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.

Background: Consumption of aristolochic acids (AA) from the plant Aristolochia results in the formation of bulky aristolactam-dA (dA-AL) and aristolactam-dG (dG-AL) adducts in cellular DNA ultimately leading to the development of urothelial cancer. Intriguingly, the dA-AL adducts induce A•T→T•A transversions in tumor cells preferentially in CpA*→TpG context. The human mismatch-specific thymine-DNA glycosylase (TDG) protects cells against mutagenesis induced by spontaneous deamination of 5-methylcytosine (5mC) by removing thymine opposite to guanine in a CpG context in the base excision repair (BER) pathway. Nevertheless, challenges for DNA glycosylases to the faithful discrimination between non-damaged and damaged DNA strands do exist, such as mismatched pairs between two canonical bases, which may result due to DNA polymerase errors during replication. Previously, we demonstrated that TDG is prone to aberrant excision of T opposite to damaged adenine in duplex DNA in CpA*/TpG context. Methods: In the present work, using in vitro reconstitution assays, we investigated whether TDG participates in the aberrant removal of thymine opposite to dA-AL adducts in duplex DNA. Results: We have demonstrated that TDG either does not excise thymine or does so with extremely low efficiency when it is paired with dA-AL or dG-ALII adducts in duplex DNA. At the same time, TDG excises with high efficiency thymine opposite to guanine and hypoxanthine in T•G and T•Hx mispairs. Discussion: These findings strongly suggest that the human TDG is not involved in the aberrant DNA repair of AA-induced DNA damage.

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 not its truncated catalytic domain TDG (TDG ^cat ) 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 ( k obs) of TDG ^FL -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, TDG ^FL , but not TDG ^cat , exhibits prolonged enzyme stability under 37°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, TDG ^FL 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.

The Escherichia coli adenine-DNA glycosylase (MutY) and its human homologue, MUTYH, protect cells against oxygen-free radical-induced mutagenesis by excising regular adenine impaired with 8-oxo-7,8-dihydro-guanine (8oxoG) in the base excision repair (BER) pathway. However, removal of adenine by MutY and MUTYH from an A·8oxoG pair generated via misincorporation of an oxidized nucleotide during DNA synthesis might induce A·T→C·G transversions. Here, to examine MutY and MUTYH in vitro activities, we used short synthetic DNA duplexes in which the target adenine residue was positioned opposite a variety of DNA base modifications. MUTYH does not excise mismatched adenine in non-canonical DNA substrates, whereas MutY excises adenine mispaired with 1,3-d(GpNpG) cisplatin intra-strand crosslink. In addition, we characterized four MUTYH variants associated with cancer risk, which exhibit the following order of DNA glycosylase deficiency: WT ≥ G169D > G202E ≈ Y165C >> D222N. Human adenine-DNA glycosylase MUTYH and its mutant variants, contrary to bacterial MutY, are not prone to aberrant removal of regular adenine residues opposite modified residues in DNA duplexes. We hypothesize that E. coli MutY is prone to aberrant repair under certain conditions and that this may prevent incorporation of adenine opposite blocking lesions in the template strand.

Тимин ДНК-гликозилаза (TDG) привлекла к себе внимание благодаря своей способности удалять тимин, т.е. нормальное основание ДНК, из G:T неправильных пар. Это указывало на его функцию в эксцизионной репарации оснований ДНК в восстановлении пар оснований G:C после дезаминирования 5-метилцитозина. Из-за очевидной каталитической неэффективности, некоторые считали TDG плохим ферментом репарации ДНК без важной биологической функции. Однако было продемонстрировано, что этот фермент может действовать как ключевой игрок в транскрипционной регуляции через взаимодействие с различными ядерными рецепторами и факторами транскрипции что указывает на ее функцию в регуляции генов, которая, по-видимому, имеет решающее значение в процессах развития. Другие показали, что TDG участвует в эпигенетической регуляции экспрессии генов защищая CpG-богатые промоторы от de novo ДНК метилирования и может инициировать аберрантную репарацию. Недавние исследования на животных выявили связь между потерей TDG и началом и началом опухолеобразования. Таким образом целью данного обзора является собрать воедино результаты различных экспериментов, в которых исследовалась тимин ДНК-гликозилаза с момента ее открытия, и оценить их влияние ля определения возможных физиологических ролей этого фермента.

Cellular organisms store heritable information in two forms, genetic and epigenetic, the latter being largely dependent on cytosine methylation (5mC). Chargaff’s Second Parity Rule (CSPR) describes the nucleotide composition of cellular genomes in terms of intra-strand DNA symmetry. However, it remains unknown whether DNA methylation patterns display intra-strand DNA symmetry. Computational analysis was conducted of the DNA methylation patterns observed in human cell lines and in tissue samples from healthy donors. Analysis of 5mC marks in mutually reverse-complementary pairs of short oligomers, containing CpG dinucleotide in the middle, revealed deviations from CSPR and methylation asymmetry that can be observed for two non-overlapping mirror groups defined by CpG methylation values. Deviations from CSPR, together with combinatorial probabilities of pattern distributions and computer simulations, highlight the non-random nature of methylation processes and enabled us to identify specific cell types as outliers. Further analysis revealed a compensatory methylation asymmetry that reduces deviations from intra-strand symmetry and implies the existence of strand-specific methylation during cell differentiation. Among six pairs of reverse-complementary tetranucleotides, four pairs with specific sequence motifs display pronounced methylation asymmetry. This mirror asymmetry may be associated with chromosome folding and the formation of a complex three-dimensional landscape.