Graduate School of Medical Life Science, Yokohama City University, 1-7-29 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa 230-0045, Japan.
Bioorganic Research Institute, Suntory Foundation for Life Sciences, 8-1-1 Seikadai, Seika, Soraku, Kyoto 619-0284, Japan.
Nucleic Acids Res. 2021 Jan 25;49(2):1152-1162. doi: 10.1093/nar/gkaa1210.
Modification of cytosine plays an important role in epigenetic regulation of gene expression and genome stability. Cytosine is converted to 5-methylcytosine (5mC) by DNA methyltransferase; in turn, 5mC may be oxidized to 5-hydroxymethylcytosine (5hmC) by ten-eleven translocation enzyme. The structural flexibility of DNA is known to affect the binding of proteins to methylated DNA. Here, we have carried out a semi-quantitative analysis of the dynamics of double-stranded DNA (dsDNA) containing various epigenetic modifications by combining data from imino 1H exchange and imino 1H R1ρ relaxation dispersion NMR experiments in a complementary way. Using this approach, we characterized the base-opening (kopen) and base-closing (kclose) rates, facilitating a comparison of the base-opening and -closing process of dsDNA containing cytosine in different states of epigenetic modification. A particularly striking result is the increase in the kopen rate of hemi-methylated dsDNA 5mC/C relative to unmodified or fully methylated dsDNA, indicating that the Watson-Crick base pairs undergo selective destabilization in 5mC/C. Collectively, our findings imply that the epigenetic modulation of cytosine dynamics in dsDNA mediates destabilization of the GC Watson-Crick base pair to allow base-flipping in living cells.
碱基的修饰在基因表达的表观遗传调控和基因组稳定性中起着重要作用。胞嘧啶由 DNA 甲基转移酶转化为 5-甲基胞嘧啶（5mC）；反过来，5mC 可被 ten-eleven 易位酶氧化为 5-羟甲基胞嘧啶（5hmC）。已知 DNA 的结构灵活性会影响蛋白质与甲基化 DNA 的结合。在这里，我们通过将来自亚氨基 1H 交换和亚氨基 1H R1ρ 弛豫分散 NMR 实验的数据以互补的方式结合起来，对含有各种表观遗传修饰的双链 DNA（dsDNA）的动力学进行了半定量分析。通过这种方法，我们表征了碱基开放（kopen）和碱基关闭（kclose）的速率，从而可以比较含有不同表观遗传修饰的 dsDNA 中胞嘧啶的碱基开放和关闭过程。一个特别引人注目的结果是半甲基化 dsDNA 5mC/C 的 kopen 速率相对于未修饰或完全甲基化的 dsDNA 增加，表明沃森-克里克碱基对在 5mC/C 中发生了选择性的去稳定化。总的来说，我们的研究结果表明，dsDNA 中胞嘧啶动力学的表观遗传调节介导了 GC 沃森-克里克碱基对的去稳定化，从而允许在活细胞中碱基翻转。
