DNA polymerase fidelity: comparing direct competition of right and wrong dNTP substrates with steady state and pre-steady state kinetics.
DNA polymerase fidelity: comparing direct competition of right and wrong dNTP substrates with steady state and pre-steady state kinetics.
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DOI：
10.1021/bi901653g
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发表时间：
2010-01-12
期刊：
影响因子：
2.9
通讯作者：
Goodman MF
中科院分区：
文献类型：
--
作者：
Bertram JG;Oertell K;Petruska J;Goodman MF
DNA聚合酶保真度定义为当dRTP和dWTP底物在聚合酶-引物-模板DNA复合物中的相同位点以相等浓度竞争引物延伸时正确（R）与错误（W）核苷酸重复的比率。通常，即使在没有3′-核酸外切酶校正的情况下，R掺入也比W有利103 - 105。简单地说，直接竞争保真度测量在实践中难以执行，因为少量W的检测被大量R掩盖。作为替代方案，基于Fersht的稳态推导，广泛使用酶动力学测量来评估R和W在单独反应中的kcat/Km，以间接测量聚合酶保真度。直接竞争与动力学之间的系统比较至今尚未完成。通过使用电泳分离R和W产物，我们成功地对12个天然碱基错配中的9个直接竞争R和W dNTP底物进行了精确的保真度测量。我们比较我们的直接竞争结果与稳态和presteady状态动力学测量保真度在同一模板网站，使用校正缺陷突变体的Klenow片段（KF−）DNA聚合酶。所有的数据在数量上是一致的。
DNA polymerase fidelity is defined as the ratio of right (R) to wrong (W) nucleotide incorporations when dRTP and dWTP substrates compete at equal concentrations for primer extension at the same site in the polymerase-primer-template DNA complex. Typically, R incorporation is favored over W by 103 – 105, even in the absence of 3′-exonuclease proofreading. Straightforward in principal, a direct competition fidelity measurement is difficult to perform in practice because detection of a small amount of W is masked by a large amount of R. As an alternative, enzyme kinetics measurements to evaluate kcat/Km for R and W in separate reactions are widely used to measure polymerase fidelity indirectly, based on a steady-state derivation by Fersht. A systematic comparison between direct competition and kinetics has not been made until now. By separating R and W products using electrophoresis, we have successfully made accurate fidelity measurements for directly competing R and W dNTP substrates for 9 of the 12 natural base mispairs. We compare our direct competition results with steady state and presteady state kinetic measurements of fidelity at the same template site, using the proofreading-deficient mutant of Klenow Fragment (KF−) DNA polymerase. All the data are in quantitative agreement.
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影响因子：
4.8
作者：
Bloom, LB;Chen, XL;Goodman, MF
通讯作者：
Goodman, MF
影响因子：
4.8
作者：
Fiala, Kevin A.;Suo, Zucai
通讯作者：
Suo, Zucai
影响因子：
2.9
作者：
EGER, BT;BENKOVIC, SJ
通讯作者：
BENKOVIC, SJ
DOI：
10.1101/sqb.1968.033.01.039
发表时间：
1968-01-01
期刊：
COLD SPRING HARBOR SYMPOSIA ON QUANTITATIVE BIOLOGY
影响因子：
--
作者：
DRAKE, JW;ALLEN, EF
通讯作者：
ALLEN, EF
影响因子：
2.9
作者：
BLOOM, LB;OTTO, MR;BEECHEM, JM
通讯作者：
BEECHEM, JM