Visualizing DNA replication in a catalytically active Bacillus DNA polymerase crystal

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Abstract

DNA polymerases copy DNA templates with remarkably high fidelity, checking for correct base-pair formation both at nucleotide insertion and at subsequent DNA extension steps1,2,3. Despite extensive biochemical, genetic and structural studies2,4 the mechanism by which nucleotides are correctly incorporated is not known. Here we present high-resolution crystal structures of a thermostable bacterial (Bacillus stearothermophilus) DNA polymerase I large fragment5 with DNA primer templates bound productively at the polymerase active site. The active site retains catalytic activity, allowing direct observation of the products of several rounds of nucleotide incorporation. The polymerase also retains its ability to discriminate between correct and incorrectly paired nucleotides in the crystal. Comparison of the structures of successively translocated complexes allows the structural features for the sequence-independent molecular recognition of correctly formed base pairs to be deduced unambiguously. These include extensive interactions with the first four to five base pairs in the minor groove, location of the terminal base pair in a pocket of excellent steric complementarity favouring correct base-pair formation, and a conformational switch from B-form to underwound A-form DNA at the polymerase active site.

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Figure 1: Structure of the Bacillus fragment with duplex DNA bound at the polymerase active site.
Figure 2: Electron-density maps showing catalysis in the crystal.
Figure 3: Polymerase active site.
Figure 4: Polymerase active site with observed DNA and modelled dTTP.

References

  1. 1

    Johnson, K. A. Conformational coupling in DNA polymerase fidelity. Annu. Rev. Biochem. 62, 685–713 (1993).

  2. 2

    Joyce, C. M. & Steitz, T. A. Function and structure relationships in DNA polymerases. Annu. Rev. Biochem. 63, 777–822 (1994).

  3. 3

    Echols, H. Fidelity mechanisms in DNA replication. Annu. Rev. Biochem. 60, 477–511 (1991).

  4. 4

    Joyce, C. M. & Steitz, T. A. Polymerase structures and function: variations on a theme? J. Bacteriol. 177, 6321–6329 (1995).

  5. 5

    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).

  6. 6

    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).

  7. 7

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

  8. 8

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

  9. 9

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

  10. 10

    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).

  11. 11

    Steitz, T. A. Structural studies of protein-nucleic acid interaction: the sources of sequence-specific binding. Q. Rev. Biophys. 23, 205–280 (1990).

  12. 12

    Harrison, S. C. Astructural taxonomy of DNA-binding proteins. Nature 353, 715–719 (1991).

  13. 13

    Steitz, T. A., Beese, L. S., Freemont, P. S., Friedman, J. M. & Sanderson, M. R. Structural studies of Klenow fragment: an enzyme with two active sites. Cold Spring Harb. Symp. Quant. Biol. 52, 465–471 (1987).

  14. 14

    Carroll, S. S., Cowart, M. & Benkovic, S. J. Amutant of DNA polymerase I (Klenow Fragment) with reduced fidelity. Biochemistry 30, 804–813 (1991).

  15. 15

    Bell, J. B., Eckert, K. A., Joyce, C. M. & Kunkel, T. A. Base miscoding and strand misalignment errors by mutator Klenow polymerases with amino acid substitutions at tyrosine 766 in the O helix of the fingers subdomain. J. Biol. Chem. 272, 7345–7351 (1997).

  16. 16

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

  17. 17

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

  18. 18

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

  19. 19

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

  20. 20

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

  21. 21

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

  22. 22

    Kuchta, R. D., Benkovic, P. & Benkovic, S. J. Kinetic mechanism whereby DNA polymerase I (Klenow) replicates DNA with high fidelity. Biochemistry 27, 6716–6725 (1988).

  23. 23

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

  24. 24

    Brünger, A. T. X-PLOR version 3.1: A System for X-ray Crystallography and NMR (Yale Univ. Press, New Haven, CT, (1992)).

  25. 25

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

  26. 26

    Parkinson, G., Vojtechovsky, J., Clowney, L., Brünger, A. T. & Berman, H. M. New parameters for the refinement of nucleic acid containing structures. Acta Crystallogr. D 52, 57–64 (1996).

  27. 27

    Collaborative Computational Project No. 4. Acta Crystallogr. D 50, 607 (1994).

  28. 28

    Lavery, R. & Sklenar, H. Defining the structure of irregular nucleic acids: Conventions and principles. J. Biomol. Struct. Dyn. 6, 655–667 (1989).

  29. 29

    Dickerson, D. E. in Oxford Handbook of Nucleic Acid Structure (ed Neidle, S.) (Oxford University Press, Oxford, UK, (1997)).

  30. 30

    Saenger, W. Principles of Nucleic Acid Structure (Springer-Verlag, New York, (1984)).

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Acknowledgements

We thank S. Johnson and A. Chapin Rodriguez for assistance in data collection and crystallization, and H. W. Hellinga for discussions. This work was supported by grants to L.S.B. from the American Cancer Society, North Carolina Biotechnology Center, and the Searle Scholars Program.

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Correspondence to Lorena S. Beese.

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