Impact of CS-cytosine methylation on the solution structure of d(GAAAACGTTTTC)2 -: An NMR and molecular modelling investigation
Impact of CS-cytosine methylation on the solution structure of d(GAAAACGTTTTC)2 -: An NMR and molecular modelling investigation
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DOI：
10.1046/j.1432-1327.1999.00819.x
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发表时间：
1999-11-01
期刊：
影响因子：
--
通讯作者：
Dodin, G
中科院分区：
文献类型：
--
作者：
Marcourt, L;Cordier, C;Dodin, G
d（GAAAACGTTTTC）（2）及其甲基化衍生物d（GAAAAMe（5）CGTTTTC）（2）的溶液结构已通过NMR和分子模拟确定，以检查胞嘧啶甲基化对中心CpG构象的影响。详细的H-1 NMR和P-31 NMR研究包括定量NOESY，二维homopolyHartmann-Hahn光谱，双量子滤波COSY和杂原子H-1-P-31相关。对NOESY谱进行了反算，并对双量子滤波COSY谱进行了模拟，以获得关于质子间距离和糖相位角的准确信息。实验约束下的分子模型生成的能量最小化的分子力学程序JUMNA。MORASS软件用于迭代地精炼所获得的结构。甲基化后，寡聚体仍具有B-DNA构象。然而，与未甲基化的分子相比，在结构参数和热稳定性方面存在差异。仔细的结构分析表明，甲基化后CpG偏离了在具有不同环境的其他ACGT四聚体中观察到的通常构象。由大沟内的两个甲基的空间相互作用施加的碱基、糖和主链的微妙位移伴随着小沟在C-G残基处的严重挤压。
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 H-1 NMR and P-31 NMR investigation of the two oligomers includes quantitative NOESY, 2D homonuclear Hartmann-Hahn spectroscopy, double-quantum-filtered COSY and heteronuclear H-1-P-31 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.