Volume 30, No 3, Mar 2020

ISSN: 1001-0602 
EISSN: 1748-7838 2018 
impact factor 17.848* 
(Clarivate Analytics, 2019)

Volume 30 Issue 3, March 2020: 197-210   |  Open Access

ORIGINAL ARTICLES

Mammalian ALKBH1 serves as an N6-mA demethylase of unpairing DNA

Min Zhang¹, Shumin Yang¹, Raman Nelakanti², Wentao Zhao¹, Gaochao Liu¹, Zheng Li², Xiaohui Liu³, Tao Wu ^2,4 , Andrew Xiao² and Haitao Li ¹

¹ 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; ²Department of Genetics and Yale Stem Cell Center, Yale School of Medicine, New Haven, CT 06520, USA and ³National Protein Science Technology Center, School of Life Sciences, Tsinghua University, 100084 Beijing, China
Correspondence: Andrew Xiao (andrew.xiao@yale.edu) or Haitao Li (lht@tsinghua.edu.cn)4 Present address: Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030, USAThese authors contributed equally: Min Zhang, Shumin Yang, Ram

N6-methyladenine (N6-mA) of DNA is an emerging epigenetic mark in mammalian genome. Levels of N6-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 N6-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 N6-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 N6-mA in mouse genome. Collectively, our biochemical, structural and genomic studies suggest that ALKBH1 is an important DNA demethylase that regulates genome N6-mA turnover of unpairing regions associated with dynamic chromosome regulation.

https://doi.org/10.1038/s41422-019-0237-5

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