Structural adaptations in the interaction of EcoRI endonuclease with methylated GAATTC sites.
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.
Jen-Jacobson
L
L
Department of Biological Sciences, University of Pittsburgh, PA 15260, USA.
Engler
L E
LE
Lesser
D R
DR
Kurpiewski
M R
MR
Yee
C
C
McVerry
B
B
D001665:Binding Sites; D003198:Computer Simulation; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D015246:Deoxyribonuclease EcoRI / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology; D008745:Methylation; D008958:Models, Molecular; D011485:Protein Binding; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D004268:DNA-Binding Proteins; D004247:DNA; D015246:Deoxyribonuclease EcoRI