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. 1993 Aug 15;90(16):7548–7552. doi: 10.1073/pnas.90.16.7548

Facilitated distortion of the DNA site enhances EcoRI endonuclease-DNA recognition.

D R Lesser 1, M R Kurpiewski 1, T Waters 1, B A Connolly 1, L Jen-Jacobson 1
PMCID: PMC47179  PMID: 8356054

Abstract

We have measured the binding of EcoRI endonuclease to a complete set of purine-base analogue sites, each of which deletes one functional group that forms a hydrogen bond with the endonuclease in the canonical GAATTC complex. For five of six functional group deletions, the observed penalty in binding free energy is +1.3 to +1.7 kcal/mol. For two of these cases (replacement of adenine N7 with carbon) a single protein-base hydrogen bond is removed without deleting an interstrand Watson-Crick hydrogen bond or causing structural "adaptation" in the complex. This observation establishes that the incremental energetic contribution of one protein-base hydrogen bond is about -1.5 kcal/mol. By contrast, deletion of the N6-amino group of the inner adenine in the site improves binding by -1.0 kcal/mol because the penalty for deleting a protein-base hydrogen bond is outweighed by facilitation of the required DNA distortion ("kinking") in the complex. This result provides direct evidence that the energetic cost of distorting a DNA site can make an unfavorable contribution to protein-DNA binding.

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Selected References

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

  1. Aggarwal A. K., Rodgers D. W., Drottar M., Ptashne M., Harrison S. C. Recognition of a DNA operator by the repressor of phage 434: a view at high resolution. Science. 1988 Nov 11;242(4880):899–907. doi: 10.1126/science.3187531. [DOI] [PubMed] [Google Scholar]
  2. Aiken C. R., McLaughlin L. W., Gumport R. I. The highly homologous isoschizomers RsrI endonuclease and EcoRI endonuclease do not recognize their target sequence identically. J Biol Chem. 1991 Oct 5;266(28):19070–19078. [PubMed] [Google Scholar]
  3. Brennan C. A., Van Cleve M. D., Gumport R. I. The effects of base analogue substitutions on the cleavage by the EcoRI restriction endonuclease of octadeoxyribonucleotides containing modified EcoRI recognition sequences. J Biol Chem. 1986 Jun 5;261(16):7270–7278. [PubMed] [Google Scholar]
  4. Clore G. M., Oschkinat H., McLaughlin L. W., Benseler F., Happ C. S., Happ E., Gronenborn A. M. Refinement of the solution structure of the DNA dodecamer 5'd(CGCGPATTCGCG)2 containing a stable purine-thymine base pair: combined use of nuclear magnetic resonance and restrained molecular dynamics. Biochemistry. 1988 May 31;27(11):4185–4197. doi: 10.1021/bi00411a042. [DOI] [PubMed] [Google Scholar]
  5. Connolly B. A. Synthetic oligodeoxynucleotides containing modified bases. Methods Enzymol. 1992;211:36–53. doi: 10.1016/0076-6879(92)11005-4. [DOI] [PubMed] [Google Scholar]
  6. Delcourt S. G., Blake R. D. Stacking energies in DNA. J Biol Chem. 1991 Aug 15;266(23):15160–15169. [PubMed] [Google Scholar]
  7. Diekmann S., McLaughlin L. W. DNA curvature in native and modified EcoRI recognition sites and possible influence upon the endonuclease cleavage reaction. J Mol Biol. 1988 Aug 20;202(4):823–834. doi: 10.1016/0022-2836(88)90561-x. [DOI] [PubMed] [Google Scholar]
  8. Ha J. H., Spolar R. S., Record M. T., Jr Role of the hydrophobic effect in stability of site-specific protein-DNA complexes. J Mol Biol. 1989 Oct 20;209(4):801–816. doi: 10.1016/0022-2836(89)90608-6. [DOI] [PubMed] [Google Scholar]
  9. Jen-Jacobson L., Lesser D., Kurpiewski M. The enfolding arms of EcoRI endonuclease: role in DNA binding and cleavage. Cell. 1986 May 23;45(4):619–629. doi: 10.1016/0092-8674(86)90294-1. [DOI] [PubMed] [Google Scholar]
  10. Kim Y. C., Grable J. C., Love R., Greene P. J., Rosenberg J. M. Refinement of Eco RI endonuclease crystal structure: a revised protein chain tracing. Science. 1990 Sep 14;249(4974):1307–1309. doi: 10.1126/science.2399465. [DOI] [PubMed] [Google Scholar]
  11. Koudelka G. B., Harbury P., Harrison S. C., Ptashne M. DNA twisting and the affinity of bacteriophage 434 operator for bacteriophage 434 repressor. Proc Natl Acad Sci U S A. 1988 Jul;85(13):4633–4637. doi: 10.1073/pnas.85.13.4633. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Lesser D. R., Grajkowski A., Kurpiewski M. R., Koziolkiewicz M., Stec W. J., Jen-Jacobson L. Stereoselective interaction with chiral phosphorothioates at the central DNA kink of the EcoRI endonuclease-GAATTC complex. J Biol Chem. 1992 Dec 5;267(34):24810–24818. [PubMed] [Google Scholar]
  13. Lesser D. R., Kurpiewski M. R., Jen-Jacobson L. The energetic basis of specificity in the Eco RI endonuclease--DNA interaction. Science. 1990 Nov 9;250(4982):776–786. doi: 10.1126/science.2237428. [DOI] [PubMed] [Google Scholar]
  14. Lu A. L., Jack W. E., Modrich P. DNA determinants important in sequence recognition by Eco RI endonuclease. J Biol Chem. 1981 Dec 25;256(24):13200–13206. [PubMed] [Google Scholar]
  15. McLaughlin L. W., Benseler F., Graeser E., Piel N., Scholtissek S. Effects of functional group changes in the EcoRI recognition site on the cleavage reaction catalyzed by the endonuclease. Biochemistry. 1987 Nov 17;26(23):7238–7245. doi: 10.1021/bi00397a007. [DOI] [PubMed] [Google Scholar]
  16. Mossing M. C., Record M. T., Jr Thermodynamic origins of specificity in the lac repressor-operator interaction. Adaptability in the recognition of mutant operator sites. J Mol Biol. 1985 Nov 20;186(2):295–305. doi: 10.1016/0022-2836(85)90106-8. [DOI] [PubMed] [Google Scholar]
  17. Record M. T., Jr, Ha J. H., Fisher M. A. Analysis of equilibrium and kinetic measurements to determine thermodynamic origins of stability and specificity and mechanism of formation of site-specific complexes between proteins and helical DNA. Methods Enzymol. 1991;208:291–343. doi: 10.1016/0076-6879(91)08018-d. [DOI] [PubMed] [Google Scholar]
  18. Reich N. O., Danzitz M. J., Jr EcoRI DNA methyltransferase-DNA interactions. Biochemistry. 1992 Feb 25;31(7):1937–1945. doi: 10.1021/bi00122a006. [DOI] [PubMed] [Google Scholar]
  19. Yanagi K., Privé G. G., Dickerson R. E. Analysis of local helix geometry in three B-DNA decamers and eight dodecamers. J Mol Biol. 1991 Jan 5;217(1):201–214. doi: 10.1016/0022-2836(91)90620-l. [DOI] [PubMed] [Google Scholar]

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