Laboratory of Molecular Modeling and Drug Discovery , Istituto Italiano di Tecnologia , Via Morego 30 , 16163 Genoa , Italy.
Computational Biophysics, German Research School for Simulation Sciences, and Computational Biomedicine, Institute for Advanced Simulation IAS-5 and Institute of Neuroscience and Medicine INM-9 , Forschungszentrum Jülich , 52425 Jülich , Germany.
J Am Chem Soc. 2018 Mar 7;140(9):3312-3321. doi: 10.1021/jacs.7b12446. Epub 2018 Feb 15.
Polymerases (Pols) synthesize the double-stranded nucleic acids in the Watson-Crick (W-C) conformation, which is critical for DNA and RNA functioning. Yet, the molecular basis to catalyze the W-C base pairing during Pol-mediated nucleic acids biosynthesis remains unclear. Here, through bioinformatics analyses on a large data set of Pol/DNA structures, we first describe the conserved presence of one positively charged residue (Lys or Arg), which is similarly located near the enzymatic two-metal active site, always interacting directly with the incoming substrate (d)NTP. Incidentally, we noted that some Pol/DNA structures showing the alternative Hoogsteen base pairing were often solved with this specific residue either mutated, displaced, or missing. We then used quantum and classical simulations coupled to free-energy calculations to illustrate how, in human DNA Pol-η, the conserved Arg61 favors W-C base pairing through defined interactions with the incoming nucleotide. Taken together, these structural observations and computational results suggest a structural framework in which this specific residue is critical for stabilizing the incoming (d)NTP nucleotide and base pairing during Pol-mediated nucleic acid biosynthesis. These results may benefit enzyme engineering for nucleic acid processing and encourage new drug discovery strategies to modulate Pols function.
聚合酶（Pols）以沃森-克里克（W-C）构象合成双链核酸，这对 DNA 和 RNA 的功能至关重要。然而，在 Pol 介导的核酸生物合成过程中催化 W-C 碱基配对的分子基础仍不清楚。在这里，我们通过对大量 Pol/DNA 结构数据集进行生物信息学分析，首先描述了一个带正电荷的保守残基（赖氨酸或精氨酸）的存在，该残基通常位于酶的两个金属活性位点附近，直接与进入的底物（d）NTP 相互作用。顺便说一句，我们注意到，一些显示替代 Hoogsteen 碱基配对的 Pol/DNA 结构通常使用具有这种特定残基的突变、位移或缺失的方法来解决。然后，我们使用量子和经典模拟结合自由能计算来阐明，在人类 DNA Pol-η 中，保守的 Arg61 如何通过与进入的核苷酸的定义相互作用来促进 W-C 碱基配对。总之，这些结构观察和计算结果提出了一个结构框架，其中该特定残基对于稳定进入的（d）NTP 核苷酸和 Pol 介导的核酸生物合成过程中的碱基配对至关重要。这些结果可能有利于核酸处理的酶工程，并鼓励新的药物发现策略来调节 Pols 的功能。
