Abstract
Thymine-DNA glycosylase (TDG) plays critical roles in DNA base excision repair and DNA demethylation. It has been proposed, based on structural studies and in vitro biochemistry, that sumoylation is required for efficient TDG enzymatic turnover following base excision. However, whether sumoylation is required for TDG activity in vivo has not previously been tested. We have developed an in vivo assay for TDG activity that takes advantage of its recently discovered role in DNA demethylation and selective recognition and repair of 5-carboxylcytosine. Using this assay, we investigated the role of sumoylation in regulating TDG activity through the use of TDG mutants defective for sumoylation and Small Ubiquitin-like Modifier (SUMO) binding and by altering TDG sumoylation through SUMO and SUMO protease overexpression experiments. Our findings indicate that sumoylation and SUMO binding are not essential for TDG-mediated excision and repair of 5-carboxylcytosine bases. Moreover, in vitro assays revealed that apurinic/apyrimidinic nuclease 1 provides nearly maximum stimulation of TDG processing of G·caC substrates. Thus, under our assay conditions, apurinic/apyrimidinic nuclease 1-mediated stimulation or other mechanisms sufficiently alleviate TDG product inhibition and promote its enzymatic turnover in vivo.
Highlights
Regulation and coordination of DNA repair mechanisms is essential for maintaining genome integrity and proper cell function
These results suggest that endogenous Thymine-DNA glycosylase (TDG) activity is insufficient to repair and effectively reduce the levels of 5caC induced by tagged catalytic domain of human TET1 (TETCD) expression
Relieving TDG AP Site Product Inhibition—We developed an assay that takes advantage of the unique specificity of TDG for excising 5caC to investigate whether sumoylation is required for efficient TDG-mediated base excision repair (BER) activity in vivo
Summary
Regulation and coordination of DNA repair mechanisms is essential for maintaining genome integrity and proper cell function. Taking advantage of TDG specificity for 5caC, we developed an in vivo assay to monitor TDG activity and explore requirements for TDG sumoylation and SUMO binding in BER. Our findings reveal that neither sumoylation of TDG nor SUMO binding by TDG are essential for its enzymatic activity under our in vivo assay conditions and raise questions about other mechanisms for relieving product inhibition and other possible functions for TDG sumoylation.
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