Advertisement

Methods for Determining Activity and Specificity of DNA Binding and DNA Cleavage by Class II Restriction Endonucleases

Protocol
Part of the Methods in Molecular Biology™ book series (MIMB, volume 160)

Abstract

Restriction endonucleases coupled with DNA methyltransferases form the restriction-modification (RM) systems that occur ubiquitously among bacteria. They protect bacterial cells against bacteriophage infection by cleaving incoming foreign DNA highly specifically if it contains the recognition sequence. Cellular DNA is protected from cleavage by a specific methylation within the recognition sequence, which is introduced by the methyltransferase (for review, see refs. 1,2). Restriction endonucleases recognize palindromic recognition sites, 4–8 base pairs in length. These enzymes are indispensable tools for genetic engineering. The biology and biochemistry of type II restriction endonucleases has been reviewed recently (3,4) and will be summarized only briefly here. Type IIS restriction enzymes differ from type II enzymes in that they recognize an asymmetric recognition sequence (for review, see ref. 5). Monomeric in solution, these enzymes consist of a DNA recognition domain and a catalytic domain (6).

Keywords

Fluorescence Resonance Energy Transfer Electrophoretic Mobility Shift Assay Cleavage Reaction Cleavage Rate Fluorescence Depolarization 
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.
This is a preview of subscription content, log in to check access.

References

  1. 1.
    Noyer-Weidner, M. and Trautner, T. A. (1993) Methylation of DNA in prokaryotes, in DNA Methylation: Molecular Biology and Biological Significance (Jost, J. P. and Saluz, H. P., eds.), Birkhauser Verlag, Basel, pp. 40–108.Google Scholar
  2. 2.
    Cheng, X. (1995) Structure and function of DNA methyltransferases. Annu. Rev. Biophys. Biomol. Struct. 24, 293–318.PubMedCrossRefGoogle Scholar
  3. 3.
    Roberts, R. J. and Halford, S. E. (1993) Type II restriction enzymes, in Nucleases (Linn, S. M., Lloyd, R. S., and Roberts, R. J., eds.), Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, pp. 35–88.Google Scholar
  4. 4.
    Pingoud, A. and Jeltsch, A. (1997) Recognition and cleavage of DNA by type-II restriction endonucleases. Eur. J. Biochem. 246, 1–22.PubMedCrossRefGoogle Scholar
  5. 5.
    Szybalski, W., Kim, S. C., Hasan, N., and Podhajska, A. J. (1991) Class-IIS restriction enzymes—a review. Gene 100, 13–26.PubMedCrossRefGoogle Scholar
  6. 6.
    Li, L., Wu, L. P., and Chandrasegaran, S. (1992) Functional domains in FokI restriction endonuclease. Proc. Natl. Acad. Sci. USA 89, 4275–4279.PubMedCrossRefGoogle Scholar
  7. 7.
    Alves, J., Urbanke, C., Fliess, A., Maass, G., and Pingoud, A. (1989) Fluorescence stopped-flow kinetics of the cleavage of synthetic oligodeoxynucleotides by the EcoRI restriction endonuclease. Biochemistry 28, 7879–7888.PubMedCrossRefGoogle Scholar
  8. 8.
    Baldwin, G. S., Vipond, I. B., and Halford, S. E. (1995) Rapid reaction analysis of the catalytic cycle of the EcoRV restriction endonuclease. Biochemistry 34, 705–714.PubMedCrossRefGoogle Scholar
  9. 9.
    Jack, W. E., Terry, B. J., and Modrich, P. (1982) Involvement of outside DNA sequences in the major kinetic path by which EcoRI endonuclease locates and leaves its recognition sequence. Proc. Natl. Acad. Sci. USA 79, 4010–4014.PubMedCrossRefGoogle Scholar
  10. 10.
    Terry, B. J., Jack, W. E., and Modrich, P. (1985) Facilitated diffusion during catalysis by EcoRI endonuclease: nonspecific interactions in EcoRI catalysis. J. Biol. Chem. 260, 13,130–13,137.PubMedGoogle Scholar
  11. 11.
    Ehbrecht, H.-J., Pingoud, A., Urbanke, C., Maass, G., and Gualerzi, C. (1985) Linear diffusion of restriction endonucleases on DNA. J. Biol. Chem. 260, 6160–6166.PubMedGoogle Scholar
  12. 12.
    Jeltsch, A., Alves, J., Wolfes, H., Maass, G., and Pingoud, A. (1994) Pausing of the restriction endonuclease EcoRI during linear diffusion on DNA. Biochemistry 33, 10,215–10,219.PubMedCrossRefGoogle Scholar
  13. 13.
    Jeltsch, A., Wenz, C., Stahl, F., and Pingoud, A. (1996) Linear diffusion of the restriction endonuclease EcoRV on DNA is essential for the in vivo function of the enzyme. EMBO J. 15, 5104–5111.PubMedGoogle Scholar
  14. 14.
    Jeltsch, A. and Pingoud, A. (1998) Kinetic characterization of linear diffusion of the restriction endonuclease EcoRV on DNA. Biochemistry 37, 2160–2169.PubMedCrossRefGoogle Scholar
  15. 15.
    Schulze, C., Jeltsch, A., Franke, I., Urbanke, C., and Pingoud, A. (1998) Crosslinking the EcoRV restriction endonuclease across the DNA-binding site reveals transient intermediates and conformational changes of the enzyme during DNA binding and catalytic turnover. EMBO J. 17, 6757–6766.PubMedCrossRefGoogle Scholar
  16. 16.
    Kim, Y., Grable, J. C., Love, R., Greene, P. J., and Rosenberg, J. M. (1990) Refinement of EcoRI endonuclease crystal structure: a revised protein chain tracing. Science 249, 1307–1309.PubMedCrossRefGoogle Scholar
  17. 17.
    Winkler, F. K., Banner, D. W., Oefner, C., Tsernoglou, D., Brown, R. S., Heathman, S. P., Bryan, R. K., Martin, P. D., Petratos, K., and Wilson, K. S. (1993) The crystal structure of EcoRV endonuclease and of its complexes with cognate and non-cognate DNA fragments. EMBO J. 12, 1781–1795.PubMedGoogle Scholar
  18. 18.
    Athanasiadis, A., Vlassi, M., Kotsifaki, D., Tucker, P. A., Wilson, K. S., and Kokkinidis, M. (1994) Crystal structure of PvuII endonuclease reveals extensive structural homologies to EcoRV. Nat. Struct. Biol. 1, 469–475.PubMedCrossRefGoogle Scholar
  19. 19.
    Cheng, X., Balendiran, K., Schildkraut, I., and Anderson, J. E. (1994) Structure of PvuII endonuclease with cognate DNA. EMBO J. 13, 3927–3935.PubMedGoogle Scholar
  20. 20.
    Newman, M., Strzelecka, T., Dorner, L. F., Schildkraut, I., and Aggarwal, A. K. (1994) Structure of restriction endonuclease BamHI and its relationship to EcoRI. Nature 368, 660–664.PubMedCrossRefGoogle Scholar
  21. 21.
    Newman, M., Strzelecka, T., Dorner, L. F., Schildkraut, I., and Aggarwal, A. K. (1995) Structure of BamHI endonuclease bound to DNA: partial folding and unfolding on DNA binding. Science 269, 656–663.PubMedCrossRefGoogle Scholar
  22. 22.
    Bozic, D., Grazulis, S., Siksnys, V., and Huber, R. (1996) Crystal structure of Citrobacter freundii restriction endonuclease Cfr10I at 2.15 Å resolution. J. Mol. Biol. 255, 176–186.PubMedCrossRefGoogle Scholar
  23. 23.
    Newman, M., Lunnen, K., Wilson, G., Greci, J., Schildkraut, I., and Phillips, S. E. V. (1998) Crystal structure of restriction endonuclease BglI bound to its interrupted DNA recognition sequence. EMBO J. 17, 5466–5476.PubMedCrossRefGoogle Scholar
  24. 24.
    Venclovas, C., Timinskas, A., and Siksnys, V. (1994) Five-stranded beta-sheet sandwiched with two a-helices: a structural link between restriction endonucleases EcoRI and EcoRV. Proteins 20, 279–282.PubMedCrossRefGoogle Scholar
  25. 25.
    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
  26. 26.
    Wah, D. A., Bitinaite, J., Schildkraut, I., and Aggarwal, A. K. (1998) Structure of FokI has implications for DNA cleavage. Proc. Natl. Acad. Sci. USA 95, 10,564–10,569.PubMedCrossRefGoogle Scholar
  27. 27.
    Aggarwal, A. K. (1995) Structure and function of restriction endonucleases. Curr. Opin. Struct. Biol. 5, 11–19.PubMedCrossRefGoogle Scholar
  28. 28.
    Rosenberg, J. M. (1991) Structure and function of restriction endonucleases. Curr. Opin. Struct. Biol. 1, 104–113.CrossRefGoogle Scholar
  29. 29.
    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
  30. 30.
    Thielking, V., Alves, J., Fliess, A., Maass, G., and Pingoud, A. (1990) Accuracy of the EcoRI restriction endonuclease: Binding and cleavage studies with oligodeoxynucleotide substrates containing degenerate recognition sequences. Biochemistry 29, 4682–4691.PubMedCrossRefGoogle Scholar
  31. 31.
    Alves, J., Selent, U., and Wolfes, H. (1995) Accuracy of the EcoRV restriction endonuclease: Binding and cleavage studies with oligodeoxynucleotide substrates containing degenerate recognition sequences. Biochemistry 34, 11,191–11,197.PubMedCrossRefGoogle Scholar
  32. 32.
    Jeltsch, A., Alves, J., Oelgeschlager, T., Wolfes, H., Maass, G. M., and Pingoud, A. (1993) Mutational analysis of the function of Gln115 in the EcoRI restriction endonuclease, a critical amino acid for recognition of the inner thymidine residue in the sequence-GAATTC-and for coupling specific DNA binding to catalysis. J. Mol. Biol. 229, 221–234.PubMedCrossRefGoogle Scholar
  33. 33.
    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
  34. 34.
    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
  35. 35.
    Stahl, F., Wende, W., Wenz, C., Jeltsch, A., and Pingoud, A. (1998) Intra-vs intersubunit communication in the homodimeric restriction enzyme EcoRV: Thr37 and Lys38 involved in indirect readout are only important for the catalytic activity of their own subunit. Biochemistry 37, 5682–5688.PubMedCrossRefGoogle Scholar
  36. 36.
    Smith, H. O. and Wilcox, K. W. (1970) A restriction enzyme from Haemophilus influenzae. I. Purification and general properties. J. Mol. Biol. 51, 379–391.PubMedCrossRefGoogle Scholar
  37. 37.
    Connolly, B. A., Eckstein, F., and Pingoud, A. (1984) The Stereochemical course of the restriction endonuclease EcoRI-catalyzed reaction. J. Biol. Chem. 259, 10,760–10,763.PubMedGoogle Scholar
  38. 38.
    Grasby, J. A. and Connolly, B. A. (1992) Stereochemical outcome of the hydrolysis reaction catalyzed by the EcoRV restriction endonuclease. Biochemistry 31, 7855–7861.PubMedCrossRefGoogle Scholar
  39. 39.
    Jeltsch, A., Alves, J., Maass, G., and Pingoud, A. (1992) On the catalytic mechanism of EcoRI and EcoRV. A detailed proposal based on biochemical results, structural data and molecular modelling. FEBS Lett. 304, 4–8.PubMedCrossRefGoogle Scholar
  40. 40.
    Thielking, V., Selent, U., Köhler, E., Wolfes, H., Pieper, U., Geiger, R., Urbanke, C., Winkler, F. K., and Pingoud, A. (1991) Site-directed mutagenesis studies with EcoRV restriction endonuclease to identify regions involved in recognition and catalysis. Biochemistry 30, 6416–6422.PubMedCrossRefGoogle Scholar
  41. 41.
    Winkler, F. K. (1992) Structure and function of restriction endonucleases. Curr. Opin. Struct. Biol. 2, 93–99.CrossRefGoogle Scholar
  42. 42.
    Jeltsch, A., Alves, J., Wolfes, H., Maass, G., and Pingoud, A. (1993) Substrate-assisted catalysis in the cleavage of DNA by the EcoRI and EcoRV restriction enzymes. Proc. Natl. Acad. Sci. USA 90, 8499–8503.PubMedCrossRefGoogle Scholar
  43. 43.
    Vipond, I. B., Baldwin, G. S., and Halford, S. E. (1995) Divalent metal ions at the active sites of the EcoRV and EcoRI restriction endonucleases. Biochemistry 34, 697–704.PubMedCrossRefGoogle Scholar
  44. 44.
    Kostrewa, D. and Winkler, F. K. (1995) Mg2+ binding to the active site of EcoRV endonuclease: a crystallographic study of complexes with substrate and product DNA at 2 Angstrom resolution. Biochemistry 34, 683–696.PubMedCrossRefGoogle Scholar
  45. 45.
    Riggs, A. D., Suzuki, H., and Bourgeois, S. (1970) Lac repressor-operator interaction. I. Equilibrium studies. J. Mol. Biol. 48, 67–83.PubMedCrossRefGoogle Scholar
  46. 46.
    Riggs, A. D., Bourgeois, S., and Cohn, M. (1970) The lac repressor-operator interaction. 3. Kinetic studies. J. Mol. Biol. 53, 401–417.PubMedCrossRefGoogle Scholar
  47. 47.
    Hinkle, D. C. and Chamberlin, M. J. (1972) Studies of the binding of Escherichia coli RNA polymerase to DNA. II. The kinetics of the binding reaction. J. Mol. Biol. 70, 187–195.PubMedCrossRefGoogle Scholar
  48. 48.
    Hinkle, D. C. and Chamberlin, M. J. (1972) Studies of the binding of Escherichia coli RNA polymerase to DNA. I. The role of sigma subunit in site selection. J. Mol. Biol. 70, 157–185.PubMedCrossRefGoogle Scholar
  49. 49.
    Wong, I. and Lohman, T. M. (1993) A double-filter method for nitrocellulose-filter binding: Application to protein-nucleic acid interactions. Proc. Natl. Acad. Sci. USA 90, 5428–5432.PubMedCrossRefGoogle Scholar
  50. 50.
    Fried, M. and Crothers, D. M. (1981) Equilibria and kinetics of lac repressor-operator interactions by polyacrylamide gel electrophoresis. Nucleic Acids Res. 9, 6505–6525.PubMedCrossRefGoogle Scholar
  51. 51.
    Garner, M. M. and Revzin, A. (1981) A gel electrophoresis method for quantifying the binding of proteins to specific DNA regions: application to components of the Escherichia coli lactose operon regulatory system. Nucleic Acids Res. 9, 3047–3060.PubMedCrossRefGoogle Scholar
  52. 52.
    Stöver, T., Köhler, E., Fagin, U., Wende, W., Wolfes, H., and Pingoud, A. (1993) Determination of the DNA bend angle induced by the restriction endonuclease EcoRV in the presence of Mg2+. J. Biol. Chem. 268, 8645–8650.PubMedGoogle Scholar
  53. 53.
    Withers, B. E. and Dunbar, J. C. (1993) The endonuclease isoschizomers, SmaI and XmaI bend DNA in opposite orientations. Nucleic Acids Res. 21, 2571–2577.PubMedCrossRefGoogle Scholar
  54. 54.
    Vipond, I. B. and Halford, S. E. (1995) Specific DNA recognition by EcoRV restriction endonuclease induced by calcium ions. Biochemistry 34, 1113–1119.PubMedCrossRefGoogle Scholar
  55. 55.
    Cal, S. and Connolly, B. A. (1996) The EcoRV modification methylase causes considerable bending of DNA upon binding to its recognition sequence GATATC. J. Biol. Chem. 271, 1008–1015.PubMedCrossRefGoogle Scholar
  56. 56.
    Engler, L. E., Welch, K. K., and Jen-Jacobson, L. (1997) Specific binding by EcoRV endonuclease to its DNA recognition site GATATC. J. Mol. Biol. 269, 82–101.PubMedCrossRefGoogle Scholar
  57. 57.
    Zhang, X., Duggan, L. J., and Gottlieb, P. A. (1993) Loading dyes used in gel electrophoresis alter the apparent thermodynamic equilibrium of the lac repressor-operator complex. Anal. Biochem 214, 580–582.PubMedCrossRefGoogle Scholar
  58. 58.
    Hassanain, H. H., Dai, W., and Gupta, S. L. (1993) Enhanced gel mobility shift assay for DNA-binding factors. Anal. Biochem 213, 162–167.PubMedCrossRefGoogle Scholar
  59. 59.
    Erskine, S. G. and Halford, S. E. (1998) Reactions of the EcoRV restriction endonuclease with fluorescent oligodeoxynucleotides: identical equilibrium constants for binding to specific and non-specific DNA. J. Mol. Biol. 275, 759–772.PubMedCrossRefGoogle Scholar
  60. 60.
    Taylor, J. D., Badcoe, I. G., Clarke, A. R., and Halford, S. E. (1991) EcoRV restriction endonuclease binds all DNA sequences with equal affinity. Biochemistry 30, 8743–8753.PubMedCrossRefGoogle Scholar
  61. 61.
    Thielking, V., Selent, U., Köhler, E., Landgraf, Z., Wolfes, H., Alves, J., and Pingoud, A. (1992) Mg2+ confers DNA binding specificity to the EcoRV restriction endonuclease. Biochemistry 31, 3727–3732.PubMedCrossRefGoogle Scholar
  62. 62.
    Jeltsch, A., Maschke, H., Selent, U., Wenz, C., Köhler, E., Connolly, B. A., Thorogood, H., and Pingoud, A. (1995) DNA binding specificity of the EcoRV restriction endonuclease is increased by Mg2+ binding to a metal ion binding site distinct from the catalytic center of the enzyme. Biochemistry 34, 6239–6246.PubMedCrossRefGoogle Scholar
  63. 63.
    Szczelkun, M. D. and Connolly, B. A. (1995) Sequence-specific binding of DNA by the EcoRV restriction and modification enzymes with nucleic acid and cofactor analogues. Biochemistry 34, 10,724–10,733.PubMedCrossRefGoogle Scholar
  64. 64.
    Halford, S. E. and Goodall, A. J. (1988) Modes of DNA cleavage by the EcoRV restriction endonuclease. Biochemistry 27, 1771–1777.PubMedCrossRefGoogle Scholar
  65. 65.
    Brownlee, G. G. and Sanger, F. (1969) Chromatography of 32P-labeled oligonucleotides on thin layers of DEAE-cellulose. Eur. J. Biochem. 11, 395–3999.PubMedCrossRefGoogle Scholar
  66. 66.
    Fierke, C. A. and Hammes, G. G. (1995) Transient kinetic approaches to enzyme mechanisms. Methods Enzymol. 249, 3–37.PubMedCrossRefGoogle Scholar
  67. 67.
    Johnson, K. A. (1995) Rapid quench kinetic analysis of polymerases, adenosinetri-phosphatases, and enzyme intermediates. Methods Enzymol. 249, 38–61.PubMedCrossRefGoogle Scholar
  68. 68.
    Zebala, J., Choi, J., and Barany, F. (1992) Characterization of steady state, single-turnover, and binding kinetics of the TaqI restriction endonuclease. J. Biol. Chem. 267, 8097–8105.PubMedGoogle Scholar
  69. 69.
    Groll, D. H., Jeltsch, A., Selent, U., and Pingoud, A. (1997) Does the restriction endonuclease EcoRV employ a two-metal-ion mechanism for DNA cleavage? Biochemistry 36, 11,389–11,401.PubMedCrossRefGoogle Scholar
  70. 70.
    Erskine, S. G., Baldwin, G. S., and Halford, S. E. (1997) Rapid-reaction analysis of plasmid DNA cleavage by the EcoRV restriction endonuclease. Biochemistry 36, 7567–7576.PubMedCrossRefGoogle Scholar
  71. 71.
    Jeltsch, A., Fritz, A., Alves, J., Wolfes, H., and Pingoud, A. (1993) A fast and accurate enzyme-linked immunosorbent assay for the determination of the DNA cleavage activity of restriction endonucleases. Anal. Biochem. 213, 234–240.PubMedCrossRefGoogle Scholar
  72. 72.
    Wenz, C., Hahn, M., and Pingoud, A. (1998) Engineering of variants of the restriction endonuclease EcoRV that depend in their cleavage activity on the flexibility of sequences flanking the recognition site. Biochemistry 37, 2234–2242.PubMedCrossRefGoogle Scholar
  73. 73.
    Glasner, W., Merkl, R., Schmidt, S., Cech, D., and Fritz, H. J. (1992) Fast quantitative assay of sequence-specific endonuclease activity based on DNA sequencer technology. Biol. Chem. Hoppe Seyler 373, 1223–1225.PubMedCrossRefGoogle Scholar
  74. 74.
    van Holde, K. E., Johnson, W. C., and Ho, P. S. (1998) Principles of Physical Biochemistry. Prentice Hall, Upper Saddle River, NJ.Google Scholar

Copyright information

© Humana Press Inc. 2001

Authors and Affiliations

  1. 1.Fachbereich Biologie, Institut für BiochemieJustus-Liebig-UniversitätGiessenGermany

Personalised recommendations

Cite protocol

Buy options