During active DNA demethylation, 5‐methylcytosine (5mC) is oxidized by TET proteins to 5‐formyl‐/5‐carboxylcytosine (5fC/5caC) for replacement by unmethylated C by TDG‐initiated DNA base excision repair (BER). Base excision generates fragile abasic sites (AP‐sites) in DNA and has to be coordinated with subsequent repair steps to limit accumulation of genome destabilizing secondary DNA lesions. Here, we show that 5fC/5caC is generated at a high rate in genomes of differentiating mouse embryonic stem cells and that SUMOylation and the BER protein XRCC1 play critical roles in orchestrating TDG‐initiated BER of these lesions. SUMOylation of XRCC1 facilitates physical interaction with TDG and promotes the assembly of a TDG‐BER core complex. Within this TDG‐BERosome, SUMO is transferred from XRCC1 and coupled to the SUMO acceptor lysine in TDG, promoting its dissociation while assuring the engagement of the BER machinery to complete demethylation. Although well‐studied, the biological importance of TDG SUMOylation has remained obscure. Here, we demonstrate that SUMOylation of TDG suppresses DNA strand‐break accumulation and toxicity to PARP inhibition in differentiating mESCs and is essential for neural lineage commitment.

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