Department of Pharmacology and Chemical Biology, State Key Laboratory of Oncogenes and Related Genes, Shanghai Jiao Tong University School of Medicine, Shanghai, China.
Research Centre for Marine Drugs, State Key Laboratory of Oncogene and Related Genes, Department of Pharmacy, Ren Ji Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China.
Nat Chem Biol. 2022 Jul;18(7):733-741. doi: 10.1038/s41589-022-01041-3. Epub 2022 Jun 2.
N-methyladenosine (6mA) is a DNA modification that has recently been found to play regulatory roles during mammalian early embryo development and mitochondrial transcription. We found that a dioxygenase CcTet from the fungus Coprinopsis cinerea is also a dsDNA 6mA demethylase. It oxidizes 6mA to the intermediate N-hydroxymethyladenosine (6hmA) with robust activity of 6mA-containing duplex DNA (dsDNA) as well as isolated genomics DNA. Structural characterization revealed that CcTet utilizes three flexible loop regions and two key residues-D337 and G331-in the active pocket to preferentially recognize substrates on dsDNA. A CcTet D337F mutant protein retained the catalytic activity on 6mA but lost activity on 5-methylcytosine. Our findings uncovered a 6mA demethylase that works on dsDNA, suggesting potential 6mA demethylation in fungi and elucidating 6mA recognition and the catalytic mechanism of CcTet. The CcTet D337F mutant protein also provides a chemical biology tool for future functional manipulation of DNA 6mA in vivo.
N6-甲基腺嘌呤（6mA）是一种 DNA 修饰，最近发现它在哺乳动物早期胚胎发育和线粒体转录过程中发挥调控作用。我们发现，真菌毛栓孔菌（Coprinopsis cinerea）中的一种双加氧酶 CcTet 也是一种双链 DNA（dsDNA）6mA 去甲基酶。它将 6mA 氧化为中间产物 N-羟甲基腺嘌呤（6hmA），对含 6mA 的双链 DNA（dsDNA）以及分离的基因组 DNA 具有很强的活性。结构特征表明，CcTet 在活性口袋中利用三个柔性环区和两个关键残基-D337 和 G331-来优先识别 dsDNA 上的底物。CcTet D337F 突变蛋白保留了对 6mA 的催化活性，但对 5-甲基胞嘧啶失去活性。我们的发现揭示了一种作用于 dsDNA 的 6mA 去甲基酶，表明真菌中可能存在 6mA 去甲基化，并阐明了 CcTet 的 6mA 识别和催化机制。CcTet D337F 突变蛋白也为未来在体内对 DNA 6mA 进行功能操作提供了一种化学生物学工具。
