Skip to main content
PMC home page
Protein Science : A Publication of the Protein Society logoLink to Protein Science : A Publication of the Protein Society
. 1998 May;7(5):1116–1123. doi: 10.1002/pro.5560070505

Crystal structures of the Klenow fragment of Thermus aquaticus DNA polymerase I complexed with deoxyribonucleoside triphosphates.

Y Li 1, Y Kong 1, S Korolev 1, G Waksman 1
PMCID: PMC2144016  PMID: 9605316

Abstract

The crystal structures of the Klenow fragment of the Thermus aquaticus DNA polymerase I (Klentaq1) complexed with four deoxyribonucleoside triphosphates (dNTP) have been determined to 2.5 A resolution. The dNTPs bind adjacent to the O helix of Klentaq1. The triphosphate moieties are at nearly identical positions in all four complexes and are anchored by three positively charged residues, Arg659, Lys663, and Arg587, and by two polar residues, His639 and Gln613. The configuration of the base moieties in the Klentaq1/dNTP complexes demonstrates variability suggesting that dNTP binding is primarily determined by recognition and binding of the phosphate moiety. However, when superimposed on the Taq polymerase/blunt end DNA complex structure (Eom et al., 1996), two of the dNTP/Klentaq1 structures demonstrate appropriate stacking of the nucleotide base with the 3' end of the DNA primer strand, suggesting that at least in these two binary complexes, the observed dNTP conformations are functionally relevant.

Full Text

The Full Text of this article is available as a PDF (8.0 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Astatke M., Grindley N. D., Joyce C. M. Deoxynucleoside triphosphate and pyrophosphate binding sites in the catalytically competent ternary complex for the polymerase reaction catalyzed by DNA polymerase I (Klenow fragment). J Biol Chem. 1995 Jan 27;270(4):1945–1954. doi: 10.1074/jbc.270.4.1945. [DOI] [PubMed] [Google Scholar]
  2. Barnes W. M. PCR amplification of up to 35-kb DNA with high fidelity and high yield from lambda bacteriophage templates. Proc Natl Acad Sci U S A. 1994 Mar 15;91(6):2216–2220. doi: 10.1073/pnas.91.6.2216. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Beese L. S., Friedman J. M., Steitz T. A. Crystal structures of the Klenow fragment of DNA polymerase I complexed with deoxynucleoside triphosphate and pyrophosphate. Biochemistry. 1993 Dec 28;32(51):14095–14101. doi: 10.1021/bi00214a004. [DOI] [PubMed] [Google Scholar]
  4. Catalano C. E., Allen D. J., Benkovic S. J. Interaction of Escherichia coli DNA polymerase I with azidoDNA and fluorescent DNA probes: identification of protein-DNA contacts. Biochemistry. 1990 Apr 17;29(15):3612–3621. doi: 10.1021/bi00467a004. [DOI] [PubMed] [Google Scholar]
  5. Cheng S., Fockler C., Barnes W. M., Higuchi R. Effective amplification of long targets from cloned inserts and human genomic DNA. Proc Natl Acad Sci U S A. 1994 Jun 7;91(12):5695–5699. doi: 10.1073/pnas.91.12.5695. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Delarue M., Poch O., Tordo N., Moras D., Argos P. An attempt to unify the structure of polymerases. Protein Eng. 1990 May;3(6):461–467. doi: 10.1093/protein/3.6.461. [DOI] [PubMed] [Google Scholar]
  7. Hamlin R. Multiwire area X-ray diffractometers. Methods Enzymol. 1985;114:416–452. doi: 10.1016/0076-6879(85)14029-2. [DOI] [PubMed] [Google Scholar]
  8. Howard A. J., Nielsen C., Xuong N. H. Software for a diffractometer with multiwire area detector. Methods Enzymol. 1985;114:452–472. doi: 10.1016/0076-6879(85)14030-9. [DOI] [PubMed] [Google Scholar]
  9. Jones T. A., Thirup S. Using known substructures in protein model building and crystallography. EMBO J. 1986 Apr;5(4):819–822. doi: 10.1002/j.1460-2075.1986.tb04287.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Joyce C. M., Steitz T. A. Function and structure relationships in DNA polymerases. Annu Rev Biochem. 1994;63:777–822. doi: 10.1146/annurev.bi.63.070194.004021. [DOI] [PubMed] [Google Scholar]
  11. Kim Y., Eom S. H., Wang J., Lee D. S., Suh S. W., Steitz T. A. Crystal structure of Thermus aquaticus DNA polymerase. Nature. 1995 Aug 17;376(6541):612–616. doi: 10.1038/376612a0. [DOI] [PubMed] [Google Scholar]
  12. Korolev S., Nayal M., Barnes W. M., Di Cera E., Waksman G. Crystal structure of the large fragment of Thermus aquaticus DNA polymerase I at 2.5-A resolution: structural basis for thermostability. Proc Natl Acad Sci U S A. 1995 Sep 26;92(20):9264–9268. doi: 10.1073/pnas.92.20.9264. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Lawyer F. C., Stoffel S., Saiki R. K., Chang S. Y., Landre P. A., Abramson R. D., Gelfand D. H. High-level expression, purification, and enzymatic characterization of full-length Thermus aquaticus DNA polymerase and a truncated form deficient in 5' to 3' exonuclease activity. PCR Methods Appl. 1993 May;2(4):275–287. doi: 10.1101/gr.2.4.275. [DOI] [PubMed] [Google Scholar]
  14. Merritt E. A., Murphy M. E. Raster3D Version 2.0. A program for photorealistic molecular graphics. Acta Crystallogr D Biol Crystallogr. 1994 Nov 1;50(Pt 6):869–873. doi: 10.1107/S0907444994006396. [DOI] [PubMed] [Google Scholar]
  15. Pandey V. N., Kaushik N., Sanzgiri R. P., Patil M. S., Modak M. J., Barik S. Site directed mutagenesis of DNA polymerase I (Klenow) from Escherichia coli. The significance of Arg682 in catalysis. Eur J Biochem. 1993 May 15;214(1):59–65. doi: 10.1111/j.1432-1033.1993.tb17896.x. [DOI] [PubMed] [Google Scholar]
  16. Pandey V. N., Modak M. J. Affinity labeling of Escherichia coli DNA polymerase I by 5'-fluorosulfonylbenzoyladenosine. Identification of the domain essential for polymerization and Arg-682 as the site of reactivity. J Biol Chem. 1988 May 5;263(13):6068–6073. [PubMed] [Google Scholar]
  17. Suzuki M., Baskin D., Hood L., Loeb L. A. Random mutagenesis of Thermus aquaticus DNA polymerase I: concordance of immutable sites in vivo with the crystal structure. Proc Natl Acad Sci U S A. 1996 Sep 3;93(18):9670–9675. doi: 10.1073/pnas.93.18.9670. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Tabor S., Richardson C. C. A single residue in DNA polymerases of the Escherichia coli DNA polymerase I family is critical for distinguishing between deoxy- and dideoxyribonucleotides. Proc Natl Acad Sci U S A. 1995 Jul 3;92(14):6339–6343. doi: 10.1073/pnas.92.14.6339. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Wyatt J. L., Colman R. F. Affinity labeling of rabbit muscle pyruvate kinase by 5'-p-fluorosulfonylbenzoyladenosine. Biochemistry. 1977 Apr 5;16(7):1333–1342. doi: 10.1021/bi00626a015. [DOI] [PubMed] [Google Scholar]

Articles from Protein Science : A Publication of the Protein Society are provided here courtesy of The Protein Society

RESOURCES