Structural Basis Of Polymerase Fidelity

工作总结：广泛的遗传学研究已经确定，复制DNA聚合酶及其相关的校对3'-外切酶的保真度是突变率的主要决定因素。现在许多这些酶的结构信息是可用的，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，可以在体内快速分析突变聚合酶的保真度，而不需要酶过表达、纯化和体外保真度分析（尽管后者仍然可用）。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（其结构已描述）可替代正常噬菌体T4 DNA聚合酶。在这个系统中，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，我们利用体内的逆转和正向突变试验以及体外的正向突变试验来描述突变率的最终变化。无论是使校对外切酶失活，还是改变聚合酶活性位点的关键酪氨酸残基，都会大大增加突变率。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，但在许多特定位点容易观察到很大的影响，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 (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.