STUDIES ON EUKARYOTIC DNA REPLICATION

虽然在阐明真核复制如何发生的大致轮廓方面已经取得了相当大的进展，但我们对这一复杂的大分子事件的细节的了解还远远不清楚。许多已被证明对DNA复制至关重要的酶被发现在DNA修复和重建中发挥作用。DNA修复和复制都是许多抗癌药物的重要靶点，但我们对它们的作用方式知之甚少。如果我们要防止异常生长，我们必须了解控制DNA复制及其细胞周期调控的关键信号。我们的目标是研究真核生物复制机制的关键酶组分的功能。特别是，我们克隆并表达了复制因子C(RFC)，这是一种关键的附属五亚单位复合体，需要将增殖细胞核抗原(PCNA)装载到DNA上，这是进行DNA聚合酶活动所必需的。我们计划a)确定RFC打开增殖细胞核抗原环并在拓扑上将PCNA与DNA连接的机制；b)检测RFC在其从启动的DNA模板中置换Pola-Primase复合体中的作用(聚合酶切换)；c)研究RFC与RFC之间的相互作用对RFC酶性质的影响。RAD17是检查点调节途径的一个组成部分；d)检测来自裂殖酵母的DNA聚合酶Delta的作用机制，它至少包含四个不同的亚基，已被发现是一个二聚体聚合酶。我们已经克隆并表达了所有四个亚基，并计划研究和比较全酶和各种亚基的性质。E)我们已经分离了S.pombe起源识别复合体(ORC)，并计划克隆和表达所有六个亚基，以便对其与起源序列以及与其他关键蛋白质的相互作用进行实验，这些蛋白质参与了对启动DNA合成至关重要的复合体的形成。

Though considerable progress has been made in elucidating the broad outline of how eukaryotic replication occurs, our knowledge of the details of this complicated macromolecular event is far from clear. Many of the enzymes that have been shown to be critical for DNA replication have been found to play roles in DNA repair and reconstruction. Both DNA repair and replication are important targets of many anticancer agents but we have little information about their mode of action. If we are to prevent aberrant growth, we must understand the critical signals governing DNA replication and its cell cycle regulation. Our goal is to study the function of key enzymatic components of the eukaryotic replication machinery. In particular, we have cloned and expressed replication factor C (RFC), a key accessory five subunit complex required to load proliferating cell nuclear antigen (PCNA) onto DNA which is required for processive DNA polymerase activities. We plan to a) determine the mechanism by which RFC opens the PCNA ring and topologically links PCNA to DNA; b) examine the role of RFC in its displacement of the pol alpha-primase complex from primed DNA templates (polymerase switching); c) study the influence of the interaction between Rad17 with RFC on the enzymatic properties of RFC. Rad17 is a component of the checkpoint regulation pathway; d) examine the mechanism of action of DNA polymerase delta from Schizosaccharomyces pombe which contains at least four distinct subunits, and has been found to be a dimeric polymerase. We have cloned and expressed all four subunits and plan to study and compare the properties of the holoenzyme and various subcomplexes. e) We have isolated the S. pombe origin recognition complex (ORC) and plan to clone and express all six subunits in order to carry out experiments on its interactions with origin sequences and with other key proteins that are involved in the formation of complexes critical for the initiation of DNA synthesis.