Defining the impact of sumoylation on substrate binding and catalysis by thymine DNA glycosylase

Thymine DNA glycosylase (TDG) excises thymine from mutagenic G·T mispairs generated by deamination of 5-methylcytosine (mC) and it removes two mC derivatives, 5−formylcytosine (fC) and 5−carboxylcytosine (caC), in a multistep pathway for DNA demethylation. TDG is modified by small ubiquitin-like modifier (SUMO) proteins, but the impact of sumoylation on TDG activity is poorly defined and the functions of TDG sumoylation remain unclear. We determined the effect of TDG sumoylation, by SUMO-1 or SUMO-2, on substrate binding and catalytic parameters. Single turnover experiments reveal that sumoylation dramatically impairs TDG base-excision activity, such that G·T activity is reduced by ≥45-fold and fC and caC are excised slowly, with a reaction half-life of ≥9 min (37°C). Fluorescence anisotropy studies reveal that unmodified TDG binds tightly to G·fC and G·caC substrates, with dissociation constants in the low nanomolar range. While sumoylation of TDG weakens substrate binding, the residual affinity is substantial and is comparable to that of biochemically-characterized readers of fC and caC. Our findings raise the possibility that sumoylation enables TDG to function, at least transiently, as reader of fC and caC. Notably, sumoylation could potentially facilitate TDG recruitment of other proteins, including transcription factors or epigenetic regulators, to these sites in DNA.