N⁶-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 N⁶-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.

N ⁶ -甲基腺苷 (6mA) 是一种 DNA 修饰，最近被发现在哺乳动物早期胚胎发育和线粒体转录过程中发挥调节作用。我们发现来自真菌Coprinopsis cinerea的双加氧酶 CcTet也是 dsDNA 6mA 去甲基化酶。它将6mA氧化成中间体N ⁶-羟甲基腺苷 (6hmA)，对含有 6mA 的双链 DNA (dsDNA) 以及分离的基因组 DNA 具有强大的活性。结构表征表明，CcTet 利用活性口袋中的三个柔性环区域和两个关键残基——D337 和 G331——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，但失去了对 5-甲基胞嘧啶的活性。我们的研究结果揭示了一种作用于 dsDNA 的 6mA 去甲基化酶，表明真菌中可能存在 6mA 去甲基化，并阐明了 6mA 识别和 CcTet 的催化机制。CcTet D337F 突变蛋白还为未来体内 DNA 6mA 的功能操作提供了一种化学生物学工具。