ENZYMATIC MECHANISMS OF DNA REPLICATION

我们研究的一个工作模型是复制叉子在 酵母染色体起源的复制发生在多个阶段： DNA聚合酶对DNA合成的识别、解离和启动 Primase，以及Pol Delta和/或Pol epsilon的招募。在一个 复杂的多组分过程，如DNA复制，甚至是最 对单个蛋白质的详尽描述不能描述 实际生理事件的动力学。为了做到这一点， 组件之间的交互，无论有多少，都必须 学习。因此，这项提案的实验主题是设计 生物化学和遗传学实验来描述这种动态 复制蛋白之间的相互作用，特别是解旋酶 解开形成原始体和伸长的聚合酶 仪器。最终，我们想要重建一个解体和 在具有纯化蛋白质的原点上的起始事件。对中国传统文化的研究 这里描述的蛋白质将提供基本的背景和材料 感谢你这么做。 该提案本身分为两部分，第一部分涉及 放松。我们已经鉴定出一种新的酵母突变株，它会影响一种基因 编码一种具有DNA解旋酶活性的蛋白质。它的独特性 解旋酶提示它可能与dna的起始有关。 复制，因为这样的解旋酶可能形成复制的关键 分叉组装，我们将表征解旋酶及其与 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人的进一步描述 复制聚合酶。两种特性最差的聚合酶POL 德尔塔和波尔埃西隆，将被强调。我们的目标是隔离 每一个复制DNA聚合酶的“全酶”，然后调查 他们如何在身体上和功能上相互作用。没有 原核系统中两个的范例，更不用说三个了 不同的聚合酶在DNA复制过程中相互作用，所以非常基本 研究是有必要的。将对Pole进行单抗亲和纯化 被执行。此外，该基因将被用来建立功能 蛋白的C-末端部分，这可能参与 蛋白质/蛋白质相互作用。对于POL三角洲的研究，我们将采用 我们表达催化亚基的能力不受其他因素的影响 在大肠杆菌中纯化55 kDa亚基并测定其 功能。我们将继续使用该基因来鉴定polDelta的 与增殖细胞核抗原的相互作用，增殖细胞核抗原是连接聚合酶的关键蛋白 DNA。我们认为解旋酶将聚合酶导向分叉。 这在提案的两个部分之间形成了联系。

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.