Structural Basis of Polymerase Fidelity

聚合酶保真度的结构基础

• 批准号: 6106738
• 负责人: JOHN W. DRAKE
• 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/6106738
• 关键词: DNA directed DNA polymerase; DNA replication; bacteriophage T4; enzyme activity; enzyme structure; gene mutation; plasmids; protein structure function

Summary of Work: Previous genetic studies have shown that the fidelities of replicative DNA polymerases and their associated proofreading 3'-exonucleases are the main determinants of mutation rates. Structural information is now available for many of these enzymes, prompting structure-driven mutational analyses of fidelity. We have in hand a newly developed bacteriophage system that permits the rapid analysis of the fidelities of many mutant polymerases in vivo without a requirement for prior enzyme over-expression, purification, and difficult fidelity analysis in vivo. The distantly related bacteriophage RB69 DNA polymerase supplied from a plasmid can replace the normal T4 DNA polymerase when the latter is mutationally inactivated, and yet retain high fidelity during T4 DNA replication. Because the RB69 polymerase can be crystallized but the T4 DNA polymerase cannot, the structure of the RB69 enzyme is now available. We will formulate structure-based hypotheses of polymerase and exonuclease function and test them by mutating critical amino acids and measuring the resulting mutation rates using both reversion and forward- mutation tests.

工作总结：以前的基因研究已经