Enhanced thermal stability enables human mismatch-specific thymine–DNA glycosylase to catalyse futile DNA repair
Manapkyzy D. Joldybayeva B. Ishchenko A.A. Matkarimov B.T. Zharkov D.O. Taipakova S. Saparbaev M.K.
October 2024Public Library of Science
PLoS ONE
2024#19Issue 10 October
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 neither its isolated catalytic domain (TDGcat) 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 TDGFL-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, TDGFL, but not TDGcat, exhibits prolonged enzyme survival at 37̊C 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, TDGFL 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.
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Department of Molecular Biology and Genetics, Faculty of Biology and Biotechnology, Al-Farabi Kazakh National University, Almaty, Kazakhstan
Scientific Research Institute of Biology and Biotechnology Problems, Al-Farabi Kazakh National University, Almaty, Kazakhstan
Group «Mechanisms of DNA Repair and Carcinogenesis», CNRS UMR9019, Université Paris-Saclay, Gustave Roussy Cancer Campus, Villejuif, Cedex, France
National Laboratory Astana, Nazarbayev University, Astana, Kazakhstan
SB RAS Institute of Chemical Biology and Fundamental Medicine, Novosibirsk, Russian Federation
Department of Natural Sciences, Novosibirsk State University, Novosibirsk, Russian Federation
Department of Molecular Biology and Genetics
Scientific Research Institute of Biology and Biotechnology Problems
Group «Mechanisms of DNA Repair and Carcinogenesis»
National Laboratory Astana
SB RAS Institute of Chemical Biology and Fundamental Medicine
Department of Natural Sciences
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