Impact of CS-cytosine methylation on the solution structure of d(GAAAACGTTTTC)(2) - An NMR and molecular modelling investigation

The solution structures of d(GAAAACGTTTTC)(2) and of its methylated derivat ive d(GAAAAMe(5)CGTTTTC)(2) have been determined by NMR and molecular model ling in order to examine the impact of cytosine methylation on the central CpG conformation. Detailed H-1 NMR and P-31 NMR investigation of the two ol igomers includes quantitative NOESY, 2D homonuclear Hartmann-Hahn spectrosc opy, double-quantum-filtered COSY and heteronuclear H-1-P-31 correlation. B ack-calculations of NOESY spectra and simulations of double-quantum-filtere d COSY patterns were performed to gain accurate information on interproton distances and sugar phase angles. Molecular models under experimental const raints were generated by energy minimization by means of the molecular mech anics program JUMNA. The MORASS software was used to iteratively refine the structures obtained. After methylation, the oligomer still has a B-DNA con formation. However, there are differences in the structural parameters and the thermal stability as compared to the unmethylated molecule. Careful str uctural analysis shows that after methylation CpG departs from the usual co nformation observed in other ACGT tetramers with different surroundings. Su btle displacements of bases, sugars and backbone imposed by the steric inte raction of the two methyl groups inside the major groove are accompanied by severe pinching of the minor groove at the C-G residues.