Advertisement

Mechanism of Action of Restriction Endonuclease EcoRV

  • S. E. Halford
  • J. D. Taylor
  • C. L. M. Vermote
  • I. B. Vipond
Part of the Nucleic Acids and Molecular Biology book series (NUCLEIC, volume 7)

Abstract

Type II restriction/modification (R/M) systems, such as EcoRV, consist of two enzymes that act at the same DNA sequence; a modification methyltransferase and a restriction endonuclease (Bennett and Halford 1989; Wilson and Murray 1991). For EcoRV, the recognition sequence is GATATC (Kholmina et al. 1980). The EcoRV modification enzyme methylates the first adenine within this sequence (Nwosu et al. 1988) while, in the presence of Mg2+ ions, the EcoRV restriction enzyme cleaves DNA specifically at this site (Schildkraut et al. 1984). The endonuclease cuts both strands at the centre of the sequence, to leave blunt-ended DNA fragments. However, prior methylation of the recognition site blocks restriction activity. The basic tenet of R/M systems is that, in vivo, the restriction enzyme cuts only DNA molecules that have not been exposed previously to the methyltransferase. Hence, they enable the cell to degrade foreign DNA entering the cell without destroying host DNA. E. coli strains carrying the EcoRV system show this phenotype (Bougueleret et al. 1984).

Keywords

Recognition Site Recognition Sequence Specific Complex Base Analogue Scissile Bond 
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.

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. Aiken CR, Fisher EW, Gumport RI (1991a) The specific binding, bending and unwinding of DNA by RsrI endonuclease, an isoschizomer of EcoRI endonuclease. J Biol Chem 266: 19063–19069PubMedGoogle Scholar
  2. Aiken CR, McLaughlin LW, Gumport RI (1991b) The highly homologous isoschizomers Rsrl endonuclease and EcoRl endonuclease do not recognize their target sequences identically. J Biol Chem 266: 19070–19078PubMedGoogle Scholar
  3. Alves J, Riiter T, Geiger R, Fliess A, Maass G, Pingoud A (1989) 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: 2678–2684PubMedCrossRefGoogle Scholar
  4. Badcoe IG (1992) A fast algorithm for counting the arrangements for packing identical items on a one dimensional grid with applications in DNA/protein and similar interactions. Comput Appl Biosci 8: 323–330PubMedGoogle Scholar
  5. Bennett SP, Halford SE (1989) Recognition of DNA by type II restriction enzymes. Curr Top Cell Regul 30: 57–104PubMedGoogle Scholar
  6. Bougueleret L, Schwarzstein M, Tsugita A, Zabeau M (1984) Characterization of the genes coding for the EcoKV restriction and modification system of Escherichia coli. Nucleic Acids Res 12: 3659–3676PubMedCrossRefGoogle Scholar
  7. Bougueleret L, Tenchini ML, Botterman J, Zabeau M (1985) Overproduction of the EcoKV endonuclease and methylase. Nucleic Acids Res 13: 3823–3839PubMedCrossRefGoogle Scholar
  8. Brennan CA, Van Cleve MD, Gumport RI (1986) The effects of base analogue substitutions on the cleavage by the EcoRI restriction endonuclease of octadeoxyribonucleotides containing modified EcoRI recognition sequences. J Biol Chem 261: 7270–7278PubMedGoogle Scholar
  9. Connolly BA, Eckstein F, Pingoud A (1984) The stereochemical course of the restriction enzyme EcoRI-catalysed reaction. J Biol Chem 259: 10760–10763PubMedGoogle Scholar
  10. D’Arcy A, Brown RS, Zabeau M, Van Resandt RW, Winkler FK (1985) Purification and characterization of the EcoKV restriction endonuclease. J Biol Chem 252: 1987–1990Google Scholar
  11. Diekmann S, McLaughlin LW (1988) DNA curvature in native and modified EcoRI recognition sites and possible influence upon the endonuclease cleavage reaction. J Mol Biol 202: 823–834PubMedCrossRefGoogle Scholar
  12. Freemont PS, Lane AN, Sanderson MR (1991) Structural aspects of protein-DNA re¬cognition. Biochem J 278: 1–23PubMedGoogle Scholar
  13. Grasby JA, Connolly BA (1992) The stereochemical outcome of the hydrolysis reaction catalysed by the EcoKV restriction endonuclease. Biochemistry 31: 7855–7861PubMedCrossRefGoogle Scholar
  14. Halford SE, Goodall AJ (1988) Modes of DNA cleavage by the EcoKV restriction endonuclease. Biochemistry 27: 1771–1777PubMedCrossRefGoogle Scholar
  15. Halford SE, Lovelady BM, McCallum SA (1986) Relaxed specificity of the EcoRV restriction endonuclease. Gene 41: 173–181PubMedCrossRefGoogle Scholar
  16. Jeltsch A, Alves J, Maass G, Pingoud A (1992) On the catalytic mechanism of EcoRI and EcoKV: a detailed proposal based on biochemical results, structural data and molecular modelling. FEBS Lett 304: 4–8PubMedCrossRefGoogle Scholar
  17. Jencks WP (1975) Binding energy, specificity, and enzyme catalysis; the Circe effect. Adv Enzymol Relat Areas Mol Biol 43: 219–410PubMedGoogle Scholar
  18. Kholmina GV, Regentish BA, Skoblov YS, Mironov AA, Yankovsky NK, Kozlov YI, Glatmen LI, Moroz AF, Debabov VG (1980) Isolation and characterization of a new site-specific endonuclease EcoRV. Dokl Akad Nauk 253: 495–497Google Scholar
  19. King K, Benkovic SJ, Mödlich P (1989) Glu-111 is required for activation of the DNA cleavage center of EcoRI endonuclease. J Biol Chem 264: 11807–11815PubMedGoogle Scholar
  20. Lauster R, Kriebardis A, Guschlbauer W (1987) The GATATC-modification enzyme EcoRV is closely related to the GATC-recognizing methyltransferases Dpnll and dam from E. coli and phage T4. FEBS Lett 220: 167–176CrossRefGoogle Scholar
  21. Lehman IR (1974) DNA ligase: structure, mechanism and function. Science 186: 790–797PubMedCrossRefGoogle Scholar
  22. Lesser DR, Kurpiewski MR, Jen-Jacobson L (1990) The energetic basis of specificity in the EcoRl endonuclease-DNA interaction. Science 250: 776–786PubMedCrossRefGoogle Scholar
  23. Luke PA, McCallum SA, Haiford SE (1987) The EcoRV restriction endonuclease. In: Chirikjian JG (ed) Gene amplification and analysis, vol 5. Restriction endonucleases and methylases. Elsevier, Amsterdam, pp 183–205Google Scholar
  24. McGhee JD, von Hippel PH (1974) Theoretical aspects of DNA-protfcin interactions: co-operative and non-co-operative binding of large ligands to a one-dimensional homogeneous lattice. J Mol Biol 86: 469–489PubMedCrossRefGoogle Scholar
  25. Newman PC, Wilhams DM, Cosstick R, Seela F, Connolly BA (1990a) Interaction of the EcoRV restriction endonuclease with the deoxyadenosine and thymidine bases in its recognition hexamer d(GATATC). Biochemistry 29: 9902–9910PubMedCrossRefGoogle Scholar
  26. Newman PC, Nwosu VU, Williams DM, Cosstick R, Seela F, Connolly BA (1990b) Incorporation of a complete set of deoxyadenosine and thymidine analogues suitable for the study of protein nucleic acid interactions into oligodeoxynucleotides. Biochemistry 29: 9891–9901PubMedCrossRefGoogle Scholar
  27. Nwosu VU, Connolly BA, Halford SE, Garnett J (1988) The cloning, purification and characterization of the EcoRV modification methylase. Nucleic Acids Res 16: 3705–3720PubMedCrossRefGoogle Scholar
  28. Record MT Jr, deHaseth PL, Lohman TM (1977) Interpretation of monovalent and divalent cation effects on the lac repressor-operator interaction. Biochemistry 16: 4791–4796PubMedCrossRefGoogle Scholar
  29. Rosenberg JM (1991) Structure and function of restriction endonucleases. Curr Opinion Struct Biol 1: 104–113CrossRefGoogle Scholar
  30. Schildkraut I, Banner CD, Rhodes CS, Parekh S (1984) The cleavage site for restriction endonuclease EcoRV is 5′-GAT/ATC-3\ Gene 27: 327–329PubMedCrossRefGoogle Scholar
  31. Selent U, Rüter T, Köhler E, Liedtke M, Thielking V, Alves J, Oelgeschläger T, Wolfes H, Peters F, Pingoud A (1992) A site-directed mutagenesis study to identify amino acid residues involved in the catalytic function of the restriction endonuclease EcoRV. Biochemistry 31: 4808–4815PubMedCrossRefGoogle Scholar
  32. Suck D (1992) Nuclease structure and catalytic function. Curr Opinion Struct Biol 2: 84–92CrossRefGoogle Scholar
  33. Tao T, Bourne JC, Blumenthal RM (1991) A family of regulatory genes associated with type II restriction-modification systems. J Bacteriol 173: 1367–1375PubMedGoogle Scholar
  34. Taylor JD (1991) Sequence specificity of the EcoRV restriction endonuclease. PhD Thesis, University of BristolGoogle Scholar
  35. Taylor JD, Halford SE (1989) Discrimination between DNA sequences by the EcoRV restriction endonuclease. Biochemistry 28: 6198–6207PubMedCrossRefGoogle Scholar
  36. Taylor JD, Halford SE (1992) The activity of the EcoRV restriction endonuclease is influenced by DNA sequences both inside and outside the DNA-protein complex. Biochemistry 31: 90–97PubMedCrossRefGoogle Scholar
  37. Taylor JD, Goodall AJ, Vermote CL, Halford SE (1990) Fidelity of DNA recognition by the EcoRV restriction/modification system in vivo. Biochemistry 29: 10727–10733PubMedCrossRefGoogle Scholar
  38. Taylor JD, Badcoe IG, Clarke AR, Halford SE (1991) EcoRV restriction endonuclease binds all DNA sequences with equal affinity. Biochemistry 30: 8743–8753PubMedCrossRefGoogle Scholar
  39. Terry BJ, Jack WE, Modrich P (1987) Thermodynamic parameters governing interaction of EcoRI endonuclease with specific and nonspecific DNA sequences. In: Chirikjian JG (ed) Gene amplification and analysis, vol 5. Restriction endonucleases and methylases. Elsevier, Amsterdam, pp 103–118Google Scholar
  40. Thielking V, Selent U, Köhler E, Wolfes H, Pieper U, Geiger R, Urbanke C, Winkler FK, Pingoud A (1991) Site-directed mutagenesis studies with EcoRV restriction endonuclease to identify regions involved in recognition and catalysis. Biochemistry 30: 6416–6422PubMedCrossRefGoogle Scholar
  41. Thielking V, Selent U, Kohler E, Landgraf A, Wolfes H, Alves J, Pingoud A (1992) Mg2+ confers binding specificity to the EcoRV restriction endonuclease. Biochemistry 31: 3727–3732PubMedCrossRefGoogle Scholar
  42. Travers AA (1991) DNA bending and kinking - sequence dependence and function. Curr Opinion Struct Biol 1: 114–122CrossRefGoogle Scholar
  43. Vermote CLM, Halford SE (1992) EcoRV restriction endonuclease: communication between catalytic metal ions and DNA recognition. Biochemistry 31: 6082–6089PubMedCrossRefGoogle Scholar
  44. Vermote CLM, Vipond IB, Halford SE (1992) EcoRV restriction endonuclease: communication between DNA recognition and catalysis. Biochemistry 31: 6089–6097PubMedCrossRefGoogle Scholar
  45. von Hippel PH, Berg OG (1986) On the specificity of DNA-protein interactions. Proc Natl Acad Sci USA 83: 1608–1612CrossRefGoogle Scholar
  46. von Hippel PH, Berg OG (1989) Facilitated target location in biological systems. J Biol Chem 264: 675–678Google Scholar
  47. Wilson GG, Murray NE (1991) Restriction and modification systems. Annu Rev Genet 25: 585–627PubMedCrossRefGoogle Scholar
  48. Winkler FK (1992) Structure and function of restriction endonucleases. Curr Opinion Struct Biol 2: 93–99CrossRefGoogle Scholar
  49. Winkler FK, D’Arcy A, Blocker H, Frank R, van Boom JH (1991) Crystallization of complexes of EcoRV endonuclease with cognate and non-cognate DNA fragments. J Mol Biol 217: 235–238PubMedCrossRefGoogle Scholar
  50. Zebala J A, Choi J, Barany F (1992a) Characterization of steady-state, single-turnover and binding kinetics of the Taql restriction endonuclease. J Biol Chem 267: 8097–8105PubMedGoogle Scholar
  51. Zebala JA, Choi J, Trainor G, Barany F (1992b) DNA recognition of base analogues and chemically modified substrates by the Taql restriction endonuclease. J Biol Chem 267: 8106–8116PubMedGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 1993

Authors and Affiliations

  • S. E. Halford
    • 1
  • J. D. Taylor
    • 2
  • C. L. M. Vermote
    • 1
  • I. B. Vipond
    • 1
  1. 1.Department of Biochemistry, Centre for Molecular RecognitionUniversity of BristolBristolUK
  2. 2.Pfizer Central ResearchSandwich, KentUK

Personalised recommendations