The Structure of a High Fidelity DNA Polymerase Bound to a Mismatched Nucleotide Reveals an "Ajar" Intermediate Conformation in the Nucleotide Selection Mechanism
The Structure of a High Fidelity DNA Polymerase Bound to a Mismatched Nucleotide Reveals an "Ajar" Intermediate Conformation in the Nucleotide Selection Mechanism
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DOI：
10.1074/jbc.m110.191130
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发表时间：
2011-06-03
影响因子：
4.8
通讯作者：
Beese, Lorena S.
中科院分区：
文献类型：
--
作者：
Wu, Eugene Y.;Beese, Lorena S.
为了实现准确的DNA合成，DNA聚合酶必须快速取样并区分不正确的核苷酸。在这里，我们报道了一个高保真DNA聚合酶I的晶体结构，该酶与DNA引物模板结合，以结合错配的（dG: dTTP）核苷三磷酸。聚合酶采用先前建立的“开放”和“封闭”状态之间的构象。在这种“ajar”构象中，模板碱基已经移动到插入位点，但相对于引物末端错配了一个不正确的核苷酸。模板碱基取代了插入位点上的保守活性位点酪氨酸，这是由聚合酶O螺旋上的独特扭结所调节的，从而形成部分开放的三元配合物。我们认为ajar构象允许模板在底物周围的酶关闭之前探测进入的核苷酸的互补性。根据本文报道的野生型和突变型聚合酶的溶液荧光、动力学和晶体学分析，我们提出了一种三态反应途径，其中核苷酸要么通过中间构象进入封闭构象并催化，要么在中间构象中错位，导致封闭构象的不稳定。
To achieve accurate DNA synthesis, DNA polymerases must rapidly sample and discriminate against incorrect nucleotides. Here we report the crystal structure of a high fidelity DNA polymerase I bound to DNA primer-template caught in the act of binding a mismatched (dG: dTTP) nucleoside triphosphate. The polymerase adopts a conformation in between the previously established "open" and "closed" states. In this "ajar" conformation, the template base has moved into the insertion site but misaligns an incorrect nucleotide relative to the primer terminus. The displacement of a conserved active site tyrosine in the insertion site by the template base is accommodated by a distinctive kink in the polymerase O helix, resulting in a partially open ternary complex. We suggest that the ajar conformation allows the template to probe incoming nucleotides for complementarity before closure of the enzyme around the substrate. Based on solution fluorescence, kinetics, and crystallographic analyses of wild-type and mutant polymerases reported here, we present a three-state reaction pathway in which nucleotides either pass through this intermediate conformation to the closed conformation and catalysis or are misaligned within the intermediate, leading to destabilization of the closed conformation.