Restriction Endonucleases and Their Uses

  • Raymond J. Williams
Part of the Methods in Molecular Biology™ book series (MIMB, volume 160)


Restriction endonucleases, which cleave DNA in a site-specific manner, are a fundamental tool of molecular biology. The discovery of endonucleases began in the 1960s and led to commercial availability in the early 1970s. The number of characterized enzymes continues to grow, as does the number of vendors and the size of their product lines. Although many similarities exist among endonucleases in terms of their structures, mechanisms, and uses, important differences remain. Now a staple of molecular biology, restriction endonucleases are an area of active research as models of site-specific DNA recognition, cleavage mechanism, in vivo function, and evolutionary origins. New enzymes continue to be discovered or developed by using protein engineering to modify the specificity of existing enzymes.


Recognition Site Star Activity Homing Endonuclease Cleavage Domain Noncatalytic Site 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


  1. 1.
    Roberts, R. J. and Halford, S. E. (1993) Type II restriction endonucleases, in Nucleases, 2nd ed. (Linn, S. M., Lloyd, S. R., and Roberts, R. J., eds.), Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, pp. 35–88.Google Scholar
  2. 2.
    Roberts, R. J. and Macelis, D. (1998) REBASE—restriction enzymes and methylases. Nucleic Acids Res. 26, 338–350.PubMedCrossRefGoogle Scholar
  3. 3.
    Wilson, G. G. and Murray, N. E. (1991) Restriction and modification systems. Annu. Rev. Genet. 25, 585–627.PubMedCrossRefGoogle Scholar
  4. 4.
    Stahl, F., Wende, W., Jeltsch, A., and Pingoud, A. (1998) The mechanism of DNA cleavage by the Type II restriction enzyme EcoRV: Asp36 is not directly involved in DNA cleavage but serves to couple indirect readout to catalysis. Biol. Chem. 379, 467–473.PubMedCrossRefGoogle Scholar
  5. 5.
    Smith, H. O., Annau, T. M., and Chandrasegaran, S. (1990) Finding sequence motifs in groups of functionally related proteins. Proc. Natl. Acad. Sci. USA. 87, 826–830.PubMedCrossRefGoogle Scholar
  6. 6.
    Szomolanyi, E., Kiss, A., and Venetianer, P. (1980) Cloning the modification methylase gene of Bacillus sphaericus R in Escherichia coli. Gene. 10, 219–225.PubMedCrossRefGoogle Scholar
  7. 7.
    Tao, T., Bourne, J. C., and Blumenthal, R. M. (1991) A family of regulatory genes associated with Type II restriction-modification systems. J. Bact. 173, 1367–1375.PubMedGoogle Scholar
  8. 8.
    Ives, C. L., Sohail, A., and Brooks, J. E. (1995) The regulatory C proteins from different restriction-modification systems can cross-complement. J. Bact. 177, 6313–6315.PubMedGoogle Scholar
  9. 9.
    Smith, H. O. and Nathans, D. (1973) A suggested nomenclature for bacterial host modification and restriction systems and their enzymes. J. Mol. Biol 81, 419–423.PubMedCrossRefGoogle Scholar
  10. 10.
    Bickle, T. A. (1993) The ATP-dependent restriction enzymes, in Nucleases, 2nd ed. (Linn, S. M., Lloyd, S. R., and Roberts, R. J., eds.), Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, pp. 35–88.Google Scholar
  11. 11.
    Pingoud, A. and Jeltsch, A. (1997) Recognition and cleavage of DNA by type-II restriction endonucleases. Eur. J. Biochem. 246, 1–22.PubMedCrossRefGoogle Scholar
  12. 12.
    Kong, H. (1998) Analyzing the functional organization of a novel restriction modification system, the BcgI system. J. Mol. Biol. 279, 823–832.PubMedCrossRefGoogle Scholar
  13. 13.
    Sears, L. E., Zhou, B., Aliotta, J. M., Morgan, R. D., and Kong, H. (1996) BaeI, another unusual BcgI-like restriction endonuclease. Nucleic Acids Res 24, 3590–3592.PubMedCrossRefGoogle Scholar
  14. 14.
    Reuter, M., Kupper, D., Pein, C. D., Petrusyte, M., Siksnys, V., Frey, B., and Kruger, D. H. (1993) Use of specific oligonucleotide duplexes to stimulate cleavage of refractory DNA sites by restriction endonucleases. Anal. Biochem. 209, 232–237.PubMedCrossRefGoogle Scholar
  15. 15.
    Oller, A. R., Broek, W. V., Conrad, M., and Topal, M. (1991) Ability of DNA and spermidine to affect the activity of restriction endonucleases from several bacterial species. Biochem. 30, 2543–2549.CrossRefGoogle Scholar
  16. 16.
    Szybalski, W., Kim, S. C., Hasan, N., and Podhajska, A. J. (1991) Class-IIS restriction enzymes—a review. Gene 100, 13–26.PubMedCrossRefGoogle Scholar
  17. 17.
    Janulaitis, A., Petrusyte, M., Maneliene, Z., Klimasauskas, S., and Butkus, V. (1992) Purification and properties of the Eco57I restriction endonuclease and methylase—prototypes of a new class (type IV). Nucleic Acids Res. 20, 6043–6049.PubMedCrossRefGoogle Scholar
  18. 18.
    Belfort, M. and Roberts, R. J. (1997) Homing endonucleases: keeping the house in order. Nucleic Acids Res. 25, 3379–3388.PubMedCrossRefGoogle Scholar
  19. 19.
    Meisel, A., Mackeldanz, P., Bickle, T. A., Kruger, D. H., and Schroeder, C. (1995) Type III restriction endonucleases translocate DNA in a reaction driven by recognition sitespecific ATP hydrolysis. EMBO J. 14, 2958–2966.PubMedGoogle Scholar
  20. 20.
    Kruger, D. H., Kupper, D., Meisel, A., Reuter, M., and Schroeder, C. (1995) The significance of distance and orientation of restriction endonuclease recognition sites in viral DNA genomes. FEMS Microbiol. Rev. 17, 177–184.PubMedCrossRefGoogle Scholar
  21. 21.
    Mise, K. and Nakajima, K. (1985) Purification of a new restriction endonuclease, StyI, from Escherichia coli carrying the hsd+ minplasmid. Gene 33, 357–361.PubMedCrossRefGoogle Scholar
  22. 22.
    Sam, M. D. and Perona, J. J. (1999) Catalytic roles of divalent metal ions in phosphoryl transfer by EcoRV endonuclease. Biochem. 38, 6576–6586.CrossRefGoogle Scholar
  23. 23.
    Wentzell, L. M., Nobbs, T. J., and Halford, S. E. (1995) The SfiI restriction endonuclease makes a 4-strand DNA break at two copies of its recognition sequence. J. Mol. Biol. 248, 581–595.PubMedCrossRefGoogle Scholar
  24. 24.
    Sato, H., Suzuki, T., and Yamada, Y. (1990) Purification of restriction endonuclease from Acetobacter aceti IFO 3281 (AatII) and its properties. Agric. Biol. Chem. 54, 3319–3325.PubMedGoogle Scholar
  25. 25.
    Nobbs, T. J. and Halford, S. E. (1995) DNA cleavage at two recognition sites by the SfiI restriction endonuclease: salt dependence of cis and trans interactions between distand DNA sites. J. Mol. Biol. 252, 399–411.PubMedCrossRefGoogle Scholar
  26. 26.
    Nobbs, T. J., Williams, S. A., Connolly, B. A., and Halford, S. E. (1998) Phosphorothioate substrates for the SfiI restriction endonuclease. Biol. Chem. 379, 599–604.PubMedGoogle Scholar
  27. 27.
    Kong, H. and Smith, C. L. (1998) Does BcgI, a uniquerestriction endonuclease, require two recognition sites for cleavage? Biol. Chem. 379, 605–609.PubMedGoogle Scholar
  28. 28.
    Kong, H. and Smith, C. L. (1997) Substrate DNA and cofactor regulate the activities of a multi-functional restriction-modification enzyme, Bcg I. Nucl. Acids Res. 25, 3687–3692.PubMedCrossRefGoogle Scholar
  29. 29.
    Wah, D. A., Hirsch, J. A., Dorner, L. F., Schildkraut, I., and Aggarwal, A. K. (1997) Structure of the multimodular endonuclease FokI bound to DNA. Nature 388, 97–100.PubMedCrossRefGoogle Scholar
  30. 30.
    Bitinaite, J., Wah, D. A., Aggarwal, A. K., and Schildkraut, I. (1998) FokI dimerization is required for DNA cleavage. Proc. Natl. Acad. Sci. 95, 10,570–10,575.PubMedCrossRefGoogle Scholar
  31. 31.
    Reuter, M., Kupper, D., Meisel, A., Schroeder, C., and Kruger, D. H. (1998) Cooperative binding properties of restriction endonuclease EcoRII with DNA recognition sites. J. Biol. Chem. 273, 8294–8300.PubMedCrossRefGoogle Scholar
  32. 32.
    Pein, C. D., Reuter, M., Meisel, A., Cech, D., and Kruger, D. H. (1991) Activation of restriction endonuclease EcoRII does not depend on the cleavage of stimulator DNA. Nucleic Acids Res. 19, 5139–5142.PubMedCrossRefGoogle Scholar
  33. 33.
    Conrad, M. and Topal, M. (1992) Modified DNA fragments activate NaeI cleavage of refractory DNA sites. Nucleic Acids Res. 20, 5127–5130.PubMedCrossRefGoogle Scholar
  34. 34.
    Senesac, J. H. and Allen, J. R. (1995) Oligonucleaotide activation of the Type IIe restriction enzyme NaeI for digestion of refractory sites. BioTechniques 19, 990–993.PubMedGoogle Scholar
  35. 35.
    Jo, K. and Topal, M. D. (1995) DNA topoisomerase and recombinase activities in NaeI restriciton endonuclease. Science 267, 1817–1820.PubMedCrossRefGoogle Scholar
  36. 36.
    Jo, K. and Topal, M. D. (1998) Step-wise DNA relaxation and decatenation by NaeI-43K. Nucleic Acids Res. 26, 2380–2384.PubMedCrossRefGoogle Scholar
  37. 37.
    Yoo, O. J. and Agarwal, K. L. (1980) Cleavage of single strand oligonucleotides and bacteriophage phiX174 DNA by Msp I endonuclease. J. Biol. Chem. 255, 10,559–10,562.PubMedGoogle Scholar
  38. 38.
    Blakesley, R. W., Dodgson, J. B., Nes, I. F., and Wells, R. D. (1977) Duplex regions in “single-stranded” phiX174 DNA are cleaved by a restriction endonuclease from Haemophilus aegypius. J. Biol. Chem. 252, 7300–7306.PubMedGoogle Scholar
  39. 39.
    Shaw, P. C. and Mok, Y. K. (1993) XcmI as a universal restriction enzyme for singlestranded DNA. Gene 133, 85–89.PubMedCrossRefGoogle Scholar
  40. 40.
    Robinson, C. R. and Sligar, S. G. (1998) Changes in solvation during DNA binding and cleavage are critical to altered specificity of the EcoRI endonuclease. Proc. Natl. Acad. Sci. USA 95, 2186–2191.PubMedCrossRefGoogle Scholar
  41. 41.
    Lesser, D. R., Kurpiewski, M. R., and Jen-Jacobson, L. (1990) The energetic basis of specificity in the EcoRI endonuclease-DNA interaction. Science 250, 776–786.PubMedCrossRefGoogle Scholar
  42. 42.
    Vermote, C. L. M. and Halford, S. E. (1992) EcoRV restriction endonuclease: communication between catalytic metal ions and DNA recognition. Biochemistry 31, 6082–6089.PubMedCrossRefGoogle Scholar
  43. 43.
    Wenz, C., Selent, U., Wende, W., Jeltsch, A., Wolfes, H., and Pingoud, A. (1994) 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. Biochim. Biophys. Acta. 1219, 73–80.PubMedGoogle Scholar
  44. 44.
    Lanio, T., Selent, U., Wnez, C., Wende, W., Schulz, A., Adiraj, M., Katti, S. B., and Pingoud, A. (1996) EcoRV-T94V: a mutant restriction endonuclease with an altered substrate specificity towards modified oligodeoxynucleotides. Protein Eng. 9, 1005–1010.PubMedCrossRefGoogle Scholar
  45. 45.
    Schottler, S., Wenz, C., Lanio, A., Jeltsch, A., and Pingoud, A. (1998) Protein engineering of the restriction endonuclease EcoRV: structure-guided design of enzyme variants that recognize the base pairs flanking the recognition site. Eur. J. Biochem. 258, 184–191.PubMedCrossRefGoogle Scholar
  46. 46.
    Lanio, T., Jeltsch, A., and Pingoud, A. (1998) Towards the design of rare cutting restriction endonucleases: using directed evolution to generate variants of EcoRV differing in their substrate specificity by two orders of magnitude. J. Mol. Biol. 283, 59–69.PubMedCrossRefGoogle Scholar
  47. 47.
    Stahl, F., Wende, W., Jeltsch, A., and Pingoud, A. (1996) Introduction of asymmetry in the naturally symmetric restriction endonuclease EcoRV to investigate intersubunit communication in the homodimeric protein. Proc. Natl. Acad. Sci. USA 93, 6175–6180.PubMedCrossRefGoogle Scholar
  48. 48.
    Kim, Y. G. and Chandrasegaran, S. (1994) Chimeric restriction endonuclease. Proc. Natl. Acad. Sci USA 91, 883–887.PubMedCrossRefGoogle Scholar
  49. 49.
    Kim, Y. G., Shi, Y., Berg, J. M., and Chandrasegaran, S. (1997) Site-specific cleavage of DNA-RNA hybrids by zinc finger/FokI cleavage domain fusions. Gene 203, 43–49.PubMedCrossRefGoogle Scholar
  50. 50.
    Kim, Y. G., Smith, J., Durgesha, M., and Chandrasegaran, S. (1998) Chimeric restriction enzyme: Gal4 fusion to FokI cleavage domain. Biol. Chem. 379, 489–495.PubMedCrossRefGoogle Scholar
  51. 51.
    Dervan, P. B. (1992) Reagents for the site-specific cleavage of megabase DNA. Nature 359, 87,88.CrossRefGoogle Scholar
  52. 52.
    Ebright, Y. W., Chen, Y., Pendergrast, P. S., and Ebright, R. H. (1992) Incorporation of an EDTA-metal complex at a rationally selected site within a protein: application to EDTA-iron DNA affinity cleaving with catabolite gene activator protein (CAP) and Cro. Biochem. 31, 10,664–10,670.CrossRefGoogle Scholar
  53. 53.
    Pendergrast, P. S., Ebright, Y. W., and Ebright, R. H. (1994) High-specificity DNA cleavage agent: design and application to kilobase and megabase DNA substrates. Science 265, 959–962.Google Scholar
  54. 54.
    Shang, Z., Ebright, Y. W., Iler, N., Pendergrast, P. S., Echelard, Y., McMahon, A. P., Ebright, R. H., and Abate, C. (1994) DNA affinity cleaving analysis of homeodomain-DNA interaction: identification of homeodomain consensus sites in genomic DNA. Proc. Natl. Acad. Sci. USA 91, 118–122.PubMedCrossRefGoogle Scholar
  55. 55.
    Koob, M., Burkiewicz, A., Kur, J., and Szybalski, W. (1992) RecA-AC: single-site cleavage of plasmids and chromosones at any predetermined restriction site. Nucleic Acids. Res. 20, 5831–5836.PubMedCrossRefGoogle Scholar
  56. 56.
    Schoenfeld, T., Harper, T., and Slater, M. (1995) RecA cleavage and protection for genomic mapping and subcloning. Promega Notes 50, 9–14.Google Scholar
  57. 57.
    Strobel, S. A. and Dervan, P. B. (1991) Single-site enzymatic cleavage of yeast genomic DNA mediated by triple helix formation. Nature 350, 172–174.PubMedCrossRefGoogle Scholar
  58. 58.
    Hung, L., Murray, E., Murray, W., Bandziulis, R., Lowery, R., Williams, R., and Noble, R. (1991) A blue/white cloning assay for quality control of DNA restriction and modifying enzymes. Promega Notes 33, 12,13.Google Scholar
  59. 59.
    Murray, E., Singer, K., Cash, K., and Williams, R. (1993) Cloning-qualified blunt end restriction enzymes: causes and cures for light blue colonies. Promega Notes 41, 1–5.Google Scholar
  60. 60.
    Turbett, G. R. and Sellner, L. N. (1996) Digestion of PCR and RT-PCR products with restriction endonucleases without prior purification or precipitation. Promega Notes 60, 23–27.Google Scholar
  61. 61.
    New England BioLabs 1998/99 Catalog (1998) New England BioLabs, 258,259.Google Scholar
  62. 62.
    Dallas-Yang, Q., Jiang, G., and Sladek, F. M., (1998) Digestion of terminal restriction endonuclease recognition sites on PCR products. BioTechniques 24, 582–584.Google Scholar
  63. 63.
    Moreira, R. F. and Noren, C. J. (1995) Minimum duplex requirements for restriction enzyme cleavage near the termini of linear DNA fragments. BioTechniques 19, 57–59.Google Scholar
  64. 64.
    Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Gel electrophoresis of DNA, in Molecular Cloning, A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, p. 65.Google Scholar

Copyright information

© Humana Press Inc. 2001

Authors and Affiliations

  • Raymond J. Williams
    • 1
  1. 1.Promega Corp.Madison

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