Biochemical characterization of translesion synthesis by Sulfolobus acidocaldarius DNA polymerases
Biochemical characterization of translesion synthesis by Sulfolobus acidocaldarius DNA polymerases
复制标题
酸热硫化叶菌 DNA 聚合酶跨损伤合成的生化表征
DOI：
10.1007/s40242-016-5337-x
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发表时间：
2016-01
影响因子：
3.1
通讯作者：
Liu Xipeng
中科院分区：
文献类型：
--
作者：
Peng Li;Xia Xu;Liu Xipeng
为研究泉古菌嗜热硫化叶菌（Sulfolobus acidocaldarius）的DNA合成机制，对两种B家族DNA聚合酶（polB 1和polB 3）和一种Y家族DNA聚合酶（polIV）进行了重组表达、纯化和生化分析。DNA聚合酶polB 1（Saci_1537）和polB 3（Saci_0074）均具有DNA聚合酶和3'至5'核酸外切酶活性，但B3的活性在体外均非常低。polIV（Saci_0554）是一种聚合酶，而不是核酸外切酶。三种DNA聚合酶的活性均依赖于二价金属离子Mn ~（2+）和Mg ~（2+）。它们在pH值为8.0至9.5时表现出最高的活性。高浓度的KCl对它们的活性有抑制作用。三种DNA聚合酶的最佳反应温度为60 - 70 °C。DNA模板上脱氨基的碱基dU和dI强烈地阻碍了B家族的两种DNA聚合酶的引物延伸，而不是Y家族的DNA聚合酶。Y家族的DNA聚合酶比B家族的DNA聚合酶更容易绕过模板上的两个AP位点类似物dSpacer和propane。结果表明，在酸热硫化叶菌细胞中，三种DNA聚合酶协同完成多种DNA合成。
To study the DNA synthesis mechanism of Sulfolobus acidocaldarius, a thermophilic species from Crenarchaeota, two DNA polymerases of B family(polB1 and polB3), and one DNA polymerase of Y family(polIV) were recombinantly expressed, purified and biochemically characterized. Both DNA polymerases polB1(Saci_1537) and polB3(Saci_0074) possessed DNA polymerase and 3’ to 5’ exonuclease activities; however, both the activities of B3 were very inefficient in vitro. The polIV(Saci_0554) was a polymerase, not an exonuclease. The activities of all the three DNA polymerases were dependent on divalent metal ions Mn2+ and Mg2+. They showed the highest activity at pH values ranging from 8.0 to 9.5. Their activities were inhibited by KCl with high concentration. The optimal reaction temperatures for the three DNA polymerases were between 60 and 70 °C. Deaminated bases dU and dI on DNA template strongly hindered primer extension by the two DNA polymerases of B family, not by the DNA polymerase of Y family. DNA polymerase of Y Family bypassed the two AP site analogues dSpacer and propane on template more easily than DNA polymerases of B family. Our results suggest that the three DNA polymerases coordinate to fulfill various DNA synthesis in Sulfolobus acidocaldarius cell.
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