Skip to main content
PMC home page
The EMBO Journal logoLink to The EMBO Journal
. 1996 Jun 3;15(11):2870–2882.

Structural adaptations in the interaction of EcoRI endonuclease with methylated GAATTC sites.

L Jen-Jacobson 1, L E Engler 1, D R Lesser 1, M R Kurpiewski 1, C Yee 1, B McVerry 1
PMCID: PMC450225  PMID: 8654385

Abstract

We have studied the interaction of EcoRI endonuclease with oligonucleotides containing GAATTC sites bearing one or two adenine-N6-methyl groups, which would be in steric conflict with key protein side chains involved in recognition and/or catalysis in the canonical complex. Single-strand methylation of either adenine produces small penalties in binding free energy (deltadeltaG0(S) approximately +1.4 kcal/mol), but elicits asymmetric structural adaptations in the complex, such that cleavage rate constants are strongly inhibited and unequal in the two DNA strands. The dependences of cleavage rate constants on the concentration of the Mg2+ cofactor are unaltered. When either adenine is methylated on both DNA strands, deltadeltaG0(S) (approximately +4 kcal/mol) is larger than the expected sum of the deltadeltaG0(S) values for the single-strand methylations, because the asymmetric adaptations cannot occur. Cleavage rate constants are reduced by 600 000-fold for the biologically relevant GAmATTC/CTTmAAG site, but the GmAATTC/CTTAmAG site forms only a non-specific complex that cannot be cleaved. These observations provide a detailed thermodynamic and kinetic explanation of how single-strand and double-strand methylation protect against endonuclease cleavage in vivo. We propose that non-additive effects on binding and structural 'adaptations' are important in understanding how DNA methylation modulates the biological activities of non-catalytic DNA binding proteins.

Full text

PDF
2873

Images in this article

2872Image
on p.2872
2874Image
on p.2874
2877Image
on p.2877
2880Image
on p.2880

Selected References

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

  1. Barras F., Marinus M. G. The great GATC: DNA methylation in E. coli. Trends Genet. 1989 May;5(5):139–143. doi: 10.1016/0168-9525(89)90054-1. [DOI] [PubMed] [Google Scholar]
  2. Becker M. M., Lesser D., Kurpiewski M., Baranger A., Jen-Jacobson L. "Ultraviolet footprinting" accurately maps sequence-specific contacts and DNA kinking in the EcoRI endonuclease-DNA complex. Proc Natl Acad Sci U S A. 1988 Sep;85(17):6247–6251. doi: 10.1073/pnas.85.17.6247. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bestor T. H., Coxon A. Cytosine methylation: the pros and cons of DNA methylation. Curr Biol. 1993 Jun 1;3(6):384–386. doi: 10.1016/0960-9822(93)90209-7. [DOI] [PubMed] [Google Scholar]
  4. Bird A. The essentials of DNA methylation. Cell. 1992 Jul 10;70(1):5–8. doi: 10.1016/0092-8674(92)90526-i. [DOI] [PubMed] [Google Scholar]
  5. Braaten B. A., Nou X., Kaltenbach L. S., Low D. A. Methylation patterns in pap regulatory DNA control pyelonephritis-associated pili phase variation in E. coli. Cell. 1994 Feb 11;76(3):577–588. doi: 10.1016/0092-8674(94)90120-1. [DOI] [PubMed] [Google Scholar]
  6. Chaillet J. R. Genomic imprinting: lessons from mouse transgenes. Mutat Res. 1994 Jun 1;307(2):441–449. doi: 10.1016/0027-5107(94)90255-0. [DOI] [PubMed] [Google Scholar]
  7. Frederick C. A., Grable J., Melia M., Samudzi C., Jen-Jacobson L., Wang B. C., Greene P., Boyer H. W., Rosenberg J. M. Kinked DNA in crystalline complex with EcoRI endonuclease. Nature. 1984 May 24;309(5966):327–331. doi: 10.1038/309327a0. [DOI] [PubMed] [Google Scholar]
  8. Frederick C. A., Quigley G. J., van der Marel G. A., van Boom J. H., Wang A. H., Rich A. Methylation of the EcoRI recognition site does not alter DNA conformation: the crystal structure of d(CGCGAm6ATTCGCG) at 2.0-A resolution. J Biol Chem. 1988 Nov 25;263(33):17872–17879. doi: 10.2210/pdb4dnb/pdb. [DOI] [PubMed] [Google Scholar]
  9. Heitman J., Zinder N. D., Model P. Repair of the Escherichia coli chromosome after in vivo scission by the EcoRI endonuclease. Proc Natl Acad Sci U S A. 1989 Apr;86(7):2281–2285. doi: 10.1073/pnas.86.7.2281. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Hsieh C. L., Lieber M. R. CpG methylated minichromosomes become inaccessible for V(D)J recombination after undergoing replication. EMBO J. 1992 Jan;11(1):315–325. doi: 10.1002/j.1460-2075.1992.tb05054.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Jen-Jacobson L., Kurpiewski M., Lesser D., Grable J., Boyer H. W., Rosenberg J. M., Greene P. J. Coordinate ion pair formation between EcoRI endonuclease and DNA. J Biol Chem. 1983 Dec 10;258(23):14638–14646. [PubMed] [Google Scholar]
  12. 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]
  13. Jen-Jacobson L. Structural-perturbation approaches to thermodynamics of site-specific protein-DNA interactions. Methods Enzymol. 1995;259:305–344. doi: 10.1016/0076-6879(95)59050-1. [DOI] [PubMed] [Google Scholar]
  14. 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]
  15. Kuhn I., Stephenson F. H., Boyer H. W., Greene P. J. Positive-selection vectors utilizing lethality of the EcoRI endonuclease. Gene. 1986;42(3):253–263. doi: 10.1016/0378-1119(86)90229-5. [DOI] [PubMed] [Google Scholar]
  16. Lahue R. S., Au K. G., Modrich P. DNA mismatch correction in a defined system. Science. 1989 Jul 14;245(4914):160–164. doi: 10.1126/science.2665076. [DOI] [PubMed] [Google Scholar]
  17. 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]
  18. 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]
  19. Lesser D. R., Kurpiewski M. R., Waters T., Connolly B. A., Jen-Jacobson L. Facilitated distortion of the DNA site enhances EcoRI endonuclease-DNA recognition. Proc Natl Acad Sci U S A. 1993 Aug 15;90(16):7548–7552. doi: 10.1073/pnas.90.16.7548. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Li E., Bestor T. H., Jaenisch R. Targeted mutation of the DNA methyltransferase gene results in embryonic lethality. Cell. 1992 Jun 12;69(6):915–926. doi: 10.1016/0092-8674(92)90611-f. [DOI] [PubMed] [Google Scholar]
  21. Lin S. Y., Riggs A. D. Lac repressor binding to non-operator DNA: detailed studies and a comparison of eequilibrium and rate competition methods. J Mol Biol. 1972 Dec 30;72(3):671–690. doi: 10.1016/0022-2836(72)90184-2. [DOI] [PubMed] [Google Scholar]
  22. Malyguine E., Vannier P., Yot P. Alteration of the specificity of restriction endonucleases in the presence of organic solvents. Gene. 1980 Jan;8(2):163–177. doi: 10.1016/0378-1119(80)90035-9. [DOI] [PubMed] [Google Scholar]
  23. McClarin J. A., Frederick C. A., Wang B. C., Greene P., Boyer H. W., Grable J., Rosenberg J. M. Structure of the DNA-Eco RI endonuclease recognition complex at 3 A resolution. Science. 1986 Dec 19;234(4783):1526–1541. doi: 10.1126/science.3024321. [DOI] [PubMed] [Google Scholar]
  24. McClelland M., Nelson M., Raschke E. Effect of site-specific modification on restriction endonucleases and DNA modification methyltransferases. Nucleic Acids Res. 1994 Sep;22(17):3640–3659. doi: 10.1093/nar/22.17.3640. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Messer W., Bellekes U., Lother H. Effect of dam methylation on the activity of the E. coli replication origin, oriC. EMBO J. 1985 May;4(5):1327–1332. doi: 10.1002/j.1460-2075.1985.tb03780.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Migeon B. R. X-chromosome inactivation: molecular mechanisms and genetic consequences. Trends Genet. 1994 Jul;10(7):230–235. doi: 10.1016/0168-9525(94)90169-4. [DOI] [PubMed] [Google Scholar]
  27. Raleigh E. A., Wilson G. Escherichia coli K-12 restricts DNA containing 5-methylcytosine. Proc Natl Acad Sci U S A. 1986 Dec;83(23):9070–9074. doi: 10.1073/pnas.83.23.9070. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Razin A., Cedar H. DNA methylation and gene expression. Microbiol Rev. 1991 Sep;55(3):451–458. doi: 10.1128/mr.55.3.451-458.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Riggs A. D., Pfeifer G. P. X-chromosome inactivation and cell memory. Trends Genet. 1992 May;8(5):169–174. doi: 10.1016/0168-9525(92)90219-t. [DOI] [PubMed] [Google Scholar]
  30. Robinson C. R., Sligar S. G. Molecular recognition mediated by bound water. A mechanism for star activity of the restriction endonuclease EcoRI. J Mol Biol. 1993 Nov 20;234(2):302–306. doi: 10.1006/jmbi.1993.1586. [DOI] [PubMed] [Google Scholar]
  31. Rubin R. A., Modrich P. EcoRI methylase. Physical and catalytic properties of the homogeneous enzyme. J Biol Chem. 1977 Oct 25;252(20):7265–7272. [PubMed] [Google Scholar]
  32. Smith D. W., Garland A. M., Herman G., Enns R. E., Baker T. A., Zyskind J. W. Importance of state of methylation of oriC GATC sites in initiation of DNA replication in Escherichia coli. EMBO J. 1985 May;4(5):1319–1326. doi: 10.1002/j.1460-2075.1985.tb03779.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES