MOE Key Laboratory of Protein Sciences, Beijing Advanced Innovation Center for Structural Biology, Beijing Frontier Research Center for Biological Structure, Tsinghua-Peking Joint Center for Life Sciences, Department of Basic Medical Sciences, School of Medicine, Tsinghua University, 100084, Beijing, China.
Department of Genetics and Yale Stem Cell Center, Yale School of Medicine, New Haven, CT, 06520, USA.
Cell Res. 2020 Mar;30(3):197-210. doi: 10.1038/s41422-019-0237-5. Epub 2020 Feb 12.
N-methyladenine (N-mA) of DNA is an emerging epigenetic mark in mammalian genome. Levels of N-mA undergo drastic fluctuation during early embryogenesis, indicative of active regulation. Here we show that the 2-oxoglutarate-dependent oxygenase ALKBH1 functions as a nuclear eraser of N-mA in unpairing regions (e.g., SIDD, Stress-Induced DNA Double Helix Destabilization regions) of mammalian genomes. Enzymatic profiling studies revealed that ALKBH1 prefers bubbled or bulged DNAs as substrate, instead of single-stranded (ss-) or double-stranded (ds-) DNAs. Structural studies of ALKBH1 revealed an unexpected "stretch-out" conformation of its "Flip1" motif, a conserved element that usually bends over catalytic center to facilitate substrate base flipping in other DNA demethylases. Thus, lack of a bending "Flip1" explains the observed preference of ALKBH1 for unpairing substrates, in which the flipped N-mA is primed for catalysis. Co-crystal structural studies of ALKBH1 bound to a 21-mer bulged DNA explained the need of both flanking duplexes and a flipped base for recognition and catalysis. Key elements (e.g., an ALKBH1-specific α1 helix) as well as residues contributing to structural integrity and catalytic activity were validated by structure-based mutagenesis studies. Furthermore, ssDNA-seq and DIP-seq analyses revealed significant co-occurrence of base unpairing regions with N-mA in mouse genome. Collectively, our biochemical, structural and genomic studies suggest that ALKBH1 is an important DNA demethylase that regulates genome N-mA turnover of unpairing regions associated with dynamic chromosome regulation.
DNA 中的 N6-甲基腺嘌呤（N6-methyladenine，N6-mA）是哺乳动物基因组中新兴的表观遗传标记。N6-mA 的水平在早期胚胎发生过程中经历剧烈波动，表明其受到活跃调控。在这里，我们发现 2-氧戊二酸依赖的加氧酶 ALKBH1 可作为哺乳动物基因组中非配对区域（例如，SIDD、应激诱导的 DNA 双螺旋不稳定区域）中 N6-mA 的核清除酶。酶谱分析研究表明，ALKBH1 优先选择起泡或膨出的 DNA 作为底物，而不是单链（ss-）或双链（ds-）DNA。ALKBH1 的结构研究揭示了其“Flip1”基序的出乎意料的“伸展”构象，这是一个保守元件，通常在催化中心上方弯曲，以促进其他 DNA 去甲基酶中的底物碱基翻转。因此，缺乏弯曲的“Flip1”解释了观察到的 ALKBH1 对非配对底物的偏好，在这种底物中，翻转的 N6-mA 已准备好进行催化。ALKBH1 与 21 -mer 膨出 DNA 结合的共晶结构研究解释了识别和催化所需的侧翼双链体和翻转碱基。通过基于结构的诱变研究验证了关键元件（例如，ALKBH1 特异性的α1 螺旋）以及对结构完整性和催化活性有贡献的残基。此外，ssDNA-seq 和 DIP-seq 分析显示，碱基非配对区域与小鼠基因组中的 N6-mA 显著共存。总的来说，我们的生化、结构和基因组研究表明，ALKBH1 是一种重要的 DNA 去甲基酶，可调节与动态染色体调控相关的非配对区域的基因组 N6-mA 周转。
