Cloning, Expression, and Characterization of a Family B-Type DNA Polymerase from the Hyperthermophilic Crenarchaeon Pyrobaculum arsenaticum and Its Application to PCR

  • SHIN HEA-JIN (Department of Genetic Engineering, Sungkyunkwan University) ;
  • LEE SUNG-KYOUNG (Department of Genetic Engineering, Sungkyunkwan University) ;
  • CHOI JEONG JIN (Department of Genetic Engineering, Sungkyunkwan University) ;
  • KOH SUK-HOON (Genome Research Center, Korea Research Institute of Bioscience and Biotechnology) ;
  • LEE JUNG-HYUN (Marine Biotechnology Research Centre, Korea Ocean Research and Development Institute) ;
  • KIM SANG-JIN (Marine Biotechnology Research Centre, Korea Ocean Research and Development Institute) ;
  • KWON SUK-TAE (Department of Genetic Engineering, Sungkyunkwan University)
  • Published : 2005.12.01

Abstract

The gene encoding Pyrobaculum arsenaticum DNA polymerase (Par DNA polymerase) was cloned and sequenced. The gene consists of 2,361 bp coding for a protein with 786 amino acid residues. The deduced amino acid sequence of Par DNA polymerase showed a high similarity to archaeal family B-type DNA polymerases (Group I), and contained all of the motifs conserved in the family B-type DNA polymerases for $3'{\rightarrow}5'$ exonuclease and polymerase activities. The Par DNA polymerase gene was expressed under the control of the T7lac promoter on the expression vector pET-22b(+) in Escherichia coli BL21-CodonPlus(DE3)-RP. The expressed enzyme was purified by heat treatment, and Cibacron blue 3GA and $Hirap^{TM}$ Heparin HP column chromatographies. The optimum pH of the purified enzyme was 7.5. The enzyme activity was activated by divalent cations, and was inhibited by EDTA and monovalent cations. The half-life of the enzyme at $95^{\circ}C$ was 6 h. Par DNA polymerase possessed associated $3'{\rightarrow}5'$ proofreading exonuclease activity, which is consistent with its deduced amino acid sequence. PCR experiment with Par DNA polymerase showed an amplified product, indicating that this enzyme might be useful in DNA amplification and PCR-based applications.

Keywords

References

  1. Bae, J.-D., Y.-J. Cho, D.-I. Kim, D.-S. Lee, and H.-J. Shin. 2003. Purification and biochemical characterization of recombinant alanine dehydrogenase from Thermus caldophilus GK24. J. Microbiol. Biotechnol. 13: 628-631
  2. Barnes, W. M. 1994. PCR amplification of up to 35-kb DNA with high fidelity and high yield from$\lambda$ bacteriophage templates. Proc. Natl. Acad. Sci. USA 91: 2216-2220 https://doi.org/10.1073/pnas.91.6.2216
  3. Barns, S. M., C. F. Delwiche, J. D. Palmer, and N. R. Pace. 1996. Perspectives on archaeal diversity, thermophily and monophyly from environmental rRNA sequences. Proc. Natl. Acad. Sci. USA 93: 9188-9193
  4. Blanco, L., A. Bernad, M. A. Blasco, and M. Salas. 1991. A general structure for DNA-dependent DNA polymerases. Gene 100: 27-38 https://doi.org/10.1016/0378-1119(91)90346-D
  5. Braithwaite, D. K. and J. Ito. 1993. Compilation, alignment, and phylogenetic relationships of DNA polymerases. Nucleic Acids Res. 21: 787-802 https://doi.org/10.1093/nar/21.4.787
  6. Brown, J. R. and W. F. Doolittle. 1995. Root of the universal tree of life based on ancient aminoacyl-tRNA synthetase gene duplications. Proc. Natl. Acad. Sci. USA 92: 2441- 2445
  7. Cann, I. K. O., S. Ishino, N. Nomura, Y. Sako, and Y. Ishino. 1999. Two family B DNA polymerases from Aeropyrum pernix, an aerobic hyperthermophilic crenarchaeote. J. Bacteriol. 181: 5984-5992
  8. Cann, I. K. O. and Y. Ishino. 1999. Archaeal DNA replication: identifying the pieces to solve a puzzle. Genetics 152: 1249- 1267
  9. Chien, A., D. B. Edgar, and J. M. Trela. 1976. Deoxyribonucleic acid polymerase from the extreme thermophile Thermus aquaticus. J. Bacteriol. 127: 1550-1557
  10. Choi, J. J., S. E. Jung, H.-K. Kim, and S.-T. Kwon. 1999. Purification and properties of Thermus filiformis DNA polymerase expressed in Escherichia coli. Biotechnol. Appl. Biochem. 30: 19-25
  11. Choi, J. J. and S.-T. Kwon. 2004. Cloning, expression, and characterization of DNA polymerase from hyperthermophilic bacterium Aquifex pyrophilus. J. Microbiol. Biotechnol. 14: 1022-1030
  12. Corpet, F. 1988. Multiple sequence alignment with hierarchical clustering. Nucleic Acids Res. 16: 10881-10890 https://doi.org/10.1093/nar/16.22.10881
  13. Dennis, P. P. 1997. Ancient ciphers: Translation in Archaea. Cell 89: 1007-1010 https://doi.org/10.1016/S0092-8674(00)80288-3
  14. Erlich, H. A. 1989. PCR Technology: Principles and Applications for DNA Amplification. Stockton Press, New York, U.S.A
  15. Fitz-Gibbon, S. T., H. Ladner, U.-J. Kim, K. O. Stetter, M. I. Simon, and J. H. Miller. 2002. Genome sequence of the hyperthermophilic crenarchaeon Pyrobaculum aerophilum. Proc. Natl. Acad. Sci. USA 99: 984-989
  16. Hanahan, D. and M. Meselson. 1980. Plasmid screening at high colony density. Gene 10: 63-67 https://doi.org/10.1016/0378-1119(80)90144-4
  17. Hoe, H.-S., S.-K. Lee, D.-S. Lee, and S.-T. Kwon. 2003. Cloning, analysis, and expression of the gene for thermostable polyphosphate kinase of Thermus caldophilus GK24 and properties of the recombinant enzyme. J. Microbiol. Biotechnol. 13: 139-145
  18. Huber, H., M. J. Hohn, R. Rachel, T. Fuchs, V. C. Wimmer, and K. O. Stetter. 2002. A new phylum of Archaea represented by a nanosized hyperthermophilic symbiont. Nature 417: 63-67 https://doi.org/10.1038/417063a
  19. Huber, R., M. Sacher, A. Vollmann, H. Huber, and D. Rose. 2000. Respiration of arsenate and selenate by hyperthermophilic archaea. Syst. Appl. Microbiol. 23: 305- 314 https://doi.org/10.1016/S0723-2020(00)80058-2
  20. Jung, S. E., J. J. Choi, H. K. Kim, and S.-T. Kwon. 1997. Cloning and analysis of the DNA polymerase-encoding gene from Thermus filiformis. Mol. Cells 7: 769-776
  21. Kahler, M. and G. Antranikian. 2000. Cloning and characterization of a family B DNA polymerase from the hyperthermophilic crenarchaeon Pyrobaculum islandicum. J. Bacteriol. 182: 655-663 https://doi.org/10.1128/JB.182.3.655-663.2000
  22. Kornberg, A. and T. Baker. 1992. DNA Replication, 2nd Ed. Freeman and Company, New York, U.S.A
  23. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680-685 https://doi.org/10.1038/227680a0
  24. Lee, J.-H., Y.-D. Cho, J. J. Choi, Y.-J. Lee, H.-S. Hoe, H.-K. Kim, and S.-T. Kwon. 2003. High-level expression in Escherichia coli of alkaline phosphatase from Thermus caldophilus GK24 and purification of the recombinant enzyme. J. Microbiol. Biotechnol. 13: 660-665
  25. Lowry, O. H., N. J. Rosebrough, A. J. Farr, and R. J. Randall. 1951. Protein measurement with folin phenol reagent. J. Biol. Chem. 193: 265-275
  26. Lundberg, K. S., D. D. Shoemaker, M. W. W. Adams, J. M. Short, J. A. Sorge, and E. J. Mathur. 1991. Highfidelity amplification using a thermostable DNA polymerase isolated from Pyrococcus furiosus. Gene 108: 1-6 https://doi.org/10.1016/0378-1119(91)90480-Y
  27. Marmur, J. 1961. A procedure for the isolation of deoxyribonucleic acid from micro-organisms. J. Mol. Biol. 3: 208-218 https://doi.org/10.1016/S0022-2836(61)80047-8
  28. Mattila, P., J. Korpela, T. Tenkanen, and K. Pitkanen. 1991. Fidelity of DNA synthesis by the Thermococcus litoralis DNA polymerase, an extremely heat stable enzyme with proofreading activity. Nucleic Acids Res. 19: 4967-4973 https://doi.org/10.1093/nar/19.18.4967
  29. Nishioka, M., H. Mizuguchi, S. Fujiwara, S. Komatsubara, M. Kitabayashi, H. Uemura, M. Takagi, and T. Imanaka. 2001. Long and accurate PCR with a mixture of KOD DNA polymerase and its exonuclease deficient mutant enzyme. J. Biotechnol. 88: 141-149 https://doi.org/10.1016/S0168-1656(01)00275-9
  30. Reiter, W., U. Hudepohl, and W. Zillig. 1990. Mutational analysis of an archaebacterial promoter: Essential role of a TATA box for transcription efficiency and start-site selection in vitro. Proc. Natl. Acad. Sci. USA 87: 9509-9513 https://doi.org/10.1073/pnas.87.24.9509
  31. Saiki, R. K., D. H. Gelfand, S. Stoffel, S. J. Scharf, R. Higuchi, G. T. Horn, K. B. Mullis, and H. A. Erlich. 1988. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239: 487-491 https://doi.org/10.1126/science.2448875
  32. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular Cloning: A Laboratory Manual, 2nd Ed. Cold Spring Harbor Laboratory Press, New York, U.S.A
  33. Sanger, F., A. R. Coulson, G. F. Hong, D. F. Hill, and G. B. Petersen. 1982. Nucleotide sequence of bacteriophage lambda DNA. J. Mol. Biol. 162: 729-773 https://doi.org/10.1016/0022-2836(82)90546-0
  34. Silhavy, T. J., M. L. Berman, and L. W. Enquist. 1984. Experiments with Gene Fusions. Cold Spring Harbor Laboratory Press, New York, U.S.A
  35. Studier, F. W. and B. A. Moffatt. 1986. Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J. Mol. Biol. 189: 113-130 https://doi.org/10.1016/0022-2836(86)90385-2
  36. Truniger, V., J. M. Lazaro, M. Salas, and L. Blanco. 1996. A DNA binding motif coordinating synthesis and degradation in proofreading DNA polymerases. EMBO J. 15: 3430- 3441
  37. Uemori, T., Y. Ishino, H. Doi, and I. Kato. 1995. The hyperthermophilic archaeon Pyrodictium occultum has two $\alpha$-like DNA polymerases. J. Bacteriol. 177: 2164-2177 https://doi.org/10.1128/jb.177.8.2164-2177.1995
  38. Uemori, T., Y. Ishino, H. Toh, K. Asada, and I. Kato. 1993. Organization and nucleotide sequence of the DNA polymerase gene from the archaeon Pyrococcus furiosus. Nucleic Acids Res. 21: 259-265 https://doi.org/10.1093/nar/21.2.259
  39. Woese, C. R., O. Kandler, and M. L. Wheelis. 1990. Towards a natural system of organisms: Proposal for the domains Archaea, Bacteria, and Eucarya. Proc. Natl. Acad. Sci. USA 87: 4576-4579