期刊
CELL RESEARCH
卷 30, 期 3, 页码 197-210出版社
SPRINGERNATURE
DOI: 10.1038/s41422-019-0237-5
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资金
- National Natural Science Foundation of China [91753203, 31725014]
- National Key RD Program [2016YFA0500700]
- NIH Medical Scientist Training Program Training Grant [T32GM007205]
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, 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-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 alpha 1 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.
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