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. 1976 Sep;127(3):1550–1557. doi: 10.1128/jb.127.3.1550-1557.1976

Deoxyribonucleic acid polymerase from the extreme thermophile Thermus aquaticus.

A Chien, D B Edgar, J M Trela
PMCID: PMC232952  PMID: 8432

Abstract

A stable deoxyribonucleic acid (DNA) polymerase (EC 2.7.7.7) with a temperature optimum of 80 degrees C has been purified from the extreme thermophile Thermus aquaticus. The enzyme is free from phosphomonoesterase, phosphodiesterase and single-stranded exonuclease activities. Maximal activity of the enzyme requires all four deoxyribonucleotides and activated calf thymus DNA. An absolute requirement for divalent cation cofactor was satisfied by Mg2+ or to a lesser extent by Mn2+. Monovalent cations at concentrations as high as 0.1 M did not show a significant inhibitory effect. The pH optimum was 8.0 in tris(hydroxymethyl)aminomethane-hydrochloride buffer. The molecular weight of the enzyme was estimated by sucrose gradient centrifugation and gel filtrations on Sephadex G-100 to be approximately 63,000 to 68,000. The elevated temperature requirement, small size, and lack of nuclease activity distinguish this polymerase from the DNA polymerase of Escherichia coli.

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Selected References

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  1. Andrews P. The gel-filtration behaviour of proteins related to their molecular weights over a wide range. Biochem J. 1965 Sep;96(3):595–606. doi: 10.1042/bj0960595. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. BESSMAN M. J., LEHMAN I. R., SIMMS E. S., KORNBERG A. Enzymatic synthesis of deoxyribonucleic acid. II. General properties of the reaction. J Biol Chem. 1958 Jul;233(1):171–177. [PubMed] [Google Scholar]
  3. Brown D. D., Stern R. Methods of gene isolation. Annu Rev Biochem. 1974;43(0):667–693. doi: 10.1146/annurev.bi.43.070174.003315. [DOI] [PubMed] [Google Scholar]
  4. Brutlag D., Atkinson M. R., Setlow P., Kornberg A. An active fragment of DNA polymerase produced by proteolytic cleavage. Biochem Biophys Res Commun. 1969 Dec 4;37(6):982–989. doi: 10.1016/0006-291x(69)90228-9. [DOI] [PubMed] [Google Scholar]
  5. Buttin G., Kornberg A. Enzymatic synthesis of deoxyribonucleic acid. XXI. Utilization of deoxyribonucleoside triphosphates by Escherichia coli cells. J Biol Chem. 1966 Nov 25;241(22):5419–5427. [PubMed] [Google Scholar]
  6. DAVIS B. J. DISC ELECTROPHORESIS. II. METHOD AND APPLICATION TO HUMAN SERUM PROTEINS. Ann N Y Acad Sci. 1964 Dec 28;121:404–427. doi: 10.1111/j.1749-6632.1964.tb14213.x. [DOI] [PubMed] [Google Scholar]
  7. De Lucia P., Cairns J. Isolation of an E. coli strain with a mutation affecting DNA polymerase. Nature. 1969 Dec 20;224(5225):1164–1166. doi: 10.1038/2241164a0. [DOI] [PubMed] [Google Scholar]
  8. Deutscher M. P., Kornberg A. Enzymatic synthesis of deoxyribonucleic acid. 28. The pyrophosphate exchange and pyrophosphorolysis reactions of deoxyribonucleic acid polymerase. J Biol Chem. 1969 Jun 10;244(11):3019–3028. [PubMed] [Google Scholar]
  9. Deutscher M. P., Kornberg A. Enzymatic synthesis of deoxyribonucleic acid. 28. The pyrophosphate exchange and pyrophosphorolysis reactions of deoxyribonucleic acid polymerase. J Biol Chem. 1969 Jun 10;244(11):3019–3028. [PubMed] [Google Scholar]
  10. Englund P. T., Kelly R. B., Kornberg A. Enzymatic synthesis of deoxyribonucleic acid. XXXI. Binding of deoxyribonucleic acid to deoxyribonucleic acid polymerase. J Biol Chem. 1969 Jun 10;244(11):3045–3052. [PubMed] [Google Scholar]
  11. Falaschi A., Kornberg A. Biochemical studies of bacterial sporulation. II. Deoxy- ribonucleic acid polymerase in spores of Bacillus subtilis. J Biol Chem. 1966 Apr 10;241(7):1478–1482. [PubMed] [Google Scholar]
  12. Gefter M. L. DNA replication. Annu Rev Biochem. 1975;44:45–78. doi: 10.1146/annurev.bi.44.070175.000401. [DOI] [PubMed] [Google Scholar]
  13. Karkas J. D. Reverse transcription by Escherichia coli DNA polymerase I. Proc Natl Acad Sci U S A. 1973 Dec;70(12):3834–3838. doi: 10.1073/pnas.70.12.3834. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Kornberg T., Gefter M. L. Deoxyribonucleic acid synthesis in cell-free extracts. IV. Purification and catalytic properties of deoxyribonucleic acid polymerase III. J Biol Chem. 1972 Sep 10;247(17):5369–5375. [PubMed] [Google Scholar]
  15. Kornberg T., Gefter M. L. Purification and DNA synthesis in cell-free extracts: properties of DNA polymerase II. Proc Natl Acad Sci U S A. 1971 Apr;68(4):761–764. doi: 10.1073/pnas.68.4.761. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  17. Lehman I. R., Zimmerman S. B., Adler J., Bessman M. J., Simms E. S., Kornberg A. ENZYMATIC SYNTHESIS OF DEOXYRIBONUCLEIC ACID. V. CHEMICAL COMPOSITION OF ENZYMATICALLY SYNTHESIZED DEOXYRIBONUCLEIC ACID. Proc Natl Acad Sci U S A. 1958 Dec 15;44(12):1191–1196. doi: 10.1073/pnas.44.12.1191. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Litman R. M. A deoxyribonucleic acid polymerase from Micrococcus luteus (Micrococcus lysodeikticus) isolated on deoxyribonucleic acid-cellulose. J Biol Chem. 1968 Dec 10;243(23):6222–6233. [PubMed] [Google Scholar]
  19. Livingston D. M., Hinkle D. C., Richardson C. C. Deoxyribonucleic acid polymerase III of Escherichia coli. Purification and properties. J Biol Chem. 1975 Jan 25;250(2):461–469. [PubMed] [Google Scholar]
  20. Livingston D. M., Richardson C. C. Deoxyribonucleic acid polymerase III of Escherichia coli. Characterization of associated exonuclease activities. J Biol Chem. 1975 Jan 25;250(2):470–478. [PubMed] [Google Scholar]
  21. Loeb L. A., Fansler B., Williams R., Mazia D. Sea urchin nuclear DNA polymerase. I. Localization in nuclei during rapid DNA synthesis. Exp Cell Res. 1969 Oct;57(2):298–304. doi: 10.1016/0014-4827(69)90153-0. [DOI] [PubMed] [Google Scholar]
  22. Loeb L. A. Purification and properties of deoxyribonucleic acid polymerase from nuclei of sea urchin embryos. J Biol Chem. 1969 Apr 10;244(7):1672–1681. [PubMed] [Google Scholar]
  23. MARTIN R. G., AMES B. N. A method for determining the sedimentation behavior of enzymes: application to protein mixtures. J Biol Chem. 1961 May;236:1372–1379. [PubMed] [Google Scholar]
  24. Reuben R., Gefter M., Enquist L., Skalka A. New method for large-scale preparation of covalently closed lambda DNA molecules. J Virol. 1974 Nov;14(5):1104–1107. doi: 10.1128/jvi.14.5.1104-1107.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Sedwick W. D., Wang T. S., Korn D. Purification and properties of nuclear and cytoplasmic deoxyribonucleic acid polymerases from human KB cells. J Biol Chem. 1972 Aug 25;247(16):5026–5033. [PubMed] [Google Scholar]
  26. Suzuki Y., Gage L. P., Brown D. D. The genes for silk fibroin in Bombyx mori. J Mol Biol. 1972 Oct 14;70(3):637–649. doi: 10.1016/0022-2836(72)90563-3. [DOI] [PubMed] [Google Scholar]
  27. Taylor J. M., Faras A. J., Varmus H. E., Levinson W. E., Bishop J. M. Ribonucleic acid directed deoxyribonucleic acid synthesis by the purified deoxyribonucleic acid polymerase of Rous sarcoma virus. Characterization of the enzymatic product. Biochemistry. 1972 Jun 6;11(12):2343–2351. doi: 10.1021/bi00762a021. [DOI] [PubMed] [Google Scholar]
  28. Verma I. M., Temple G. F., Fan H., Baltimore D. In vitro synthesis of DNA complementary to rabbit reticulocyte 10S RNA. Nat New Biol. 1972 Feb 9;235(58):163–167. doi: 10.1038/newbio235163a0. [DOI] [PubMed] [Google Scholar]
  29. Wickner R. B., Ginsberg B., Berkower I., Hurwitz J. Deoxyribonucleic acid plymerase II. of Escherichia coli. I. The purification and characterization of the enzyme. J Biol Chem. 1972 Jan 25;247(2):489–497. [PubMed] [Google Scholar]

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