Impact of C5-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 derivative d(GAAAAMe5CGTTTTC)2 have been determined by NMR and molecular modelling in order to examine the impact of cytosine methylation on the central CpG conformation. Detailed 1H NMR and 31P NMR investigation of the two oligomers includes quantitative NOESY, 2D homonuclear Hartmann-Hahn spectroscopy, double-quantum-filtered COSY and heteronuclear 1H-31P correlation. Back-calculations of NOESY spectra and simulations of double-quantum-filtered COSY patterns were performed to gain accurate information on interproton distances and sugar phase angles. Molecular models under experimental constraints were generated by energy minimization by means of the molecular mechanics program JUMNA. The MORASS software was used to iteratively refine the structures obtained. After methylation, the oligomer still has a B-DNA conformation. However, there are differences in the structural parameters and the thermal stability as compared to the unmethylated molecule. Careful structural analysis shows that after methylation CpG departs from the usual conformation observed in other ACGT tetramers with different surroundings. Subtle displacements of bases, sugars and backbone imposed by the steric interaction of the two methyl groups inside the major groove are accompanied by severe pinching of the minor groove at the C-G residues.
Marcourt
L
L
Institut de Topologie et de Dynamique des Systèmes, associé au CNRS, Université D. Diderot (Paris 7), Paris, France.
Cordier
C
C
Couesnon
T
T
Dodin
G
G
D044503:5-Methylcytosine; D001483:Base Sequence; D018899:CpG Islands; D003596:Cytosine / Q000031:analogs & derivatives / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008745:Methylation; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides / Q000737:chemistry*; D012996:Solutions
D009838:Oligodeoxyribonucleotides; D012996:Solutions; D044503:5-Methylcytosine; D003596:Cytosine