Direct observation and analysis of TET-mediated oxidation processes in a DNA origami nanochip
Direct observation and analysis of TET-mediated oxidation processes in a DNA origami nanochip
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DNA 折纸纳米芯片中 TET 介导的氧化过程的直接观察和分析
DOI：
10.1093/nar/gkaa137
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发表时间：
2020-03
影响因子：
14.9
通讯作者：
Masayuki Endo
中科院分区：
文献类型：
--
作者：
Xiwen Xing;Shinsuke Sato;Nai-Kei Wong;Kumi Hidaka;Hiroshi Sugiyama;Masayuki Endo
摘要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，由10 - 11易位（泰特）酶介导的一系列5-甲基胞嘧啶（5-methyl cytosine，5 mC）氧化反应驱动的去甲基化过程尚未被发现。为了阐明DNA的氧化过程和结构因素之间的关系，我们分析了TET介导的5 mC氧化的行为，通过使用DNA折纸纳米芯片将结构应力结合到底物双链DNA（dsDNA）上。利用原子力显微镜（AFM）对泰特酶的反应和行为进行了系统的生化分析和单分子观察。建立了一种改进的框架样DNA折纸，以允许将dsDNA作为含5 mC的底物以平行方向掺入。我们测试了DNA纳米芯片内以紧张和松弛状态存在的dsDNA对泰特氧化的潜在影响。基于酶结合和DNA纳米芯片内的氧化反应的检测，揭示了泰特偏好松弛的底物，而不管5-氧化甲基胞嘧啶的修饰类型。引人注目的是，当采用多5 mCG位点模型进一步表征泰特的底物偏好时，泰特优选完全甲基化位点而不是半甲基化位点。这种分析模式还允许直接观察泰特的动态运动，如滑动和股间转移的高速AFM。此外，胸腺嘧啶DNA糖基化酶介导的碱基切除修复过程中的DNA纳米芯片的特点。因此，我们已经令人信服地建立了系统的物理调节酶反应的能力，这可能被证明是有用的观察和表征协调的DNA去甲基化过程在纳米级。
Abstract 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.
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影响因子：
15
作者：
Rajendran A;Endo M;Hidaka K;Tran PL;Mergny JL;Gorelick RJ;Sugiyama H
通讯作者：
Sugiyama H
影响因子：
64.8
作者：
Wang L;Zhou Y;Xu L;Xiao R;Lu X;Chen L;Chong J;Li H;He C;Fu XD;Wang D
通讯作者：
Wang D
影响因子：
14.9
作者：
D'Annessa I;Coletta A;Sutthibutpong T;Mitchell J;Chillemi G;Harris S;Desideri A
通讯作者：
Desideri A
影响因子：
--
作者：
G. Raghavan;Kumi Hidaka;H. Sugiyama;Masayuki Endo
通讯作者：
G. Raghavan;Kumi Hidaka;H. Sugiyama;Masayuki Endo
影响因子：
56.9
作者：
Bostick, Magnolia;Kim, Jong Kyong;Jacobsen, Steven E.
通讯作者：
Jacobsen, Steven E.