Structural Basis Of Polymerase Fidelity

聚合酶保真度的结构基础

• 批准号: 7169968
• 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/7169968
• 关键词: DNA directed DNA polymerase; DNA replication; active sites; aminoacid; bacterial virus; bacteriophage T4; enzyme activity; enzyme structure; exonuclease; gene mutation; genetic manipulation; high throughput technology; method development; microorganism genetics; mutagen testing; nucleic acid sequence; plasmids; polymerase chain reaction; protein purification; protein structure function

Summary of Work: Extensive genetic studies have established that the fidelities of replicative DNA polymerases and their associated proofreading 3'-exonucleases are the primary determinants of mutation rates. Structural information is now available for many of these enzymes, prompting structure-driven mutational analyses of fidelity. We have developed a bacteriophage system that permits the rapid analysis of the fidelities of mutant polymerases in vivo without a requirement for enzyme over-expression, purification, and fidelity analysis in vitro (although the latter option remains available). Bacteriophage RB69 DNA polymerase (whose structure has been described) supplied from a plasmid can replace the normal bacteriophage T4 DNA polymerase when the latter is mutationally inactivated. In this system, high fidelity is retained during T4 DNA replication. Using mutations that alter critical polymerase and exonuclease amino acids, we are characterizing the resulting changes in mutation rates using both reversion and forward-mutation tests in vivo and forward-mutation tests in vitro. Either inactivating the proofreading exonuclease or altering a key tyrosine residue in the polymerase active site strongly increases mutation rates. When the fidelities of the mutant polymerases are examined in vitro with or without the support of accessory proteins (the SSB and the clamp), overall impacts of the accessory proteins on fidelity are nil or small but large impacts are readily observed at numerous specific sites, sometimes increasing and sometimes decreasing mutation rates.

工作摘要：广泛的遗传学研究已经证实，复制 DNA 聚合酶及其相关校对 3'-核酸外切酶的保真度是突变率的主要决定因素。现在许多酶的结构信息都可用，促进了结构驱动的保真度突变分析。我们开发了一种噬菌体系统，可以在体内快速分析突变聚合酶的保真度，而不需要在体外​​进行酶过度表达、纯化和保真度分析（尽管后一种选择仍然可用）。当正常噬菌体 T4 DNA 聚合酶突变失活时，由质粒提供的噬菌体 RB69 DNA 聚合酶（其结构已被描述）可以替代正常噬菌体 T4 DNA 聚合酶。在此系统中，T4 DNA 复制过程中保持了高保真度。利用改变关键聚合酶和核酸外切酶氨基酸的突变，我们使用体内回复和正向突变测试以及体外正向突变测试来表征突变率的变化。灭活校对核酸外切酶或改变聚合酶活性位点中的关键酪氨酸残基都会大大增加突变率。当在体外检查突变聚合酶的保真度时，有或没有辅助蛋白（SSB 和钳）的支持，辅助蛋白对保真度的总体影响为零或很小，但在许多特定位点很容易观察到较大的影响，有时会增加有时会降低突变率。