Mammalian DNA Polymerase Alpha Primase Complex

哺乳动物 DNA 聚合酶 Alpha 引物酶复合物

• 批准号: 6535120
• 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/6535120
• 关键词: DNA directed DNA polymerase; DNA primase; DNA repair; DNA replication; Escherichia coli; active sites; aminoacid; enzyme activity; enzyme complex; 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. 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 understanding the role of conserved amino acids in the active site of the catalytic polymerase subunit in the fidelity of DNA replication. Alignment of characteristic family A and B polymerases over motif A suggested that Y865 of human pol a may function as E710 in E. coli DNA pol I which is critical for ribose discrimination. The effect of this change, Y865S, has been analyzed for the incorporation of ribonucleotides. Y865 has been changed to alanine and two additional mutant proteins, Y957A and Y957F, were produced. Tyrosine-957 is functionally analogous to Tyr766 in E. coli DNA pol I which is important for fidelity. These mutant proteins are being overproduced in our baculovirus system, purified and characterized for their polymerase function and DNA synthetic fidelity.

工作总结：人类 DNA 复制的启动需要聚合酶复合物以及伴侣蛋白和其他酶的高度调节组装。具有相关引物酶活性的聚合酶α是参与该起始过程的唯一聚合酶。该酶复合物由四种蛋白质组成：一种含有 DNA 聚合酶活性的 180 kDa 磷酸糖蛋白、一种功能未知的 70 kDa 磷蛋白以及含有引物酶活性的两个较小的 49 和 58 kDa 亚基。目前的研究重点是了解催化聚合酶亚基活性位点的保守氨基酸在 DNA 复制保真度中的作用。特征家族 A 和 B 聚合酶与基序 A 的比对表明，人 pol a 的 Y865 可能充当大肠杆菌 DNA pol I 中的 E710，这对于核糖辨别至关重要。已经分析了这种变化 Y865S 的影响，以了解核糖核苷酸的掺入。 Y865 已更改为丙氨酸，并产生了另外两种突变蛋白 Y957A 和 Y957F。 Tyrosine-957 在功能上类似于大肠杆菌 DNA pol I 中的 Tyr766，这对于保真度很重要。这些突变蛋白在我们的杆状病毒系统中过量产生，并对其聚合酶功能和 DNA 合成保真度进行纯化和表征。