Crystal structure of a bacteriophage T7 DNA replication complex at 2.2 Å resolution

Article metrics


DNA polymerases change their specificity for nucleotide substrates with each catalytic cycle, while achieving error frequencies in the range of 10−5to 10−6. Here we present a 2.2 Å crystal structure of the replicative DNA polymerase from bacteriophage T7 complexed with a primer–template and a nucleoside triphosphate in the polymerase active site. The structure illustrates how nucleotides are selected in a template-directed manner, and provides a structural basis for a metal-assisted mechanism of phosphoryl transfer by a large group of related polymerases.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Figure 1: Topology and overall fold of T7 DNA polymerase.
Figure 2: Polymerase contacts to the primer–template.
Figure 3: Two-metal ligation of the bound nucleotide.
Figure 4: Selectivity for 2′-deoxyribonucleotides.
Figure 5: Open and closed conformations of polymerase active sites.
Figure 6: Misincorporation and proofreading.


  1. 1

    Tabor, S., Huber, H. E. & Richardson, C. C. Escherichia coli thioredoxin confers processivity on the DNA polymerase activity of the gene 5 protein of bacteriophage T7. J. Biol. Chem. 262, 16212– 16223 (1987).

  2. 2

    Modrich, P. & Richardson, C. C. Bacteriophage T7 deoxyribonucleic acid replication in vitro. A protein of Escherichia coli required for bacteriophage T7 DNA polymerase activity. J. Biol. Chem. 250, 5508–5514 (1975).

  3. 3

    Debyser, Z., Tabor, S. & Richardson, C. C. Coordination of leading and lagging strand DNA synthesis at the replication fork of bacteriophage T7. Cell 77 , 157–166 (1994).

  4. 4

    Delarue, M., Poch, O., Tordo, N., Moras, D. & Argos, P. An attempt to unify the structure of polymerases. Protein Eng. 3, 461–467 (1990).

  5. 5

    Braithwaite, D. K. & Ito, J. Compilation, alignment, and phylogenetic relationships of DNA polymerases. Nucleic Acids Res. 21, 787–802 ( 1993).

  6. 6

    Pelletier, H., Sawaya, M. R., Kumar, A., Wilson, S. H. & Kraut, J. Structures of ternary complexes of rat DNA polymerase beta, a DNA template-primer, and ddCTP. Science 264 , 1891–1903 (1994).

  7. 7

    Tabor, S. & Richardson, C. C. Selective inactivation of the exonuclease activity of bacteriophage T7 DNA polymerase by in vitro mutagenesis. J. Biol. Chem. 264, 6447 –6458 (1989).

  8. 8

    Steitz, T. A. DNA- and RNA-dependent DNA polymerases. Curr. Opin. Struct. Biol. 3, 31–38 (1993 ).

  9. 9

    Polesky, A. H., Steitz, T. A., Grindley, N. D. & Joyce, C. M. Identificaiton of residues critical for the polymerase activity of the Klenow fragment of DNA polymerase I from Escherichia coli. J. Biol. Chem. 265, 14579–14591 ( 1990).

  10. 10

    Polesky, A. H., Dahlberg, M. E., Benkovic, S. J., Grindley, N. D. & Joyce, C. M. Side chains involved in catalysis of the polymerase reaction of DNA polymerase I from Escherichia coli. J. Biol. Chem. 267, 8417– 8428 (1992).

  11. 11

    Ollis, D. L., Brick, P., Hamlin, R., Xuong, N. G. & Steitz, T. A. Structure of large fragment of Escherichia coli DNA polymerase I complexed with dTMP. Nature 313 , 762–766 (1985).

  12. 12

    Beese, L. S., Derbyshire, V. & Steitz, T. A. Structure of DNA polymerase I Klenow fragment bound to duplex DNA. Science 260, 352– 355 (1993).

  13. 13

    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-Å resolution: structural basis for thermostability. Proc. Natl Acad. Sci. USA 92, 9264–9268 (1995).

  14. 14

    Kiefer, J. R. et al. Crystal structure of a thermostable Bacillus DNA polymerase I large fragment at 2.1 Å resolution. Structure 5, 95–108 (1997).

  15. 15

    Kim, Y. et al. Crystal structure of Thermus aquaticus DNA polymerase. Nature 376, 612–616 ( 1995).

  16. 16

    Eom, S. H., Wang, J. & Steitz, T. A. Structure of Taq polymerase with DNA at the polymerase active site. Nature 382, 278– 281 (1996).

  17. 17

    Sawaya, M. R., Prasad, R., Wilson, S. H., Kraut, J. & Pelletier, H. Crystal structures of human DNA polymerase β complexed with gapped and nicked DNA: Evidence for an induced fit mechanism. Biochemistry 36, 11205– 11215 (1997).

  18. 18

    Minnick, D. T., Astatke, M., Joyce, C. M. & Kunkel, T. A. Athumb subdomain mutant of the large fragment of Escherichia coli DNA polymerase I with reduced DNA binding affinity, processivity, and frameshift fidelity. J. Biol. Chem. 271, 24954– 24961 (1996).

  19. 19

    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. 270, 1945–1954 ( 1995).

  20. 20

    Burgers, P. M. & Eckstein, F. Astudy of the mechanism of DNA polymerase I from Escherichia coli with diastereomeric phosphorothioate analogs of deoxyadenosine triphosphate. J. Biol. Chem. 254, 6889–6893 ( 1979).

  21. 21

    Beese, L. S. & Steitz, T. A. Structural basis for the 3′-5′ exonuclease activity of Escherichia coli DNA polymerase I: a two metal ion mechanism. EMBO J. 10, 25– 33 (1991).

  22. 22

    Kaushik, N., Pandey, V. N. & Modak, M. J. Significance of the O-helix residues of Escherichia coli DNA polymerase I in DNA synthesis: dynamics of the dNTP binding pocket. Biochemistry 35, 7256– 7266 (1996).

  23. 23

    Joyce, C. M. Choosing the right sugar: how polymerases select a nucleotide substrate. Proc. Natl Acad. Sci. USA 94, 1619– 1622 (1997).

  24. 24

    Tabor, S. & Richardson, C. C. Asingle residue in DNA polymerases of the Escherichia coli DNA polymerase I family is critical for distinguishing between deoxy- and dideoxyribonucleotides. Proc. Natl Acad. Sci. USA 92, 6339–6343 ( 1995).

  25. 25

    Patel, S. S., Wong, I. & Johnson, K. A. Pre-steady-state kinetic analysis of processive DNA replication including complete characterization of an exonuclease-deficient mutant. Biochemistry 30, 511– 525 (1991).

  26. 26

    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 32, 14095– 14101 (1993).

  27. 27

    Bryant, F. R., Johnson, K. A., & Benkovic, S. J. Elementary steps in the DNA polymerase I reaction pathway. Biochemistry 22, 3537– 3546 (1983).

  28. 28

    Wong, I., Patel, S. S. & Johnson, K. A. An induced-fit kinetic mechanism for DNA replcation fidelity: direct measurement by single-turnover kinetics. Biochemistry 30, 526–537 ( 1991).

  29. 29

    Donlin, M. J., Patel, S. S. & Johnson, K. A. Kinetic partitioning between the exonuclease and polymerase sites in DNA error correction. Biochemistry 30, 538–546 (1991).

  30. 30

    Seeman, N. C., Rosenberg, J. M. & Rich, A. Sequence-specific recognition of double helical nucleic acids by proteins. Proc. Natl Acad. Sci. USA 73, 804–808 (1976).

  31. 31

    Hunter, W. N., Brown, T., Anand, N. N. & Kennard, O. Structure of adenine·cytosine base pair in DNA and its implications for mismatch repair. Nature 320, 552–555 ( 1986).

  32. 32

    Kong, X. P., Onrust, R., O'Donnell, M. & Kuriyan, J. Three-dimensional structure of the beta subunit of E. coli DNA polymerase III holoenzyme: a sliding DNA clamp. Cell 69, 425–437 (1992).

  33. 33

    Himawan, J. S. & Richardson, C. C. Amino acid residues critical for the interaction between bacteriophage T7 DNA polymerase and Escherichia coli thioredoxin. J. Biol. Chem. 271, 19999–20008 (1996).

  34. 34

    Yang, X. M. & Richardson, C. C. Amino acid changes in a unique sequence of bacteriophage T7 DNA polymerase alter the processivity of nucleotide polymerization. J. Biol. Chem. 272, 6599 –6606 (1997).

  35. 35

    Adler, S. & Modrich, P. T7-induced DNA polymerase. Requirement for thioredoxin sulfhydryl groups. J. Biol. Chem. 258 , 6956–6962 (1983).

  36. 36

    Huber, H. E., Russel, M., Model, P. & Richardson, C. C. Interaction of mutant thioredoxins of Escherichia coli with the gene 5 protein of phage T7. The redox capacity of thioredoxin is not required for stimulation of DNA polymerase activity. J. Biol. Chem. 261, 15006–15012 (1986).

  37. 37

    Qin, J., Clore, G. M., Kennedy, W. M., Huth, J. R. & Gronenborn, A. M. Solution structure of human thioredoxin in a mixed disulfide intermediate complex with its target peptide from the transcription factor NF kappa B. Structure 3, 289–297 (1995).

  38. 38

    Bedford, E., Tabor, S. & Richardson, C. C. The thioredoxin binding domain of bacteriophage T7 DNA polymerase confers processivity on Escherichia coli DNA polymerase I. Proc. Natl Acad. Sci. USA 94, 479– 484 (1997).

  39. 39

    Doublié, S. Preparation of selenomethionyl proteins for phase determination. Methods Enzymol. 276, 523–530 (1997).

  40. 40

    Carter, C. W. J in Crystallization of Nucleic Acids and Proteins: A Practical Approach (eds Ducruix, A. & Giegé, R.) 47– 71 (IRL Press, New York, (1992)).

  41. 41

    Otwinowski, Z. & Minor, W. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol 276, 307–326 ( 1997).

  42. 42

    Rould, M. A. Screening for heavy-atom derivatives and obtaining accurate isomorphous differences. Methods Enzymol. 276, 461– 472 (1997).

  43. 43

    Sheldrick, G. M. Phase annealing in SHELX-90: direct methods for larger structures. Acta Crystallogr. A 46, 467–473 (1990).

  44. 44

    Bailey, S. The CCP4 Suite-programs for protein crystallography. Acta Crystallogr. D 50, 760–763 ( 1994).

  45. 45

    Jones, T. A., Zou, J. Y., Cowan, S. W. & Kjeldgaard. Improved methods for binding protein models in electron density maps and the location of errors in these models. Acta Crystallogr. A 47, 110– 119 (1991).

  46. 46

    Brünger, A. T. XPLOR Version 3.1: A system for X-ray crystallography and NMR.(Yale University Press, New Haven, CT, (1992)).

  47. 47

    Laskowski, R. A., McArthur, M. W., Moss, D. S. & Thornton, J. M. PROCHECK — a program to check the stereochemical quality of protein structures. J. Appl. Crystallogr. 26, 283 –291 (1993).

  48. 48

    Evans, S. V. SETOR: Hardware lighted three-dimensional solid model representations of macromolecules. J. Mol. Graphics 11, 134– 138 (1993).

  49. 49

    Nicholls,, Charp, K. A. & Honig, B. Protein folding and association: insights from interfacial and thermodynamics properties of hydrocarbons. Proteins Struct. Funct. Genet. 11, 281–296 (1991).

  50. 50

    Merritt, E. A. & Bacon, D. J. Raster3D: photorealistic molecular graphics. Methods Enzymol 276, 505–524 ((1997)).

Download references


We thank R. M. Sweet and F. W. Studier for access and assistance with the use of beamline X12C (NSLS, Brookhaven National Laboratory). We thank H. J. Kwon, A. Lau and M. Rould for experimental help at the synchrotron, members of the Ellenberger, Richardson and Hogle groups for many discussions and M. Rould for programs and discussions. This work was supported by the National Institutes of Health, the Lucille Markey Charitable Trust, the Department of Energy, the Harvard Center for Structural Biology and the Armenise-Harvard Foundation for Advanced Scientific Research. C.C.R. and S.T. are consultants to Amersham Life Science Inc., which has licenses from Harvard University to commercialize DNA polymerases for use in DNA sequencing.

Author information

Correspondence to Tom Ellenberger.

Rights and permissions

Reprints and Permissions

About this article

Further reading


By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.