Discrimination against purine-pyrimidine mispairs in the polymerase active site of DNA polymerase I: A structural explanation
Discrimination against purine-pyrimidine mispairs in the polymerase active site of DNA polymerase I: A structural explanation
复制标题
DOI：
10.1073/pnas.032457899
复制
发表时间：
2002-02-05
影响因子：
11.1
通讯作者：
Joyce, CM
中科院分区：
文献类型：
--
作者：
Minnick, DT;Liu, LX;Joyce, CM
我们先前鉴定了五个Klenow片段DNA聚合酶的衍生物，它们的保真度较低，因为聚合酶活性部位的氨基酸替换。其中之一的丙氨酸取代了不变的Glu-710，Glu-710的侧链与传入dNTP的脱氧核糖相互作用。在这里，我们展示了E710a酶已经降低了12个可能的错配中的5个的保真度。除了一个以外，所有这些都涉及嘧啶的错误插入，包括两个转换错配A-dCTP和G-dTTP。相反，C-dATP和T-dGTP转换错配的E710a聚合酶错配错配率与野生型酶相似。野生型和E710a聚合酶形成正确碱基对和转换错配的动力学，结合DNA聚合酶活性部位结构和摆动碱基对的不对称性的信息，为E710a突变对保真度的不同影响提供了一个可信的解释。这些数据表明，Glu-710侧链在通过空间碰撞排除模板嘧啶和三磷酸嘌呤之间的摆动碱基对方面起着关键作用。此外，相同的侧链增强了引入的正确dNTPs的稳定性，使得在移除侧链时失去这种相互作用导致对涉及引入的嘧啶的错配的较低的选择性。
We previously identified five derivatives of Klenow fragment DNA polymerase that have lower fidelity because of amino acid substitutions in the polymerase active site. One of these has alanine substituted for the invariant Glu-710, whose side chain interacts with the deoxyribose of the incoming dNTP. Here we show that the E710A enzyme has reduced fidelity for five of the 12 possible mismatches. All but one of these involve misinsertion of pyrimidines, including two transition mismatches A-dCTP and G-dTTP. In contrast, E710A polymerase error rates for the reciprocal C-dATP and T-dGTP transition mismatches were similar to those of the wild-type enzyme. The kinetics of formation of correct base pairs and transition mismatches by the wild-type and E710A polymerases, combined with information on the structure of the DNA polymerase active site and the asymmetry of wobble base pairs, provides a plausible explanation for the differential effects of the E710A mutation on fidelity. The data suggest that the Glu-710 side chain plays a pivotal role in excluding wobble base pairs between template pyrimidines and purine triphosphates by steric clash. Moreover, this same side chain enhances the stability of incoming correct dNTPs, such that loss of this interaction on removal of the side chain leads to lower selectivity against mismatches involving incoming pyrimidines.