ENZYMATIC MECHANISMS OF DNA REPLICATION

我们研究的一个工作模型是DNA复制在 酵母染色体起源的复制发生在多个阶段： 解开DNA聚合酶α和叉子对DNA合成的启动 用POL增量和/或POL epsilon组装。这项提案本身就是 分为两部分，第一部分涉及DNA聚合酶。 酵母中有三种基本的dna聚合酶，pola，poldelta. 和polepsilon，DNA需要polalpha和poldelta. 复制。我们必须通过证明 Polepsilon的基本功能是复制或修复。这个 我们研究聚合酶的方法的性质将与过去不同 强调反向遗传学对催化核心亚单位的更多 生化方法。我们将越来越重视刑警组织的作用 (Alpha，因为它显然在耦合解开和 叉子组件。长期目标是分离出每一个的全酶。 复制的DNA聚合酶，然后研究它们是如何相互作用的 在身体上相互联系，以及每个人是如何刺激 另一种是由DNA聚合酶催化的各种模型反应。 在原核生物系统中，没有两个人的范例，更不用说三个了， 基因上不同的聚合酶在DNA复制过程中相互作用，因此 非常基础的研究是有必要的。 赠款的第二部分利用了酵母是 唯一具有复制染色体起源的真核生物 得到了很好的表征，可以进行分离和详细研究。我们 将在ARS上研究蛋白质/DNA和蛋白质/蛋白质的相互作用 元素，酵母复制的起源。ARS结合因子I、ABFI、 我们已经证明它参与了质粒复制，SSB， Rp-A，以及我们已经鉴定的一种新的解旋酶，它也可以与ARS DNA结合 将会被研究。如果发现了其他关键的启动蛋白， 我们还将把这些添加到我们的工作中。我们建议一个或多个 DNA结合蛋白将DNA聚合酶定向到 复制，而正是这种拟议的交互形成了联系 在提案的两个部分之间。 最终，我们想重建一个解体和启蒙 在ARS上用纯化的蛋白质举行的活动。所描述的蛋白质的研究 将提供有用的背景资料。

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.