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

Abstract N 6 -methyladenine ( N 6 -mA) of DNA is an emerging epigenetic mark in mammalian genome. Levels of N 6 -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 6 -mA in unpairing regions (e.g., SIDD, S tress- I nduced D NA Double Helix D estabilization 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 6 -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 6 -mA in mouse genome. Collectively, our biochemical, structural and genomic studies suggest that ALKBH1 is an important DNA demethylase that regulates genome N 6 -mA turnover of unpairing regions associated with dynamic chromosome regulation.