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.

Computational redesign of endonuclease DNA binding and cleavage specificity

Abstract

The reprogramming of DNA-binding specificity is an important challenge for computational protein design that tests current understanding of protein–DNA recognition, and has considerable practical relevance for biotechnology and medicine1,2,3,4,5,6. Here we describe the computational redesign of the cleavage specificity of the intron-encoded homing endonuclease I-MsoI7 using a physically realistic atomic-level forcefield8,9. Using an in silico screen, we identified single base-pair substitutions predicted to disrupt binding by the wild-type enzyme, and then optimized the identities and conformations of clusters of amino acids around each of these unfavourable substitutions using Monte Carlo sampling10. A redesigned enzyme that was predicted to display altered target site specificity, while maintaining wild-type binding affinity, was experimentally characterized. The redesigned enzyme binds and cleaves the redesigned recognition site 10,000 times more effectively than does the wild-type enzyme, with a level of target discrimination comparable to the original endonuclease. Determination of the structure of the redesigned nuclease-recognition site complex by X-ray crystallography confirms the accuracy of the computationally predicted interface. These results suggest that computational protein design methods can have an important role in the creation of novel highly specific endonucleases for gene therapy and other applications.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Figure 1: Comparison of the predicted interactions in cognate and non-cognate binding complexes, illustrating the designed specificity switch.
Figure 2: Switch in nuclease cleavage specificity.
Figure 3: Crystal structure of the designed enzyme–DNA complex.

References

  1. 1

    Uil, T. G., Haisma, H. J. & Rots, M. G. Therapeutic modulation of endogenous gene function by agents with designed DNA-sequence specificities. Nucleic Acids Res. 31, 6064–6078 (2003)

    CAS  Article  Google Scholar 

  2. 2

    Bibikova, M. et al. Stimulation of homologous recombination through targeted cleavage by chimeric nucleases. Mol. Cell. Biol. 21, 289–297 (2001)

    CAS  Article  Google Scholar 

  3. 3

    Porteus, M. H. & Baltimore, D. Chimeric nucleases stimulate gene targeting in human cells. Science 300, 763 (2003)

    Article  Google Scholar 

  4. 4

    Wickelgren, I. Molecular biology. Spinning junk into gold. Science 300, 1646–1649 (2003)

    CAS  Article  Google Scholar 

  5. 5

    Stoddard, B. L. Homing endonuclease structure and function. Q. Rev. Biophys. 38, 1–47 (2005)

    Google Scholar 

  6. 6

    Urnov, F. D. et al. Highly efficient endogenous human gene correction using designed zinc-finger nucleases. Nature 435, 646–651 (2005)

    ADS  CAS  Article  Google Scholar 

  7. 7

    Lucas, P., Otis, C., Mercier, J. P., Turmel, M. & Lemieux, C. Rapid evolution of the DNA-binding site in LAGLIDADG homing endonucleases. Nucleic Acids Res. 29, 960–969 (2001)

    CAS  Article  Google Scholar 

  8. 8

    Rohl, C. A., Strauss, C. E., Misura, K. M. & Baker, D. Protein structure prediction using Rosetta. Methods Enzymol. 383, 66–93 (2004)

    CAS  Article  Google Scholar 

  9. 9

    Havranek, J. J., Duarte, C. M. & Baker, D. A simple physical model for the prediction and design of protein–DNA interactions. J. Mol. Biol. 344, 59–70 (2004)

    CAS  Article  Google Scholar 

  10. 10

    Voigt, C. A., Gordon, D. B. & Mayo, S. L. Trading accuracy for speed: A quantitative comparison of search algorithms in protein sequence design. J. Mol. Biol. 299, 789–803 (2000)

    CAS  Article  Google Scholar 

  11. 11

    Kono, H. & Sarai, A. Structure-based prediction of DNA target sites by regulatory proteins. Proteins 35, 114–131 (1999)

    CAS  Article  Google Scholar 

  12. 12

    Pabo, C. O. & Nekludova, L. Geometric analysis and comparison of protein–DNA interfaces: why is there no simple code for recognition? J. Mol. Biol. 301, 597–624 (2000)

    CAS  Article  Google Scholar 

  13. 13

    Luscombe, N. M., Laskowski, R. A. & Thornton, J. M. Amino acid-base interactions: a three-dimensional analysis of protein–DNA interactions at an atomic level. Nucleic Acids Res. 29, 2860–2874 (2001)

    CAS  Article  Google Scholar 

  14. 14

    Morozov, A. V., Havranek, J. J., Baker, D. & Siggia, E. D. Protein–DNA binding specificity predictions with structural models. Nucleic Acids Res. 33, 5781–5798 (2005)

    CAS  Article  Google Scholar 

  15. 15

    Seligman, L. M. et al. Mutations altering the cleavage specificity of a homing endonuclease. Nucleic Acids Res. 30, 3870–3879 (2002)

    CAS  Article  Google Scholar 

  16. 16

    Chevalier, B., Turmel, M., Lemieux, C., Monnat, R. J. Jr & Stoddard, B. L. Flexible DNA target site recognition by divergent homing endonuclease isoschizomers I-CreI and I-MsoI. J. Mol. Biol. 329, 253–269 (2003)

    CAS  Article  Google Scholar 

  17. 17

    Heath, P. J., Stephens, K. M., Monnat, R. J. Jr & Stoddard, B. L. The structure of I–Crel, a group I intron-encoded homing endonuclease. Nature Struct. Biol. 4, 468–476 (1997)

    CAS  Article  Google Scholar 

  18. 18

    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)

    ADS  CAS  Article  Google Scholar 

  19. 19

    Sussman, D. et al. Isolation and characterization of new homing endonuclease specificities at individual target site positions. J. Mol. Biol. 342, 31–41 (2004)

    CAS  Article  Google Scholar 

  20. 20

    Doyon, J. B., Pattanayak, V., Meyer, C. B. & Liu, D. R. Directed evolution and substrate specificity profile of homing endonuclease I-SceI. J. Am. Chem. Soc. 128, 2477–2484 (2006)

    CAS  Article  Google Scholar 

  21. 21

    Gouble, A. et al. Efficient in toto targeted recombination in mouse liver by meganuclease-induced double-strand break. J. Gene Med. published online 13 February 2006 (doi:10.1002/jgm.879) (2006)

  22. 22

    Arnould, S. et al. Engineering of large numbers of highly specific homing endonucleases that induce recombination on novel DNA targets. J. Mol. Biol. 355, 443–458 (2006)

    CAS  Article  Google Scholar 

  23. 23

    Dunbrack, R. L. Jr & Cohen, F. E. Bayesian statistical analysis of protein side-chain rotamer preferences. Protein Sci. 6, 1661–1681 (1997)

    CAS  Article  Google Scholar 

  24. 24

    Onufriev, A., Bashford, S. D. & Case, D. A. Exploring protein native states and large-scale conformational changes with a modified generalized Born model. Proteins 55, 383–394 (2004)

    CAS  Article  Google Scholar 

  25. 25

    Press, W. H., Flannery, B. P., Teukolsky, S. A. & Vetterling, W. T. Numerical Recipes in C: The Art of Scientific Computing (Cambridge Univ. Press, New York, 1992)

    MATH  Google Scholar 

  26. 26

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

    CAS  Article  Google Scholar 

Download references

Acknowledgements

We thank J. L. Eklund for assistance with binding assays, and B. W. Shen for assistance with data collection and refinement. This work was supported by fellowships from the Jane Coffin Childs Memorial Fund (J.J.H.), the National Science Foundation (C.M.D.), and grants from the National Institute of Health (R.J.M. and B.L.S.), the Howard Hughes Medical Institute (D.B.), and the Gates Foundation Grand Challenges Program (B.L.S., D.B., R.J.M.). Author Contributions J.J.H. and C.M.D. developed the original protein–DNA interface design methods and code. J.A. made further code and method developments, generated and assessed the computational predictions, and performed mutagenesis, biochemical characterization, and crystallization. D.S. collected and processed the crystallographic data, and aided in protein purification and structure refinement.

Author information

Affiliations

Authors

Corresponding authors

Correspondence to Justin Ashworth or David Baker.

Ethics declarations

Competing interests

The atomic coordinates of the redesigned I-MsoI endonuclease bound to its cognate DNA have been deposited in the Protein Data Bank with the accession number 2FLD. Reprints and permissions information are available at npg.nature.com/reprintsandpermissions. The authors declare no competing financial interests.

Supplementary information

Supplementary Notes

This file contains Supplementary Figures 1–6, Supplementary Tables, Supplementary Methods and additional references. (PDF 1471 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Ashworth, J., Havranek, J., Duarte, C. et al. Computational redesign of endonuclease DNA binding and cleavage specificity. Nature 441, 656–659 (2006). https://doi.org/10.1038/nature04818

Download citation

Further reading

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

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