Marine Science & Technology Institute, College of Environmental Science and Engineering, Yangzhou University, Jiangsu Province 225127, China; Guangling College, Yangzhou University, China.
Marine Science & Technology Institute, College of Environmental Science and Engineering, Yangzhou University, Jiangsu Province 225127, China.
Int J Biol Macromol. 2020 Aug 1;156:217-224. doi: 10.1016/j.ijbiomac.2020.03.204. Epub 2020 Mar 27.
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.
产γ高温古菌是一种严格厌氧菌，最适生长温度为 88°C，其基因组编码 B 家族 DNA 聚合酶（Tga PolB）。本文中，我们克隆了 Tga PolB 基因，表达并纯化了该基因产物，并对该酶进行了生化特性分析。重组 Tga PolB 能在高温下高效合成 DNA，在 95°C 加热 1.0 h 后仍保留 93%的活性，表明该酶具有热稳定性。此外，该酶的最适 pH 值范围为 7.0-9.0。Tga PolB 活性依赖于二价阳离子，其中镁离子最佳。低浓度的 NaCl 能刺激酶活性，而高浓度的 NaCl 则抑制酶活性。有趣的是，Tga PolB 能够有效地绕过 DNA 中的尿嘧啶，这与其他古菌 B 家族 DNA 聚合酶不同。相比之下，Tga PolB 像其他古菌 B 家族 DNA 聚合酶一样，在 DNA 中的 AP 位点处停滞。此外，Tga PolB 以较低的效率延伸错配末端。该酶具有 3'-5'外切核酸酶活性，且该活性被 dNTPs 抑制。DNA 结合实验表明，Tga PolB 能有效地结合 ssDNA 和引物延伸的 DNA，并且对引物延伸的 DNA 具有明显的偏好。最后，Tga PolB 可用于常规 PCR。
