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

1996; Springer Nature; Volume: 15; Issue: 11 Linguagem: Inglês

10.1002/j.1460-2075.1996.tb00648.x

1460-2075

Linda Jen‐Jacobson, Lisa E. Engler, David Lesser, Michael R. Kurpiewski, Cindy J. Yee, Bryan J. McVerry,

RNA Interference and Gene Delivery

Research Article3 June 1996free access Structural adaptations in the interaction of EcoRI endonuclease with methylated GAATTC sites. L. Jen-Jacobson L. Jen-Jacobson Department of Biological Sciences, University of Pittsburgh, PA 15260, USA. Search for more papers by this author L. E. Engler L. E. Engler Department of Biological Sciences, University of Pittsburgh, PA 15260, USA. Search for more papers by this author D. R. Lesser D. R. Lesser Department of Biological Sciences, University of Pittsburgh, PA 15260, USA. Search for more papers by this author M. R. Kurpiewski M. R. Kurpiewski Department of Biological Sciences, University of Pittsburgh, PA 15260, USA. Search for more papers by this author C. Yee C. Yee Department of Biological Sciences, University of Pittsburgh, PA 15260, USA. Search for more papers by this author B. McVerry B. McVerry Department of Biological Sciences, University of Pittsburgh, PA 15260, USA. Search for more papers by this author L. Jen-Jacobson L. Jen-Jacobson Department of Biological Sciences, University of Pittsburgh, PA 15260, USA. Search for more papers by this author L. E. Engler L. E. Engler Department of Biological Sciences, University of Pittsburgh, PA 15260, USA. Search for more papers by this author D. R. Lesser D. R. Lesser Department of Biological Sciences, University of Pittsburgh, PA 15260, USA. Search for more papers by this author M. R. Kurpiewski M. R. Kurpiewski Department of Biological Sciences, University of Pittsburgh, PA 15260, USA. Search for more papers by this author C. Yee C. Yee Department of Biological Sciences, University of Pittsburgh, PA 15260, USA. Search for more papers by this author B. McVerry B. McVerry Department of Biological Sciences, University of Pittsburgh, PA 15260, USA. Search for more papers by this author Author Information L. Jen-Jacobson1, L. E. Engler1, D. R. Lesser1, M. R. Kurpiewski1, C. Yee1 and B. McVerry1 1Department of Biological Sciences, University of Pittsburgh, PA 15260, USA. The EMBO Journal (1996)15:2870-2882https://doi.org/10.1002/j.1460-2075.1996.tb00648.x PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info 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. Previous ArticleNext Article Volume 15Issue 111 June 1996In this issue RelatedDetailsLoading ...