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Structure of the multimodular endonuclease FokI bound to DNA

Abstract

FokI is a member of an unusual class of bipartite restriction enzymes that recognize a specific DNA sequence and cleave DNA nonspecifically a short distance away from that sequence1,2,3. Because of its unusual bipartite nature, FokI has been used to create artificial enzymes with new specificities4,5,6,7. We have determined the crystal structure at 2.8 Å resolution of the complete FokI enzyme bound to DNA. As anticipated, the enzyme contains amino- and carboxy-terminal domains corresponding to the DNA-recognition and cleavage functions, respectively. The recognition domain is made of three smaller subdomains (D1, D2 and D3) which are evolutionarily related to the helix–turn–helix-containing DNA-binding domain of the catabolite gene activator protein CAP8. The CAP core has been extensively embellished in the first two subdomains, whereas in the third subdomain it has been co-opted for protein–protein interactions. Surprisingly, the cleavage domain contains only a single catalytic centre, raising the question of how monomeric FokI manages to cleave both DNA strands. Unexpectedly, the cleavage domain is sequestered in a ‘piggyback’ fashion by the recognition domain. The structure suggests a new mechanism for nuclease activation and provides a framework for the design of chimaeric enzymes with altered specificities.

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Figure 1: Sequence of the 20-bp DNA fragment used to co-crystallize FokI.
Figure 2: The complete FokI enzyme (579 amino acids) bound to DNA.
Figure 3: Comparison of FokI recognition subdomains D1, D2 and D3 to the DNA-binding domains of gene regulatory proteins CAP8, HNF-3γ10 and BirA11.
Figure 4: Comparison of FokI cleavage domain to a monomer of endonuclease BamHI.
Figure 5: The new class of FokI mutants (labelled in white) identified by Waugh and Sauer19 map to the interdomain interface between the recognition domain and the cleavage domain.

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References

  1. Sugisaki, H. & Kanazawa, S. New restriction endonucleases from Flavobacterium okeanokoites (FokI) and Micrococcus luteus (MluI). Gene 16, 73–78 (1981).

    Article  CAS  Google Scholar 

  2. Li, L., Wu, L. P. & Chandrasegarran, S. Functional domains of FokI restriction endonuclease. Proc. Natl Acad. Sci. USA 89, 4275–4279 (1992).

    Article  ADS  CAS  Google Scholar 

  3. Skowron, P., Kaczorowski, T., Tucholski, J. & Podhajska, A. J. Atypical DNA-binding properties of class-IIS restriction endonucleases: evidence for recognition of the cognate sequence by a FokI monomer. Gene 125, 1–10 (1993).

    Article  CAS  Google Scholar 

  4. Kim, Y.-G. & Chandrasegarran, S. Chimeric restriction endonuclease. Proc. Natl Acad. Sci. USA 91, 883–887 (1994).

    Article  ADS  CAS  Google Scholar 

  5. Kim, Y.-G., Cha, J. & Chandrasegarran, S. Hybrid restriction enzymes: Zinc finger fusions to FokI cleavage domain. Proc. Natl Acad. Sci. USA 93, 1156–1160 (1996).

    Article  ADS  CAS  Google Scholar 

  6. Huang, B., Schaeffer, C. J., Li, Q. & Tsai, M.-D. Splase: a new class IIs zinc-finger restriction endonuclease with specificity for SP1 binding sites. J. Prot. Chem. 15, 481–489 (1996).

    Article  CAS  Google Scholar 

  7. Podhajska, A. J. & Szybalski, W. Conversion of the FokI endonuclease to a universal restriction enzyme: cleavage of phase M13mp7 DNA at predetermined sites. Gene 40, 175–182 (1985).

    Article  CAS  Google Scholar 

  8. Schultz, S. C., Shields, G. C. & Steitz, T. A. Crystal structure of a CAP–DNA complex: the DNA is bent by 90 degrees. Science 253, 1001–1007 (1991).

    Article  ADS  CAS  Google Scholar 

  9. Ramakrishnan, V., Finch, J. T., Graziano, V., Lee, P. L. & Sweet, R. M. Crystal structure of globular domain histone H5 and its implications for nucleosome binding. Nature 362, 219–223 (1993).

    Article  ADS  CAS  Google Scholar 

  10. Clark, K. L., Halay, E. D., Lai, E. & Burley, S. K. Co-crystal structure of the HNF-3γ DNA recognition motif resembles histone H5. Nature 364, 412–420 (1993).

    Article  ADS  CAS  Google Scholar 

  11. Wilson, K. S., Shewchuk, L. M., Brennan, R. G., Otsuka, A. J. & Matthews, B. W. Escherichia coli biotin holoenzyme synthetase/biorepressor crystal structure delineates biotin- and DNA-binding domains. Proc. Natl Acad. Sci USA 89, 9257–9261 (1992).

    Article  ADS  CAS  Google Scholar 

  12. Harrison, S. C. & Aggarwal, A. K. DNA recognition by proteins with the helix-turn-helix motif. Annu. Rev. Biochem. 59, 933–969 (1990).

    Article  CAS  Google Scholar 

  13. Pavletich, N. P. & Pabo, C. O. Zinc finger–DNA recognition: crystal structure of a zif268–DNA complex at 2.1 Å resolution. Science 252, 809–817 (1991).

    Article  ADS  CAS  Google Scholar 

  14. Klemm, J. D., Rould, M. A., Aurora, R., Herr, W. & Pabo, C. O. Crystal structure of Oct-1 POU domain bound to an octamer site: DNA recognition with tethered DNA-binding modules. Cell 77, 21–32 (1994).

    Article  CAS  Google Scholar 

  15. Newman, M., Strzelecka, T., Dorner, L. F., Schildkraut, I. & Aggarwal, A. K. Structure of restriction endonuclease BamHI and its relationship to EcoRI. Nature 268, 660–664 (1994).

    Article  ADS  Google Scholar 

  16. Waugh, D. S. & Sauer, R. T. Single amino acid substitutions uncouple the DNA binding and strand scission activities of FokI endonuclease. Proc. Natl Acad. Sci. USA 90, 9596–9600 (1993).

    Article  ADS  CAS  Google Scholar 

  17. Yonezawa, A. & Sugiura, Y. DNA binding mode of class-IIS restriction endonuclease FokI revealed by DNA footprinting analysis. Biochem. Biophys. Acta 1219, 369–379 (1994).

    CAS  PubMed  Google Scholar 

  18. Li, L., Wu, L. P., Clarke, R. & Chandrasegarran, S. C-terminal deletion mutants of the FokI restriction endonuclease. Gene 133, 79–84 (1993).

    Article  CAS  Google Scholar 

  19. Waugh, D. S. & Sauer, R. T. Anovel class of FokI restriction endonuclease mutants that cleave hemi-methylated substrates. J. Biol. Chem. 269, 12298–12303 (1994).

    CAS  PubMed  Google Scholar 

  20. Heitman, J. & Model, P. Mutants of the EcoRI endonuclease with promiscuous substrate specificity implicate residues involved in substrate recognition. EMBO J. 9, 3369–3378 (1990).

    Article  CAS  Google Scholar 

  21. Holm, L. & Sander, C. J. Protein structural comparison by alignment of distance matrices. J. Mol. Biol. 233, 123–138 (1993).

    Article  CAS  Google Scholar 

  22. Churchill, M. E., Tullius, T. D. & Klug, A. Mode of interaction of the zinc finger protein TFIIIA with a 5S RNA gene of Xenopus. Proc. Natl Acad. Sci. USA 87, 5528–5532 (1990).

    Article  ADS  CAS  Google Scholar 

  23. Clemens, K. R., Liao, X., Wolf, V., Wright, P. E. & Gottesfeld, J. M. Definition of the binding sites of individual zinc fingers in the transcription factor IIIA-5S RNA gene complex. Proc. Natl Acad. Sci. USA 89, 10822–10826 (1992).

    Article  ADS  CAS  Google Scholar 

  24. Pavletich, N. P. & Pabo, C. O. Crystal structure of a five-finger GL1-DNA complex: new perspectives on Zn fingers. Science 261, 1701–1707 (1993).

    Article  ADS  CAS  Google Scholar 

  25. Hirsch, J. A., Wah, D. A., Dorner, L. F., Schildkraut, I. & Aggarwal, A. K. Crystallization and preliminary X-ray analysis of restriction endonuclease FokI bound to DNA. FEBS Lett. 403, 136–138 (1997).

    Article  CAS  Google Scholar 

  26. Furey, W. Phases—A Program Package for the Processing and Analysis of Diffraction Data for Macromolecules(VA Medical Center, Pittsburgh, PA, (1993)).

    Google Scholar 

  27. Abrahams, J. P. & Leslie, A. G. W. Methods used in the structure determination of bovine mitochondrial F1ATPase. Acta Crystallogr. D 52, 30–42 (1996).

    Article  CAS  Google Scholar 

  28. Brunger, A. T. X-PLOR, Version 3.1, A System for X-ray Crystallography and NMR(Yale University, New Haven, CT, (1992)).

    Google Scholar 

  29. Jones, T. A., Zou, J.-Y. & Cowan, S. W. Improved methods for building models in electron density maps and the location of errors in these models. Acta Crystallogr. A 47, 110–119 (1991).

    Article  Google Scholar 

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Acknowledgements

We thank the staff at CHESS and C. Escalante, E. Jacobson, M. Newman and H. Viadiu for help with data collection; R. Knott for DNA synthesis; and T. Bestor, W. Hendrickson, R. Mann and R. Roberts for comments on the manuscript. This work was supported by an NIH grant (A.K.A.) and NIH training grants (J.A.H. and D.A.W.).

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Correspondence to Aneel K. Aggarwal.

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Wah, D., Hirsch, J., Dorner, L. et al. Structure of the multimodular endonuclease FokI bound to DNA. Nature 388, 97–100 (1997). https://doi.org/10.1038/40446

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