Thermococcus gammatolerans is anaerobic euryarchaeon which grows optimally at 88 °C and its genome encodes a family B DNA polymerase (Tga PolB). Herein, we cloned the gene of Tga PolB, expressed and purified the gene product, and characterized the enzyme biochemically. The recombinant Tga PolB can efficiently synthesize DNA at high temperature, and retain 93% activity after heated at 95 °C for 1.0 h, suggesting that the enzyme is thermostable. Furthermore, the optimal pH for the enzyme activity was measured to be 7.0-9.0. Tga PolB activity is dependent on a divalent cation, among which magnesium ion is optimal. NaCl at low concentration stimulates the enzyme activity but at high concentration inhibits enzyme activity. Interestingly, Tga PolB is able to efficiently bypass uracil in DNA, which is distinct from other archaeal family B DNA pols. By contrast, Tga PolB is halted by an AP site in DNA, as observed in other archaeal family B DNA polymerases. Furthermore, Tga PolB extends the mismatched ends with reduced efficiencies. The enzyme possesses 3'-5' exonuclease activity and this activity is inhibited by dNTPs. The DNA binding assays showed that Tga PolB can efficiently bind to ssDNA and primed DNA, and have a marked preference for primed DNA. Last, Tga PolB can be used in routine PCR.

中文翻译：

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嗜热和耐辐射性euryarchaeon嗜热球菌γ家族的B族DNA聚合酶的表征和应用。

嗜热热球菌是厌氧性euryarchaeon，在88°C时最佳生长，其基因组编码B族DNA聚合酶（Tga PolB）。在这里，我们克隆了Tga PolB基因，表达并纯化了基因产物，并对其生化特性进行了表征。重组Tga PolB可以在高温下有效合成DNA，并在95°C加热1.0 h后仍保留93％的活性，这表明该酶是热稳定的。此外，测得的酶活性的最适pH为7.0-9.0。Tga PolB活性取决于二价阳离子，其中镁离子是最佳的。低浓度的NaCl会刺激酶的活性，而高浓度的NaCl会抑制酶的活性。有趣的是，Tga PolB能够有效绕过DNA中的尿嘧啶，这与其他古细菌家族B DNA pols不同。相反，如在其他古细菌家族B DNA聚合酶中所观察到的，Tga PolB被DNA中的AP位点终止。此外，Tga PolB降低了不匹配末端的效率。该酶具有3'-5'核酸外切酶活性，该活性被dNTPs抑制。DNA结合试验表明，Tga PolB可以有效结合ssDNA和引发的DNA，并且对引发的DNA有明显的偏爱。最后，Tga PolB可用于常规PCR。并且对引物DNA有明显的偏爱。最后，Tga PolB可用于常规PCR。并且对引物DNA有明显的偏爱。最后，Tga PolB可用于常规PCR。