Structural Basis of Polymerase Fidelity

工作总结：以前的遗传学研究 表明复制型DNA聚合酶及其 相关的校对3 '-核酸外切酶是主要的决定因素 突变率。结构信息现在可用于许多 这些酶，促进结构驱动的突变分析 忠诚我们手上有一种新开发的噬菌体 一个系统，允许快速分析的结晶性， 体内突变聚合酶而不需要先前的酶 过表达、纯化和难以进行体内保真度分析。 噬菌体RB 69 DNA聚合酶 由质粒提供的DNA可以取代正常的T4 DNA 当后者突变失活时， 在T4 DNA复制过程中保持高保真度。因为RB 69 聚合酶可以结晶但T4 DNA聚合酶不能， RB 69酶的结构现在是可用的。我们将 提出基于结构的聚合酶假说， 核酸外切酶的功能，并通过突变关键氨基酸来测试它们 并使用回复突变和 正向突变试验。

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.