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:

Structural and energetic origins of indirect readout in site-specific DNA cleavage by a restriction endonuclease

Nature Structural Biology volume 6, pages 269–277 (1999)Cite this article

Abstract

Specific recognition by EcoRV endonuclease of its cognate, sharply bent GATATC site at the center TA step occurs solely via hydrophobic interaction with thymine methyl groups. Mechanistic kinetic analyses of base analog-substituted DNAs at this position reveal that direct readout provides 5 kcal mol–1 toward specificity, with an additional 6–10 kcal mol–1 arising from indirect readout. Crystal structures of several base analog complexes show that the major-groove hydrophobic contacts are crucial to forming required divalent metal-binding sites, and that indirect readout operates in part through the sequence-dependent free-energy cost of unstacking the center base-pair step of the DNA.

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: a, Ribbon representation of the EcoRV dimer showing the dimerization domain at bottom (orange), the DNA-binding/catalytic domains in yellow and the four flexible linkers I–IV in each subunit13.
Figure 2: a, Single-turnover reaction for the determination of kc for the TA substrate.
Figure 3: a, Superposition of the wild-type (green), CI (red) and MI (blue) structures using all atoms in the R-loop (residues 182–188) for one of the two subunits (top).
Figure 4: a, Superposition of the structures of EcoRV bound to the wild-type site in the presence of Ca2+ (green), and bound to CI (red).
Figure 5: Superposition of the structures of EcoRV bound to TA (green), MI (blue) and CI (red).
Figure 6: Free-energy component analysis for the sub-strates TA, UA, MI and CI.

Similar content being viewed by others

Accession codes

Accessions

Protein Data Bank

References

  1. Rhodes, D., Schwabe, J.W., Chapman, L. & Fairall, L. Towards an understanding of protein–DNA recognition. Phil. Trans. Royal Soc. B 351, 501–509 (1996).

    Article  CAS  Google Scholar 

  2. 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).

    Article  CAS  Google Scholar 

  3. Otwinowski, Z. et al. Crystal structure of trp repressor/operator complex at atomic resolution. Nature 335, 321–329 (1988).

    Article  CAS  Google Scholar 

  4. Koudelka, G.B., Harrison, S.C. & Ptashne, M. Effect of noncontacted bases on the affinity of 434 operator for 434 repressor and Cro. Nature 326, 886–888 (1987).

    Article  CAS  Google Scholar 

  5. Rosenberg, J.M. Structure and function of restriction endonucleases. Curr. Opin. Struct. Biol. 1, 104–113 (1991).

    Article  CAS  Google Scholar 

  6. Lesser, D.R., Kurpiewski, M.R., Waters, T., Connolly, B.A. & Jen-Jacobson L. Facilitated distortion of the DNA site enhances EcoRI endonuclease–DNA recognition. Proc. Natl. Acad. Sci. USA 90, 7548–7552 (1993).

    Article  CAS  Google Scholar 

  7. Draper, D.E. Protein–DNA complexes: the cost of recognition. Proc. Natl. Acad. Sci. USA 90, 7429–7430 (1993).

    Article  CAS  Google Scholar 

  8. Jen-Jacobson, L. Structural-perturbation approaches to thermodynamics of site-specific protein–DNA interactions. Meth. Enz. 259, 305–344 (1995).

    Article  CAS  Google Scholar 

  9. Schildkraut, I., Banner, C.D.B., Rhodes, C.S. & Parekh, S. The cleavage site for the restriction enzyme EcoRV is 5'-GAT/ATC-3'. Gene 27, 327–329 (1984).

    Article  CAS  Google Scholar 

  10. Roberts, R.J. & Halford, S.E. In Nucleases (eds Linn, S.M., Lloyd, R.S. & Roberts, R.J.) 35–88 (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York; 1993).

    Google Scholar 

  11. Vipond, I.B. & Halford, S.E. Structure–function correlation for the EcoRV restriction enzyme: from non-specific binding to specific DNA cleavage. Mol. Microbiol. 9, 225–231 (1993).

    Article  CAS  Google Scholar 

  12. Engler, L.E., Welch, K.K. & Jen-Jacobson, L. Specific binding by EcoRV endonuclease to its DNA recognition site GATATC. J. Mol. Biol. 269, 82–101 (1997).

    Article  CAS  Google Scholar 

  13. Winkler, F.K. et al. The crystal structure of the EcoRV endonuclease and of its complexes with cognate and non-cognate DNA fragments. EMBO J. 12, 1781–1795 (1993).

    Article  CAS  Google Scholar 

  14. Kostrewa, D. & Winkler, F.K. Mg2+ binding to the active site of EcoRV endonuclease—A crystallographic study of complexes with substrate and product DNA at 2 Å resolution. Biochemistry 34, 683–696 (1995).

    Article  CAS  Google Scholar 

  15. Perona, J.J. & Martin, A.M. Conformational transitions and structural deformability of EcoRV restriction endonuclease revealed by crystallographic analysis. J. Mol. Biol. 273, 207–225 (1997).

    Article  CAS  Google Scholar 

  16. Horton, N.C. & Perona, J.J. Role of protein-induced bending in the specificity of DNA recognition: crystal structure of EcoRV endonuclease complexed with d(AAAGAT) + d(ATCTT). J. Mol. Biol. 277, 779–787 (1998).

    Article  CAS  Google Scholar 

  17. Cheng, X., Balendiran, K., Schildkraut, I. & Anderson, J.E. Structure of PvuII endonuclease with cognate DNA. EMBO J. 13, 3927–3935 (1994).

    Article  CAS  Google Scholar 

  18. Wah, D.A., Hirsch, J.A., Dorner, L.F., Schildkraut, I. & Aggarwal, A.K. Structure of the multimodular endonuclease FokI bound to DNA. Nature 388, 97–100 (1997).

    Article  CAS  Google Scholar 

  19. Newman, M., Strzelecka, T., Dorner, L.F., Schildkraut, I. & Aggarwal, A.K. Structure of BamHI endonuclease bound to DNA: partial folding and unfolding on DNA binding. Science 269, 656–663 (1995).

    Article  CAS  Google Scholar 

  20. Vipond, I.B. & Halford, S.E. Specific DNA recognition by EcoRV restriction endonuclease induced by calcium ions. Biochemistry 34, 1113–1119 (1995).

    Article  CAS  Google Scholar 

  21. Lesser, D.R., Kurpiewski, M.R. & Jen-Jacobson, L. The energetic basis of specificity in the EcoRI endonuclease–DNA interaction. Science 250, 776–786 (1990).

    Article  CAS  Google Scholar 

  22. Fersht, A.R. Enzyme structure and mechanism (W.H. Freeman & Co., New York; 1985).

    Google Scholar 

  23. Alves, J., Selent, U. & Wolfes, H. Accuracy of the EcoRV restriction endonuclease: binding and cleavage studies with oligodeoxynucleotide substrates containing degenerate recognition sequences. Biochemistry 34, 11191–11197 (1995).

    Article  CAS  Google Scholar 

  24. Taylor, J.D. & Halford, S.E. Discrimination between DNA sequences by the EcoRV restriction endonuclease. Biochemistry 28, 6198–6207 (1989).

    Article  CAS  Google Scholar 

  25. Lipanov, A., Kopka, M.L., Kaczor-Grzeskowiak, M., Quintana, J. & Dickerson, R.E. Structure of the B-DNA decamer C-C-A-A-C-I-T-T-G-G in two different space groups: conformational flexibility of B-DNA. Biochemistry 32, 1373–1389 (1993).

    Article  CAS  Google Scholar 

  26. Xuan, J.C. & Weber, I.T. Crystal structure of a B-DNA dodecamer containing inosine, d(CGCIAATTCGCG), at 2.4 A resolution and its comparison with other B-DNA dodecamers. Nucleic Acids Res. 20, 5457–5464 (1992).

    Article  CAS  Google Scholar 

  27. Selent, U. et al. A site-directed mutagenesis study to identify amino acid residues involved in the catalytic function of the restriction endonuclease EcoRV. Biochemistry 31, 4808–4815 (1992).

    Article  CAS  Google Scholar 

  28. Horton, N.C., Newberry, K.J. & Perona, J.J. Metal ion-mediated substrate-assisted catalysis in type II restriction endonucleases. Proc. Natl. Acad. Sci. USA, 95, 13489–13494 (1998).

    Article  CAS  Google Scholar 

  29. Wenz, C., Jeltsch, A. & Pingoud, A. Probing the indirect readout of the restriction enzyme EcoRV. J. Biol. Chem. 271, 5565–5573 (1996).

    Article  CAS  Google Scholar 

  30. Hancox, E.L. & Halford, S.E. Kinetic analysis of a mutational hot spot in the EcoRV restriction endonuclease. Biochemistry 36, 7577–7585 (1997).

    Article  CAS  Google Scholar 

  31. Goodsell, D.S., Kaczor-Grzeskowiak, M. & Dickerson, R.E. The crystal structure of CCATTAATGG: implications for bending of B-DNA at TA steps. J. Mol. Biol. 239, 79–96 (1994).

    Article  CAS  Google Scholar 

  32. El Hassan, M.A. & Calladine, C.R. Propeller-twisting of base-pairs and the conformational mobility of dinucleotide steps in DNA. J. Mol. Biol. 259, 95–103 (1996).

    Article  CAS  Google Scholar 

  33. Hunter, C.A. Sequence-dependent DNA structure. The role of base stacking interactions. J. Mol. Biol. 230, 1025–1054 (1993).

    Article  CAS  Google Scholar 

  34. Hunter, C.A. & Lu, X-J. DNA base-stacking interactions: a comparison of theoretical calculations with oligonucleotide X-ray crystal structures. J. Mol. Biol. 265, 603–619 (1997).

    Article  CAS  Google Scholar 

  35. Santa Lucia, J., Jr., Allawi, H.T. & Seneviratne, P.A. Improved nearest-neighbor parameters for predicting DNA duplex stability. Biochemistry 35, 3555–3562 (1996).

    Article  CAS  Google Scholar 

  36. Doktycz, M.J., Morris, M.D., Dormady, S.J., Beattie, K.L. & Jacobson, K.B. Optical melting of 128 octamer DNA duplexes. J. Biol. Chem. 270, 8439–8445 (1995).

    Article  CAS  Google Scholar 

  37. Jen-Jacobson, L. Protein–DNA recognition complexes: conservation of structure and binding energy in the transition state. Biopolymers 44, 153–180 (1997).

    Article  CAS  Google Scholar 

  38. Aggarwal, A.K. Crystallization of DNA binding proteins with oligodeoxynucleotides. Meth. Enz. 1, 83–90 (1990).

    Article  CAS  Google Scholar 

  39. Surby, M.A. & Reich, N.O. Facilitated diffusion of the EcoRI DNA methyltransferase is described by a novel mechanism. Biochemistry 35, 2209–2217 (1996).

    Article  CAS  Google Scholar 

  40. van Zoelen, E.J.J. Analysis of receptor binding displacement curves by a nonhomologous ligand, on the basis of an equivalent competition principle. Anal. Biochem. 200, 393–399 (1992).

    Article  CAS  Google Scholar 

  41. Sack, J.S. CHAIN—A crystallographic modelling program. J. Mol. Graphics 6, 224–225 (1988).

    Article  Google Scholar 

  42. Brunger, A.T., Kuriyan, J. & Karplus, M. Crystallographic R-factor refinement by molecular dynamics. Science 235, 458–460 (1987).

    Article  CAS  Google Scholar 

  43. Parkinson, G. et al. New parameters for the refinement of nucleic acid-containing structures. Acta Crystallogr. D 52, 57–64 (1996).

    Article  CAS  Google Scholar 

  44. Baldwin, G.S., Vipond, I.B. & Halford, S.E. Rapid reaction analysis of the catalytic cycle of the EcoRV restriction endonuclease. Biochemistry 34, 705–714 (1995).

    Article  CAS  Google Scholar 

  45. Newman, P.C., Williams, D.M., Cosstick, R., Seela, F. & Connolly, B.A. Interaction of the EcoRV restriction endonuclease with the deoxyadenosine and thymidine bases in its recognition hexamer d(GATATC). Biochemistry 29, 9902–9910 (1990).

    Article  CAS  Google Scholar 

  46. Evans, S.V. SETOR: hardware lighted three-dimensional solid model representations of macromolecules. J. Mol. Graphics 11, 134–138 (1993)..

Download references

Acknowledgements

We thank N. Horton for insightful discussions and J. Kohn for assistance with protein purification. This work was supported by grants from the NIH and ACS-PRF (to J.J.P.).

Author information

Authors and Affiliations

  1. Department of Chemistry and Interdepartmental Program in Biochemistry and Molecular Biology, University of California at Santa Barbara, Santa Barbara, 93106-9510, California, USA

    Amy M. Martin, My D. Sam, Norbert O. Reich & John J. Perona

Authors
  1. Amy M. Martin
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. My D. Sam
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Norbert O. Reich
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. John J. Perona
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to John J. Perona.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Martin, A., Sam, M., Reich, N. et al. Structural and energetic origins of indirect readout in site-specific DNA cleavage by a restriction endonuclease. Nat Struct Mol Biol 6, 269–277 (1999). https://doi.org/10.1038/6707

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1038/6707

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