A DNA polymerase with specificity for five base pairs
A DNA polymerase with specificity for five base pairs
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DOI：
10.1021/ja005758x
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发表时间：
2001-02-28
影响因子：
15
通讯作者：
Tsai, MD
中科院分区：
文献类型：
--
作者：
Showalter, AK;Tsai, MD
非洲猪瘟病毒X家族DNA聚合酶（Pol X）是目前已知的最小的核苷酸转移酶，在DNA修复中的作用类似于其哺乳动物同源序列DNA聚合酶β（Pol β）。在这项研究中，Pol X的深入动力学分析，包括所有可能碱基对的催化效率和保真度测量，表明Pol X是迄今为止研究的所有聚合酶中最不忠实或最容易出错的，我们的结论是Pol X是最不忠实的聚合酶，这是基于前聚合酶，稳态动力学，使用模型DNA底物（图1）。我们首先测量了单营业额（酶超过DNA底物）饱和动力学的所有16个可能的碱基对在单缺口的DNA底物。先前已经表明，Pol X仅在作用于有缺口的底物时才是进行性酶，1并且我们观察到仅用有缺口的DNA观察到爆发动力学（数据未显示）证实了这一点，并表明有缺口的DNA可能是酶的天然底物，Pol β也是如此。单周转实验允许直接测定核苷酸掺入的主要动力学参数kpol（伪一级催化速率常数）和Kd，app（从酶DNA复合物中解离三磷酸核苷酸的表观平衡常数）。比率kpol/Kd是底物特异性的定义（它也被称为“催化效率”），因此正确和不正确的纯化的该值的比较给出了聚合酶保真度的定量测量。表1所示的结果表明与修复功能不相容的活性。该酶具有相对较低的催化效率，平均为Pol β（一种已知在碱基切除修复或BER 2中起作用的酶）的1/5000，用于正确的碱基对切除。[3]更引人注目的是，Pol X的同源性非常低，从C：C碱基对的7700到G：G碱基对的1.9。由于该酶的底物特异性定义为[（kpol/Kd，app）cor+（kpol/Kd，app）inc]/（kpol/Kd，app）inc（其中下标“cor”和“inc”分别指正确和不正确的掺入），解为错误频率的倒数，这表明该酶对正确的碱基对（G：C）相对于相应的不正确的碱基对（G：G）没有底物特异性。虽然Pol X的整个保真度谱非常低，但G：C和G：G碱基对之间的这种不区分代表了对于模板指导的核苷酸聚合酶所观察到的最低核苷酸掺入特异性。人Pol η，一种最近被确定为最容易出错的聚合酶的酶，4在这种情况下比Pol X至少10倍更忠实。所示
The X family DNA polymerase from African swine fever virus (Pol X) has recently been characterized as the smallest known nucleotidyl transferase and has been suggested to play a role in DNA repair analogous to that of its mammalian sequence homologue, DNA polymerase β (Pol β). 1 In this study an indepth kinetic analysis of Pol X, including catalytic efficiency and fidelity measurements for all possible base pairs, demonstrates that Pol X is the least faithful, or most error-prone, of all polymerases studied to date, with a specific preference for five base pairs including the four Watson-Crick base pairs plus one mismatched pair.Our conclusion that Pol X is the least faithful polymerase is based on pre-steady-state kinetics, using model DNA substrates (Figure 1). We have first measured single turnover (with enzyme in excess of DNA substrate) saturation kinetics for all 16 possible base pairs in single-gapped DNA substrates. It has previously been shown that Pol X is a processive enzyme only when acting on gapped substrate, 1 and our observation that burst kinetics are observed only with gapped DNA (data not shown) confirm this and suggest that gapped DNA is likely to be the enzyme’s natural substratesas is the case for Pol β. Single turnover experiments allow direct determination of the principal kinetic parameters kpol (the pseudo-first-order catalytic rate constant) and Kd, app (the apparent equilibrium constant for dissociation of nucleotide triphosphate from the enzyme ‚DNA complex) of nucleotide incorporation. The ratio kpol/Kd is the definition of substrate specificity (it is also termed “catalytic efficiency”), and thus comparison of this value for correct and incorrect incorporations gives a quantitative measurement of the fidelity for a polymerase. The results shown in Table 1 indicate an activity which is incompatible with a repair function. The enzyme has relatively low catalytic efficiency, on average 1/5000th that of Pol β (an enzyme known to function in base excision repair, or BER2) for correct base-pair incorporations. 3 More strikingly, Pol X has exceptionally low fidelities, ranging from 7700 for the C: C base pair to 1.9 for the G: G base pair. As the fidelitysdefined as [(kpol/Kd, app) cor+(kpol/Kd, app) inc]/(kpol/Kd, app) inc where the subscripts “cor” and “inc” refer to the correct and incorrect incorporation, respectivelysis the inverse of the error frequency, this indicates that the enzyme has no substrate specificity for a correct base pair (G: C) relative to the corresponding incorrect base pair (G: G). While the entire fidelity spectrum for Pol X is remarkably low, this absence of discrimination between the G: C and G: G base pairs represents the lowest nucleotide incorporation specificity ever observed for a templatedirected nucleotide polymerase. Human Pol η, an enzyme recently determined to be the most error-prone polymerase, 4 is at least 10 times more faithful than Pol X in this instance. As illustrated in