TY - JOUR

T1 - Enhanced thermal stability enables human mismatch-specific thymine-DNA glycosylase to catalyse futile DNA repair

AU - Manapkyzy, Diana

AU - Joldybayeva, Botagoz

AU - Ishchenko, Alexander A

AU - Matkarimov, Bakhyt T

AU - Zharkov, Dmitry O

AU - Taipakova, Sabira

AU - Saparbaev, Murat K

N1 - This work was supported by grants from the Committee of Science of the Ministry of Science and Higher Education of the Republic of Kazakhstan grants 13067762 to S.T. and AP19676334 to S.T. and B.T.M.; French National Research Agency (ANR-22-CE12-0034-01) and Electricit\u00E9 de France RB 2021-05 to M.S.; Fondation ARC PJA-2021060003796 to A.A.I.; Russian Ministry of Higher Education and Science [FSUS-2020-0035] to D.O.Z.; D.M. was supported by fellowship Abai-Vern, Kazakhstan. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. There was no additional external funding received for this study. We would like to thank Dr Viktoryia Sidorenko and Arthur Grollman (State University of New York at Stony Brook, Stony Brook, U.S.A.) for their important role in initiating this work.

PY - 2024/10

Y1 - 2024/10

N2 - 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 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, 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.

AB - 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 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, 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.

KW - Thymine DNA Glycosylase/metabolism

KW - Humans

KW - DNA Repair

KW - Enzyme Stability

KW - 5-Methylcytosine/metabolism

KW - DNA/metabolism

KW - Catalytic Domain

KW - Temperature

KW - Base Pair Mismatch

KW - Endodeoxyribonucleases

UR - https://www.scopus.com/record/display.uri?eid=2-s2.0-85206808557&origin=inward&txGid=640d929e0e486091b0c7139a6a66bff9

U2 - 10.1371/journal.pone.0304818

DO - 10.1371/journal.pone.0304818

M3 - Article

C2 - 39423202

VL - 19

JO - PLoS ONE

JF - PLoS ONE

SN - 1932-6203

IS - 10

M1 - e0304818

ER -