...
  • 主题
高级搜索  >
历史搜索 清空历史
首页> 外文期刊>Biochemistry >A Computational Study of the Hydrolysis of dGTP Analogues with Halomethylene-Modified Leaving Groups in Solution: Implications for the Mechanism of DNA Polymerases
【24h】

A Computational Study of the Hydrolysis of dGTP Analogues with Halomethylene-Modified Leaving Groups in Solution: Implications for the Mechanism of DNA Polymerases

机译：溶液中带有卤亚甲基修饰的离去基团水解dGTP类似物的计算研究：对DNA聚合酶机理的启示

获取原文
获取原文并翻译 | 示例
           
页面导航
  • 摘要
  • 著录项
  • 相似文献
  • 相关主题

摘要

DNA polymerases make up a family of enzymes responsible for regulating DNA replication andnrepair, which in turn maintains the integrity of the genome. However, despite intensive kinetic, crystal-nlographic, and computational studies, elucidation of the detailed enzymatic mechanism still presentsna significant challenge.We recently developed an alternative strategy for exploring the fidelity andmechanismnof DNA polymerases, by probing leaving group effects on nucleotidyl transfer using a series of dGTPnbisphosphonate analogues in which the β,γ-bridging oxygen was replaced by a series of substitutedmethylenengroups (X = CYZ, where Y and Z = H, halogen, or another substituent). Pre-steady state kinetic mea-nsurements of DNA polymerase-catalyzed incorporation of correctly base paired (R) and mispaired (W)nanalogues demonstrated a strong linear free energy relationship (LFER) between the polymerase ratenconstant (kpol) and the highest pKa of the free bisphosphonic acid corresponding to the leaving group.nHowever, unexpectedly, the data segregated into two distinctly different linear correlations depending on thennature of the substituent. The discrepancy between the two lines was considerably greater when the dGTPnanalogue formed an incorrect (G3nT) rather than a correct (G3nC) base pair, although the reason for thisnphenomenon remains unexplained. Here, we have evaluated the complete free energy surfaces for bispho-nsphonate hydrolysis in aqueous solution and evaluated the corresponding LFER. Our study, which employsnseveral alternative solvation models, finds a split of the calculated LFER for the mono- and dihalogenncompounds into two parallel lines, reflecting their behavior in the polymerase-catalyzed condensationnreaction. We suggest that the division into two linear subsets may be a generalized solvation phenomenonninvolving the overall electrostatic interaction between the substrates and their surroundings and would also benobserved in polar solvents in the absence of the enzyme, if the reaction in solvent is in fact identical to that ofnthe enzyme.However, the amplified differences between the LFER lines for the incorporation ofmatched andnmismatched deoxynucleotides probably reflects the differences in the electrostatic interaction between the TSncharges in the polymerase active site. An understanding of the mechanism of this reaction in solution couldnthereby provide a steppingstone for understanding the factors governing the fidelity of DNA polymerases.
机译：DNA聚合酶组成了一个酶家族，负责调节DNA复制和修复，从而维护了基因组的完整性。然而，尽管进行了大量的动力学，晶体学和计算学研究，详细的酶促机理的阐明仍然是一个严峻的挑战。我们最近开发了另一种策略来探索DNA聚合酶的保真度和机制，方法是使用一系列方法探索离开基团对核苷酸转移的影响。的dGTPn双膦酸酯类似物，其中β，γ桥接氧被一系列取代的亚甲基取代（X = CYZ，其中Y和Z = H，卤素或其他取代基）。 DNA聚合酶催化的正确配对的碱基配对（R）和错配对（W）的类似物的稳态前动力学测量表明，聚合酶速率常数（kpol）和最高pKa之间存在很强的线性自由能关系（LFER）。然而，出乎意料的是，根据取代基的性质，数据分离为两个截然不同的线性相关性。当dGTPnanalogue形成一个错误的（G3nT）碱基而不是一个正确的（G3nC）碱基对时，两行之间的差异要大得多，尽管这种现象的原因仍然无法解释。在这里，我们评估了双膦酸酯在水溶液中水解的完整自由能表面，并评估了相应的LFER。我们的研究采用了多种替代溶剂化模型，发现单卤代化合物和二卤代化合物的LFER计算值分为两条平行线，反映了它们在聚合酶催化的缩合反应中的行为。我们建议将其分为两个线性子集可能是一种泛化的溶剂化现象，涉及底物与其周围环境之间的整体静电相互作用，并且如果在溶剂中的反应实际上与溶剂中的反应相同，则在不存在酶的情况下也可在极性溶剂中观察到。然而，用于掺入匹配的和错配的脱氧核苷酸的LFER品系之间的扩增差异可能反映了聚合酶活性位点中TSn电荷之间的静电相互作用的差异。因此，对溶液中该反应机理的理解不能为理解控制DNA聚合酶保真度的因素提供垫脚石。

著录项

相似文献

  • 外文文献
  • 中文文献
  • 专利
获取原文

客服邮箱：kefu@zhangqiaokeyan.com

京公网安备：11010802029741号 ICP备案号：京ICP备15016152号-6 六维联合信息科技 (北京) 有限公司©版权所有

  • 客服微信

  • 服务号