6isi

Structure of 9N-I DNA polymerase incorporation with 3'-CL in the active site

spinqualitylabelsall models 6isi, resolution 3.20Å Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image   Print3D Structural highlights 6isi is a 3 chain structure with sequence from Thermococcus sp. 9oN-7 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 3.2Å Ligands: B0U, CA, PPV Resources: FirstGlance, OCA, PDBe, RCSB, PDBsum, ProSAT Function DPOL_THES9 Publication Abstract from PubMed It was reported in 1995 that T7 and Taq DNA polymerases possess 3'-esterase activity, but without follow-up studies. Here we report that the 3'-esterase activity is intrinsic to the Thermococcus sp. 9 degrees N DNA polymerase, and that it can be developed into a continuous method for DNA sequencing with dNTP analogs carrying a 3'-ester with a fluorophore. We first show that 3'-esterified dNTP can be incorporated into a template-primer DNA, and solve the crystal structures of the reaction intermediates and products. Then we show that the reaction can occur continuously, modulated by active site residues Tyr409 and Asp542. Finally, we use 5'-FAM-labeled primer and esterified dNTP with a dye to show that the reaction can proceed to ca. 450 base pairs, and that the intermediates of many individual steps can be identified. The results demonstrate the feasibility of a 3'-editing based DNA sequencing method that could find practical applications after further optimization. Thermococcus sp. 9 degrees N DNA polymerase exhibits 3'-esterase activity that can be harnessed for DNA sequencing.,LinWu SW, Tu YH, Tsai TY, Maestre-Reyna M, Liu MS, Wu WJ, Huang JY, Chi HW, Chang WH, Chiou CF, Wang AH, Lee J, Tsai MD Commun Biol. 2019 Jun 20;2:224. doi: 10.1038/s42003-019-0458-7. eCollection 2019. PMID:31240262 From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. See Also DNA polymerase 3D structures

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