Journal Article

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

Ishchenko, Alexander A., Taipakova, Sabira, Manapkyzy, Diana, Zharkov, Dmitry O., Joldybayeva, Botagoz, Matkarimov, Bakhyt T., Saparbaev, Murat K.

2024

Overview

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 (TDG FL ), but neither its isolated catalytic domain (TDG cat ) nor methyl-CpG binding domain-containing protein 4 (MBD4) DNA glycosylase, exhibits 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 accumulation under single-turnover conditions shows that the apparent rate constant for TDG FL -catalysed excision of T from T•A base pairs (0.0014–0.0069 min −1 ) is 85–330-fold lower than for the excision of T from T•G mispairs (0.47–0.61 min −1 ). Unexpectedly, TDG FL , but not TDG cat , exhibits prolonged enzyme survival at 37°C when incubated in the presence of equimolar concentrations of a 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. Notably, TDG FL was 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 from regular DNA duplexes. We propose that in vivo the TDG-initiated futile DNA BER may lead to formation of persistent single-strand breaks in non-methylated or hydroxymethylated chromatin regions.

• Publisher: Public Library of Science
• Subject: 5-Methylcytosine - analogs & derivatives / 5-Methylcytosine - metabolism / Base excision repair / Base Pair Mismatch / Biochemistry / Biochemistry, Molecular Biology / Catalytic Domain / Chromatin / Context / CpG islands / Demethylation / Deoxyribonucleic acid / DNA / DNA - metabolism / DNA damage / DNA glycosylase / DNA glycosylases / DNA methylation / DNA Repair / E coli / Endodeoxyribonucleases / Enzyme Stability / Epigenetics / Gene expression / Genomics / Humans / Kinases / Life Sciences / Methods / Mutation / Nucleotide sequence / Oxidation / Physiological aspects / Protein structure / Proteins / Residues / Temperature / Thermal properties / Thermal stability / Thymine / Thymine DNA Glycosylase - chemistry / Thymine DNA Glycosylase - genetics / Thymine DNA Glycosylase - metabolism