Skip to main content
PMC home page
The EMBO Journal logoLink to The EMBO Journal
. 1996 Apr 15;15(8):2003–2009.

Restriction by EcoKI is enhanced by co-operative interactions between target sequences and is dependent on DEAD box motifs.

J L Webb 1, G King 1, D Ternent 1, A J Titheradge 1, N E Murray 1
PMCID: PMC450119  PMID: 8617247

Abstract

One subunit of both type I and type III restriction and modification enzymes contains motifs characteristic of DEAD box proteins, which implies that these enzymes may be DNA helicases. This subunit is essential for restriction, but not modification. The current model for restriction by both types of enzyme postulates that DNA cutting is stimulated when two enzyme complexes bound to neighbouring target sequences meet as the consequence of ATP-dependent DNA translocation. For type I enzymes, this model is supported by in vitro experiments, but the predicted co-operative interactions between targets have not been detected by assays that monitor restriction in vivo. The experiments reported here clearly establish the required synergistic effect but, in contrast to earlier experiments, they use Escherichia coli K-12 strains deficient in the restriction alleviation function associated with the Rac prophage. In bacteria with elevated levels of EcoKI the co-operative interactions are obscured, consistent with co-operation between free enzyme and that bound at target sites. We have made changes in three of the motifs characteristic of DEAD box proteins, including motif III, which in RecG is implicated in the migration of Holliday junctions. Conservative changes in each of the three motifs impair restriction.

Full text

PDF

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Appleyard R K. Segregation of New Lysogenic Types during Growth of a Doubly Lysogenic Strain Derived from Escherichia Coli K12. Genetics. 1954 Jul;39(4):440–452. doi: 10.1093/genetics/39.4.440. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bickle T. A., Brack C., Yuan R. ATP-induced conformational changes in the restriction endonuclease from Escherichia coli K-12. Proc Natl Acad Sci U S A. 1978 Jul;75(7):3099–3103. doi: 10.1073/pnas.75.7.3099. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Brammar W. J., Murray N. E., Winton S. Restriction of lambda trp bacteriophages by Escherichia coli K. J Mol Biol. 1974 Dec 25;90(4):633–647. doi: 10.1016/0022-2836(74)90529-4. [DOI] [PubMed] [Google Scholar]
  4. Cooper L. P., Dryden D. T. The domains of a type I DNA methyltransferase. Interactions and role in recognition of DNA methylation. J Mol Biol. 1994 Mar 4;236(4):1011–1021. doi: 10.1016/0022-2836(94)90008-6. [DOI] [PubMed] [Google Scholar]
  5. Dartois V., De Backer O., Colson C. Sequence of the Salmonella typhimurium StyLT1 restriction-modification genes: homologies with EcoP1 and EcoP15 type-III R-M systems and presence of helicase domains. Gene. 1993 May 15;127(1):105–110. doi: 10.1016/0378-1119(93)90623-b. [DOI] [PubMed] [Google Scholar]
  6. Fuller-Pace F. V., Murray N. E. Two DNA recognition domains of the specificity polypeptides of a family of type I restriction enzymes. Proc Natl Acad Sci U S A. 1986 Dec;83(24):9368–9372. doi: 10.1073/pnas.83.24.9368. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Fuller-Pace F. V. RNA helicases: modulators of RNA structure. Trends Cell Biol. 1994 Aug;4(8):271–274. doi: 10.1016/0962-8924(94)90210-0. [DOI] [PubMed] [Google Scholar]
  8. Gough J. A., Murray N. E. Sequence diversity among related genes for recognition of specific targets in DNA molecules. J Mol Biol. 1983 May 5;166(1):1–19. doi: 10.1016/s0022-2836(83)80047-3. [DOI] [PubMed] [Google Scholar]
  9. Heitman J. On the origins, structures and functions of restriction-modification enzymes. Genet Eng (N Y) 1993;15:57–108. doi: 10.1007/978-1-4899-1666-2_4. [DOI] [PubMed] [Google Scholar]
  10. Kannan P., Cowan G. M., Daniel A. S., Gann A. A., Murray N. E. Conservation of organization in the specificity polypeptides of two families of type I restriction enzymes. J Mol Biol. 1989 Oct 5;209(3):335–344. doi: 10.1016/0022-2836(89)90001-6. [DOI] [PubMed] [Google Scholar]
  11. King G., Murray N. E. Restriction alleviation and modification enhancement by the Rac prophage of Escherichia coli K-12. Mol Microbiol. 1995 May;16(4):769–777. doi: 10.1111/j.1365-2958.1995.tb02438.x. [DOI] [PubMed] [Google Scholar]
  12. Loenen W. A., Daniel A. S., Braymer H. D., Murray N. E. Organization and sequence of the hsd genes of Escherichia coli K-12. J Mol Biol. 1987 Nov 20;198(2):159–170. doi: 10.1016/0022-2836(87)90303-2. [DOI] [PubMed] [Google Scholar]
  13. Loenen W. A., Murray N. E. Modification enhancement by the restriction alleviation protein (Ral) of bacteriophage lambda. J Mol Biol. 1986 Jul 5;190(1):11–22. doi: 10.1016/0022-2836(86)90071-9. [DOI] [PubMed] [Google Scholar]
  14. Low B. Rapid mapping of conditional and auxotrophic mutations in Escherichia coli K-12. J Bacteriol. 1973 Feb;113(2):798–812. doi: 10.1128/jb.113.2.798-812.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Meisel A., Bickle T. A., Krüger D. H., Schroeder C. Type III restriction enzymes need two inversely oriented recognition sites for DNA cleavage. Nature. 1992 Jan 30;355(6359):467–469. doi: 10.1038/355467a0. [DOI] [PubMed] [Google Scholar]
  16. Meisel A., Mackeldanz P., Bickle T. A., Krüger D. H., Schroeder C. Type III restriction endonucleases translocate DNA in a reaction driven by recognition site-specific ATP hydrolysis. EMBO J. 1995 Jun 15;14(12):2958–2966. doi: 10.1002/j.1460-2075.1995.tb07296.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Murray N. E., Batten P. L., Murray K. Restriction of bacteriophage lambda by Escherichia coli K. J Mol Biol. 1973 Dec 15;81(3):395–407. doi: 10.1016/0022-2836(73)90149-6. [DOI] [PubMed] [Google Scholar]
  18. Murray N. E., Daniel A. S., Cowan G. M., Sharp P. M. Conservation of motifs within the unusually variable polypeptide sequences of type I restriction and modification enzymes. Mol Microbiol. 1993 Jul;9(1):133–143. doi: 10.1111/j.1365-2958.1993.tb01675.x. [DOI] [PubMed] [Google Scholar]
  19. Murray N. E., De Ritis P. M., Foster L. A. DNA targets for the Escherichia coli K restriction system analysed genetically in recombinants between phages phi80 and lambda. Mol Gen Genet. 1973 Feb 2;120(3):261–281. doi: 10.1007/BF00267157. [DOI] [PubMed] [Google Scholar]
  20. Pause A., Sonenberg N. Mutational analysis of a DEAD box RNA helicase: the mammalian translation initiation factor eIF-4A. EMBO J. 1992 Jul;11(7):2643–2654. doi: 10.1002/j.1460-2075.1992.tb05330.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Price C., Lingner J., Bickle T. A., Firman K., Glover S. W. Basis for changes in DNA recognition by the EcoR124 and EcoR124/3 type I DNA restriction and modification enzymes. J Mol Biol. 1989 Jan 5;205(1):115–125. doi: 10.1016/0022-2836(89)90369-0. [DOI] [PubMed] [Google Scholar]
  22. RICKENBERG H. V., LESTER G. The preferential synthesis of beta-galactosidase in Escherichia coli. J Gen Microbiol. 1955 Oct;13(2):279–284. doi: 10.1099/00221287-13-2-279. [DOI] [PubMed] [Google Scholar]
  23. Saha S., Rao D. N. ATP hydrolysis is required for DNA cleavage by EcoPI restriction enzyme. J Mol Biol. 1995 Apr 7;247(4):559–567. doi: 10.1016/s0022-2836(05)80137-8. [DOI] [PubMed] [Google Scholar]
  24. Sain B., Murray N. E. The hsd (host specificity) genes of E. coli K 12. Mol Gen Genet. 1980;180(1):35–46. doi: 10.1007/BF00267350. [DOI] [PubMed] [Google Scholar]
  25. Schmid S. R., Linder P. D-E-A-D protein family of putative RNA helicases. Mol Microbiol. 1992 Feb;6(3):283–291. doi: 10.1111/j.1365-2958.1992.tb01470.x. [DOI] [PubMed] [Google Scholar]
  26. Sharp P. M., Kelleher J. E., Daniel A. S., Cowan G. M., Murray N. E. Roles of selection and recombination in the evolution of type I restriction-modification systems in enterobacteria. Proc Natl Acad Sci U S A. 1992 Oct 15;89(20):9836–9840. doi: 10.1073/pnas.89.20.9836. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Sharples G. J., Whitby M. C., Ryder L., Lloyd R. G. A mutation in helicase motif III of E. coli RecG protein abolishes branch migration of Holliday junctions. Nucleic Acids Res. 1994 Feb 11;22(3):308–313. doi: 10.1093/nar/22.3.308. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Studier F. W., Bandyopadhyay P. K. Model for how type I restriction enzymes select cleavage sites in DNA. Proc Natl Acad Sci U S A. 1988 Jul;85(13):4677–4681. doi: 10.1073/pnas.85.13.4677. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Studier F. W., Moffatt B. A. Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J Mol Biol. 1986 May 5;189(1):113–130. doi: 10.1016/0022-2836(86)90385-2. [DOI] [PubMed] [Google Scholar]
  30. Sung P., Higgins D., Prakash L., Prakash S. Mutation of lysine-48 to arginine in the yeast RAD3 protein abolishes its ATPase and DNA helicase activities but not the ability to bind ATP. EMBO J. 1988 Oct;7(10):3263–3269. doi: 10.1002/j.1460-2075.1988.tb03193.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Umezu K., Nakayama K., Nakayama H. Escherichia coli RecQ protein is a DNA helicase. Proc Natl Acad Sci U S A. 1990 Jul;87(14):5363–5367. doi: 10.1073/pnas.87.14.5363. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Weiserova M., Janscak P., Benada O., Hubácek J., Zinkevich V. E., Glover S. W., Firman K. Cloning, production and characterisation of wild type and mutant forms of the R.EcoK endonucleases. Nucleic Acids Res. 1993 Feb 11;21(3):373–379. doi: 10.1093/nar/21.3.373. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Whittaker P. A., Campbell A. J., Southern E. M., Murray N. E. Enhanced recovery and restriction mapping of DNA fragments cloned in a new lambda vector. Nucleic Acids Res. 1988 Jul 25;16(14B):6725–6736. doi: 10.1093/nar/16.14.6725. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Willcock D. F., Dryden D. T., Murray N. E. A mutational analysis of the two motifs common to adenine methyltransferases. EMBO J. 1994 Aug 15;13(16):3902–3908. doi: 10.1002/j.1460-2075.1994.tb06701.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Wilson G. G., Murray N. E. Restriction and modification systems. Annu Rev Genet. 1991;25:585–627. doi: 10.1146/annurev.ge.25.120191.003101. [DOI] [PubMed] [Google Scholar]
  36. Yuan R., Hamilton D. L., Burckhardt J. DNA translocation by the restriction enzyme from E. coli K. Cell. 1980 May;20(1):237–244. doi: 10.1016/0092-8674(80)90251-2. [DOI] [PubMed] [Google Scholar]

Articles from The EMBO Journal are provided here courtesy of Nature Publishing Group

RESOURCES