Impact of C5‐cytosine methylation on the solution structure of d(GAAAACGTTTTC)₂

Abstract

The solution structures of d(GAAAACGTTTTC) 2 and of its methylated derivative d(GAAAAMe 5 CGTTTTC) 2 have been determined by NMR and molecular modelling in order to examine the impact of cytosine methylation on the central CpG conformation. Detailed 1 H NMR and 31 P NMR investigation of the two oligomers includes quantitative NOESY, 2D homonuclear Hartmann±Hahn spectroscopy, double-quantum-filtered COSY and heteronuclear 1 H-31 P correlation. Back-calculations of NOESY spectra and simulations of double-quantumfiltered 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.Keywords: DNA; 2D NMR; modelling; cytosine methylation; CpG; adenine tract.DNA methylation at cytosines in CpG steps is recognized as an important event in biological processes that control gene expression. Methylation turns off the transcription of genes involved in mammal development [1] and genomic imprinting [2], and abolishes the immune response induced by oligonucleotide motifs containing a CpG step flanked by two purines and two pyrimidines in the 5 H and 3 H directions, respectively [3]. The hypothesis that gene silencing stems from blocking the binding of transcription factors at methylated CpG has led to several investigations designed to unveil possible structural singularities of the methylated sequence as compared to the unmodified one. Crystallographic data show that cytosine methylation in the CMe 5 CTG oligomer only induces a slight structural alteration [4]. NMR analysis has underlined the marked conformational difference between the octamers d(CATCGATG) 2 and d(CTTCGAAG) 2 , despite the same central four-base sequence, and their specific structural response to the methylation of the central cytosine [5]. The 1 H NMR results show that cytosine methylation induces more significant structural effects in the first oligomer than in the other. The conclusions reached by 1 H NMR have been corroborated by 1 H-31 P coupling constant and 31 P chemical shift investigation. These studies have shown that the CpG backbone is more sensitive to methylation in d(CATCGATG) 2 than in the second octamer. The observation that the conformation of the central tetranucleotide is strongly affected by its 5 H and 3 H environment is in line wit...

The Influence of the Thymine C5 Methyl Group on Spontaneous Base Pair Breathing in DNA

“…Furthermore, only small differences are discernable when structures with and without the C5 methyl group are compared in crystals (40,43) as well as in solution (41,42,44,45). The NOESY spectra of the d(CATA 2 T 2 ATG) 2 and d(CATA 2 U 2 ATG) 2 oligomers that have been investigated in the present study are in agreement with these earlier studies.…”

“…The lack of structural impact of C5 methylation as evidenced by Fig. 4 and previous studies (41,42,44,45) is therefore compatible with the large dynamical effects.…”

“…4). These spectral features indicate that the C5 methyl group exerts only a minor influence on the helix structure in keeping with crystal (40,43) as well as solution structure studies (41,42,44,45).…”

Methylation of Cytosine at C5 in a CpG Sequence Context Causes a Conformational Switch of a Benzo[a]pyrene diol epoxide-N2-guanine Adduct in DNA from a Minor Groove Alignment to Intercalation with Base Displacement

“…41 Finally, structural analysis by solution NMR and molecular modeling methods shows that replacement of cytosine by meC in a 12-mer oligonucleotide duplex with a single, central CpG dinucleotide step gives rise to a local narrowing of the minor groove, while the major groove becomes more shallow. 42 In summary, the reported physical and structural differences in CpG and meCpG sequence contexts do not provide any simple explanations for the unusual conformational switch of the 10R (−)-trans-anti- [BP]G adduct when the cytosine residue flanking the adduct is replaced by 5-methylcytosine in the 11-mer duplex studied in this work.…”

“…36,37 The impact of the methyl group at the 5-position of cytosine on the characteristics of double-stranded DNA have been studied by a variety of approaches. [38][39][40][41][42] Cyclization of ligated mixed-sequence oligonucleotides followed by analysis of electrophoretic mobilities showed that the changes in helical twist or intrinsic flexibility are not altered significantly by cytosine methylation. 40 However, in the case of an EcoRI decamer, methylation of the cytosine residue at the CpG dinucleotide steps lowers flexibility by ~30% and causes an underwinding by ~0.5 base-pair per turn.…”