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:

Understanding the immutability of restriction enzymes: crystal structure of BglII and its DNA substrate at 1.5 Å resolution

Nature Structural Biology volume 7, pages 134–140 (2000)Cite this article

Abstract

Restriction endonucleases are remarkably resilient to alterations in their DNA binding specificity. To understand the basis of this immutability, we have determined the crystal structure of endonuclease BglII bound to its recognition sequence (AGATCT), at 1.5 Å resolution. We compare the structure of BglII to endonuclease BamHI, which recognizes a closely related DNA site (GGATCC). We show that both enzymes share a similar α/β core, but in BglII, the core is augmented by a β-sandwich domain that encircles the DNA to provide extra specificity. Remarkably, the DNA is contorted differently in the two structures, leading to different protein–DNA contacts for even the common base pairs. Furthermore, the BglII active site contains a glutamine in place of the glutamate at the general base position in BamHI, and only a single metal is found coordinated to the putative nucleophilic water and the phosphate oxygens. This surprising diversity in structures shows that different strategies can be successful in achieving site-specific recognition and catalysis in restriction endonucleases.

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: Overall structure.
Figure 2: Experimental electron density of the BglII–DNA complex from MAD phasing.
Figure 3: DNA parameters.
Figure 4: DNA recognition.
Figure 5: Active site.

Similar content being viewed by others

Accession codes

Accessions

Protein Data Bank

References

  1. Roberts, R.J. & Halford, S.E. Type II restriction endonucleases . 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 

  2. Aggarwal, A.K. Structure and function of restriction endonucleases. Curr. Opin. Struct. Biol. 5, 11–19 ( 1995).

    Article  CAS  Google Scholar 

  3. Pingoud, A. & Jeltsch, A. Recognition and cleavage of DNA by type-II restriction endonucleases. Eur. J. Biochem. 246, 1–22 (1997).

    Article  CAS  Google Scholar 

  4. Newman, M., Strzelecka, T., Dorner, L.F., Schildkraut, I. & Aggarwal, A.K. Structure of restriction endonuclease BamHI and its relationship to EcoRI. Nature 368, 660–664 (1994).

    Article  CAS  Google Scholar 

  5. Newman, M., Strzelecka, T., Dorner, L.F., Schildkraut, I. & Aggarwal, A.K. Structure of restriction endonuclease BamHI phased at 1.95Å resolution by MAD analysis. Structure 2, 439–452 ( 1994).

    Article  CAS  Google Scholar 

  6. Kim, Y.C., Grable, J.C., Love, R., Greene, P.J. & Rosenberg, J.M. Refinement of EcoRI endonuclease crystal structure: a revised protein chain tracing. Science 249, 1307–1309 (1990).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  8. 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 

  9. Athanasiadis, A. et al. Crystal structure of PvuII endonuclease reveals extensive structural homologies to EcoRV. Nature Struct. Biol. 1, 469–475 (1994).

    Article  CAS  Google Scholar 

  10. Alves, J. et al. Changing the hydrogen-bonding potential in the DNA binding site of EcoRI by site-directed mutagenesis drastically reduces the enzymatic activity, not, however, the preference of this restriction endonuclease for cleavage within the site -GAATTC-. Biochemistry 28 (1989).

  11. Heitman, J. & Model, P. Substrate recognition by the Eco RI endonuclease. Proteins 7, 185– 197 (1990).

    Article  CAS  Google Scholar 

  12. Osuna, J., Flores, H. & Soberon, X. Combinatorial mutagenesis of three major groove-contacting residues of EcoRI: single and double amino acid replacements retaining methyltransferase-sensitive activities. Gene 106, 7–12 (1991).

    Article  CAS  Google Scholar 

  13. Xu, S. & Schildkraut, I. Isolation of BamHI variants with reduced cleavage activities. J. Biol. Chem. 266 , 4425–4429 (1991).

    CAS  PubMed  Google Scholar 

  14. Wenz, C. et al. Protein engineering of the restriction endonuclease EcoRV: replacement of an amino acid residue in the DNA binding site leads to an altered selectivity towards unmodified and modified substrates. Biochem. Biophys. Acta 1219, 73–80 (1994).

    CAS  PubMed  Google Scholar 

  15. Dorner, L.F., Bitinaite, J., Whitaker, R.D. & Schildraut, I. Genetic analysis of the base-specific contacts of BamHI restriction endonuclease. J. Mol. Biol. 285, 1515– 1523 (1999).

    Article  CAS  Google Scholar 

  16. 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 

  17. Viadiu, H. & Aggarwal, A.K. The role of metals in catalysis by the restriction endonuclease BamHI. Nature Struct. Biol. 5, 910–916 ( 1998).

    Article  CAS  Google Scholar 

  18. Anton, B.P. et al. Cloning and characterization of the BglII restriction-modification system reveals a possible evolutionary footprint. Gene 187, 19–27 (1997).

    Article  CAS  Google Scholar 

  19. Schneider, B., Neidle, S. & Berman, H.M. Conformations of the sugar-phosphate backbone in helical DNA crystal structures. Biopolymers 42, 113–124 (1997).

    Article  CAS  Google Scholar 

  20. Beamer, L.J. & Pabo, C.O. Refined 1.8 Angstrom crystal structure of the λ-repressor operator complex. J. Mol. Biol. 227, 177–196 (1992).

    Article  CAS  Google Scholar 

  21. Dorner, L.F. & Schildkraut, I. Direct selection of binding proficient/catalytic deficient variants of BamHI endonuclease. Nucleic Acids Res. 22, 1068–1074 (1994).

    Article  CAS  Google Scholar 

  22. Reber, E.J. & Pabo, C.O. Zinc finger phage: affinity selection of fingers with new DNA-binding specificities. Science 263, 671–673 (1994).

    Article  Google Scholar 

  23. Choo, Y. & Klug, A. Toward a code for the interactions of zinc fingers with DNA: selection of randomized fingers displayed on phage . Proc. Natl. Acad. Sci. 91, 11163– 11167 (1994).

    Article  CAS  Google Scholar 

  24. Wharton, R.P. & Ptashne, M. Changing the binding specificity of a repressor by redesigning an alpha-helix. Nature 316, 601–605 (1985).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  26. Vipond, I.B., Baldwin, G.S. & Halford, S.E. Divalent metal ions at the active sites of Eco RV and EcoRI restriction endonucleases. Biochemistry 34, 697–704 ( 1995).

    Article  CAS  Google Scholar 

  27. Jeltsch, A., Alves, J., Wolfes, H., Maass, G. & Pingoud, A. Substrate-assisted catalysis in the cleavage of DNA by the EcoRI and EcoRV restriction enzymes. Proc. Natl. Acad. Sci. USA 90, 8499–8503 (1993).

    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. Biochemistry 95, 13489–13494 (1998).

    CAS  Google Scholar 

  29. Jurica, M.S., Monnat, J., R.J. & Stoddard, B.L. DNA recognition and cleavage by the LAGLIDADG homing endonuclease I-CreI. Mol. Cell 2, 469–476 (1998).

    Article  CAS  Google Scholar 

  30. Hendrickson, W.A. Determination of macromolecular structures from anomalous diffraction of synchrotron radiation. Science 254, 51– 58 (1991).

    Article  CAS  Google Scholar 

  31. Otwinowski, Z. & Minor, W. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307–326 ( 1997).

    Article  CAS  Google Scholar 

  32. Furey, W. & Swaminathan, S. PHASES-95: a program package for the processing and analysis of diffraction data from macromolecules. Methods Enzymol. 277, 590–629 (1997).

    Article  CAS  Google Scholar 

  33. Tong, L. PATSOL - v1.2. (1993).

  34. de La Fortelle, E. & Bricogne, G. Maximum-likelihood heavy-atom parameter refinement for multiple isomorphous replacement and multiwavelength anomalous diffraction methods. Methods Enzymol. 276 , 472–494 (1997).

    Article  CAS  Google Scholar 

  35. Collaborative Computational Project Number 4. CCP4 Suite: programs for protein crystallography. Acta Crystallogr. D 50, 760–763 (1994).

  36. Jones, A.T., 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).

    Article  Google Scholar 

  37. Adams, P.D., Pannu, N.S., Read, R.J. & Brunger, A.T. Cross-validated manximum likelihood enhances crystallographic simulated annealing refinement . Proc. Natl. Acad. Sci. USA 94, 5018– 5023 (1997).

    Article  CAS  Google Scholar 

  38. Kraulis, P.J. MOLSCRIPT: a program to produce both detailed and schematic plots of protein structures. J. Appl. Crystallogr. 24, 946 –950 (1991).

    Article  Google Scholar 

  39. Merritt, E.A. & Bacon, D.J. Raster3D photorealistic molecular graphics. Methods Enzymol 277, 505– 524 (1997).

    Article  CAS  Google Scholar 

  40. Nicholls, A., Sharp, K. & Honig, B. Protein folding and association: insights from the interfacial and thermodynamic properties of hydrocarbons. Proteins Struct. Funct. Genet. 11, 281–296 (1991).

    Article  CAS  Google Scholar 

  41. Laverly, R. & Sklenar, H. The definition of generalized helicoidal parameters and of axis curvature for irregular nucleic acids. J. Biomol. Struct. Dyn. 6, 63–91 (1988).

    Article  Google Scholar 

Download references

Acknowledgements

We are grateful to P. Weber and C. Lesburg (Schering-Plough Research Institute) for use of APS synchrotron time and for assistance with data collection, respectively. We thank L. Berman and H. Lewis for facilitating data collection at NSLS. We also thank H. Viadiu for stimulating discussion and analysis. A.K.A. is supported by a grant from the NIH, and C.M.L. is supported by the Damon Runyon-Walter Winchell Cancer Research Fund.

Author information

Authors and Affiliations

  1. Department of Physiology and Biophysics, Structural Biology Program, Mt. Sinai School of Medicine, 1425 Madison Avenue , New York, 10029, New York, USA

    Christine M. Lukacs & Aneel K. Aggarwal

  2. New England Biolabs, 32 Tozer Road, Massachusetts, Beverly, 01915-5599, USA

    Rebecca Kucera & Ira Schildkraut

Authors
  1. Christine M. Lukacs
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Rebecca Kucera
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Ira Schildkraut
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Aneel K. Aggarwal
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Aneel K. Aggarwal.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Lukacs, C., Kucera, R., Schildkraut, I. et al. Understanding the immutability of restriction enzymes: crystal structure of BglII and its DNA substrate at 1.5 Å resolution. Nat Struct Mol Biol 7, 134–140 (2000). https://doi.org/10.1038/72405

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1038/72405

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