How E-coli DNA polymerase I (Klenow fragment) distinguishes between deoxy- and dideoxynucleotides
How E-coli DNA polymerase I (Klenow fragment) distinguishes between deoxy- and dideoxynucleotides
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DOI：
10.1006/jmbi.1998.1672
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发表时间：
1998-04-24
影响因子：
5.6
通讯作者：
Joyce, CM
中科院分区：
文献类型：
--
作者：
Astatke, M;Grindley, NDF;Joyce, CM
脱氧和双脱氧核苷酸的区别仅在于它们是否在核糖部分的C-3‘有羟基取代基，而Klenow片段DNA聚合酶对天然(DNTP)底物的偏好是天然的几千倍。我们使用这一偏好来研究Klenow片段如何与传入dNTP的糖部分相互作用。我们筛选了Klenow片段的突变衍生物，以确定在区分dNTPs和ddNTPs中起重要作用的氨基酸残基。用Ala或Tyr取代Phe762导致对ddNTPs的识别能力显著下降，而Tyr766和Glu710的突变影响较小，这表明这两个侧链在dNTPs的选择中起次要作用。为了了解酶-DNA-dNTP三元复合体中的相互作用，我们测定了野生型Klenow片段和突变的dNTP/ddNTP识别改变的dNTPs和ddNTPs的稳态前动力学参数。根据元素效应测量，我们推断针对双脱氧核苷酸的选择发生在磷酸转移之前的构象变化的过渡态。Phe762侧链的关键作用似乎是约束dNTP分子，以便3‘-OH可以与三元络合物中的另一个基团相互作用。当Tyr在762位被取代时，同样的相互作用可以发生来定位dNTP，但对ddNTP的特异性失去了，因为酚羟基可以弥补核苷酸缺失的3‘-羟基。取代较小的ALA侧链会导致特异性的丧失，因为dNTP不再受到适当的限制。对反应速率随镁离子浓度的变化的测量表明，dNTP3‘-OH与Glu710侧链的相互作用可能涉及金属离子，最简单的情况是Glu710的3’-OH和羧酸盐都是同一金属离子的配体。(C)1998年学术出版社有限公司。
Deoxy- and dideoxynucleotides differ only in whether they have a hydroxyl substituent at C-3' of the ribose moiety, and yet the Klenow fragment DNA polymerase prefers the natural (dNTP) substrate by several thousandfold. We have used this preference in order to investigate how Klenow fragment interacts with the sugar portion of an incoming dNTP. We screened mutant derivatives of Klenow fragment so as to identify those amino acid residues that play important roles in distinguishing between dNTPs and ddNTPs. Substitution of Phe762 with Ala or Tyr caused a dramatic decrease in the discrimination against ddNTPs, while mutations in Tyr766 and Glu710 had a smaller effect, suggesting that these two side-chains Flay secondary roles in the selection of dNTPs over ddNTPs.In order to understand the interactions in the enzyme-DNA-dNTP ternary complex, pre-steady-state kinetic parameters for the incorporation of dNTPs and ddNTPs were determined for wild-type Klenow fragment and for mutant derivatives that showed changes in dNTP/ddNTP discrimination. From elemental effect measurements we infer that selection against dideoxynucleotides takes place in the transition state for the conformational change that precedes phosphoryl transfer. The crucial role of the Phe762 side-chain appears to be to constrain the dNTP molecule so that the 3'-OH can make an interaction with another group within the ternary complex. When Tyr is substituted at position 762, the same interactions can take place to position the dNTP, but specificity against the ddNTP is lost because the phenolic OH can compensate for the missing 3'-OH of the nucleotide. Substitution of the smaller Ala side-chain results in a loss in specificity because the dNTP is no longer appropriately constrained. Measurement of reaction rates as a function of magnesium ion concentration suggests that the interaction made with the dNTP 3'-OH may involve a metal ion and the Glu710 side-chain, the simplest scenario being that both the 3'-OH and the carboxylate of Glu710 are ligands to the same metal ion. (C) 1998 Academic Press Limited.