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.

工作摘要：人类DNA复制的启动需要高度调节聚合酶复合物以及伴侣蛋白和其他酶的组装。及其相关的培根活性的聚合酶α是参与此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磷酸甘氨酸蛋白，一种没有已知功能的70 kDa磷酸蛋白，以及两个含有原始酶活性的49和58 kDa的较小亚基。当前的研究集中在理解保守氨基酸在催化聚合酶亚基的活性位点中的作用，在DNA复制的忠诚度中。特征家族A和B聚合酶在基序上的比对表明，人类pol a的Y865在大肠杆菌DNA pol I中可以用作E710，这对于核糖歧视至关重要。已分析了这种变化的Y865S的效果，以掺入核糖核苷酸。 Y865已更改为丙氨酸，并产生了另外两个突变蛋白Y957A和Y957F。酪氨酸-957在大肠杆菌DNA pol I中的功能类似于Tyr766，这对于忠诚度很重要。这些突变蛋白在我们的杆状病毒系统中被过量生产，以其聚合酶功能和DNA合成保真度的纯化和特征。