Chemical Research in Chinese Universities ›› 2016, Vol. 32 ›› Issue (2): 226-233.doi: 10.1007/s40242-016-5337-x

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Biochemical Characterization of Translesion Synthesis by Sulfolobus acidocaldarius DNA Polymerases

PENG Li, XIA Xu, LIU Xipeng   

  1. State Key Laboratory of Microbial Metabolism, School of Life Sciences & Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, P. R. China
  • Received:2015-08-21 Revised:2015-12-08 Online:2016-04-01 Published:2016-01-04
  • Contact: LIU Xipeng E-mail:xpliu@sjtu.edu.cn
  • Supported by:

    Supported by the National Natural Science Foundation of China(No.31371260) and the Natural Science Foundation of Shanghai City, China(No.12ZR1413700).

Abstract:

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℃. 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.

Key words: Archaeota, Sulfolobus acidocaldarius, DNA polymerase, DNA damage, DNA replication