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°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.

中文翻译：

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热球菌属 sp. 9°N DNA 聚合酶具有 3'-酯酶活性，可用于 DNA 测序。


1995年报道T7和Taq DNA聚合酶具有3'-酯酶活性，但没有后续研究。在这里，我们报告 3'-酯酶活性是热球菌属物种固有的。 9°N DNA 聚合酶，并且可以将其开发为一种使用带有荧光团的 3'-酯的 dNTP 类似物进行 DNA 测序的连续方法。我们首先证明3'-酯化的dNTP可以掺入模板引物DNA中，并解析反应中间体和产物的晶体结构。然后我们证明该反应可以在活性位点残基 Tyr409 和 Asp542 的调节下连续发生。最后，我们使用 5'-FAM 标记的引物和用染料酯化的 dNTP，以表明反应可以进行到约。 450 个碱基对，并且可以识别许多单独步骤的中间体。结果证明了基于3'编辑的DNA测序方法的可行性，经过进一步优化可以找到实际应用。