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

Cell Res. 2020 Mar;30(3):197-210. doi: 10.1038/s41422-019-0237-5. Epub 2020 Feb 12.


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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenosine / analogs & derivatives*
  • AlkB Homolog 1, Histone H2a Dioxygenase* / chemistry
  • AlkB Homolog 1, Histone H2a Dioxygenase* / physiology
  • Animals
  • Cells, Cultured
  • DNA / chemistry*
  • DNA Demethylation*
  • Embryonic Stem Cells
  • Mice
  • Molecular Structure
  • Protein Binding


  • DNA
  • N-methyladenosine
  • AlkB Homolog 1, Histone H2a Dioxygenase
  • Alkbh1 protein, mouse
  • Adenosine