Key Laboratory of Bio-Resource and Eco-Environment of Ministry of Education, College of Life Sciences, Sichuan University, Chengdu 610064, China.
Henan Institute of Medical and Pharmaceutical Sciences, Zhengzhou University, Zhengzhou 450000, China.
Int J Mol Sci. 2023 Oct 30;24(21):15758. doi: 10.3390/ijms242115758.
DNA synthesis catalyzed by DNA polymerase is essential for all life forms, and phosphodiester bond formation with phosphorus center inversion is a key step in this process. Herein, by using a single-selenium-atom-modified dNTP probe, we report a novel strategy to visualize the reaction stereochemistry and catalysis. We capture the before- and after-reaction states and provide explicit evidence of the center inversion and in-line attacking S2 mechanism of DNA polymerization, while solving the diastereomer absolute configurations. Further, our kinetic and thermodynamic studies demonstrate that in the presence of Mg ions (or Mn), the binding affinity () and reaction selectivity (/) of dGTPαSe-Rp were 51.1-fold (or 19.5-fold) stronger and 21.8-fold (or 11.3-fold) higher than those of dGTPαSe-Sp, respectively, indicating that the diastereomeric Se-Sp atom was quite disruptive of the binding and catalysis. Our findings reveal that the third metal ion is much more critical than the other two metal ions in both substrate recognition and bond formation, providing insights into how to better design the polymerase inhibitors and discover the therapeutics.
DNA 聚合酶催化的 DNA 合成对于所有生命形式都是必不可少的，而磷中心反转时磷酸二酯键的形成是该过程的关键步骤。在此，我们通过使用单个硒原子修饰的 dNTP 探针，报告了一种可视化反应立体化学和催化的新策略。我们捕捉到了反应前后的状态，并提供了 DNA 聚合中中心反转和直线进攻 S2 机制的明确证据，同时解决了非对映异构体的绝对构型。此外，我们的动力学和热力学研究表明，在存在 Mg 离子（或 Mn）的情况下，dGTPαSe-Rp 的结合亲和力（）和反应选择性（/）分别比 dGTPαSe-Sp 强 51.1 倍（或 19.5 倍）和高 21.8 倍（或 11.3 倍），表明非对映异构体的 Se-Sp 原子对结合和催化有很大的干扰。我们的发现表明，第三个金属离子在底物识别和键形成中比另外两个金属离子都更为关键，为如何更好地设计聚合酶抑制剂和发现治疗方法提供了思路。
