PROBING STRUCTURE-FUNCTION RELATIONSHIPS WITH DNA POLYMERASES

我们正在使用通过重组DNA技术获得的突变酶来 考察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聚合酶已被检测为反应pH的函数。总括 当PH值从7.6降低到6.2时，保真度增加了10倍以上， 但并不是所有的错误都会对比率产生同样的影响。这些非随机的 这种影响与稳态动力学分析相一致，表明 降低pH会降低错对延伸合成的效率。 终止概率的变化超过20倍是 在沿着模板DNA的各个位置观察到作为pH的函数， 更高的终止率与更高的保真度相关。这些数据 表明在低pH值下的高保真度可能是由于两者的降低所致 由于加工性增加而形成的突变前中间产物 由于聚合酶的减少和由于减少而导致的突变固定减少 错位或错配中间体的延伸。我们还扩展了 新发现的766位酪氨酸残基突变体的分析 聚合酶活性部位的“O螺旋”被改变为丙氨酸或 丝氨酸。两个突变体的保真度都较低，这表明酪氨酸 已知的与dNTP底物结合的残基对碱基的确定很重要 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。我们还在检查变种人 包括HIV-1在内的其他几种重组DNA聚合酶的衍生物 逆转录酶、DNA聚合酶β、T7DNA聚合酶和T4DNA 聚合酶。对这些酶的分析，对其相当大的结构 和/或动力学数据，应该会提高我们对 准确的DNA合成以及DNA加合物对保真度的影响。

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.