L. Marcourt et al., Impact of CS-cytosine methylation on the solution structure of d(GAAAACGTTTTC)(2) - An NMR and molecular modelling investigation, EUR J BIOCH, 265(3), 1999, pp. 1032-1042
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.