Department of Biotechnology, Key Laboratory of Virology of Guangzhou, College of Life Science and Technology, Jinan University, Guangzhou 510632, China.
Department of Chemistry, Graduate School of Science, Kyoto University, Kitashirakawa-oiwakecho, Sakyo-ku, Kyoto 606-8502, Japan.
Nucleic Acids Res. 2020 May 7;48(8):4041-4051. doi: 10.1093/nar/gkaa137.
DNA methylation and demethylation play a key role in the epigenetic regulation of gene expression; however, a series of oxidation reactions of 5-methyl cytosine (5mC) mediated by ten-eleven translocation (TET) enzymes driving demethylation process are yet to be uncovered. To elucidate the relationship between the oxidative processes and structural factors of DNA, we analysed the behavior of TET-mediated 5mC-oxidation by incorporating structural stress onto a substrate double-stranded DNA (dsDNA) using a DNA origami nanochip. The reactions and behaviors of TET enzymes were systematically monitored by biochemical analysis and single-molecule observation using atomic force microscopy (AFM). A reformative frame-like DNA origami was established to allow the incorporation of dsDNAs as 5mC-containing substrates in parallel orientations. We tested the potential effect of dsDNAs present in the tense and relaxed states within a DNA nanochip on TET oxidation. Based on enzyme binding and the detection of oxidation reactions within the DNA nanochip, it was revealed that TET preferred a relaxed substrate regardless of the modification types of 5-oxidated-methyl cytosine. Strikingly, when a multi-5mCG sites model was deployed to further characterize substrate preferences of TET, TET preferred the fully methylated site over the hemi-methylated site. This analytical modality also permits the direct observations of dynamic movements of TET such as sliding and interstrand transfer by high-speed AFM. In addition, the thymine DNA glycosylase-mediated base excision repair process was characterized in the DNA nanochip. Thus, we have convincingly established the system's ability to physically regulate enzymatic reactions, which could prove useful for the observation and characterization of coordinated DNA demethylation processes at the nanoscale.
DNA 甲基化和去甲基化在基因表达的表观遗传调控中起着关键作用；然而，TET 酶介导的一系列 5-甲基胞嘧啶（5mC）氧化反应驱动去甲基化过程尚未被揭示。为了阐明氧化过程与 DNA 的结构因素之间的关系，我们通过在底物双链 DNA（dsDNA）上引入结构应力，利用 DNA 折纸纳米芯片分析了 TET 介导的 5mC 氧化反应。通过生化分析和原子力显微镜（AFM）的单分子观察，系统地监测了 TET 酶的反应和行为。建立了一种创新性的框状 DNA 折纸结构，允许以平行取向掺入含有 5mC 的 dsDNA 作为底物。我们测试了 DNA 纳米芯片中紧张和松弛状态下的 dsDNA 对 TET 氧化的潜在影响。基于酶结合和 DNA 纳米芯片内氧化反应的检测，结果表明 TET 优先选择松弛的底物，而不管 5-氧化甲基胞嘧啶的修饰类型如何。引人注目的是，当采用多 5mCG 位点模型进一步表征 TET 的底物偏好时，TET 优先选择完全甲基化的位点而不是半甲基化的位点。这种分析方法还允许通过高速 AFM 直接观察 TET 的动态运动，如滑动和链间转移。此外，还在 DNA 纳米芯片中表征了胸腺嘧啶 DNA 糖基化酶介导的碱基切除修复过程。因此，我们已经令人信服地建立了该系统物理调节酶反应的能力，这对于在纳米尺度上观察和表征协调的 DNA 去甲基化过程可能非常有用。
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