STUDIES ON EUKARYOTIC DNA REPLICATION

真核DNA复制研究

• 批准号: 6476456
• 负责人: Jerard Hurwitz
• 金额: $ 71.03万
• 依托单位: SLOAN-KETTERING INST CAN RESEARCH
• 依托单位国家: 美国
• 财政年份: 1984
• 资助国家: 美国
• 起止时间: 1984-07-01 至 2003-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6476456
• 关键词: Baculoviridae; DNA binding protein; DNA directed DNA polymerase; DNA primase; DNA repair; DNA replication; DNA replication origin; Schizosaccharomyces pombe; enzyme mechanism; eukaryote; fungal genetics; genetic manipulation; intermolecular interaction; molecular cloning; proliferating cell nuclear antigen; protein biosynthesis; protein reconstitution; simian virus 40; translation factor; virus DNA

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.

尽管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，但我们对这一复杂大分子事件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标，但我们对其作用方式的信息知之甚少。 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节的关键信号。我们的目标是研究真核复制机制关键酶组分的功能。 特别是，我们克隆并表达了复制因子 C (RFC)，这是将增殖细胞核抗原 (PCNA) 加载到 DNA 上所需的关键辅助五亚基复合物，而 DNA 聚合酶活性是持续进行的。 我们计划 a) 确定 RFC 打开 PCNA 环并将 PCNA 与 DNA 拓扑连接的机制； b) 检查 RFC 在从引物 DNA 模板中置换 pol α-引物酶复合物中的作用（聚合酶转换）； c) 研究Rad17与RFC之间的相互作用对RFC酶学性质的影响。 Rad17 是检查点调节通路的一个组成部分； d) 检查来自粟酒裂殖酵母的 DNA 聚合酶 δ 的作用机制，该酶至少包含四个不同的亚基，并且已被发现是一种二聚体聚合酶。 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复合物的特性。 e) 我们已经分离了粟酒裂殖酵母起源识别复合物（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参与关键复合物形成的其他关键蛋白质的相互作用进行实验。用于启动 DNA 合成。