N6-methyladenosine in DNA promotes genome stability

DNA base lesions, such as incorporation of uracil into DNA or base mismatches, can be mutagenic and toxic to replicating cells. To discover factors in repair of genomic uracil, we performed a CRISPR knockout screen in the presence of floxuridine, a chemotherapeutic agent that incorporates uracil and fluorouracil into DNA. We identified known factors, such as uracil DNA N-glycosylase (UNG), and unknown factors, such as the N6-adenosine methyltransferase, METTL3, as required to overcome floxuridine-driven cytotoxicity. Visualized with immunofluorescence, the product of METTL3 activity, N6-methyladenosine, formed nuclear foci in cells treated with floxuridine. The observed N6-methyladenosine was embedded in DNA, called 6mA, and these results were confirmed using an orthogonal approach, liquid chromatography coupled to tandem mass spectrometry. METTL3 and 6mA were required for repair of lesions driven by additional base-damaging agents, including raltitrexed, gemcitabine, and hydroxyurea. Our results establish a role for METTL3 and 6mA in promoting genome stability in mammalian cells, especially in response to base damage.