9fmf

KlenTaq DNA polymerase in a ternary complex with primer/template and a fluorobenzofuran-modified dUTP (FBFdUTP)

spinqualitylabelsall models 9fmf, resolution 2.10Å Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image   Print3D Structural highlights 9fmf is a 3 chain structure with sequence from Thermus aquaticus and Synthetic construct. Full crystallographic information is available from OCA. For a guided tour on the structure components use FirstGlance. Method: X-ray diffraction, Resolution 2.1Å Ligands: A1IDW, DOC, GOL, MG Resources: FirstGlance, OCA, PDBe, RCSB, PDBsum, ProSAT Function DPO1T_THET8 In addition to polymerase activity, this DNA polymerase exhibits 5'-3' exonuclease activity.[UniProtKB:P52026] Publication Abstract from PubMed 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 (19)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 (19)F NMR spectroscopy. Comparative analysis of incorporation profiles, fluorescence, (19)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. Structures of a DNA Polymerase Caught while Incorporating Responsive Dual-Functional Nucleotide Probes.,Ghosh P, Betz K, Gutfreund C, Pal A, Marx A, Srivatsan SG Angew Chem Int Ed Engl. 2025 Jan 10;64(2):e202414319. doi: , 10.1002/anie.202414319. Epub 2024 Nov 22. PMID:39428682 From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.

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