Prechemistry nucleotide selection checkpoints in the reaction pathway of DNA polymerase I and roles of glu710 and tyr766.
Prechemistry nucleotide selection checkpoints in the reaction pathway of DNA polymerase I and roles of glu710 and tyr766.
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DNA 聚合酶 I 反应途径中的化学前核苷酸选择检查点以及 glu710 和 tyr766 的作用。
DOI：
10.1021/bi400837k
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发表时间：
2013
期刊：
影响因子：
2.9
通讯作者：
Joyce,CatherineM
中科院分区：
文献类型：
--
作者：
Bermek,Oya;Grindley,NigelDF;Joyce,CatherineM
高保真DNA聚合酶的准确性，如DNA聚合酶I(Klenow片段)，由反应路径早期的构象变化决定，这些构象变化充当保真度检查点，识别不适当的模板-核苷酸配对。闭合手指的转变(通过基于荧光共振能量转移的分析检测到)是与正确的传入核苷酸结合的独特结果，该核苷酸与模板碱基和脱氧核糖(而不是核糖)结构互补。与错配的dNTPs或互补的rNTPs的络合物在早期被阻止，对应于部分闭合的手指构象，其中DNA和核苷酸的弱结合促进底物池的解离和重采样。DNA模板上的2-氨基嘌呤荧光探针提供了有关合指前步骤的进一步信息。无论糖是脱氧核糖还是核糖，在与互补核苷酸结合时都能观察到特征的2-氨基嘌呤信号。然而，错配的dNTP表现出完全不同的行为。因此，手指合拢之前的保真度检查点负责区分互补核苷酸和非互补核苷酸，并将它们发送到不同的结果。E710a突变聚合酶在早期的保真度检查点中存在缺陷，以至于一些互补的dNTP被视为错配。在Y766A突变体中，早期检查点功能正常，但一些正确配对的dNTP不能有效地闭合手指。因此，这两个突变等位基因导致正确和不正确碱基对之间的区别变得模糊，并导致更大比例的错误通过化学前保真度检查点。
The accuracy of high-fidelity DNA polymerases such as DNA polymerase I (Klenow fragment) is governed by conformational changes early in the reaction pathway that serve as fidelity checkpoints, identifying inappropriate template–nucleotide pairings. The fingers-closing transition (detected by a fluorescence resonance energy transfer-based assay) is the unique outcome of binding a correct incoming nucleotide, both complementary to the templating base and with a deoxyribose (rather than ribose) sugar structure. Complexes with mispaired dNTPs or complementary rNTPs are arrested at an earlier stage, corresponding to a partially closed fingers conformation, in which weak binding of DNA and nucleotide promote dissociation and resampling of the substrate pool. A 2-aminopurine fluorescence probe on the DNA template provides further information about the steps preceding fingers closing. A characteristic 2-aminopurine signal is observed on binding a complementary nucleotide, regardless of whether the sugar is deoxyribose or ribose. However, mispaired dNTPs show entirely different behavior. Thus, a fidelity checkpoint ahead of fingers closing is responsible for distinguishing complementary from noncomplementary nucleotides and routing them toward different outcomes. The E710A mutator polymerase has a defect in the early fidelity checkpoint such that some complementary dNTPs are treated as if they were mispaired. In the Y766A mutant, the early checkpoint functions normally, but some correctly paired dNTPs do not efficiently undergo fingers closing. Thus, both mutator alleles cause a blurring of the distinction between correct and incorrect base pairs and result in a larger fraction of errors passing through the prechemistry fidelity checkpoints.