Department of Chemistry, Lomonosov Moscow State University, Leninskie Gory 1, 119991 Moscow, Russia.
Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Leninskie Gory 1, 119991 Moscow, Russia.
Int J Mol Sci. 2022 Sep 6;23(18):10226. doi: 10.3390/ijms231810226.
In mammals, de novo methylation of cytosines in DNA CpG sites is performed by DNA methyltransferase Dnmt3a. Changes in the methylation status of CpG islands are critical for gene regulation and for the progression of some cancers. Recently, the potential involvement of DNA G-quadruplexes (G4s) in methylation control has been found. Here, we provide evidence for a link between G4 formation and the function of murine DNA methyltransferase Dnmt3a and its individual domains. As DNA models, we used (i) an isolated G4 formed by oligonucleotide capable of folding into parallel quadruplex and (ii) the same G4 inserted into a double-stranded DNA bearing several CpG sites. Using electrophoretic mobility shift and fluorescence polarization assays, we showed that the Dnmt3a catalytic domain (Dnmt3a-CD), in contrast to regulatory PWWP domain, effectively binds the G4 structure formed in both DNA models. The G4-forming oligonucleotide displaced the DNA substrate from its complex with Dnmt3a-CD, resulting in a dramatic suppression of the enzyme activity. In addition, a direct impact of G4 inserted into the DNA duplex on the methylation of a specific CpG site was revealed. Possible mechanisms of G4-mediated epigenetic regulation may include Dnmt3a sequestration at G4 and/or disruption of Dnmt3a oligomerization on the DNA surface.
在哺乳动物中，DNA 中 CpG 位点的胞嘧啶的从头甲基化是由 DNA 甲基转移酶 Dnmt3a 完成的。CpG 岛甲基化状态的变化对于基因调控和某些癌症的进展至关重要。最近，已经发现 DNA G-四链体 (G4s) 可能参与了甲基化调控。在这里，我们提供了证据表明 G4 形成与鼠类 DNA 甲基转移酶 Dnmt3a 及其各个结构域的功能之间存在联系。作为 DNA 模型，我们使用了 (i) 能够折叠成平行四链体的寡核苷酸形成的分离 G4，以及 (ii) 插入到带有几个 CpG 位点的双链 DNA 中的相同 G4。通过电泳迁移率变动和荧光偏振测定，我们表明与调节性 PWWP 结构域相比，Dnmt3a 催化结构域 (Dnmt3a-CD) 有效地结合了在两种 DNA 模型中形成的 G4 结构。形成 G4 的寡核苷酸将 DNA 底物从其与 Dnmt3a-CD 的复合物中置换出来，导致酶活性急剧抑制。此外，还揭示了插入到 DNA 双链中的 G4 对特定 CpG 位点甲基化的直接影响。G4 介导的表观遗传调控的可能机制可能包括 G4 对 Dnmt3a 的隔离和/或对 Dnmt3a 在 DNA 表面的寡聚化的破坏。
