9FM3
KlenTaq DNA polymerase in a ternary complex with primer/template and a selenophene-modified dUTP (SedUTP)
This is a non-PDB format compatible entry.
Summary for 9FM3
| Entry DOI | 10.2210/pdb9fm3/pdb |
| Descriptor | DNA polymerase I, thermostable, Primer with terminal DOC, DNA template, ... (7 entities in total) |
| Functional Keywords | dna polymerase, selenophene, fluorescent nucleotide probe, klentaq, transferase |
| Biological source | Thermus aquaticus More |
| Total number of polymer chains | 3 |
| Total formula weight | 70117.02 |
| Authors | |
| Primary citation | Ghosh, P.,Betz, K.,Gutfreund, C.,Pal, A.,Marx, A.,Srivatsan, S.G. Structures of a DNA Polymerase Caught while Incorporating Responsive Dual-Functional Nucleotide Probes. Angew.Chem.Int.Ed.Engl., :e202414319-e202414319, 2024 Cited by PubMed Abstract: 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, FBFdUTP's responsiveness enabled real-time monitoring of the binary complex formation and polymerase activity through fluorescence and 19F NMR. Comparative analysis of incorporation profiles, fluorescence, 19F NMR data, and crystal structures of ternary complexes highlights the enzyme's plasticity. Key insights are 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. PubMed: 39428682DOI: 10.1002/anie.202414319 PDB entries with the same primary citation |
| Experimental method | X-RAY DIFFRACTION (2.59 Å) |
Structure validation
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