Structural Basis of Polymerase Fidelity

工作总结：以前的基因研究已经 表明复制DNA聚合酶的保真度和它们的 相关的校对3‘-核酸外切酶是主要的决定因素 突变率。结构信息现在可供许多人使用 这些酶，促使结构驱动的突变分析 忠诚度。我们手头有一种新研制的噬菌体 一种系统，允许快速分析多个 体内突变聚合酶，不需要先前的酶 体内过表达、纯化和难以进行保真度分析。 远亲噬菌体RB69 DNA聚合酶 可以替代正常的T4DNA 当后者突变失活时，聚合酶，但 在T4 DNA复制过程中保持高保真。因为RB69 聚合酶可以结晶，但T4DNA聚合酶不能， RB69酶的结构现已可用。我们会 阐述基于结构的聚合酶和聚合酶假说 核酸外切酶的功能，并通过突变关键氨基酸来测试它们 并使用反向和反向两种方法测量产生的突变率 正向突变测试。

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.