Regulation of DNA Methylation Using Different Tensions of Double Strands Constructed in a Defined DNA Nanostructure
Regulation of DNA Methylation Using Different Tensions of Double Strands Constructed in a Defined DNA Nanostructure
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DOI：
10.1021/ja907649w
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发表时间：
2010-02-10
影响因子：
15
通讯作者：
Sugiyama, Hiroshi
中科院分区：
文献类型：
--
作者：
Endo, Masayuki;Katsuda, Yousuke;Sugiyama, Hiroshi
一种新的策略，用于调节酶的DNA修饰反应已开发通过采用设计的纳米级DNA支架。使用酶进行DNA修饰通常需要弯曲特定的DNA链以促进反应。DNA甲基化酶EcoRI甲基转移酶（M.EcoRI）在反应过程中使双螺旋DNA弯曲55度-59度，随着甲基转移反应的进行，GAATTC序列中的第二个腺嘌呤翻转。本研究创造了两种不同的双螺旋张力，即双螺旋的紧张和松弛状态，以控制M.EcoRI的甲基转移反应并考察结构对甲基化的影响。我们设计并制备了一个二维（2D）DNA支架命名为“DNA框架”使用DNA折纸的方法，容纳两种不同长度的双链DNA片段，紧张的64聚体双链和放松的74聚体双链。快速扫描原子力显微镜（AFM）成像显示了双链DNA以及M.EcoRI与64 mer和74 mer双链DNA复合物的不同动力学运动。用M.EcoRI处理DNA框架中的双链，然后用限制性内切酶EcoRI（R.EcoRI）消化，AFM分析显示，与64 mer双链DNA相比，74 mer双链DNA没有被有效地切割，表明甲基化优先发生在松弛的74 mer双链DNA中，而不是紧张的64 mer双链DNA中。甲基化的生化分析和使用实时PCR的特异性消化也支持上述结果。这些结果表明，在与M. EcoRI的甲基转移反应期间，双链体DNA的弯曲的结构柔性的重要性。因此，可以使用在DNA框架纳米结构中构建的结构受控的双链DNA来调节DNA甲基化。
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.