Stoichiometry and affinity for thymine DNA glycosylase binding to specific and nonspecific DNA

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.     

Functionality of human thymine DNA glycosylase requires SUMO-regulated changes in protein conformation

BACKGROUND: Base excision repair initiated by human thymine-DNA glycosylase (TDG) results in the generation of abasic sites (AP sites) in DNA. TDG remains bound to this unstable repair intermediate, indicating that its transmission to the downstream-acting AP endonuclease is a coordinated process. Previously, we established that posttranslational modification of TDG with Small Ubiquitin-like MOdifiers (SUMOs) facilitates the dissociation of the DNA glycosylase from the product AP site, but the underlying molecular mechanism remained unclear. RESULTS: We now show that upon DNA interaction, TDG undergoes a dramatic conformational change, which involves its flexible N-terminal domain and accounts for the nonspecific DNA binding ability of the enzyme. This function is required for efficient processing of the G.T mismatch but then cooperates with the specific DNA contacts established in the active site pocket of TDG to prevent its dissociation from the product AP site after base release. SUMO1 conjugation to the C-terminal K330 of TDG modulates the DNA binding function of the N terminus to induce dissociation of the glycosylase from the AP site while it leaves the catalytic properties of base release in the active site pocket of the enzyme unaffected. CONCLUSION: Our data provide insight into the molecular mechanism of SUMO modification mediated modulation of enzymatic properties of TDG. A conformational change, involving the N-terminal domain of TDG, provides unspecific DNA interactions that facilitate processing of a wider spectrum of substrates at the expense of enzymatic turnover. SUMOylation then reverses this structural change in the product bound TDG.     

Coordinating the initial steps of base excision repair. Apurinic/apyrimidinic endonuclease 1 actively stimulates thymine DNA glycosylase by disrupting the product complex

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.     

Excision of 5-hydroxymethyluracil and 5-carboxylcytosine by the thymine DNA glycosylase domain: its structural basis and implications for active DNA demethylation

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.     

Separating substrate recognition from base hydrolysis in human thymine DNA glycosylase by mutational analysis

Human thymine DNA glycosylase (TDG) was discovered as an enzyme that can initiate base excision repair at sites of 5-methylcytosine- or cytosine deamination in DNA by its ability to release thymine or uracil from G.T and G.U mismatches. Crystal structure analysis of an Escherichia coli homologue identified conserved amino acid residues that are critical for its substrate recognition/interaction and base hydrolysis functions. Guided by this revelation, we performed a mutational study of structure function relationships with the human TDG. Substitution of the postulated catalytic site asparagine with alanine (N140A) resulted in an enzyme that bound mismatched substrates but was unable to catalyze base removal. Mutation of Met-269 in a motif with a postulated role in protein-substrate interaction selectively inactivated stable binding of the enzyme to mismatched substrates but not so its glycosylase activity. These results establish that the structure function model postulated for the E. coli enzyme is largely applicable to the human TDG. We further provide evidence for G.U being the preferred substrate of TDG, not only at the mismatch recognition step of the reaction but also in base hydrolysis, and for the importance of stable complementary strand interactions by TDG to compensate for its comparably poor hydrolytic potential.     

Association of Dnmt3a and thymine DNA glycosylase links DNA methylation with base-excision repair

While methylcytosines serve as the fifth base encoding epigenetic information, they are also a dangerous endogenous mutagen due to their intrinsic instability. Methylcytosine undergoes spontaneous deamination, at a rate much higher than cytosine, to generate thymine. In mammals, two repair enzymes, thymine DNA glycosylase (TDG) and methyl-CpG binding domain 4 (MBD4), have evolved to counteract the mutagenic effect of methylcytosines. Both recognize G/T mismatches arising from methylcytosine deamination and initiate base-excision repair that corrects them to G/C pairs. However, the mechanism by which the methylation status of the repaired cytosines is restored has remained unknown. We show here that the DNA methyltransferase Dnmt3a interacts with TDG. Both the PWWP domain and the catalytic domain of Dnmt3a are able to mediate the interaction with TDG at its N-terminus. The interaction affects the enzymatic activity of both proteins: Dnmt3a positively regulates the glycosylase activity of TDG, while TDG inhibits the methylation activity of Dnmt3a in vitro. These data suggest a mechanistic link between DNA repair and remethylation at sites affected by methylcytosine deamination.     

Crystal structure of a thwarted mismatch glycosylase DNA repair complex

The bacterial mismatch-specific uracil-DNA glycosylase (MUG) and eukaryotic thymine-DNA glycosylase (TDG) enzymes form a homologous family of DNA glycosylases that initiate base-excision repair of G:U/T mismatches. Despite low sequence homology, the MUG/TDG enzymes are structurally related to the uracil-DNA glycosylase enzymes, but have a very different mechanism for substrate recognition. We have now determined the crystal structure of the Escherichia coli MUG enzyme complexed with an oligonucleotide containing a non-hydrolysable deoxyuridine analogue mismatched with guanine, providing the first structure of an intact substrate-nucleotide productively bound to a hydrolytic DNA glycosylase. The structure of this complex explains the preference for G:U over G:T mispairs, and reveals an essentially non-specific pyrimidine-binding pocket that allows MUG/TDG enzymes to excise the alkylated base, 3, N(4)-ethenocytosine. Together with structures for the free enzyme and for an abasic-DNA product complex, the MUG-substrate analogue complex reveals the conformational changes accompanying the catalytic cycle of substrate binding, base excision and product release.     

Cell cycle regulation as a mechanism for functional separation of the apparently redundant uracil DNA glycosylases TDG and UNG2

Human Thymine-DNA Glycosylase (TDG) is a member of the uracil DNA glycosylase (UDG) superfamily. It excises uracil, thymine and a number of chemical base lesions when mispaired with guanine in double-stranded DNA. These activities are not unique to TDG; at least three additional proteins with similar enzymatic properties are present in mammalian cells. The successful co-evolution of these enzymes implies the existence of non-redundant biological functions that must be coordinated. Here, we report cell cycle regulation as a mechanism for the functional separation of apparently redundant DNA glycosylases. We show that cells entering S-phase eliminate TDG through the ubiquitin–proteasome system and then maintain a TDG-free condition until G2. Incomplete degradation of ectopically expressed TDG impedes S-phase progression and cell proliferation. The mode of cell cycle regulation of TDG is strictly inverse to that of UNG2, which peaks in and throughout S-phase and then declines to undetectable levels until it appears again just before the next S-phase. Thus, TDG- and UNG2-dependent base excision repair alternates throughout the cell cycle, and the ubiquitin–proteasome pathway constitutes the underlying regulatory system.     

Thymine DNA glycosylase can rapidly excise 5-formylcytosine and 5-carboxylcytosine: potential implications for active demethylation of CpG sites

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.     

Modification of the human thymine-DNA glycosylase by ubiquitin-like proteins facilitates enzymatic turnover

DNA glycosylases initiate base excision repair (BER) through the generation of potentially harmful abasic sites (AP sites) in DNA. Human thymine-DNA glycosylase (TDG) is a mismatch-specific uracil/thymine-DNA glycosylase with an implicated function in the restoration of G*C base pairs at sites of cytosine or 5-methylcytosine deamination. The rate-limiting step in the action of TDG in vitro is its dissociation from the product AP site, suggesting the existence of a specific enzyme release mechanism in vivo. We show here that TDG interacts with and is covalently modified by the ubiquitin-like proteins SUMO-1 and SUMO-2/3. SUMO conjugation dramatically reduces the DNA substrate and AP site binding affinity of TDG, and this is associated with a significant increase in enzymatic turnover in reactions with a G*U substrate and the loss of G*T processing activity. Sumoylation also potentiates the stimulatory effect of APE1 on TDG. These observations implicate a function of sumoylation in the controlled dissociation of TDG from the AP site and open up novel perspectives for the understanding of the molecular mechanisms coordinating the early steps of BER.     

Thymine DNA glycosylase exhibits negligible affinity for nucleobases that it removes from DNA

Thymine DNA Glycosylase (TDG) performs essential functions in maintaining genetic integrity and epigenetic regulation. Initiating base excision repair, TDG removes thymine from mutagenic G·T mispairs caused by 5-methylcytosine (mC) deamination and other lesions including uracil (U) and 5-hydroxymethyluracil (hmU). In DNA demethylation, TDG excises 5-formylcytosine (fC) and 5-carboxylcytosine (caC), which are generated from mC by Tet (ten–eleven translocation) enzymes. Using improved crystallization conditions, we solved high-resolution (up to 1.45 Å) structures of TDG enzyme–product complexes generated from substrates including G·U, G·T, G·hmU, G·fC and G·caC. The structures reveal many new features, including key water-mediated enzyme–substrate interactions. Together with nuclear magnetic resonance experiments, the structures demonstrate that TDG releases the excised base from its tight product complex with abasic DNA, contrary to previous reports. Moreover, DNA-free TDG exhibits no significant binding to free nucleobases (U, T, hmU), indicating a K_d >> 10 mM. The structures reveal a solvent-filled channel to the active site, which might facilitate dissociation of the excised base and enable caC excision, which involves solvent-mediated acid catalysis. Dissociation of the excised base allows TDG to bind the beta rather than the alpha anomer of the abasic sugar, which might stabilize the enzyme–product complex.     

Xeroderma pigmentosum group C protein interacts physically and functionally with thymine DNA glycosylase

The XPC-HR23B complex recognizes various helix-distorting lesions in DNA and initiates global genome nucleotide excision repair. Here we describe a novel functional interaction between XPC-HR23B and thymine DNA glycosylase (TDG), which initiates base excision repair (BER) of G/T mismatches generated by spontaneous deamination of 5-methylcytosine. XPC-HR23B stimulated TDG activity by promoting the release of TDG from abasic sites that result from the excision of mismatched T bases. In the presence of AP endonuclease (APE), XPC-HR23B had an additive effect on the enzymatic turnover of TDG without significantly inhibiting the subsequent action of APE. Our observations suggest that XPC-HR23B may participate in BER of G/T mismatches, thereby contributing to the suppression of spontaneous mutations that may be one of the contributory factors for the promotion of carcinogenesis in xeroderma pigmentosum genetic complementation group C patients.     

Repair of deaminated base damage by Schizosaccharomyces pombe thymine DNA glycosylase

Thymine DNA glycosylases (TDG) in eukaryotic organisms are known for their double-stranded glycosylase activity on guanine/uracil (G/U) base pairs. Schizosaccharomyces pombe (Spo) TDG is a member of the MUG/TDG family that belongs to a uracil DNA glycosylase superfamily. This work investigates the DNA repair activity of Spo TDG on all four deaminated bases: xanthine (X) and oxanine (O) from guanine, hypoxanthine (I) from adenine, and uracil from cytosine. Unexpectedly, Spo TDG exhibits glycosylase activity on all deaminated bases in both double-stranded and single-stranded DNA in the descending order of X > I > U  O. In comparison, human TDG only excises deaminated bases from G/U and, to a much lower extent, A/U and G/I base pairs. Amino acid substitutions in motifs 1 and 2 of Spo TDG show a significant impact on deaminated base repair activity. The overall mutational effects are characterized by a loss of glycosylase activity on oxanine in all five mutants. L157I in motif 1 and G288M in motif 2 retain xanthine DNA glycosylase (XDG) activity but reduce excision of hypoxanthine and uracil, in particular in C/I, single-stranded hypoxanthine (ss-I), A/U, and single-stranded uracil (ss-U). A proline substitution at I289 in motif 2 causes a significant reduction in XDG activity and a loss of activity on C/I, ss-I, A/U, C/U, G/U, and ss-U. S291G only retains reduced activity on T/I and G/I base pairs. S163A can still excise hypoxanthine and uracil in mismatched base pairs but loses XDG activity, making it the closest mutant, functionally, to human TDG. The relationship among amino acid substitutions, binding affinity and base recognition is discussed.