PROBING STRUCTURE-FUNCTION RELATIONSHIPS WITH DNA POLYMERASES

我们正在使用通过重组DNA技术获得的突变酶， 研究结构和动力学性质之间的关系， DNA聚合酶及其保真度 核酸外切酶缺陷的保真度 Klenow聚合酶已被检查为反应pH的函数。 当pH从7.6降低到6.2时，保真度增加超过10倍， 但是速率对于所有错误的影响并不相同。 这些非随机 影响与稳态动力学分析一致，表明 降低pH降低了错配延伸合成的效率。 终止概率的变化超过20倍， 在沿着模板DNA沿着的各个位点观察到作为pH的函数， 并且增加的终止与增加的保真度相关。 这些数据 这表明在低pH下高保真度可能是由于 由于持续合成能力增加而形成突变前中间体 聚合酶和减少固定的突变，由于减少 未对齐或错配的中间体的延伸。 我们还扩大了 分析了两个新的突变体，其中766位的酪氨酸残基， 聚合酶活性位点的“O螺旋”变为丙氨酸或 丝氨酸 两种突变体的保真度都较低，这表明酪氨酸 已知结合dNTP底物的残基对于确定碱基 选择性 我们正在定义详细的错误 这些改变的聚合酶的特异性。 我们也在研究突变体 其他几种重组DNA聚合酶的衍生物，包括HIV-1 逆转录酶、DNA聚合酶β、T7 DNA聚合酶和T4 DNA 聚合酶。 这些酶的分析，其中相当大的结构 和/或动力学数据，应该提高我们的理解 精确的DNA合成以及保真度如何受到DNA加合物的影响。

We are using mutant enzymes obtained by recombinant DNA technology to examine the relationship between the structural and kinetic properties of DNA polymerases and their fidelity. The fidelity of exonuclease-deficient Klenow polymerase has been examined as a function of reaction pH. Overall fidelity increase more than 10-fold as the pH is lowered from 7.6 to 6.2, but rates are not affected equally for all errors. These non-random effects are consistent with steady-state kinetic analyses indicating that lowering the pH lowers the efficiency of mispair extension synthesis. Variations in the termination probabilities of more than 20-fold were observed at individual sites along the template DNA as a function of pH, and increased termination correlated with increased fidelity. These data suggest that high fidelity at low pH may result from both decreased formation of premutational intermediates due to the increased processivity of the polymerase and from decreased fixation of mutations due to decreased extension of misaligned or mispaired intermediates. We also extended the analysis to two new mutants in which a tyrosine residue at position 766, in the "O helix" of the polymerase active site, was changed to an alanine or serine. Both mutants have lower fidelity, suggesting that the tyrosine residue, known to bind dNTP substrates, is important for determining base selectivity. We are in the process of defining the detailed error specificity of these altered polymerases. We are also examining mutant derivatives of several other recombinant DNA polymerases, including HIV-1 reverse transcriptase, DNA polymerase beta, T7 DNA polymerase and T4 DNA polymerase. Analyses of these enzymes, for which considerable structural and/or kinetic data are available, should improve our understanding of accurate DNA synthesis and how fidelity is affected by DNA adducts.