Institute for Integrated Cell-Material Sciences, Kyoto University, Yoshida-ushinomiyacho, Sakyo-ku, Kyoto 606-8501, Japan.
J Am Chem Soc. 2010 Feb 10;132(5):1592-7. doi: 10.1021/ja907649w.
A novel strategy for regulation of an enzymatic DNA modification reaction has been developed by employing a designed nanoscale DNA scaffold. DNA modification using enzymes often requires bending of specific DNA strands to facilitate the reaction. The DNA methylation enzyme EcoRI methyltransferase (M.EcoRI) bends double helix DNA by 55 degrees-59 degrees during the reaction with flipping out of the second adenine in the GAATTC sequence as the methyl transfer reaction proceeds. In this study, two different double helical tensions, tense and relaxed states of double helices, were created to control the methyl transfer reaction of M.EcoRI and examine the structural effect on the methylation. We designed and prepared a two-dimensional (2D) DNA scaffold named the "DNA frame" using the DNA origami method that accommodates two different lengths of the double-strand DNA fragments, a tense 64mer double strand and a relaxed 74mer double strand. Fast-scanning atomic force microscope (AFM) imaging revealed the different dynamic movement of the double-strand DNAs and complexes of M.EcoRI with 64mer and 74mer double-strand DNAs. After treatment of the double strands in the DNA frame with M.EcoRI and the subsequent digestion with restriction enzyme EcoRI (R.EcoRI), AFM analysis revealed that the 74mer double-strand DNA was not effectively cleaved compared with the 64mer double-strand DNA, indicating that the methylation preferentially occurred in the relaxed 74mer double-strand DNA compared with that in the tense 64mer double strand. Biochemical analysis of the methylation and specific digestion using a real-time PCR also supported the above results. These results indicate the importance of the structural flexibility for bending of the duplex DNA during the methyl transfer reaction with M.EcoRI. Therefore, the DNA methylation can be regulated using the structurally controlled double-strand DNAs constructed in the DNA frame nanostructure.
一种调控酶促 DNA 修饰反应的新策略是通过采用设计的纳米尺度 DNA 支架来实现的。在使用酶进行 DNA 修饰时，通常需要弯曲特定的 DNA 链，以促进反应的进行。在与 GAATTC 序列中的第二个腺嘌呤翻转的过程中，EcoRI 甲基转移酶（M.EcoRI）在反应中使双螺旋 DNA 弯曲 55 度-59 度。在这项研究中，我们创建了两种不同的双螺旋张力，即紧张和松弛状态的双螺旋，以控制 M.EcoRI 的甲基转移反应，并检查结构对甲基化的影响。我们使用 DNA 折纸方法设计并制备了一个名为“DNA 框架”的二维（2D）DNA 支架，该支架容纳了两条不同长度的双链 DNA 片段，一条是紧张的 64 碱基对双链，一条是松弛的 74 碱基对双链。快速扫描原子力显微镜（AFM）成像揭示了双链 DNA 和 M.EcoRI 与 64 碱基对和 74 碱基对双链 DNA 复合物的不同动态运动。在用 M.EcoRI 处理 DNA 框架中的双链 DNA 后，用限制酶 EcoRI（R.EcoRI）进行消化，AFM 分析表明，74 碱基对双链 DNA 没有被有效地切割，与 64 碱基对双链 DNA 相比，这表明在松弛的 74 碱基对双链 DNA 中，甲基化比在紧张的 64 碱基对双链 DNA 中更优先发生。使用实时 PCR 的甲基化和特异性消化的生化分析也支持了上述结果。这些结果表明，在 M.EcoRI 的甲基转移反应中，双链 DNA 的结构灵活性对于弯曲的重要性。因此，可以使用在 DNA 框架纳米结构中构建的结构控制的双链 DNA 来调节 DNA 甲基化。
