ENZYMATIC MECHANISMS OF DNA REPLICATION

我们研究的一个工作模型是，复制叉在 酵母染色体复制起点发生在多个阶段： 识别，解旋，启动DNA聚合酶α- 引发酶和pol δ和/或pol β的募集。中 复杂的，多组分的过程，如DNA复制，即使是最 对单个蛋白质的详尽表征不能描述 实际生理事件的动力学。为了做到这一点， 组件之间的相互作用，无论有多少，必须是 研究了因此，本提案的实验主题是设计 生物化学和遗传学实验来描述 复制蛋白之间的相互作用，特别是解旋酶 而聚合酶则形成了原始体和延伸 设备.最终，我们希望重建一个解旋， 起始事件在原点与纯化的蛋白质。研究 本文所述的蛋白质将提供必要的背景和材料 因为你这么做了 建议本身分为两部分，第一部分涉及 放松我们发现了一种新的酵母突变体 编码具有DNA解旋酶活性的蛋白质。的特异性 解旋酶表明它可能参与DNA的起始 由于这种解旋酶可以形成复制的关键， 叉组装，我们将表征解旋酶及其相互作用， 起源复制复合体ORC和DNA聚合酶。第二 提案的一部分涉及对这三个问题的进一步定性， 复制聚合酶。两种最不好表征的聚合酶，pol 三角洲和波利尼西亚，将被强调。我们的目标是隔离 每个复制DNA聚合酶的“全酶”，然后研究 它们是如何在物理和功能上相互作用的。没有 在原核系统中，两个，更不用说三个， DNA复制过程中不同的聚合酶相互作用， 研究是必要的。pol e的单克隆亲和纯化将是 贯彻此外，该基因将用于建立功能 蛋白质的C-末端部分，这可能涉及 蛋白质/蛋白质相互作用。对于PolDelta的研究，我们将 我们表达催化亚基的能力的优势， 亚基E.大肠杆菌纯化55 kDa亚基，并测定其 功能我们将继续用这个基因来描述德尔塔的 与PCNA的相互作用，PCNA是将聚合酶拴在一起的关键蛋白， DNA我们认为解旋酶将聚合酶引导到分叉处 这是建议的两个部分之间的联系。

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.