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.

  • Article
  • Published:

Structure of a bifunctional DNA primase-polymerase

Nature Structural & Molecular Biology volume 11, pages 157–162 (2004)Cite this article

Abstract

Genome replication generally requires primases, which synthesize an initial oligonucleotide primer, and DNA polymerases, which elongate the primer. Primase and DNA polymerase activities are combined, however, in newly identified replicases from archaeal plasmids, such as pRN1 from Sulfolobus islandicus. Here we present a structure-function analysis of the pRN1 primase-polymerase (prim-pol) domain. The crystal structure shows a central depression lined by conserved residues. Mutations on one side of the depression reduce DNA affinity. On the opposite side of the depression cluster three acidic residues and a histidine, which are required for primase and DNA polymerase activity. One acidic residue binds a manganese ion, suggestive of a metal-dependent catalytic mechanism. The structure does not show any similarity to DNA polymerases, but is distantly related to archaeal and eukaryotic primases, with corresponding active-site residues. We propose that archaeal and eukaryotic primases and the prim-pol domain have a common evolutionary ancestor, a bifunctional replicase for small DNA genomes.

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: Structure of the prim-pol domain.
Figure 2: Surface features.
Figure 3: Functional analysis.
Figure 4: Comparison of the prim-pol domain with Pfu archaeal primase3.

Similar content being viewed by others

Accession codes

Accessions

GenBank/EMBL/DDBJ

Protein Data Bank

References

  1. Kornberg, A. & Baker, T. DNA Replication (W.H. Freeman, New York, USA, 1991).

    Google Scholar 

  2. Frick, D.N. & Richardson, C.C. DNA primases. Annu. Rev. Biochem. 70, 39–80 (2001).

    Article  CAS  Google Scholar 

  3. Augustin, M.A., Huber, R. & Kaiser, J.T. Crystal structure of a DNA-dependent RNA polymerase (DNA primase). Nat. Struct. Biol. 8, 57–61 (2001).

    Article  CAS  Google Scholar 

  4. Keck, J.L., Roche, D.D., Lynch, A.S. & Berger, J.M. Structure of the RNA polymerase domain of E. coli primase. Science 287, 2482–2486 (2000).

    Article  CAS  Google Scholar 

  5. Hubscher, U., Maga, G. & Spadari, S. Eukaryotic DNA polymerases. Annu. Rev. Biochem. 71, 133–163 (2002).

    Article  CAS  Google Scholar 

  6. Steitz, T.A. DNA polymerases: structural diversity and common mechanisms. J. Biol. Chem. 274, 17395–81739 (1999).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  8. Lipps, G., Rother, S., Hart, C. & Krauss, G. A novel type of replicative enzyme harbouring ATPase, primase and DNA polymerase activity. EMBO J. 22, 2516–2525 (2003).

    Article  CAS  Google Scholar 

  9. De Guzman, R.N., Wu, Z.R., Stalling, C.C., Pappalardo, L., Borer, P.N. & Summers, M.F. Structure of the HIV-1 nucleocapsid protein bound to the SL3 Ψ-RNA recognition element. Science 279, 384–388 (1998).

    Article  CAS  Google Scholar 

  10. Steitz, T.A. A mechanism for all polymerases. Nature 391, 231–223 (1998).

    Article  CAS  Google Scholar 

  11. Sawaya, M.R., Pelletier, H., Kumar, A., Wilson, S.H. & Kraut, J. Crystal structure of rat DNA polymerase β: evidence for a common polymerase mechanism. Science 264, 1930–1937 (1994).

    Article  CAS  Google Scholar 

  12. Davies, J.F., Almassy, R.J., Hostomska, Z., Ferre, R.A. & Hostomsky, Z. 2.3 A crystal structure of the catalytic domain of DNA polymerase beta. Cell 76, 1123–1133 (1994).

    Article  CAS  Google Scholar 

  13. Doublie, S., Tabor, S., Long, A.M., Richardson, C.C. & Ellenberger, T. Crystal structure of a bacteriophage T7 DNA replication complex at 2.2 Å resolution. Nature 391, 251–258 (1998).

    Article  CAS  Google Scholar 

  14. 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).

    Article  CAS  Google Scholar 

  15. Kato, M., Ito, T., Wagner, G., Richardson, C.C. & Ellenberger, T. Modular architecture of the bacteriophage T7 primase couples RNA primer synthesis to DNA synthesis. Mol. Cell 11, 1349–1360 (2003).

    Article  CAS  Google Scholar 

  16. Holm, L. & Sander, C. DALI: a network tool for protein structure comparison. Trends Biochem. Sci. 20, 478–480 (1995).

    Article  CAS  Google Scholar 

  17. Koonin, E.V., Wolf, Y.I., Kondrashov, A.S., Aravind, L. Bacterial homologs of the small subunit of eukaryotic DNA primase. J. Mol. Microbiol. Biotechnol. 2, 509–512 (2000).

    CAS  PubMed  Google Scholar 

  18. Forterre, P. The origin of DNA genomes and DNA replication proteins. Curr. Opin. Microbiol. 5, 525–532 (2002).

    Article  CAS  Google Scholar 

  19. Liu, L. et al. The archaeal DNA primase: biochemical characterization of the p41-p46 complex from Pyrococcus furiosus. J. Biol. Chem. 276, 45484–45490 (2001).

    Article  CAS  Google Scholar 

  20. Bocquier, A.A. et al. Archaeal primase: bridging the gap between RNA and DNA polymerases. Curr. Biol. 11, 452–456 (2001).

    Article  CAS  Google Scholar 

  21. Meinhart, A., Silberzahn, T. & Cramer, P. The mRNA transcription/processing factor Ssu72 is a potential tyrosine phosphatase. J. Biol. Chem. 278, 15917–15921 (2003).

    Article  CAS  Google Scholar 

  22. Budisa, N. et al. High-level biosynthetic substitution of methionine in proteins by its analogs 2-aminohexanoic acid, selenomethionine, telluromethionine and ethionine in Escherichia coli. Eur. J. Biochem. 230, 788–796 (1995).

    Article  CAS  Google Scholar 

  23. Otwinowski, Z. & Minor, W. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307–326 (1996).

    Article  Google Scholar 

  24. Matthews, B.W. Solvent content of protein crystals. J. Mol. Biol. 33, 491–495 (1968).

    Article  CAS  Google Scholar 

  25. Terwilliger, T.C. & Berendzen, J. Automated MAD and MIR structure solution. Acta Crystallogr. D 55, 849–861 (1999).

    Article  CAS  Google Scholar 

  26. Brunger, A.T. et al. Crystallography & NMR system: a new software suite for macromolecular structure determination. Acta Crystallogr. D 54, 905–921 (1998).

    Article  CAS  Google Scholar 

  27. Navaza, J. AMoRe: an automated package for molecular replacement. Acta Crystallogr. A 50, 157–163 (1994).

    Article  Google Scholar 

  28. Carson, M. Ribbons. Methods Enzymol. 277, 493–505 (1997).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank A. Schmidt for excellent technical assistance, K. Zeth for data collection at ESRF, and C. Schulze-Briese and the staff of beamline X06SA of the Swiss Light Source for help. P.C. is supported by the Deutsche Forschungsgemeinschaft, the EMBO Young Investigator Programme and the Fonds der chemischen Industrie. G.L. is supported by the Deutsche Forschungsgemeinschaft. G.L. dedicates this work to G. Krauss, University of Bayreuth, on the occasion of his 60th birthday.

Author information

Authors and Affiliations

  1. Institute of Biochemistry, University of Bayreuth, Universitätsstrasse 30, Bayreuth, D-95447, Germany

    Georg Lipps & Gudrun von Scheven

  2. Institute of Biochemistry, Gene Center, University of Munich, Feodor-Lynen-Strasse 25, Munich, D-81377, Germany

    Andreas O Weinzierl, Claudia Buchen & Patrick Cramer

Authors
  1. Georg Lipps
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Andreas O Weinzierl
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Gudrun von Scheven
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Claudia Buchen
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Patrick Cramer
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Georg Lipps or Patrick Cramer.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Lipps, G., Weinzierl, A., von Scheven, G. et al. Structure of a bifunctional DNA primase-polymerase. Nat Struct Mol Biol 11, 157–162 (2004). https://doi.org/10.1038/nsmb723

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nsmb723

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