Ghosh Pulak, Betz Karin, Gutfreund Cédric, Pal Arindam, Marx Andreas, Srivatsan Seergazhi G
Department of Chemistry, Indian Institute of Science Education and Research (IISER), Pune, Dr. Homi Bhabha Road, Pune, 411008, India.
Department of Chemistry, Konstanz Research School Chemical Biology, University of Konstanz, Universitätsstraße 10, 78457, Konstanz, Germany.
Angew Chem Int Ed Engl. 2025 Jan 10;64(2):e202414319. doi: 10.1002/anie.202414319. Epub 2024 Nov 22.
Functionalizing nucleic acids using DNA polymerases is essential in biophysical and biotechnology applications. This study focuses on understanding how DNA polymerases recognize and incorporate nucleotides with diverse chemical modifications, aiming to develop advanced nucleotide probes. We present the crystal structures of ternary complexes of Thermus aquaticus DNA polymerase (KlenTaq) with C5-heterocycle-modified environment-sensitive 2'-deoxyuridine-5'-triphosphate (dUTP) probes. These nucleotides include SedUTP, BFdUTP and FBFdUTP, which bear selenophene, benzofuran and fluorobenzofuran, respectively, at the C5 position of uracil, and exhibit high conformational sensitivity. SedUTP and FBFdUTP serve as dual-app probes, combining a fluorophore with X-ray anomalous scattering Se or F NMR labels. Our study reveals that the size of the heterocycle influences how DNA polymerase families A and B incorporate these modified nucleotides during single nucleotide incorporation and primer extension reactions. Remarkably, the responsiveness of FBFdUTP enabled real-time monitoring of the binary complex formation and polymerase activity through fluorescence and F NMR spectroscopy. Comparative analysis of incorporation profiles, fluorescence, F NMR data, and crystal structures of ternary complexes highlights the plasticity of the enzyme. Key insight is provided into the role of gatekeeper amino acids (Arg660 and Arg587) in accommodating and processing these modified substrates, offering a structural basis for next-generation nucleotide probe development.
利用DNA聚合酶对核酸进行功能化修饰在生物物理和生物技术应用中至关重要。本研究聚焦于了解DNA聚合酶如何识别并掺入具有不同化学修饰的核苷酸，旨在开发先进的核苷酸探针。我们展示了嗜热水生栖热菌DNA聚合酶（KlenTaq）与C5-杂环修饰的环境敏感型2'-脱氧尿苷-5'-三磷酸（dUTP）探针形成的三元复合物的晶体结构。这些核苷酸包括SedUTP、BFdUTP和FBFdUTP，它们分别在尿嘧啶的C5位带有硒吩、苯并呋喃和氟苯并呋喃，并且表现出高构象敏感性。SedUTP和FBFdUTP作为双功能探针，将荧光团与X射线异常散射硒或氟核磁共振标记相结合。我们的研究表明，杂环的大小会影响A和B类DNA聚合酶家族在单核苷酸掺入和引物延伸反应中掺入这些修饰核苷酸的方式。值得注意的是，FBFdUTP的响应性使得能够通过荧光和氟核磁共振光谱实时监测二元复合物的形成和聚合酶活性。对掺入图谱、荧光、氟核磁共振数据以及三元复合物晶体结构的比较分析突出了该酶的可塑性。为守门氨基酸（Arg660和Arg587）在容纳和处理这些修饰底物中的作用提供了关键见解，为下一代核苷酸探针的开发提供了结构基础。
