态。为了做到这一点， 组件之间的交互，无论有多少个，都必须是 研究过。因此，本提案的实验主题是设计 生化和遗传实验来表征动态 复制蛋白之间的相互作用，尤其是解旋酶 解旋以及形成引发体和延伸的聚合酶 设备。最终我们希望重建一个放松和 纯化蛋白质的起始事件。研究的 本文描述的蛋白质将提供必要的背景和材料 因为这样做。 该提案本身分为两部分，第一部分涉及 放松。我们发现了一种影响基因的新酵母突变体 编码具有DNA解旋酶活性的蛋白质。的特殊性 解旋酶表明它可能参与 DNA 的起始 复制并且因为这样的解旋酶可能形成复制的关键 叉组装，我们将描述解旋酶及其与 起源复制复合体、ORC 和 DNA 聚合酶。第二个 该提案的一部分涉及对这三个因素的进一步定性 复制聚合酶。两种最不明确的聚合酶，pol 将强调 delta 和 pol epsilon。目标是隔离 每个复制 DNA 聚合酶的“全酶”，然后进行研究 他们如何在身体和功能上相互作用。没有 原核系统中两个，更不用说三个，遗传上的范式 DNA复制过程中不同的聚合酶相互作用，所以非常基本 研究是有必要的。 pol e的单克隆亲和纯化将是 执行。此外，该基因将用于建立功能 蛋白质的 C 末端部分，可能涉及 蛋白质/蛋白质相互作用。对于 pol delta 的研究，我们将采取 利用我们能够表达不含其他物质的催化亚基的能力 大肠杆菌中的亚基纯化 55 kDa 亚基并测定其 功能。我们将继续使用该基因来表征 pol delta 与 PCNA 相互作用，PCNA 是连接聚合酶的关键蛋白 DNA。我们建议解旋酶将聚合酶引导至叉子 这形成了提案的两个部分之间的联系。