Skip to main content
NCBI home page
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1986 Apr 25;14(8):3463–3474. doi: 10.1093/nar/14.8.3463

Role of thymidine residues in DNA recognition by the EcoRI and EcoRV restriction endonucleases.

A Fliess, H Wolfes, A Rosenthal, K Schwellnus, H Blöcker, R Frank, A Pingoud
PMCID: PMC339785  PMID: 3010238

Abstract

We have synthesized a series of oligonucleotides containing the EcoRI (GAATTC) or EcoRV (GATATC) recognition site within which or adjacent to which thymidine was substituted by uridine or derivatives of uridine. The effects of these substitutions on the rate of the EcoRI and EcoRV catalyzed cleavage reaction were investigated. Our results show that most of the substitutions within the site are quite well tolerated by EcoRI, not, however, by EcoRV. We conclude that the thymin residues most likely are not directly involved in the recognition process of the EcoRI reaction. In contrast, they are major points of contact, between substrate and enzyme in the EcoRV reaction. The effects of substitutions in the position adjacent to the recognition site is also markedly different for EcoRI and EcoRV. Here, EcoRI seems to be considerably more selective than EcoRV.

Full text

PDF
3463

Selected References

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

  1. Alves J., Pingoud A., Haupt W., Langowski J., Peters F., Maass G., Wolff C. The influence of sequences adjacent to the recognition site on the cleavage of oligodeoxynucleotides by the EcoRI endonuclease. Eur J Biochem. 1984 Apr 2;140(1):83–92. doi: 10.1111/j.1432-1033.1984.tb08069.x. [DOI] [PubMed] [Google Scholar]
  2. Armstrong K. A., Bauer W. R. Site-dependent cleavage of pBR322 DNA by restriction endonuclease HinfI. Nucleic Acids Res. 1983 Jun 25;11(12):4109–4126. doi: 10.1093/nar/11.12.4109. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Armstrong K., Bauer W. R. Preferential site-dependent cleavage by restriction endonuclease PstI. Nucleic Acids Res. 1982 Feb 11;10(3):993–1007. doi: 10.1093/nar/10.3.993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Berkner K. L., Folk W. R. EcoRI cleavage and methylation of DNAs containing modified pyrimidines in the recogintion sequence. J Biol Chem. 1977 May 25;252(10):3185–3193. [PubMed] [Google Scholar]
  5. Berkner K. L., Folk W. R. The effects of substituted pyrimidines in DNAs on cleavage by sequence-specific endonucleases. J Biol Chem. 1979 Apr 10;254(7):2551–2560. [PubMed] [Google Scholar]
  6. D'Arcy A., Brown R. S., Zabeau M., van Resandt R. W., Winkler F. K. Purification and crystallization of the EcoRV restriction endonuclease. J Biol Chem. 1985 Feb 25;260(4):1987–1990. [PubMed] [Google Scholar]
  7. Delort A. M., Neumann J. M., Molko D., Hervé M., Téoule R., Tran Dinh S. Influence of uracil defect on DNA structure: 1H NMR investigation at 500 MHz. Nucleic Acids Res. 1985 May 10;13(9):3343–3355. doi: 10.1093/nar/13.9.3343. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Frank R., Heikens W., Heisterberg-Moutsis G., Blöcker H. A new general approach for the simultaneous chemical synthesis of large numbers of oligonucleotides: segmental solid supports. Nucleic Acids Res. 1983 Jul 11;11(13):4365–4377. doi: 10.1093/nar/11.13.4365. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Goppelt M., Pingoud A., Maass G., Mayer H., Köster H., Frank R. The interaction of the EcoRI restriction endonuclease with its substrate. A physico-chemical study employing natural and synthetic oligonucleotides and polynucleotides. Eur J Biochem. 1980 Feb;104(1):101–107. doi: 10.1111/j.1432-1033.1980.tb04405.x. [DOI] [PubMed] [Google Scholar]
  10. Grotjahn L., Frank R., Blöcker H. Ultrafast sequencing of oligodeoxyribonucleotides by FAB-mass spectrometry. Nucleic Acids Res. 1982 Aug 11;10(15):4671–4678. doi: 10.1093/nar/10.15.4671. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Kaplan D. A., Nierlich D. P. Cleavage of Nonglucosylated Bacteriophage T4 deoxyribonucleic acid by Restriction Endonuclease Eco RI. J Biol Chem. 1975 Mar 25;250(6):2395–2397. [PubMed] [Google Scholar]
  12. Marchionni M. A., Roufa R. J. Digestion of 5-bromodeoxyuridine-substituted lambda-DNA by restriction endonucleases. J Biol Chem. 1978 Dec 25;253(24):9075–9081. [PubMed] [Google Scholar]
  13. Modrich P., Rubin R. A. Role of the 2-amino group of deoxyguanosine in sequence recognition by EcoRI restriction and modification enzymes. J Biol Chem. 1977 Oct 25;252(20):7273–7278. [PubMed] [Google Scholar]
  14. Ohtsuka E., Ishino Y., Ibaraki K., Ikehara M. Recognition by restriction endonuclease EcoRI of deoxyoctanucleotides containing modified sugar moieties. Eur J Biochem. 1984 Mar 15;139(3):447–450. doi: 10.1111/j.1432-1033.1984.tb08025.x. [DOI] [PubMed] [Google Scholar]
  15. Petruska J., Horn D. Sequence-specific responses of restriction endonucleases to bromodeoxyuridine substitution in mammalian DNA. Nucleic Acids Res. 1983 Apr 25;11(8):2495–2510. doi: 10.1093/nar/11.8.2495. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Remin M., Darzynkiewicz E., Ekiel I., Shugar D. Conformation in aqueous medium of the neutral, protonated and anionic forms of 9-beta-D-arabinofuranosyladenine. Biochim Biophys Acta. 1976 Jul 16;435(4):405–416. doi: 10.1016/0005-2787(76)90205-7. [DOI] [PubMed] [Google Scholar]
  17. Rosenthal A., Schwertner S., Hahn V., Hunger H. D. Solid-phase methods for sequencing of nucleic acids I. Simultaneous sequencing of different oligodeoxyribonucleotides using a new, mechanically stable anion-exchange paper. Nucleic Acids Res. 1985 Feb 25;13(4):1173–1184. doi: 10.1093/nar/13.4.1173. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Wolfes H., Fliess A., Pingoud A. A comparison of the structural requirements for DNA cleavage by the isoschizomers HaeIII, BspRI and BsuRI. Eur J Biochem. 1985 Jul 1;150(1):105–110. doi: 10.1111/j.1432-1033.1985.tb08994.x. [DOI] [PubMed] [Google Scholar]
  19. Yolov A. A., Vinogradova M. N., Gromova E. S., Rosenthal A., Cech D., Veiko V. P., Metelev V. G., Kosykh V. G., Buryanov Y. I., Bayev A. A. Interaction of EcoRII restriction and modification enzymes with synthetic DNA fragments. VI. The binding and cleavage of substrates containing nucleotide analogs. Nucleic Acids Res. 1985 Dec 20;13(24):8983–8998. doi: 10.1093/nar/13.24.8983. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Nucleic Acids Research are provided here courtesy of Oxford University Press

RESOURCES