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Spontaneous hydrolytic deamination of cytosine to uracil (U) in DNA is a constant source of genome instability in cells. This mutagenic process is greatly enhanced at high temperatures and in single-stranded DNA. If not repaired, these uracil residues give rise to C → T transitions, which are the most common spontaneous mutations occurring in living organisms and are frequently found in human tumors. In the majority of species, uracil residues are removed from DNA by specific uracil-DNA glycosylases in the base excision repair pathway. Alternatively, in certain archaeal organisms, uracil residues are eliminated by apurinic/apyrimidinic (AP) endonucleases in the nucleotide incision repair pathway. Here, we characterized the substrate specificity of the major human AP endonuclease 1, APE1, toward U in duplex DNA. APE1 cleaves oligonucleotide duplexes containing a single U⋅G base pair; this activity depends strongly on the sequence context and the base opposite to U. The apparent kinetic parameters of the reactions show that APE1 has high affinity for DNA containing U but cleaves the DNA duplex at an extremely low rate. MALDI-TOF MS analysis of the reaction products demonstrated that APE1-catalyzed cleavage of a U⋅G duplex generates the expected DNA fragments containing a 5'-terminal deoxyuridine monophosphate. The fact that U in duplex DNA is recognized and cleaved by APE1 in vitro suggests that this property of the exonuclease III family of AP endonucleases is remarkably conserved from Archaea to humans. We propose that nucleotide incision repair may act as a backup pathway to base excision repair to remove uracils arising from cytosine deamination.

Members of the microRNA-29 (miR-29) family directly target the DNA methyltransferases, DNMT3A and DNMT3B. Disturbances in the expression levels of miR-29 have been linked to tumorigenesis and tumor aggressiveness. Members of the miR-29 family are currently thought to repress DNA methylation and suppress tumorigenesis by protecting against de novo methylation. Here, we report that members of the miR-29 family repress the activities of DNA methyltransferases and DNA demethylases, which have opposing roles in control of DNA methylation status. Members of the miR-29 family directly inhibited DNA methyltransferases and two major factors involved in DNA demethylation, namely tet methylcytosine dioxygenase 1 (TET1) and thymine DNA glycosylase (TDG). Overexpression of miR-29 upregulated the global DNA methylation level in some cancer cells and downregulated DNA methylation in other cancer cells, suggesting that miR-29 suppresses tumorigenesis by protecting against changes in the existing DNA methylation status rather than by preventing de novo methylation of DNA.

A novel homogeneous fluorescence assay strategy for highly sensitive detection of thymine DNA glycosylase (TDG) enzyme activity based on the exonuclease-mediated signal amplification reaction was reported.

The mammalian thymine DNA glycosylase (TDG) excises 5-carboxylcytosine (5caC) when paired with a guanine in a CpG sequence, in addition to mismatched bases. Here we present a complex structure of the human TDG catalytic mutant, asparagine 140 to alanine (N140A), with a 28-base pair DNA containing a G:5caC pair at pH 4.6. TDG interacts with the carboxylate moiety of target nucleotide 5caC using the side chain of asparagine 230 (N230), instead of asparagine 157 (N157) as previously reported. Mutation of either N157 or N230 residues to aspartate has minimal effect on G:5caC activity while significantly reducing activity on G:U substrate. Combination of both the asparagine-to-aspartate mutations (N157D/N230D) resulted in complete loss of activity on G:5caC while retaining measurable activity on G:U, implying that 5caC can adopt alternative conformations (either N157-interacting or N230-interacting) in the TDG active site to interact with either of the two asparagine side chain for 5caC excision., (Copyright © 2013 Elsevier B.V. All rights reserved.)

What's the catch? A pyrrolysine analogue bearing a terminal alkyne and an ester functionality can be incorporated into recombinant proteins and render them amenable to capture by the click reaction and subsequent release through ester hydrolysis. The utility of this pyrrolysine-inspired technology is demonstrated for the identification of SUMOylation sites., (Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

TET dioxygenases successively oxidize 5-methylcytosine (5mC) in mammalian genomes to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC). 5fC/5caC can be excised and repaired to regenerate unmodified cytosines by thymine-DNA glycosylase (TDG) and base excision repair (BER) pathway, but it is unclear to what extent and at which part of the genome this active demethylation process takes place. Here, we have generated genome-wide distribution maps of 5hmC/5fC/5caC using modification-specific antibodies in wild-type and Tdg-deficient mouse embryonic stem cells (ESCs). In wild-type mouse ESCs, 5fC/5caC accumulates to detectable levels at major satellite repeats but not at nonrepetitive loci. In contrast, Tdg depletion in mouse ESCs causes marked accumulation of 5fC and 5caC at a large number of proximal and distal gene regulatory elements. Thus, these results reveal the genome-wide view of iterative 5mC oxidation dynamics and indicate that TET/TDG-dependent active DNA demethylation process occurs extensively in the mammalian genome., (Copyright © 2013 Elsevier Inc. All rights reserved.)

The base excision repair system is vital to the repair of endogenous and exogenous DNA damage. This pathway is initiated by one of several DNA glycosylases that recognizes and excises specific DNA lesions in a coordinated fashion. Methyl-CpG Domain Protein 4 (MBD4) and Thymine DNA Glycosylase (TDG) are the two major G:T glycosylases that remove thymine generated by the deamination of 5-methylcytosine. Both of these glycosylases also remove a variety of other base lesions, including G:U and preferentially act at CpG sites throughout the genome. Many have questioned the purpose of seemingly redundant glycosylases, but new information has emerged to suggest MBD4 and TDG have diverse biological functions. MBD4 has been closely linked to apoptosis, while TDG has been clearly implicated in transcriptional regulation. This article reviews all of these developments, and discusses the consequences of germline and somatic mutations that lead to non-synonymous amino acid substitutions on MBD4 and TDG protein function. In addition, we report the finding of alternatively spliced variants of MBD4 and TDG and the results of functional studies of a tumor-associated variant of MBD4., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Imaging single proteins within cells is challenging if the possibility of artefacts due to tagging or to recognition by antibodies is to be avoided. It is generally believed that the biological properties of proteins remain unaltered when (14)N isotopes are replaced with (15)N. (15)N-enriched proteins can be localised by dynamic Secondary Ion Mass Spectrometry (D-SIMS). We describe here a novel imaging analysis algorithm to detect a few (15)N-enriched proteins--and even a single protein--within a cell using D-SIMS. The algorithm distinguishes statistically between a low local increase in (15)N isotopic fraction due to an enriched protein and a stochastic increase due to the background. To determine the number of enriched proteins responsible for the increase in the isotopic fraction, we use sequential D-SIMS images in which we compare the measured isotopic fractions to those expected if 1, 2 or more enriched proteins are present. The number of enriched proteins is the one that gives the best fit between the measured and the expected values. We used our method to localise (15)N-enriched thymine DNA glycosylase (TDG) and retinoid X receptor α (RXRα) proteins delivered to COS-7 cells. We show that both a single TDG and a single RXRα can be detected. After 4 h incubation, both proteins were found mainly in the nucleus; RXRα as a monomer or dimer and TDG only as a monomer. After 7 h, RXRα was found in the nucleus as a monomer, dimer or tetramer, whilst TDG was no longer in the nucleus and instead formed clusters in the cytoplasm. After 24 h, RXRα formed clusters in the cytoplasm, and TDG was no longer detectable. In conclusion, single unmodified proteins in cells can be counted and localised with 50 nm resolution by combining D-SIMS with our method of analysis.

Hydroxyl radicals predominantly react with the C(8) of purines forming 7,8-dihydro-8-oxoguanine (8oxoG) and 7,8-dihydro-8-oxoadenine (8oxoA) adducts, which are highly mutagenic in mammalian cells. The majority of oxidized DNA bases are removed by DNA glycosylases in the base excision repair pathway. Here, we report for the first time that human thymine-DNA glycosylase (hTDG) and Escherichia coli mismatch-specific uracil-DNA glycosylase (MUG) can remove 8oxoA from 8oxoA•T, 8oxoA•G and 8oxoA•C pairs. Comparison of the kinetic parameters of the reaction indicates that full-length hTDG excises 8oxoA, 3,N(4)-ethenocytosine (εC) and T with similar efficiency (k(max) = 0.35, 0.36 and 0.16 min(-1), respectively) and is more proficient as compared with its bacterial homologue MUG. The N-terminal domain of the hTDG protein is essential for 8oxoA-DNA glycosylase activity, but not for εC repair. Interestingly, the TDG status had little or no effect on the proliferation rate of mouse embryonic fibroblasts after exposure to γ-irradiation. Nevertheless, using whole cell-free extracts from the DNA glycosylase-deficient murine embryonic fibroblasts and E. coli, we demonstrate that the excision of 8oxoA from 8oxoA•T and 8oxoA•G has an absolute requirement for TDG and MUG, respectively. The data establish that MUG and TDG can counteract the genotoxic effects of 8oxoA residues in vivo.

Thymine DNA glycosylase (TDG) belongs to the superfamily of uracil DNA glycosylases (UDG) and is the first enzyme in the base-excision repair pathway (BER) that removes thymine from G:T mismatches at CpG sites. This glycosylase activity has also been found to be critical for active demethylation of genes involved in embryonic development. Here we show that wild-type p53 transcriptionally regulates TDG expression. Chromatin immunoprecipitation (ChIP) and luciferase assays indicate that wild-type p53 binds to a domain of TDG promoter containing two p53 consensus response elements (p53RE) and activates its transcription. Next, we have used a panel of cell lines with different p53 status to demonstrate that TDG mRNA and protein expression levels are induced in a p53-dependent manner under different conditions. This panel includes isogenic breast and colorectal cancer cell lines with wild-type or inactive p53, esophageal squamous cell carcinoma cell lines lacking p53 or expressing a temperature-sensitive p53 mutant and normal human bronchial epithelial cells. Induction of TDG mRNA expression is accompanied by accumulation of TDG protein in both nucleus and cytoplasm, with nuclear re-localization occurring upon DNA damage in p53-competent, but not -incompetent, cells. These observations suggest a role for p53 activity in TDG nuclear translocation. Overall, our results show that TDG expression is directly regulated by p53, suggesting that loss of p53 function may affect processes mediated by TDG, thus negatively impacting on genetic and epigenetic stability.

The mammalian thymine DNA glycosylase (TDG) is implicated in active DNA demethylation via the base excision repair pathway. TDG excises the mismatched base from G:X mismatches, where X is uracil, thymine or 5-hydroxymethyluracil (5hmU). These are, respectively, the deamination products of cytosine, 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC). In addition, TDG excises the Tet protein products 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) but not 5hmC and 5mC, when paired with a guanine. Here we present a post-reactive complex structure of the human TDG domain with a 28-base pair DNA containing a G:5hmU mismatch. TDG flips the target nucleotide from the double-stranded DNA, cleaves the N-glycosidic bond and leaves the C1' hydrolyzed abasic sugar in the flipped state. The cleaved 5hmU base remains in a binding pocket of the enzyme. TDG allows hydrogen-bonding interactions to both T/U-based (5hmU) and C-based (5caC) modifications, thus enabling its activity on a wider range of substrates. We further show that the TDG catalytic domain has higher activity for 5caC at a lower pH (5.5) as compared to the activities at higher pH (7.5 and 8.0) and that the structurally related Escherichia coli mismatch uracil glycosylase can excise 5caC as well. We discuss several possible mechanisms, including the amino-imino tautomerization of the substrate base that may explain how TDG discriminates against 5hmC and 5mC.

Active DNA demethylation in mammals occurs via hydroxylation of 5-methylcytosine to 5-hydroxymethylcytosine (5hmC) by the ten-eleven translocation family of proteins (TETs). 5hmC residues in DNA can be further oxidized by TETs to 5-carboxylcytosines and/or deaminated by the Activation Induced Deaminase/Apolipoprotein B mRNA-editing enzyme complex family proteins to 5-hydromethyluracil (5hmU). Excision and replacement of these intermediates is initiated by DNA glycosylases such as thymine-DNA glycosylase (TDG), methyl-binding domain protein 4 (MBD4) and single-strand specific monofunctional uracil-DNA glycosylase 1 in the base excision repair pathway. Here, we report detailed biochemical and structural characterization of human MBD4 which contains mismatch-specific TDG activity. Full-length as well as catalytic domain (residues 426-580) of human MBD4 (MBD4(cat)) can remove 5hmU when opposite to G with good efficiency. Here, we also report six crystal structures of human MBD4(cat): an unliganded form and five binary complexes with duplex DNA containing a T•G, 5hmU•G or AP•G (apurinic/apyrimidinic) mismatch at the target base pair. These structures reveal that MBD4(cat) uses a base flipping mechanism to specifically recognize thymine and 5hmU. The recognition mechanism of flipped-out 5hmU bases in MBD4(cat) active site supports the potential role of MBD4, together with TDG, in maintenance of genome stability and active DNA demethylation in mammals.

Background: Methylation of cytosine in DNA (5mC) is an important epigenetic mark that is involved in the regulation of genome function. During early embryonic development in mammals, the methylation landscape is dynamically reprogrammed in part through active demethylation. Recent advances have identified key players involved in active demethylation pathways, including oxidation of 5mC to 5-hydroxymethylcytosine (5hmC) and 5-formylcytosine (5fC) by the TET enzymes, and excision of 5fC by the base excision repair enzyme thymine DNA glycosylase (TDG). Here, we provide the first genome-wide map of 5fC in mouse embryonic stem (ES) cells and evaluate potential roles for 5fC in differentiation., Results: Our method exploits the unique reactivity of 5fC for pulldown and high-throughput sequencing. Genome-wide mapping revealed 5fC enrichment in CpG islands (CGIs) of promoters and exons. CGI promoters in which 5fC was relatively more enriched than 5mC or 5hmC corresponded to transcriptionally active genes. Accordingly, 5fC-rich promoters had elevated H3K4me3 levels, associated with active transcription, and were frequently bound by RNA polymerase II. TDG down-regulation led to 5fC accumulation in CGIs in ES cells, which correlates with increased methylation in these genomic regions during differentiation of ES cells in wild-type and TDG knockout contexts., Conclusions: Collectively, our data suggest that 5fC plays a role in epigenetic reprogramming within specific genomic regions, which is controlled in part by TDG-mediated excision. Notably, 5fC excision in ES cells is necessary for the correct establishment of CGI methylation patterns during differentiation and hence for appropriate patterns of gene expression during development.

DNA methylation has long been considered a very stable DNA modification in mammals that could only be removed by replication in the absence of remethylation - that is, by mere dilution of this epigenetic mark (so-called passive DNA demethylation). However, in recent years, a significant number of studies have revealed the existence of active processes of DNA demethylation in mammals, with important roles in development and transcriptional regulation, allowing the molecular mechanisms of active DNA demethylation to be unraveled. In this article, we review the recent literature highlighting the prominent role played in active DNA demethylation by base excision repair and especially by TDG.

Cells with DNA repair defects have increased genomic instability and are more likely to acquire secondary mutations that bring about cellular transformation. We describe the frequency and spectrum of somatic mutations involving several tumor suppressor genes in the rectal carcinoma of a 13-year-old girl harboring biallelic, germline mutations in the DNA mismatch repair gene PMS2. Apart from microsatellite instability, the tumor DNA contained a number of C:G→T:A or G:C→A:T transitions in CpG dinucleotides, which often result through spontaneous deamination of cytosine or 5-methylcytosine. Four DNA glycosylases, UNG2, SMUG1, MBD4 and TDG, are involved in the repair of these deamination events. We identified a heterozygous missense mutation in TDG, which was associated with TDG protein loss in the tumor. The CpGs mutated in this patient's tumor are generally methylated in normal colonic mucosa. Thus, it is highly likely that loss of TDG contributed to the supermutator phenotype and that most of the point mutations were caused by deamination of 5-methylcytosine to thymine, which remained uncorrected owing to the TDG deficiency. This case provides the first in vivo evidence of the key role of TDG in protecting the human genome against the deleterious effects of 5-methylcytosine deamination., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Cytosine residues in mammalian DNA occur in at least three forms, cytosine (C), 5-methylcytosine (M; 5mC) and 5-hydroxymethylcytosine (H; 5hmC). During semi-conservative DNA replication, hemi-methylated (M/C) and hemi-hydroxymethylated (H/C) CpG dinucleotides are transiently generated, where only the parental strand is modified and the daughter strand contains native cytosine. Here, we explore the role of DNA methyltransferases (DNMT) and ten eleven translocation (Tet) proteins in perpetuating these states after replication, and the molecular basis of their recognition by methyl-CpG-binding domain (MBD) proteins. Using recombinant proteins and modified double-stranded deoxyoligonucleotides, we show that DNMT1 prefers a hemi-methylated (M/C) substrate (by a factor of >60) over hemi-hydroxymethylated (H/C) and unmodified (C/C) sites, whereas both DNMT3A and DNMT3B have approximately equal activity on all three substrates (C/C, M/C and H/C). Binding of MBD proteins to methylated DNA inhibited Tet1 activity, suggesting that MBD binding may also play a role in regulating the levels of 5hmC. All five MBD proteins generally have reduced binding affinity for 5hmC relative to 5mC in the fully modified context (H/M versus M/M), though their relative abilities to distinguish the two varied considerably. We further show that the deamination product of 5hmC could be excised by thymine DNA glycosylase and MBD4 glycosylases regardless of context.

DNA base excision repair is essential for maintaining genomic integrity and for active DNA demethylation, a central element of epigenetic regulation. A key player is thymine DNA glycosylase (TDG), which excises thymine from mutagenic G·T mispairs that arise by deamination of 5-methylcytosine (mC). TDG also removes 5-formylcytosine and 5-carboxylcytosine, oxidized forms of mC produced by Tet enzymes. Recent studies show that the glycosylase activity of TDG is essential for active DNA demethylation and for embryonic development. Our understanding of how repair enzymes excise modified bases without acting on undamaged DNA remains incomplete, particularly for mismatch glycosylases such as TDG. We solved a crystal structure of TDG (catalytic domain) bound to a substrate analog and characterized active-site residues by mutagenesis, kinetics, and molecular dynamics simulations. The studies reveal how TDG binds and positions the nucleophile (water) and uncover a previously unrecognized catalytic residue (Thr197). Remarkably, mutation of two active-site residues (Ala145 and His151) causes a dramatic enhancement in G·T glycosylase activity but confers even greater increases in the aberrant removal of thymine from normal A·T base pairs. The strict conservation of these residues may reflect a mechanism used to strike a tolerable balance between the requirement for efficient repair of G·T lesions and the need to minimize aberrant action on undamaged DNA, which can be mutagenic and cytotoxic. Such a compromise in G·T activity can account in part for the relatively weak G·T activity of TDG, a trait that could potentially contribute to the hypermutability of CpG sites in cancer and genetic disease.

Our ability to selectively manipulate gene expression by epigenetic means is limited, as there is no approach for targeted reactivation of epigenetically silenced genes, in contrast to what is available for selective gene silencing. We aimed to develop a tool for selective transcriptional activation by DNA demethylation. Here we present evidence that direct targeting of thymine-DNA-glycosylase (TDG) to specific sequences in the DNA can result in local DNA demethylation at potential regulatory sequences and lead to enhanced gene induction. When TDG was fused to a well-characterized DNA-binding domain [the Rel-homology domain (RHD) of NFκB], we observed decreased DNA methylation and increased transcriptional response to unrelated stimulus of inducible nitric oxide synthase (NOS2). The effect was not seen for control genes lacking either RHD-binding sites or high levels of methylation, nor in control mock-transduced cells. Specific reactivation of epigenetically silenced genes may thus be achievable by this approach, which provides a broadly useful strategy to further our exploration of biological mechanisms and to improve control over the epigenome.

Human thymine DNA glycosylase (hTDG) efficiently excises 5-carboxylcytosine (5caC), a key oxidation product of 5-methylcytosine in genomic DNA, in a recently discovered cytosine demethylation pathway. We present here the crystal structures of the hTDG catalytic domain in complex with duplex DNA containing either 5caC or a fluorinated analog. These structures, together with biochemical and computational analyses, reveal that 5caC is specifically recognized in the active site of hTDG, supporting the role of TDG in mammalian 5-methylcytosine demethylation.

Thymine DNA glycosylase (TDG) is involved in the repair of G:T and G:U mismatches caused by hydrolytic deamination of 5-methylcytosine and cytosine, respectively. Recent studies have shown that TDG not only has G-T/U glycosylase activities but also acts in the maintaining proper epigenetic status. In order to investigate the function of TDG in vivo, mice lacking Tdg, Tdg (-/-), were generated. Tdg mutant mice died in utero by 11.5 days post coitum (dpc), although there were no significant differences in the spontaneous mutant frequencies between wild type and Tdg (-/-) embryos. On the other hand, the levels of noradrenaline in 10.5 dpc whole embryos, which is necessary for normal embryogenesis, were dramatically reduced in Tdg (-/-) embryos. Consequently, we tested the effect of D, L-threo-3, 4-dihydroxyphenylserine (DOPS), a synthetic precursor of noradrenaline, on the survival of the Tdg (-/-) embryos. DOPS was given to pregnant Tdg (+/-) mice from 6.5 dpc through drinking water. Most of the Tdg (-/-) embryos were alive at 11.5 dpc, and they were partially rescued up to 14.5 dpc by the administration of DOPS. In contrast, the administration of L-3, 4-dihydroxyphenylalanine (L-DOPA) had marginal effects on Tdg (-/-) embryonic lethality. No embryo was alive without DOPS beyond 11.5 dpc, suggesting that the lethality in (-/-) embryos is partially due to the reduction of noradrenaline. These results suggest that embryonic lethality in Tdg (-/-) embryos is due, in part, to the reduction of noradrenaline levels.

The hepatitis B virus (HBV) genome encodes the X protein (HBx), a ubiquitous transactivator that is required for HBV replication. Expression of the HBx protein has been associated with the development of HBV infection-related hepatocellular carcinoma (HCC). Previously, we generated a 3D structure of HBx by combined homology and ab initio in silico modelling. This structure showed a striking similarity to the human thymine DNA glycosylase (TDG), a key enzyme in the base excision repair (BER) pathway. To further explore this finding, we investigated whether both proteins interfere with or complement each other's functions. Here we show that TDG does not affect HBV replication, but that HBx strongly inhibits TDG-initiated base excision repair (BER), a major DNA repair pathway. Inhibition of the BER pathway may contribute substantially to the oncogenic effect of HBV infection.

Thymine DNA glycosylase (TDG) excises T from G·T mispairs and is thought to initiate base excision repair (BER) of deaminated 5-methylcytosine (mC). Recent studies show that TDG, including its glycosylase activity, is essential for active DNA demethylation and embryonic development. These and other findings suggest that active demethylation could involve mC deamination by a deaminase, giving a G·T mispair followed by TDG-initiated BER. An alternative proposal is that demethylation could involve iterative oxidation of mC to 5-hydroxymethylcytosine (hmC) and then to 5-formylcytosine (fC) and 5-carboxylcytosine (caC), mediated by a Tet (ten eleven translocation) enzyme, with conversion of caC to C by a putative decarboxylase. Our previous studies suggest that TDG could excise fC and caC from DNA, which could provide another potential demethylation mechanism. We show here that TDG rapidly removes fC, with higher activity than for G·T mispairs, and has substantial caC excision activity, yet it cannot remove hmC. TDG excision of fC and caC, oxidation products of mC, is consistent with its strong specificity for excising bases from a CpG context. Our findings reveal a remarkable new aspect of specificity for TDG, inform its catalytic mechanism, and suggest that TDG could protect against fC-induced mutagenesis. The results also suggest a new potential mechanism for active DNA demethylation, involving TDG excision of Tet-produced fC (or caC) and subsequent BER. Such a mechanism obviates the need for a decarboxylase and is consistent with findings that TDG glycosylase activity is essential for active demethylation and embryonic development, as are mechanisms involving TDG excision of deaminated mC or hmC.

Cytotoxicity of 5-fluorouracil (FU) and 5-fluoro-2'-deoxyuridine (FdUrd) due to DNA fragmentation during DNA repair has been proposed as an alternative to effects from thymidylate synthase (TS) inhibition or RNA incorporation. The goal of the present study was to investigate the relative contribution of the proposed mechanisms for cytotoxicity of 5-fluoropyrimidines. We demonstrate that in human cancer cells, base excision repair (BER) initiated by the uracil-DNA glycosylase UNG is the major route for FU-DNA repair in vitro and in vivo. SMUG1, TDG and MBD4 contributed modestly in vitro and not detectably in vivo. Contribution from mismatch repair was limited to FU:G contexts at best. Surprisingly, knockdown of individual uracil-DNA glycosylases or MSH2 did not affect sensitivity to FU or FdUrd. Inhibitors of common steps of BER or DNA damage signalling affected sensitivity to FdUrd and HmdUrd, but not to FU. In support of predominantly RNA-mediated cytotoxicity, FU-treated cells accumulated ~3000- to 15 000-fold more FU in RNA than in DNA. Moreover, FU-cytotoxicity was partially reversed by ribonucleosides, but not deoxyribonucleosides and FU displayed modest TS-inhibition compared to FdUrd. In conclusion, UNG-initiated BER is the major route for FU-DNA repair, but cytotoxicity of FU is predominantly RNA-mediated, while DNA-mediated effects are limited to FdUrd.

The prevalent DNA modification in higher organisms is the methylation of cytosine to 5-methylcytosine (5mC), which is partially converted to 5-hydroxymethylcytosine (5hmC) by the Tet (ten eleven translocation) family of dioxygenases. Despite their importance in epigenetic regulation, it is unclear how these cytosine modifications are reversed. Here, we demonstrate that 5mC and 5hmC in DNA are oxidized to 5-carboxylcytosine (5caC) by Tet dioxygenases in vitro and in cultured cells. 5caC is specifically recognized and excised by thymine-DNA glycosylase (TDG). Depletion of TDG in mouse embyronic stem cells leads to accumulation of 5caC to a readily detectable level. These data suggest that oxidation of 5mC by Tet proteins followed by TDG-mediated base excision of 5caC constitutes a pathway for active DNA demethylation.

DNA methylation is a major epigenetic mechanism for gene silencing. Whereas methyltransferases mediate cytosine methylation, it is less clear how unmethylated regions in mammalian genomes are protected from de novo methylation and whether an active demethylating activity is involved. Here, we show that either knockout or catalytic inactivation of the DNA repair enzyme thymine DNA glycosylase (TDG) leads to embryonic lethality in mice. TDG is necessary for recruiting p300 to retinoic acid (RA)-regulated promoters, protection of CpG islands from hypermethylation, and active demethylation of tissue-specific developmentally and hormonally regulated promoters and enhancers. TDG interacts with the deaminase AID and the damage response protein GADD45a. These findings highlight a dual role for TDG in promoting proper epigenetic states during development and suggest a two-step mechanism for DNA demethylation in mammals, whereby 5-methylcytosine and 5-hydroxymethylcytosine are first deaminated by AID to thymine and 5-hydroxymethyluracil, respectively, followed by TDG-mediated thymine and 5-hydroxymethyluracil excision repair., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Repair of G·T mismatches arising from deamination of 5-methylcytosine (m(5)C) involves excision of thymine and restoration of a G·C pair via base excision repair (BER). Thymine DNA glycosylase (TDG) is one of two mammalian enzymes that can specifically remove thymine from G·T mispairs. While TDG can excise other bases, it maintains stringent specificity for a CpG context, suggesting deaminated m(5)C is an important biological substrate. Recent studies reveal TDG is essential for embryogenesis; it helps to maintain an active chromatin complex and initiates BER to counter aberrant de novo CpG methylation, which may involve excision of actively deaminated m(5)C. The relatively weak G·T activity of TDG has been implicated in the hypermutability of CpG sites, which largely involves C→T transitions arising from m(5)C deamination. Thus, it is important to understand how TDG recognizes and process substrates, particularly G·T mispairs. Here, we extend our detailed studies of TDG by examining the dependence of substrate binding and catalysis on pH, ionic strength, and temperature. Catalytic activity is relatively constant for pH 5.5-9, but falls sharply for pH>9 due to severely weakened substrate binding, and, potentially, ionization of the target base. Substrate binding and catalysis diminish sharply with increasing ionic strength, particularly for G·T substrates, due partly to effects on nucleotide flipping. TDG aggregates rapidly and irreversibly at 37°C, but can be stabilized by specific and nonspecific DNA. The temperature dependence of catalysis reveals large and unexpected differences for G·U and G·T substrates, where G·T activity exhibits much steeper temperature dependence. The results suggest that reversible nucleotide flipping is much more rapid for G·T substrates, consistent with our previous findings that steric effects limit the active-site lifetime of thymine, which may account for the relatively weak G·T activity. Our findings provide important insight into catalysis by TDG, particularly for mutagenic G·T mispairs., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Etheno (ε) DNA adducts, including 1,N(6)-ethenoadenine (εA), are formed by various bifunctional agents of exogenous and endogenous origin. The AT→TA transversion, the most frequent mutation provoked by the presence of εA in DNA, is very common in critical codons of the TP53 and RAS genes in tumours induced by exposure to carcinogenic vinyl compounds. Here, using a method that allows examination of the mutagenic potency of a metabolite of vinyl chloride, chloroacetaldehyde (CAA), but eliminates its cytotoxicity, we studied the participation of alkA, alkB and mug gene products in the repair of εA in Escherichia coli cells. The test system used comprised the pIF105 plasmid bearing the lactose operon of CC105 origin, which allowed monitoring of Lac(+) revertants that arose by AT→TA substitutions due to the modification of adenine by CAA. The plasmid was CAA-modified in vitro and replicated in E.coli of various genetic backgrounds (wt, alkA, alkB, mug, alkAalkB, alkAmug and alkBmug). To modify the levels of the AlkA and AlkB proteins, mutagenesis was studied in E.coli cells induced or not in adaptive response to alkylating agents. Considering the levels of CAA-induced Lac(+) revertants in strains harbouring the CAA-modified pIF105 plasmid and induced or not in adaptive response, we conclude that the AlkB dioxygenase plays a major role in decreasing the level of AT→TA mutations, thus in the repair of εA in E.coli cells. The observed differences of mutation frequencies in the various mutant strains assayed indicate that Mug glycosylase is also engaged in the repair of εA, whereas the role the AlkA glycosylase in this repair is negligible.

Mismatch uracil DNA glycosylase (Mug) from Escherichia coli is an initiating enzyme in the base-excision repair pathway. As with other DNA glycosylases, the abasic product is potentially more harmful than the initial lesion. Since Mug is known to bind its product tightly, inhibiting enzyme turnover, understanding how Mug binds DNA is of significance when considering how Mug interacts with downstream enzymes in the base-excision repair pathway. We have demonstrated differential binding modes of Mug between its substrate and abasic DNA product using both band shift and fluorescence anisotropy assays. Mug binds its product cooperatively, and a stoichiometric analysis of DNA binding, catalytic activity and salt-dependence indicates that dimer formation is of functional significance in both catalytic activity and product binding. This is the first report of cooperativity in the uracil DNA glycosylase superfamily of enzymes, and forms the basis of product inhibition in Mug. It therefore provides a new perspective on abasic site protection and the findings are discussed in the context of downstream lesion processing and enzyme communication in the base excision repair pathway.

Deamination of 5-methylcytosine to thymine creates mutagenic G · T mispairs, contributing to cancer and genetic disease. Thymine DNA glycosylase (TDG) removes thymine from these G · T lesions, and follow-on base excision repair yields a G · C pair. A previous crystal structure revealed TDG (catalytic domain) bound to abasic DNA product in a 2:1 complex, one subunit at the abasic site and the other bound to undamaged DNA. Biochemical studies showed TDG can bind abasic DNA with 1:1 or 2:1 stoichiometry, but the dissociation constants were unknown, as was the stoichiometry and affinity for binding substrates and undamaged DNA. We showed that 2:1 binding is dispensable for G · U activity, but its role in G · T repair was unknown. Using equilibrium binding anisotropy experiments, we show that a single TDG subunit binds very tightly to G · U mispairs and abasic (G · AP) sites, and somewhat less tightly G · T mispairs. Kinetics experiments show 1:1 binding provides full G · T activity. TDG binds undamaged CpG sites with remarkable affinity, modestly weaker than G · T mispairs, and exhibits substantial affinity for nonspecific DNA. While 2:1 binding is observed for large excess TDG concentrations, our findings indicate that a single TDG subunit is fully capable of locating and processing G · U or G · T lesions.

Thymine DNA glycosylase (TDG) is a member of the uracil DNA glycosylase (UDG) superfamily of DNA repair enzymes. Owing to its ability to excise thymine when mispaired with guanine, it was proposed to act against the mutability of 5-methylcytosine (5-mC) deamination in mammalian DNA. However, TDG was also found to interact with transcription factors, histone acetyltransferases and de novo DNA methyltransferases, and it has been associated with DNA demethylation in gene promoters following activation of transcription, altogether implicating an engagement in gene regulation rather than DNA repair. Here we use a mouse genetic approach to determine the biological function of this multifaceted DNA repair enzyme. We find that, unlike other DNA glycosylases, TDG is essential for embryonic development, and that this phenotype is associated with epigenetic aberrations affecting the expression of developmental genes. Fibroblasts derived from Tdg null embryos (mouse embryonic fibroblasts, MEFs) show impaired gene regulation, coincident with imbalanced histone modification and CpG methylation at promoters of affected genes. TDG associates with the promoters of such genes both in fibroblasts and in embryonic stem cells (ESCs), but epigenetic aberrations only appear upon cell lineage commitment. We show that TDG contributes to the maintenance of active and bivalent chromatin throughout cell differentiation, facilitating a proper assembly of chromatin-modifying complexes and initiating base excision repair to counter aberrant de novo methylation. We thus conclude that TDG-dependent DNA repair has evolved to provide epigenetic stability in lineage committed cells.

Background: The human thymine-DNA glycosylase (TDG) plays a dual role in base excision repair of G:U/T mismatches and in transcription. Regulation of TDG activity by SUMO-1 conjugation was shown to act on both functions. Furthermore, TDG can interact with SUMO-1 in a non-covalent manner., Results: Using NMR spectroscopy we have determined distinct conformational changes in TDG upon either covalent sumoylation on lysine 330 or intermolecular SUMO-1 binding through a unique SUMO-binding motif (SBM) localized in the C-terminal region of TDG. The non-covalent SUMO-1 binding induces a conformational change of the TDG amino-terminal regulatory domain (RD). Such conformational dynamics do not exist with covalent SUMO-1 attachment and could potentially play a broader role in the regulation of TDG functions for instance during transcription. Both covalent and non-covalent processes activate TDG G:U repair similarly. Surprisingly, despite a dissociation of the SBM/SUMO-1 complex in presence of a DNA substrate, SUMO-1 preserves its ability to stimulate TDG activity indicating that the non-covalent interactions are not directly involved in the regulation of TDG activity. SUMO-1 instead acts, as demonstrated here, indirectly by competing with the regulatory domain of TDG for DNA binding., Conclusions: SUMO-1 increases the enzymatic turnover of TDG by overcoming the product-inhibition of TDG on apurinic sites. The mechanism involves a competitive DNA binding activity of SUMO-1 towards the regulatory domain of TDG. This mechanism might be a general feature of SUMO-1 regulation of other DNA-bound factors such as transcription regulatory proteins.

To assess the role of lipid peroxidation-induced DNA damage and repair in colon carcinogenesis, the excision rates and levels of 1,N(6)-etheno-2'-deoxyadenosine (epsilondA), 3,N(4)-etheno-2'-deoxycytidine (epsilondC), and 1,N(2)-etheno-2'-deoxyguanosine (1,N(2)-epsilondG) were analyzed in polymorphic blood leukocytes (PBL) and resected colon tissues of 54 colorectal carcinoma (CRC) patients and PBL of 56 healthy individuals. In PBL the excision rates of 1,N(6)-ethenoadenine (epsilonAde) and 3,N(4)-ethenocytosine (epsilonCyt), measured by the nicking of oligodeoxynucleotide duplexes with single lesions, and unexpectedly also the levels of epsilondA and 1,N(2)-epsilondG, measured by LC/MS/MS, were lower in CRC patients than in controls. In contrast the mRNA levels of repair enzymes, alkylpurine- and thymine-DNA glycosylases and abasic site endonuclease (APE1), were higher in PBL of CRC patients than in those of controls, as measured by QPCR. In the target colon tissues epsilonAde and epsilonCyt excision rates were higher, whereas the epsilondA and epsilondC levels in DNA, measured by (32)P-postlabeling, were lower in tumor than in adjacent colon tissue, although a higher mRNA level was observed only for APE1. This suggests that during the onset of carcinogenesis, etheno adduct repair in the colon seems to be under a complex transcriptional and posttranscriptional control, whereby deregulation may act as a driving force for malignancy., (Copyright 2010 Elsevier Inc. All rights reserved.)

DNA methyltransferase (DNMT) 1 and 3 are primarily responsible for abnormal methylation in cancer. Unlike these DNMTs, DNA methyltransferase 3-like (DNMT3L) harbors no conserved catalytic domain, and has been shown to function as a regulatory cofactor for DNA methylation. However, it is unclear whether DNMT3L directly regulates DNA methylation in cancer cells. To address this, we investigated the methylation targets of DNMT3L by conducting methylation microarray trials after the siRNA-induced knockdown. We determined that methylation of 242 out of 1,505 CpG sites was significantly altered by DNMT3L knockdown. Among these 242 CpG sites, 204, 12, and 11 CpG sites were identified as common targets of DNMT 1/3B/3L, 1/3L, and 3B/3L, respectively; this indicates that DNMT3L participates in DNA methylation via cooperation with other DNMTs. However, we also determined that the methylation of 15 CpG sites was significantly altered by DNMT3L knockdown only. As a validation, we confirmed that thymine DNA glycosylase (TDG), an enzyme involved in the base excision repair of mismatched-DNA, was up-regulated in DNMT3L knockdown cells, but neither in DNMT1 nor 3B knockdown cells. Methylation-specific PCR (MSP) also showed that promoter methylation of TDG was decreased in DNMT3L knockdown cells. Interestingly, 5-aza-2'-deoxycitidine (5-aza-dC) re-expressed DNMT3L, leading to down-regulation of TDG. This study is the first to show that DNMT3L exerts a major effect on the transcriptional regulation of a specific target gene, such as TDG, despite the absence of enzymatic activity.

CpG dinucleotides are mutational hotspots associated with cancer and genetic diseases. Thymine DNA glycosylase (TDG) plays an integral role in CpG maintenance by excising mispaired thymine and uracil in a CpG context and also participates in transcriptional regulation via gene-specific CpG demethylation and functional interactions with the transcription machinery. Here, we report that protein kinase C alpha (PKCalpha) interacts with TDG and phosphorylates amino-terminal serine residues adjacent to lysines acetylated by CREB-binding protein (CBP) and p300 (CBP/p300). We establish that acetylation and phosphorylation are mutually exclusive, and their interplay dramatically alters the DNA mispair-processing functions of TDG. Remarkably, acetylation of the amino-terminal region abrogates high-affinity DNA binding and selectively prevents processing of G:T mispairs. In contrast, phosphorylation does not markedly alter DNA interactions, but may preserve G:T processing in vivo by preventing CBP-mediated acetylation. Mutational analysis suggests that the acetyl-acceptor lysines are not directly involved in contacting DNA, but may constitute a conformationally sensitive interface that modulates DNA interactions. These findings reveal opposing roles of CBP/p300 and PKCalpha in regulating the DNA repair functions of TDG and suggest that the interplay of these modifications in vivo may be critically important in the maintenance of CpG dinucleotides and epigenetic regulation.

The recombinant thymine-DNA glycosylase (TDG) from Aeropyrum pernix (A. pernix) was expressed in Escherichia coli. The enzymatic activity of recombinant A. pernix TDG (ApeTDG) was characterized using oligonucleotides containing a thymine/uracil base as substrate. ApeTDG had distinct glycosylase activity on T/G mismatch. The optimal temperature and pH for thymine removal were 65-70 degrees C and pH 7.0-8.5, respectively. High concentration of NaCl inhibited the thymine removal. Divalent ions had different influence on the thymine removal by ApeTDG. Ca(2+) and Mg(2+) had no inhibition on the enzymic activity, but Ni(2+), Co(2+), Cu(2+), Mn(2+), and Zn(2+) completely inhibited the excision reaction. As derived from a hyperthermophilic archaea, ApeTDG protein was heat-resistant at 75 degrees C. ApeTDG also had a relatively weak DNA glycosylase activity on uracil base, with the following order: U/C>U/G approximately U/T>U/U approximately U/I approximately U/AP approximately U/->U/A. Additional mismatch located at 3' of T/G had less inhibition on the thymine removal than that located at 5' of T/G, and two additional mismatches located at each side of T/G completely inhibited the excision of thymine. Together, these data suggest that ApeTDG is a TDG protein with weak UDG activity., ((c) 2009 Elsevier Inc. All rights reserved.)

The T:G mismatch specific DNA glycosylase (TDG) is known as an important enzyme in repairing damaged DNA. Recent studies also showed that TDG interacts with a p160 protein, steroid receptor coactivator 1 or nuclear receptor coactivator 1 (SRC1), and is involved in transcriptional activation of the estrogen receptor. However, whether other members of the p160 family are also involved in TDG-interaction and signal transduction regulation remains to be seen. In this study, we employed the mammalian two-hybrid system to investigate the interaction between TDG and another member of the p160 family, nuclear receptor coactivator 3 (NCoA-3). We found that a DXXD motif from aa 294-297 within TDG was responsible for the TDG-NCoA-3 interaction, we also found that a LLXXXL motif (X means any amino acid) from aa 1029-1037 (LLRNSL) and a merged LLXXL motif (LLDQLHTLL) from aa 1053-1061 in NCoA-3 were important for the TDG-NCoA-3 interactions. Mutation of the two aspartic acids (aa 294 and 297) into two alanines in TDG significantly affected the interaction and subsequent transcriptional activation of several steroid hormone receptors including, estrogen-, androgen- and progesterone- receptors in Huh7 cells. We also identified that mutations of NCoA-3 at either leucines 1029-1030 or 1053-1054 (replaced by alanines) also reduced the interaction activity between TDG and NCoA1. These data indicated that the TDG-NCoA-3 interaction is important for broad range activation of steroid hormone nuclear receptors, and may also contribute significantly to further understanding of TDG-related nuclear receptor regulation.

Thymine DNA glycosylase (TDG) promotes genomic integrity by excising thymine from mutagenic G.T mismatches arising by deamination of 5-methylcytosine, and follow-on base excision repair enzymes restore a G.C pair. TDG cleaves the N-glycosylic bond of dT and some other nucleotides, including 5-substituted 2'-deoxyuridine analogs, once they have been flipped from the helix into its active site. We examined the role of two strictly conserved residues; Asn(140), implicated in the chemical step, and Arg(275), implicated in nucleotide flipping. The N140A variant binds substrate DNA with the same tight affinity as wild-type TDG, but it has no detectable base excision activity for a G.T substrate, and its excision rate is vastly diminished (by approximately 10(4.4)-fold) for G.U, G.FU, and G.BrU substrates. Thus, Asn(140) does not contribute substantially to substrate binding but is essential for the chemical step, where it stabilizes the transition state by approximately 6 kcal/mol (compared with 11.6 kcal/mol stabilization provided by TDG overall). Our recent crystal structure revealed that Arg(275) penetrates the DNA minor groove, filling the void created by nucleotide flipping. We found that the R275A and R275L substitutions weaken substrate binding and substantially decrease the base excision rate for G.T and G.BrU substrates. Our results indicate that Arg(275) promotes and/or stabilizes nucleotide flipping, a role that is most important for target nucleotides that are relatively large (dT and bromodeoxyuridine) and/or have a stable N-glycosylic bond (dT). Arg(275) does not contribute substantially to the binding of TDG to abasic DNA product, and it cannot account for the slow product release exhibited by TDG.

DNA glycosylases function to remove endogenous and exogenous base damage and thus contribute to the maintenance of genomic integrity. This function gains clinical relevance when base mispairs introduced by chemotherapy or radiosensitizing drugs become their substrate. This report describes the action of DNA glycosylases on the mispairs generated by iododeoxyuridine (IUdR)-a radiosensitizer. A non-radioactive fluorescent dye-based in vitro glycosylase assay was employed to quantitatively measure the enzymatic activities of functionally related DNA glycosylases on IUdR generated mispairs including G:IU and A:IU. Thymine DNA glycosylase (TDG) and methyl binding domain protein 4 (MBD4/MED1) are found to act on G:IU (but not A:IU) mispairs and are functionally complementary to each other. However, uracil DNA glycosylase (UDG) does not show any activity on these mispairs. The methyl binding domain of MBD4/MED1 was found to specifically inhibit the activity of MBD4/MED1 as well as the glycosylase domain, when the G:IU mispairs were located in a methylated CpG context. However, inhibition of TDG activity on methylated G:IU mispairs by the methyl binding domain was not observed.

5-Fluorouracil (5-FU), a chemotherapeutic drug commonly used in cancer treatment, imbalances nucleotide pools, thereby favoring misincorporation of uracil and 5-FU into genomic DNA. The processing of these bases by DNA repair activities was proposed to cause DNA-directed cytotoxicity, but the underlying mechanisms have not been resolved. In this study, we investigated a possible role of thymine DNA glycosylase (TDG), one of four mammalian uracil DNA glycosylases (UDGs), in the cellular response to 5-FU. Using genetic and biochemical tools, we found that inactivation of TDG significantly increases resistance of both mouse and human cancer cells towards 5-FU. We show that excision of DNA-incorporated 5-FU by TDG generates persistent DNA strand breaks, delays S-phase progression, and activates DNA damage signaling, and that the repair of 5-FU-induced DNA strand breaks is more efficient in the absence of TDG. Hence, excision of 5-FU by TDG, but not by other UDGs (UNG2 and SMUG1), prevents efficient downstream processing of the repair intermediate, thereby mediating DNA-directed cytotoxicity. The status of TDG expression in a cancer is therefore likely to determine its response to 5-FU-based chemotherapy., Competing Interests: Competing interests. The authors have declared that no competing interests exist.

Whether 5-methylcytosine (meC) can be enzymatically removed from vertebrate DNA has been the subject of extensive study and also some controversy. Rai et al. (2008) now report that cytosine demethylation can be accomplished in a one-cell zebrafish embryo by the combined action of a cytidine deaminase and a thymine DNA glycosylase.

Evidence for active DNA demethylation in vertebrates is accumulating, but the mechanisms and enzymes remain unclear. Using zebrafish embryos we provide evidence for 5-methylcytosine (5-meC) removal in vivo via the coupling of a 5-meC deaminase (AID, which converts 5-meC to thymine) and a G:T mismatch-specific thymine glycosylase (Mbd4). The injection of methylated DNA into embryos induced a potent DNA demethylation activity, which was attenuated by depletion of AID or the non enzymatic factor Gadd45. Remarkably, overexpression of the deaminase/glycosylase pair AID/Mbd4 in vivo caused demethylation of the bulk genome and injected methylated DNA fragments, likely involving a G:T intermediate. Furthermore, AID or Mbd4 knockdown caused the remethylation of a set of common genes. Finally, Gadd45 promoted demethylation and enhanced functional interactions between deaminase/glycosylase pairs. Our results provide evidence for a coupled mechanism of 5-meC demethylation, whereby AID deaminates 5-meC, followed by thymine base excision by Mbd4, promoted by Gadd45.

Thymine-DNA glycosylase (TDG), a DNA repair enzyme specific for G/T mismatches, plays a role in the regulation of gene expression through its physical interaction with transcription factors. Here, we show that TDG functionally associates with members of the p53 tumor suppressor family and directly modulates their activity. Yeast two-hybrid analysis indicated a physical interaction between a region including the oligomerization domain (OD) of p73alpha (residues 345-380) or p53 (residues 319-360) and residues 123-346 of TDG, which localizes in the G/T glycosylase catalytic domain (residues 123-372). This interaction was also detected in vitro and in vivo by GST pull-down and immunoprecipitation assays, respectively. TDG over-expression promoted the p73- and p53-mediated transcriptional activation of the p21Waf1 promoter in a dose-dependent manner. Further, TDG enhanced the p53 or p73alpha-induced growth repression. These observations suggest that TDG modulates the biological function of p53 and other members of the p53 family as a transcriptional coactivator.

Myocardin is a serum response factor (SRF) co-activator that regulates transcription of many smooth muscle-specific genes and is essential for development of vascular smooth muscle. We used a yeast two-hybrid screen, with myocardin as bait in a search for factors that regulate myocardin transcriptional activity. From this screen, thymine DNA glycosylase (TDG) was identified as a myocardin-associated protein. TDG was originally identified as an enzyme involved in base excision repair of T:G mismatches caused by spontaneous deamination of methylated cytosines. However, TDG has also been shown to act as a transcriptional co-activator or co-repressor. The interaction between TDG and myocardin was confirmed in vitro by glutathione S-transferase pull down and in vivo by co-immunoprecipitation assays. We found that TDG abrogates myocardin induced expression of smooth muscle-specific genes and represses the trans-activation of the promoters of myocardin of these genes. Overexpression of TDG in SMCs down-regulated smooth muscle marker expression. Conversely, depletion of endogenous TDG in SMCs increased smooth muscle-specific myosin heavy chain (SM MHC) and Telokin gene expression. Glutathione S-transferase pull-down assays demonstrated that TDG binds to a region of myocardin that includes the SRF binding domain. Furthermore, TDG was found to compete with SRF for binding to myocardin in vitro and in vivo, suggesting that TDG can inhibit expression of smooth muscle-specific genes, at least in part, through disrupting SRF/myocardin interactions. Finally, we demonstrated that the glycosylase activity of TDG is not required for its inhibitory effects on myocardin function. This study reveals a previously unsuspected role for the repair enzyme TDG as a repressor of smooth muscle differentiation via competing with SRF for binding to myocardin.

DNA glycosylases initiate base excision repair by removing damaged or mismatched bases, producing apurinic/apyrimidinic (AP) DNA. For many glycosylases, the AP-DNA remains tightly bound, impeding enzymatic turnover. A prominent example is thymine DNA glycosylase (TDG), which removes T from G.T mispairs and recognizes other lesions, with specificity for damage at CpG dinucleotides. TDG turnover is very slow; its activity appears to reach a plateau as the [product]/[enzyme] ratio approaches unity. The follow-on base excision repair enzyme, AP endonuclease 1 (APE1), stimulates the turnover of TDG and other glycosylases, involving a mechanism that remains largely unknown. We examined the catalytic activity of human TDG (hTDG), alone and with human APE1 (hAPE1), using pre-steady-state kinetics and a coupled-enzyme (hTDG-hAPE1) fluorescence assay. hTDG turnover is exceedingly slow for G.T (k(cat)=0.00034 min(-1)) and G.U (k(cat)=0.005 min(-1)) substrates, much slower than k(max) from single turnover experiments, confirming that AP-DNA release is rate-limiting. We find unexpectedly large differences in k(cat) for G.T, G.U, and G.FU substrates, indicating the excised base remains trapped in the product complex by AP-DNA. hAPE1 increases hTDG turnover by 42- and 26-fold for G.T and G.U substrates, the first quantitative measure of the effect of hAPE1. hAPE1 stimulates hTDG by disrupting the product complex rather than merely depleting (endonucleolytically) the AP-DNA. The enhancement is greater for hTDG catalytic core (residues 111-308 of 410), indicating the N- and C-terminal domains are dispensable for stimulatory interactions with hAPE1. Potential mechanisms for hAPE1 disruption of the of hTDG product complex are discussed.

Cytosine methylation at CpG dinucleotides produces m(5)CpG, an epigenetic modification that is important for transcriptional regulation and genomic stability in vertebrate cells. However, m(5)C deamination yields mutagenic G.T mispairs, which are implicated in genetic disease, cancer, and aging. Human thymine DNA glycosylase (hTDG) removes T from G.T mispairs, producing an abasic (or AP) site, and follow-on base excision repair proteins restore the G.C pair. hTDG is inactive against normal A.T pairs, and is most effective for G.T mispairs and other damage located in a CpG context. The molecular basis of these important catalytic properties has remained unknown. Here, we report a crystal structure of hTDG (catalytic domain, hTDG(cat)) in complex with abasic DNA, at 2.8 A resolution. Surprisingly, the enzyme crystallized in a 2:1 complex with DNA, one subunit bound at the abasic site, as anticipated, and the other at an undamaged (nonspecific) site. Isothermal titration calorimetry and electrophoretic mobility-shift experiments indicate that hTDG and hTDG(cat) can bind abasic DNA with 1:1 or 2:1 stoichiometry. Kinetics experiments show that the 1:1 complex is sufficient for full catalytic (base excision) activity, suggesting that the 2:1 complex, if adopted in vivo, might be important for some other activity of hTDG, perhaps binding interactions with other proteins. Our structure reveals interactions that promote the stringent specificity for guanine versus adenine as the pairing partner of the target base and interactions that likely confer CpG sequence specificity. We find striking differences between hTDG and its prokaryotic ortholog (MUG), despite the relatively high (32%) sequence identity.

Thymine-DNA glycosylases (TDGs) initiate base excision repair by debasification of the erroneous thymine or uracil nucleotide in G.T and G.U mispairs which arise at high frequency through spontaneous or enzymatic deamination of methylcytosine and cytosine, respectively. Human TDG has furthermore been shown to have a functional role in transcription and epigenetic regulation through the interaction with transcription factors from the nuclear receptor superfamily, transcriptional coregulators, and a DNA methyltransferase. The TDG N-terminus encodes regulatory functions, as it assures both G.T versus G.U specificity and contains the sites for interaction and posttranslational modification by transcription-related activities. While the molecular function of the evolutionarily conserved central catalytic domain of TDG in base excision repair has been elucidated by determination of its three-dimensional structure, the mechanisms by which the N-terminus exerts its regulatory roles, as well as the function of TDG in transcription regulation, remain to be understood. We describe here the residual structure of the TDG N-terminus in both contexts of the isolated domain and the entire protein. These studies lead to the characterization of a small structural domain in the TDG N-terminal region preceding the catalytic core and coinciding with the region of functional regulation of TDG's activities. This regulatory domain exhibits a small degree of organization and is implicated in dynamic molecular interactions with the catalytic domain and nonselective interactions with double-stranded DNA, providing a molecular explanation for the evolutionarily acquired G.T mismatch processing activity of TDG.

Methylation of cytosine residues in CpG dinucleotides plays an important role in epigenetic regulation of gene expression and chromatin structure/stability in higher eukaryotes. DNA methylation patterns are established and maintained at CpG dinucleotides by DNA methyltransferases (Dnmt1, Dnmt3a, and Dnmt3b). In mammals and many other eukaryotes, the CpG dinucleotide is underrepresented in the genome. This loss is postulated to be the result of unrepaired deamination of cytosine and 5-methylcytosine to uracil and thymine, respectively. Two thymine glycosylases are believed to reduce the impact of 5-methylcytosine deamination. G/T mismatch-specific thymine-DNA glycosylase (Tdg) and methyl-CpG binding domain protein 4 can both excise uracil or thymine at U.G and T.G mismatches to initiate base excision repair. Here, we report the characterization of interactions between Dnmt3b and both Tdg and methyl-CpG binding domain protein 4. Our results demonstrate (1) that both Tdg and Dnmt3b are colocalized to heterochromatin and (2) reduction of T.G mismatch repair efficiency upon loss of DNA methyltransferase expression, as well as a requirement for an RNA component for correct T.G mismatch repair.