Structural Basis Of Polymerase Fidelity

工作总结：广泛的遗传学研究已经确定，复制型DNA聚合酶及其相关的校正3 '-外切核酸酶的重复性是突变率的主要决定因素。现在可以获得许多这些酶的结构信息，促进了结构驱动的保真度突变分析。我们已经开发了一种噬菌体系统，允许快速分析的突变体聚合酶在体内的结晶性，而不需要在体外的酶的过表达，纯化和保真度分析。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RB 69 DNA聚合酶（其结构已描述）可以取代正常噬菌体T4 DNA聚合酶，在T4 DNA复制期间保持高保真度。使用改变关键聚合酶和核酸外切酶氨基酸的突变，我们正在体内和体外使用回复突变和正向突变试验来表征突变率的变化。失活校正核酸外切酶或改变聚合酶活性位点中的关键酪氨酸残基强烈增加突变率。当在有或没有辅助蛋白（SSB和夹）的支持下在体外检查突变聚合酶的纯度时，辅助蛋白对保真度的总体影响为零或很小，但在许多特定位点容易观察到大的影响，有时增加，有时降低突变率。

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.