DNA damage-induced YTHDC1 O-GlcNAcylation promotes homologous recombination by enhancing m6A binding

N6-methyladenosine (m⁶A) is the most prevalent internal RNA modification, and its regulators include writers, readers and erasers. m⁶A is under stringent control and takes part in many biological events, but it is not known whether there is an interplay between m⁶A and glycosylation. Here we investigated an m⁶A reader, YTHDC1, which has been shown to be recruited to the DNA-RNA hybrid at DNA damage sites and regulate homologous recombination (HR) during DNA damage repair. We found that YTHDC1 is subject to O-linked β-N-acetylglucosamine (O-GlcNAc) modification at Ser396 upon DNA damage, which is pivotal for YTHDC1 chromatin binding and ionization radiation induced focus (IRIF) formation. RNA immunoprecipitation (RIP) and molecular dynamics (MD) simulations indicate that O-GlcNAcylation is vital for YTHDC1 to bind with m⁶A RNA. Fluorescence recovery after photo bleaching (FRAP) analysis revealed that YTHDC1 O-GlcNAcylation is essential for DNA damage-induced YTHDC1-m⁶A condensate formation. We further demonstrate that YTHDC1 O-GlcNAcylation promotes HR-mediated DNA damage repair and cell survival, probably through recruitment of Rad51 to the damage sites. We propose that YTHDC1 O-GlcNAcylation is instrumental for HR.