Structural Basis Of Polymerase Fidelity

工作总结：广泛的遗传学研究已经证实，复制DNA聚合酶及其相关的校对3‘-核酸外切酶的保真度是突变率的主要决定因素。现在这些酶中的许多都有结构信息，促使对保真度进行结构驱动的突变分析。我们已经开发了一种噬菌体系统，它允许在体内快速分析突变聚合酶的保真度，而不需要酶的过度表达、纯化和体外保真度分析。噬菌体RB69DNA聚合酶(其结构已描述)可在正常噬菌体T4DNA聚合酶突变失活时取代后者，在T4DNA复制过程中保持高保真。利用改变关键聚合酶和核酸外切酶氨基酸的突变，我们正在利用体内和体外的反向和正向突变测试来表征由此产生的突变率的变化。无论是失活校对核酸外切酶，还是改变聚合酶活性部位的一个关键酪氨酸残基，都会极大地增加突变率。当在体外检测突变聚合酶的保真度时，无论是否有辅助蛋白(SSB和夹子)的支持，辅助蛋白对保真度的总体影响为零或很小，但在许多特定位置很容易观察到较大的影响，有时增加，有时减少突变率。

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. 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, retaining high fidelity 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 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.