To study the DNA synthesis mechanism of Sulfolobus acidocaldarius, a thermophilic species from Crenarehaeota, two DNA polymerases of B family（polB1 and polB3）, and one DNA polymerase of Y family（polIV） were recomb...To study the DNA synthesis mechanism of Sulfolobus acidocaldarius, a thermophilic species from Crenarehaeota, 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 polBl（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 Mn1＋ and Mg2＋. They showed the highest activity at pH values ranging from 8.0 to 9.5. Their activities were inhibited by KC1 with high concentration. The optimal reaction temperatures for the three DNA polymerases were between 60 and 70 ℃. 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.展开更多
基金Supported by the National Natural Science Foundation of China（No.31371260） and the Natural Science Foundation of Shanghai City, China（No. 12ZR1413700）. We greatly appreciate Professor Sonja-Verena Albers for kindly providing the strain of S. acidocaldarius.
文摘To study the DNA synthesis mechanism of Sulfolobus acidocaldarius, a thermophilic species from Crenarehaeota, 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 polBl（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 Mn1＋ and Mg2＋. They showed the highest activity at pH values ranging from 8.0 to 9.5. Their activities were inhibited by KC1 with high concentration. The optimal reaction temperatures for the three DNA polymerases were between 60 and 70 ℃. 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.
