SUMO-modification and elimination of the active DNA demethylation enzyme TDG in cultured human cells

•TDG and SUMOylated TDG fluctuate during cell cycle.•TDG and SUMOylated TDG are eliminated in S phase cells.•TDG interacts with RNF4 in a SUMOylation-independent manner.•RNF4-depletion has a negligible impact on TDG elimination during S phase. Thymine DNA glycosylase (TDG) is a base excision repair enzyme that interacts with the small ubiquitin-related modifier (SUMO)-targeted ubiquitin E3 ligase RNF4 and functions in the active DNA demethylation pathway. Here we showed that both SUMOylated and non-modified forms of endogenous TDG fluctuated during the cell cycle and in response to drugs that perturbed cell cycle progression, including hydroxyurea and nocodazole. Additionally, we detected a SUMOylation-independent association between TDG and RNF4 in vitro as well as in vivo, and observed that both forms of TDG were efficiently degraded in RNF4-depleted cells when arrested at S phase. Our findings provide insights into the in vivo dynamics of TDG SUMOylation and further clarify the TDG–RNF4 interaction.