Publikation: Structures of a DNA Polymerase Caught while Incorporating Responsive Dual-Functional Nucleotide Probes
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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 19F 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 19F NMR spectroscopy. Comparative analysis of incorporation profiles, fluorescence, 19F 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.
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GHOSH, Pulak, Karin BETZ, Cedric GUTFREUND, Arindam PAL, Andreas MARX, Seergazhi G. SRIVATSAN, 2025. Structures of a DNA Polymerase Caught while Incorporating Responsive Dual-Functional Nucleotide Probes. In: Angewandte Chemie International Edition. Wiley. 2025, 64(2), e202414319. ISSN 1433-7851. eISSN 1521-3773. Verfügbar unter: doi: 10.1002/anie.202414319BibTex
@article{Ghosh2025-01-10Struc-71627, title={Structures of a DNA Polymerase Caught while Incorporating Responsive Dual-Functional Nucleotide Probes}, year={2025}, doi={10.1002/anie.202414319}, number={2}, volume={64}, issn={1433-7851}, journal={Angewandte Chemie International Edition}, author={Ghosh, Pulak and Betz, Karin and Gutfreund, Cedric and Pal, Arindam and Marx, Andreas and Srivatsan, Seergazhi G.}, note={Article Number: e202414319} }
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