Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Letter
  • Published:

Structural basis for uracil recognition by archaeal family B DNA polymerases

Nature Structural Biology volume 9, pages 922–927 (2002)Cite this article

Abstract

Deamination of cytosine to uracil in a G-C base pair is a major promutagenic event, generating G-C→A-T mutations if not repaired before DNA replication. Archaeal family B DNA polymerases are uniquely able to recognize unrepaired uracil in a template strand and stall polymerization upstream of the lesion, thereby preventing the irreversible fixation of an A-T mutation. We have now identified a 'pocket' in the N-terminal domains of archaeal DNA polymerases that is positioned to interact with the template strand and provide this ability. The structure of this pocket provides interacting groups that discriminate uracil from the four normal DNA bases (including thymine). These groups are conserved in archaeal polymerases but absent from homologous viral polymerases that are unable to recognize uracil. Using site-directed mutagenesis, we have confirmed the biological role of this pocket and have engineered specific mutations in the Pfu polymerase that confer the ability to read through template-strand uracils and carry out PCR with dUTP in place of dTTP.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: The N-terminal domain of archaeal, but not viral, family B DNA polymerases contains a uracil-binding pocket.
Figure 2: Uracil recognition by wild type (WT) and site-directed mutants of Pfu-Pol.
Figure 3: Modeling uracil into the N-terminal domain pocket of Tgo-Pol.
Figure 4: Amino acid sequence alignment of archaeal family B DNA polymerases, corresponding to residues 1–130 of Pyrococcus furiosus polymerase.

Similar content being viewed by others

Accession codes

Accessions

Protein Data Bank

References

  1. Lindahl, T. Nature 362, 709–715 (1993).

    Article  CAS  Google Scholar 

  2. Krokan, H.E., Standal, R. & Slupphaug, G. Biochem. J. 325, 1–16 (1997).

    Article  CAS  Google Scholar 

  3. Parikh, S.S., Mol, C.D., Hosfield, D.J. & Tainer, J.A. Curr. Opin. Struct. Biol. 9, 37–47 (1999).

    Article  CAS  Google Scholar 

  4. Pearl, L.H. Mutat. Res. 460, 165–181 (2000).

    Article  CAS  Google Scholar 

  5. Lindahl, T. Proc. Natl. Acad. Sci. USA 71, 3649–3653 (1974).

    Article  CAS  Google Scholar 

  6. Lindahl, T. & Nyberg, B. Biochemistry 13, 3405–3410 (1974).

    Article  CAS  Google Scholar 

  7. Grogan, D.W. Trends Microbiol. 8, 180–185 (2000).

    Article  CAS  Google Scholar 

  8. Horst, J.P. & Fritz, H.J. EMBO J. 15, 5459–5469 (1996).

    Article  CAS  Google Scholar 

  9. Yang, H. et al. J. Bacteriol. 182, 1272–1279 (2000).

    Article  CAS  Google Scholar 

  10. Sandigursky, M. & Franklin, W.A. J. Biol. Chem. 275, 19146–19149 (2000).

    Article  CAS  Google Scholar 

  11. Sartori, A.A., Schär, P., Fitz-Gibbon, S., Miller, J.E. & Jiriciny, J. J. Biol. Chem. 276, 29979–29986 (2001).

    Article  CAS  Google Scholar 

  12. Sandigursky, M. & Franklin, W.A. Curr. Biol. 9, 531–534 (1999).

    Article  CAS  Google Scholar 

  13. Sandigursky, M., Faje, A. & Franklin, W.A. Mutat. Res. 485, 187–195 (2001).

    Article  CAS  Google Scholar 

  14. Lasken, R.S., Schuster, D.M. & Rashtchian, A. J. Biol. Chem. 271, 17692–17696 (1996).

    Article  CAS  Google Scholar 

  15. Greagg, M.A. et al. Proc. Natl. Acad. Sci. USA 96, 9045–9050 (1999).

    Article  CAS  Google Scholar 

  16. Shinohara, A. & Ogawa, T. Trends Biochem. Sci. 20, 387–391 (1995).

    Article  CAS  Google Scholar 

  17. Fuchs, R.P.P. & Baynton, K. Trends Biochem. Sci. 25, 74–79 (2000).

    Article  Google Scholar 

  18. Wang, J. et al. Cell 89, 1087–1099 (1997).

    Article  CAS  Google Scholar 

  19. Franklin, M.C., Wang, J. & Steitz, T.A. Cell 105, 657–667 (2001).

    Article  CAS  Google Scholar 

  20. Hopfner, K.P. et al. Proc. Natl. Acad. Sci. USA 96, 3600–3605 (1999).

    Article  CAS  Google Scholar 

  21. Zhao, Y. et al. Structure 7, 1189–1199 (1999).

    Article  CAS  Google Scholar 

  22. Rodriguez, A.C., Park, H.-W., Mao, C. & Beese, L. J. Mol. Biol. 299, 447–462 (2000).

    Article  CAS  Google Scholar 

  23. Hashimoto, H. et al. J. Mol. Biol. 306, 469–477 (2001).

    Article  CAS  Google Scholar 

  24. Wang, J., Yu, P., Lin, T.C., Konigsberg, W.H. & Steitz, T.A. Biochemistry 35, 8110–8119 (1996).

    Article  CAS  Google Scholar 

  25. Reid, S.L., Parry, D., Liu, H.-H. & Connolly, B.A. Biochemistry 40, 2484–2484 (2001).

    Article  CAS  Google Scholar 

  26. Hogrefe, H.H., Hansen, C.J., Scott, B.R. & Nielson, K.B. Proc. Natl. Acad. Sci. USA 99, 596–602 (2002).

    Article  CAS  Google Scholar 

  27. Larson, G., Svenson, L.A. & Nyman, P.O. Nature Struct. Biol. 3, 532–536 (1996).

    Article  Google Scholar 

  28. Pearl, L.H. & Savva, R. Nature Struct. Biol. 3, 485–487 (1996).

    Article  CAS  Google Scholar 

  29. Ronen, A. & Glickman, B.W. Environ. Mol. Mutagen. 37, 241–283 (2001).

    Article  CAS  Google Scholar 

  30. Wood, R.D., Mitchell, M., Sgouros, J. & Lindahl, T. Science, 291, 1284–1289 (2001).

    Article  CAS  Google Scholar 

  31. Goodman, M.F. Trends Biochem. Sci. 25, 189–195 (2000).

    Article  CAS  Google Scholar 

  32. Evans S.J. et al. Nucleic Acids. Res. 28, 1059–1066 (2000).

    Article  CAS  Google Scholar 

  33. Ho, S.N., Hunt, H.D., Horton, R.M., Pullen, J.K. & Pease, L.R. Gene 77, 51–59 (1989).

    Article  CAS  Google Scholar 

  34. Richardson, C.C. In Procedures in Nucleic Acids Research (eds Cantoni, G.L. & Davies, D.R.) 263–276 (Harper and Row, New York; 1966).

    Google Scholar 

  35. Thompson, J.D., Gibson, T.J., Plewniak, F., Jeanmougin, F. & Higgins, D.G. Nucleic Acids Res. 24, 4876–4882 (1997).

    Article  Google Scholar 

Download references

Acknowledgements

This work was supported by the UK Biotechnology and Biological Science Research Council, Cancer Research UK and the UK Wellcome Trust.

Author information

Authors and Affiliations

  1. School of Cell and Molecular Biosciences, University of Newcastle, Newcastle upon Tyne, NE2 4HH, UK

    Mark J. Fogg & Bernard A. Connolly

  2. Cancer Research UK DNA Repair Enzyme Group, Section of Structural Biology, The Institute of Cancer Research, 237 Fulham Road, London, SW3 6JB, UK

    Laurence H. Pearl

Authors
  1. Mark J. Fogg
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Laurence H. Pearl
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Bernard A. Connolly
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Bernard A. Connolly.

Ethics declarations

Competing interests

Some of the mutants described in this paper, which may be useful in PCR have been patented.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Fogg, M., Pearl, L. & Connolly, B. Structural basis for uracil recognition by archaeal family B DNA polymerases. Nat Struct Mol Biol 9, 922–927 (2002). https://doi.org/10.1038/nsb867

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nsb867

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing