Enhanced thermal stability enables human mismatch-specific thymine-DNA glycosylase to catalyse futile DNA repair

Abstract

Human thymine-DNA glycosylase (TDG) excises T mispaired with G in a CpG context to initiate the base excision repair (BER) pathway. TDG is also involved in epigenetic regulation of gene expression by participating in active DNA demethylation. Here we demonstrate that under extended incubation time the full-length TDG (TDGFL), but not its truncated catalytic domain TDG (TDGcat) or methyl-binding protein 4 (MBD4) DNA glycosylase, exhibits a significant excision activity towards T and C in regular non-damaged DNA duplex in TpG/CpA and CpG/CpG contexts. Time course of the cleavage product generation under single-turnover conditions shows that the maximal rate of base excision (kobs) of TDGFL-catalysed excision of T in the T*A base pair (0.0014 - 0.0069 min-1) is 85 - 330-fold lower than that of T in T*G mismatch (0.47 - 0.61 min-1). Unexpectedly, TDGFL, but not TDGcat, exhibits prolonged enzyme stability under 37{degrees}C when incubated in the presence of equimolar concentration of non-specific DNA duplex, suggesting that the disordered N- and C-terminal domains of TDG can interact with DNA and stabilize the overall conformation of the protein. Noteworthy, TDGFL is able to excise 5-hydroxymethylcytosine (5hmC), but not 5-methylcytosine residues, from duplex DNA with the efficiency that could be physiologically relevant in post-mitotic cells. Our findings demonstrate that, under the experimental conditions used, TDG catalyses sequence context-dependent removal of T, C and 5hmC residues in regular DNA duplexes. We propose that in vivo this futile DNA repair may lead to formation of persistent single-strand breaks in non-methylated regions of chromosomal DNA.