Abstract
Helicases are essential enzymes for DNA replication, a fundamental process in all living organisms. The DnaB family are hexameric replicative helicases that unwind duplex DNA and coordinate with RNA primase and other proteins at the replication fork in prokaryotes. Here, we report the full-length crystal structure of G40P, a DnaB family helicase. The hexamer complex reveals an unusual architectural feature and a new type of assembly mechanism. The hexamer has two tiers: a three-fold symmetric N-terminal tier and a six-fold symmetric C-terminal tier. Monomers with two different conformations, termed cis and trans, come together to provide a topological solution for the dual symmetry within a hexamer. Structure-guided mutational studies indicate an important role for the N-terminal tier in binding primase and regulating primase-mediated stimulation of helicase activity. This study provides insights into the structural and functional interplay between G40P helicase and DnaG primase.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 12 print issues and online access
$189.00 per year
only $15.75 per issue
Rent or buy this article
Prices vary by article type
from$1.95
to$39.95
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
Schaeffer, P.M., Headlam, M.J. & Dixon, N.E. Protein–protein interactions in the eubacterial replisome. IUBMB Life 57, 5–12 (2005).
Corn, J.E. & Berger, J.M. Regulation of bacterial priming and daughter strand synthesis through helicase-primase interactions. Nucleic Acids Res. 34, 4082–4088 (2006).
Wickner, S. & Hurwitz, J. Interaction of Escherichia coli dnaB and dnaC(D) gene products in vitro. Proc. Natl. Acad. Sci. USA 72, 921–925 (1975).
Erzberger, J.P., Mott, M.L. & Berger, J.M. Structural basis for ATP-dependent DnaA assembly and replication-origin remodeling. Nat. Struct. Mol. Biol. 13, 676–683 (2006).
Clarey, M.G. et al. Nucleotide-dependent conformational changes in the DnaA-like core of the origin recognition complex. Nat. Struct. Mol. Biol. 13, 684–690 (2006).
Arai, K. & Kornberg, A. A general priming system employing only dnaB protein and primase for DNA replication. Proc. Natl. Acad. Sci. USA 76, 4308–4312 (1979).
Tougu, K., Peng, H. & Marians, K.J. Identification of a domain of Escherichia coli primase required for functional interaction with the DnaB helicase at the replication fork. J. Biol. Chem. 269, 4675–4682 (1994).
Johnson, S.K., Bhattacharyya, S. & Griep, M.A. DnaB helicase stimulates primer synthesis activity on short oligonucleotide templates. Biochemistry 39, 736–744 (2000).
Soultanas, P. The bacterial helicase-primase interaction: a common structural/functional module. Structure 13, 839–844 (2005).
Glover, B.P. & McHenry, C.S. The DNA polymerase III holoenzyme: an asymmetric dimeric replicative complex with leading and lagging strand polymerases. Cell 105, 925–934 (2001).
Benkovic, S.J., Valentine, A.M. & Salinas, F. Replisome-mediated DNA replication. Annu. Rev. Biochem. 70, 181–208 (2001).
Patel, S.S. & Picha, K.M. Structure and function of hexameric helicases. Annu. Rev. Biochem. 69, 651–697 (2000).
Lu, Y.B., Ratnakar, P.V., Mohanty, B.K. & Bastia, D. Direct physical interaction between DnaG primase and DnaB helicase of Escherichia coli is necessary for optimal synthesis of primer RNA. Proc. Natl. Acad. Sci. USA 93, 12902–12907 (1996).
Tougu, K. & Marians, K.J. The extreme C terminus of primase is required for interaction with DnaB at the replication fork. J. Biol. Chem. 271, 21391–21397 (1996).
Bird, L.E., Pan, H., Soultanas, P. & Wigley, D.B. Mapping protein-protein interactions within a stable complex of DNA primase and DnaB helicase from Bacillus stearothermophilus. Biochemistry 39, 171–182 (2000).
Thirlway, J. & Soultanas, P. In the Bacillus stearothermophilus DnaB-DnaG complex, the activities of the two proteins are modulated by distinct but overlapping networks of residues. J. Bacteriol. 188, 1534–1539 (2006).
Ayora, S., Langer, U. & Alonso, J.C. Bacillus subtilis DnaG primase stabilises the bacteriophage SPP1 G40P helicase-ssDNA complex. FEBS Lett. 439, 59–62 (1998).
Pedre, X., Weise, F., Chai, S., Luder, G. & Alonso, J.C. Analysis of cis and trans acting elements required for the initiation of DNA replication in the Bacillus subtilis bacteriophage SPP1. J. Mol. Biol. 236, 1324–1340 (1994).
Yu, X., Jezewska, M.J., Bujalowski, W. & Egelman, E.H. The hexameric E. coli DnaB helicase can exist in different quaternary states. J. Mol. Biol. 259, 7–14 (1996).
San Martin, M.C., Stamford, N.P., Dammerova, N., Dixon, N.E. & Carazo, J.M. A structural model for the Escherichia coli DnaB helicase based on electron microscopy data. J. Struct. Biol. 114, 167–176 (1995).
San Martin, C. et al. Three-dimensional reconstructions from cryoelectron microscopy images reveal an intimate complex between helicase DnaB and its loading partner DnaC. Structure 6, 501–509 (1998).
Yang, S. et al. Flexibility of the rings: structural asymmetry in the DnaB hexameric helicase. J. Mol. Biol. 321, 839–849 (2002).
Nunez-Ramirez, R. et al. Quaternary polymorphism of replicative helicase G40P: structural mapping and domain rearrangement. J. Mol. Biol. 357, 1063–1076 (2006).
Fass, D., Bogden, C.E. & Berger, J.M. Crystal structure of the N-terminal domain of the DnaB hexameric helicase. Structure 7, 691–698 (1999).
Weigelt, J., Brown, S.E., Miles, C.S., Dixon, N.E. & Otting, G. NMR structure of the N-terminal domain of E. coli DnaB helicase: implications for structure rearrangements in the helicase hexamer. Structure 7, 681–690 (1999).
Sawaya, M.R., Guo, S., Tabor, S., Richardson, C.C. & Ellenberger, T. Crystal structure of the helicase domain from the replicative helicase-primase of bacteriophage T7. Cell 99, 167–177 (1999).
Singleton, M.R., Sawaya, M.R., Ellenberger, T. & Wigley, D.B. Crystal structure of T7 gene 4 ring helicase indicates a mechanism for sequential hydrolysis of nucleotides. Cell 101, 589–600 (2000).
Gai, D., Zhao, R., Li, D., Finkielstein, C.V. & Chen, X.S. Mechanisms of conformational change for a replicative hexameric helicase of SV40 large tumor antigen. Cell 119, 47–60 (2004).
Thirlway, J. et al. DnaG interacts with a linker region that joins the N- and C-domains of DnaB and induces the formation of 3-fold symmetric rings. Nucleic Acids Res. 32, 2977–2986 (2004).
Kaito, C., Kurokawa, K., Hossain, M.S., Akimitsu, N. & Sekimizu, K. Isolation and characterization of temperature-sensitive mutants of the Staphylococcus aureus dnaC gene. FEMS Microbiol. Lett. 210, 157–164 (2002).
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).
Oakley, A.J. et al. Crystal and solution structures of the helicase-binding domain of Escherichia coli primase. J. Biol. Chem. 280, 11495–11504 (2005).
Syson, K., Thirlway, J., Hounslow, A.M., Soultanas, P. & Waltho, J.P. Solution structure of the helicase-interaction domain of the primase DnaG: a model for helicase activation. Structure 13, 609–616 (2005).
Mitkova, A.V., Khopde, S.M. & Biswas, S.B. Mechanism and stoichiometry of interaction of DnaG primase with DnaB helicase of Escherichia coli in RNA primer synthesis. J. Biol. Chem. 278, 52253–52261 (2003).
Lee, J.B. et al. DNA primase acts as a molecular brake in DNA replication. Nature 439, 621–624 (2006).
Otwinowski, Z. & Minor, W. in Methods Enzymol. xxx, 307–326 (1997).
Terwilliger, T.C. & Berendzen, J. Automated MAD and MIR structure solution. Acta Crystallogr. D Biol. Crystallogr. 55, 849–861 (1999).
Jones, T.A., Zou, J.Y., Cowan, S.W. & Kjeldgaard, M. 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).
Vonrhein, C., Blanc, E., Roversi, P. & Bricogne, G. Automated structure solution with autoSHARP. Methods Mol. Biol. 364, 215–230 (2006).
Strokopytov, B.V. et al. Phased translation function revisited: structure solution of the cofilin-homology domain from yeast actin-binding protein 1 using six-dimensional searches. Acta Crystallogr. D Biol. Crystallogr. 61, 285–293 (2005).
Cowtan, K. 'dm': an automated procedure for phase improvement by density modification. Joint CCP4 and ESF-EACBM Newsletter on Protein Crystallography 31, 34–38 (1994).
Vagin, A.A. & Teplyakov, A. MOLREP: an automated program for molecular replacement. J. Appl. Crystallogr. 30, 1022–1025 (1997).
Winn, M.D., Murshudov, G.N. & Papiz, M.Z. Macromolecular TLS refinement in REFMAC at moderate resolutions. Methods Enzymol. 374, 300–321 (2003).
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).
Brunger, A.T. et al. Crystallography & NMR system: a new software suite for macromolecular structure determination. Acta Crystallogr. D Biol. Crystallogr. 54, 905–921 (1998).
Acknowledgements
We thank the staffs at Lawrence Berkeley Laboratory's Advanced Light Source beamlines 8.2.1, 8.2.2, 4.2.2 and Argonne National Laboratory's Advanced Photon Source 19id for assistance in data collection.
Author information
Authors and Affiliations
Corresponding author
Supplementary information
Supplementary Text and Figures
Supplementary Figures 1–4 (PDF 2247 kb)
Rights and permissions
About this article
Cite this article
Wang, G., Klein, M., Tokonzaba, E. et al. The structure of a DnaB-family replicative helicase and its interactions with primase. Nat Struct Mol Biol 15, 94–100 (2008). https://doi.org/10.1038/nsmb1356
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/nsmb1356
This article is cited by
-
Understanding how the replisome works
Nature Structural & Molecular Biology (2008)