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Identification of a DNA primase template tracking site redefines the geometry of primer synthesis

Nature Structural & Molecular Biology volume 15, pages 163–169 (2008)Cite this article

Abstract

Primases are essential RNA polymerases required for the initiation of DNA replication, lagging strand synthesis and replication restart. Many aspects of primase function remain unclear, including how the enzyme associates with a moving nucleic acid strand emanating from a helicase and orients primers for handoff to replisomal components. Using a new screening method to trap transient macromolecular interactions, we determined the structure of the Escherichia coli DnaG primase catalytic domain bound to single-stranded DNA. The structure reveals an unanticipated binding site that engages nucleic acid in two distinct configurations, indicating that it serves as a nonspecific capture and tracking locus for template DNA. Bioinformatic and biochemical analyses show that this evolutionarily constrained region enforces template polarity near the active site and is required for primase function. Together, our findings reverse previous proposals for primer–template orientation and reconcile disparate studies to re-evaluate replication fork organization.

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Figure 1: Structure of the RNA polymerase domain of DnaG bound to single-stranded DNA (ssDNA).
Figure 2: Two primase–single-stranded DNA (ssDNA) complexes reveal distinct nucleic acid conformations that correlate with concerted amino acid motions.
Figure 3: 1H-15N HSQC NMR and statistical coupling analyses of the single-stranded DNA (ssDNA) binding groove.
Figure 4: Biochemical validation of template binding groove–DNA interactions and binding polarity.
Figure 5: Primer synthesis geometry.
Figure 6: Structural constraints for primer synthesis geometry in the context of the replicative helicase.

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References

  1. Rowen, L. & Kornberg, A. Primase, the dnaG protein of Escherichia coli. An enzyme which starts DNA chains. J. Biol. Chem. 253, 758–764 (1978).

    CAS  PubMed  Google Scholar 

  2. Kitani, T., Yoda, K., Ogawa, T. & Okazaki, T. Evidence that discontinuous DNA replication in Escherichia coli is primed by approximately 10 to 12 residues of RNA starting with a purine. J. Mol. Biol. 184, 45–52 (1985).

    Article  CAS  Google Scholar 

  3. Kornberg, A. & Baker, T. DNA Replication (Freeman, New York, 1992).

    Google Scholar 

  4. Heller, R.C. & Marians, K.J. Replication fork reactivation downstream of a blocked nascent leading strand. Nature 439, 557–562 (2006).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  6. Kato, M., Ito, T., Wagner, G., Richardson, 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 

  7. Godson, G.N., Schoenich, J., Sun, W. & Mustaev, A.A. Identification of the magnesium ion binding site in the catalytic center of Escherichia coli primase by iron cleavage. Biochemistry 39, 332–339 (2000).

    Article  CAS  Google Scholar 

  8. Aravind, L., Leipe, D. & Koonin, E. Toprim - a conserved catalytic domain in type IA and II topoisomerases, DnaG-type primases, OLD family nucleases and RecR proteins. Nucleic Acids Res. 26, 4205–4213 (1998).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  10. Podobnik, M., McInerney, P., O'Donnell, M. & Kuriyan, J.A. TOPRIM domain in the crystal structure of the catalytic core of Escherichia coli primase confirms a structural link to DNA topoisomerases. J. Mol. Biol. 300, 353–362 (2000).

    Article  CAS  Google Scholar 

  11. Toth, E.A., Li, Y., Sawaya, M.R., Cheng, Y. & Ellenberger, T. The crystal structure of the bifunctional primase-helicase of bacteriophage T7. Mol. Cell 12, 1113–1123 (2003).

    Article  CAS  Google Scholar 

  12. Kusakabe, T., Baradaran, K., Lee, J. & Richardson, C.C. Roles of the helicase and primase domain of the gene 4 protein of bacteriophage T7 in accessing the primase recognition site. EMBO J. 17, 1542–1552 (1998).

    Article  CAS  Google Scholar 

  13. VanLoock, M.S., Chen, Y.J., Yu, X., Patel, S.S. & Egelman, E.H. The primase active site is on the outside of the hexameric bacteriophage T7 gene 4 helicase-primase ring. J. Mol. Biol. 311, 951–956 (2001).

    Article  CAS  Google Scholar 

  14. Yuzhakov, A., Kelman, Z. & O'Donnell, M. Trading places on DNA—a three-point switch underlies primer handoff from primase to the replicative DNA polymerase. Cell 96, 153–163 (1999).

    Article  CAS  Google Scholar 

  15. Swart, J.R. & Griep, M.A. Primer synthesis kinetics by Escherichia coli primase on single-stranded DNA templates. Biochemistry 34, 16097–16106 (1995).

    Article  CAS  Google Scholar 

  16. Khopde, S., Biswas, E.E. & Biswas, S.B. Affinity and sequence specificity of DNA binding and site selection for primer synthesis by Escherichia coli primase. Biochemistry 41, 14820–14830 (2002).

    Article  CAS  Google Scholar 

  17. Corn, J.E. & Berger, J.M. FASTDXL: a generalized screen to trap disulfide-stabilized complexes for use in structural studies. Structure 15, 773–780 (2007).

    Article  CAS  Google Scholar 

  18. Verdine, G.L. & Norman, D.P. Covalent trapping of protein-DNA complexes. Annu. Rev. Biochem. 72, 337–366 (2003).

    Article  CAS  Google Scholar 

  19. Corn, J.E., Pease, P.J., Hura, G.L. & Berger, J.M. Crosstalk between primase subunits can act to regulate primer synthesis in trans. Mol. Cell 20, 391–401 (2005).

    Article  CAS  Google Scholar 

  20. Lockless, S.W. & Ranganathan, R. Evolutionarily conserved pathways of energetic connectivity in protein families. Science 286, 295–299 (1999).

    Article  CAS  Google Scholar 

  21. Sun, W., Schoneich, J. & Godson, G.N. A mutant Escherichia coli primase defective in elongation of primer RNA chains. J. Bacteriol. 181, 3761–3767 (1999).

    CAS  PubMed  PubMed Central  Google Scholar 

  22. Versalovic, J. & Lupski, J.R. The Haemophilus influenzae dnaG sequence and conserved bacterial primase motifs. Gene 136, 281–286 (1993).

    Article  CAS  Google Scholar 

  23. Brautigam, C.A. & Steitz, T.A. Structural and functional insights provided by crystal structures of DNA polymerases and their substrate complexes. Curr. Opin. Struct. Biol. 8, 54–63 (1998).

    Article  CAS  Google Scholar 

  24. Cheetham, G.M. & Steitz, T.A. Structure of a transcribing T7 RNA polymerase initiation complex. Science 286, 2305–2309 (1999).

    Article  CAS  Google Scholar 

  25. Qimron, U., Lee, S.J., Hamdan, S.M. & Richardson, C.C. Primer initiation and extension by T7 DNA primase. EMBO J. 25, 2199–2208 (2006).

    Article  CAS  Google Scholar 

  26. Lee, S.J. & Richardson, C.C. Interaction of adjacent primase domains within the hexameric gene 4 helicase-primase of bacteriophage T7. Proc. Natl. Acad. Sci. USA 99, 12703–12708 (2002).

    Article  CAS  Google Scholar 

  27. Rodina, A. & Godson, G.N. Role of conserved amino acids in the catalytic activity of Escherichia coli primase. J. Bacteriol. 188, 3614–3621 (2006).

    Article  CAS  Google Scholar 

  28. Bailey, S., Eliason, W.K. & Steitz, T.A. Structure of hexameric DnaB helicase and its complex with a domain of DnaG primase. Science 318, 459–463 (2007).

    Article  CAS  Google Scholar 

  29. MacDowell, A.A. et al. Suite of three protein crystallography beamlines with single superconducting bend magnet as the source. J. Synchrotron Radiat. 11, 447–455 (2004).

    Article  CAS  Google Scholar 

  30. Storoni, L.C., McCoy, A.J. & Read, R.J. Likelihood-enhanced fast rotation functions. Acta Crystallogr. D Biol. Crystallogr. 60, 432–438 (2004).

    Article  Google Scholar 

  31. Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D Biol. Crystallogr. 60, 2126–2132 (2004).

    Article  Google Scholar 

  32. Murshudov, G.N., Vagin, A.A. & Dodson, E.J. Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr. D Biol. Crystallogr. 53, 240–255 (1997).

    Article  CAS  Google Scholar 

  33. Davis, I.W. et al. MolProbity: all-atom contacts and structure validation for proteins and nucleic acids. Nucleic Acids Res. 35, W375–W383 (2007).

    Article  Google Scholar 

  34. DeLano, W.L. The PyMOL Molecular Graphics System. (DeLano Scientific, San Carlos, California, USA, 2002).

  35. Baker, N.A., Sept, D., Joseph, S., Holst, M.J. & McCammon, J.A. Electrostatics of nanosystems: application to microtubules and the ribosome. Proc. Natl. Acad. Sci. USA 98, 10037–10041 (2001).

    Article  CAS  Google Scholar 

  36. Mori, S., Abeygunawardana, C., Johnson, M.O. & van Zijl, P.C. Improved sensitivity of HSQC spectra of exchanging protons at short interscan delays using a new fast HSQC (FHSQC) detection scheme that avoids water saturation. J. Magn. Reson. B. 108, 94–98 (1995).

    Article  CAS  Google Scholar 

  37. Edgar, R.C. MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res. 32, 1792–1797 (2004).

    Article  CAS  Google Scholar 

  38. Shulman, A.I., Larson, C., Mangelsdorf, D.J. & Ranganathan, R. Structural determinants of allosteric ligand activation in RXR heterodimers. Cell 116, 417–429 (2004).

    Article  CAS  Google Scholar 

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Acknowledgements

We thank A. Falick for assistance with mass spectrometry, C. Fromme for advice on disulfide cross-linking, J. Holton and G. Meigs of Beamline 8.3.1 for assistance with data collection, and members of the Berger laboratory for helpful discussion. This work was supported by the G. Harold and Leila Y. Mathers Foundation, and by the US National Institute of General Medical Sciences (GM071747).

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Authors and Affiliations

  1. Department of Molecular and Cell Biology, QB3 Institute, 374D Stanley Hall no. 3220, University of California, Berkeley, Berkeley, 94720, California, USA

    Jacob E Corn, Jeffrey G Pelton & James M Berger

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  1. Jacob E Corn
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  2. Jeffrey G Pelton
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  3. James M Berger
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Contributions

J.E.C. designed and performed experiments and wrote the manuscript, J.G.P. performed NMR analysis and J.M.B. supervised the experimenter, discussed results and wrote the manuscript.

Corresponding author

Correspondence to James M Berger.

Supplementary information

Supplementary Text and Figures

Supplementary Figs 1–5, Methods (PDF 660 kb)

Supplementary Movie

Interpolated ssDNA tracking via the basic groove. (AVI 1830 kb)

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Corn, J., Pelton, J. & Berger, J. Identification of a DNA primase template tracking site redefines the geometry of primer synthesis. Nat Struct Mol Biol 15, 163–169 (2008). https://doi.org/10.1038/nsmb.1373

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