MECHANISM OF THE MAMMALIAN DNA POLYMERASE ALPHA PRIMASE COMPLEX

哺乳动物 DNA 聚合酶 α 引物酶复合物的机制

• 批准号: 6106748
• 负责人: William Copeland
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF ENVIRONMENTAL HEALTH SCIENCES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6106748
• 关键词: DNA directed DNA polymerase; DNA primase; DNA repair; DNA replication; Escherichia coli; complementary DNA; enzyme activity; enzyme complex; human genetic material tag; intermolecular interaction; molecular cloning; nucleic acid sequence

Summary of Work: Initiation of human DNA replication entails a highly regulated assembly of polymerase complexes along with chaperone proteins and other enzymes. The polymerase alpha with its associated primase activity is the only polymerase involved in this initiation process. Our research is concentrated on understanding the regulation and mechanism of the human DNA polymerase alpha - primase complex. This enzyme complex is composed of four proteins, a 180 kDa phospho-glyco protein containing the DNA polymerase activity, a 70 kDa phosphoprotein with no known function, and two smaller subunits of 49 and 58 kDa containing the primase activity. Current research is focused on the enzymatic mechanism and regulation of the two primase subunits. Previously we had cloned and overexpressed the cDNA for the two human DNA primase subunits (Hp49 and Hp58) and purified the proteins from E. coli. This overexpression system was used to study the protein interaction between the two primase subunits. We determined that protein-protein contacts between the two subunits are made over several domains within the Hp58 subunit and most of this interaction is essential for primer initiation by the Hp49 subunit. With our development of a high yield expression system we have entered into a collaboration with Dr. Silvia Onesti (Imperial College, London) to crystallize the two human primase subunits and determine the three dimensional structure. Using highly purified primase subunits in single form and as a heterodimeric complex, crystal growth conditions are being discovered for optimal X-ray diffraction. The three dimensional structure of the primase complex will help elucidate the complex chemistry of primer formation and open up the field for structure specific design of inhibitors which possibly may be used in chemotheropeutics. We have also entered into a collaboration with Dr. Rob Kuchta, Bolder, CO, using Hp58 deletion mutants to study the role of Hp58 in primer initiation.

工作摘要：人类DNA的启动 复制需要高度调节的聚合酶组装 复合物以及伴侣蛋白和其他酶。这 聚合酶α及其相关的培根酶活性是唯一的 聚合酶参与此开始过程。我们的研究是 专注于理解调节和机制 人DNA聚合酶α-原始酶复合物。这种酶 复合物由四种蛋白质组成，一种180 kDa磷酸化蛋白 含有DNA聚合酶活性的蛋白质70 kDa 没有已知功能的磷蛋白，两个较小的亚基 49和58 kDa含有原始酶活性。当前的研究 专注于两者的酶促机制和调节 原始亚基。以前我们已经克隆并过表达 两种人DNA原始酶亚基的cDNA（HP49和HP58） 并从大肠杆菌中纯化蛋白质。这个过表达系统 被用来研究两种原始酶之间的蛋白质相互作用 亚基。我们确定蛋白质 - 蛋白质接触 在HP58中的几个域中制作了两个亚基 亚基和大多数这种相互作用对于底漆启动至关重要 由HP49亚基。随着我们高产的发展 表达系统我们已经与Dr. 西尔维亚·奥特斯蒂（Silvia Onesti）（伦敦帝国学院）将两者结晶 人类原始子亚基并确定三维 结构。使用高度纯化的单一形式的原始原始子亚基和 作为异二聚体复合物，晶体生长条件正在 发现用于最佳X射线衍射。三维 原始酶复合物的结构将有助于阐明复合物 底漆形成的化学性质并打开田间的结构 可能在 化学治疗学。我们还与 Bolder，CO Rob Kuchta博士，使用HP58缺失突变体来研究 HP58在底漆启动中的作用。