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，而不需要在体内的酶的过表达，纯化和保真度分析。 当正常的T4 DNA聚合酶突变失活时，由质粒提供的噬菌体RB 69 DNA聚合酶可以取代正常的T4 DNA聚合酶，在T4 DNA复制期间保持高保真度。 描述RB 69 DNA聚合酶的结构是因为它可以结晶，而T4 DNA聚合酶不能。 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。 我们已经改变了活性位点中的一个关键残基，以产生一种强增变聚合酶，该聚合酶优先产生转换突变。 我们发现，增变基因突变引起的增加因子在体内测量时高于体内测量。 我们还发现，聚合酶突变子和核酸外切酶缺陷突变子的组合突变子活性并不比单独的突变子活性大得多。 在体内和体内的增变子特异性之间也观察到明显的差异。

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 vivo. Bacteriophage RB69 DNA polymerase supplied from a plasmid can replace the normal T4 DNA polymerase when the latter is mutationally inactivated, retaining high fidelity during T4 DNA replication. The structure of the RB69 DNA polymerase is described because it can crystallize, whereas the T4 DNA polymerase cannot. 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. We have altered a key residue in the active site to produce a strong mutator polymerase that preferentially produces transition mutations. We find that factors of increase caused by mutator mutations are higher when measured in vivo than in vivo. We also find that the combined mutator activity of a polymerase mutator and an exonuclease-defective mutator is not much greater than either alone. Clear differences are also seen between mutator specificities in vivo and in vivo.