Preferential CEBP binding to T:G mismatches and increased C-to-T human somatic mutations

DNA cytosine methylation in mammals modulates gene expression and chromatin accessibility. It also impacts mutation rates, via spontaneous oxidative deamination of 5-methylcytosine (5mC) to thymine. In most cases the resulting T:G mismatches are repaired, following T excision by one of the thymine DNA glycosylases, TDG or MBD4. We found that C-to-T mutations are enriched in the binding sites of CCAAT/enhancer binding proteins (CEBP). Within a CEBP site, the presence of a T:G mismatch increased CEBPβ binding affinity by a factor of >60 relative to the normal C:G base pair. This enhanced binding to a mismatch inhibits its repair by both TDG and MBD4 in vitro. Furthermore, repair of the deamination product of unmethylated cytosine, which yields a U:G DNA mismatch that is normally repaired via uracil DNA glycosylase, is also inhibited by CEBPβ binding. Passage of a replication fork over either a T:G or U:G mismatch, before repair can occur, results in a C-to-T mutation in one of the daughter duplexes. Our study thus provides a plausible mechanism for accumulation of C-to-T human somatic mutations.     

Initiation of strand incision at G:T and O(6)-methylguanine:T base mismatches in DNA by human cell extracts

Extracts of the human glioma cell line A1235 (lacking O⁶-methylguanine-DNA methyltransferase) are known to restore a G:T mismatch to a normal G:C pair in a G:T-containing model (45 bp) DNA substrate. Herein we demonstrate that substitution of G:T with O⁶-methylguanine:T (m6G:T) results in extract-induced intra-strand incision in the DNA at an efficiency comparable to that of complete repair of the G:T-containing substrate, although the m6G:T mispair serves as a poor substrate for later repair steps (e.g. gap filling, as judged by defective DNA repair synthesis). The A1235 extract, when supplemented with ATP and the four normal dNTPs, incises 5′ to the mismatched T, as inferred by the generation of a single-stranded 20mer fragment. Unlike its parental (A1235) counterpart, an extract of the alkylation-tolerant derivative cell line A1235-MR4 produces no 20mer fragment, even when thymine-DNA glycosylase (TDG) is added to the reaction mixture. In contrast, the A1235 extract, when augmented with TDG, catalyzes enhanced incision at m6G:T in the 45 bp DNA, yielding 5–10-fold greater 20mer than that of either extract or TDG alone. Interestingly, the absence of m6G:T incision activity in the A1235-MR4 extract is similar to that seen for extracts of several known mismatch repair-deficient cell lines of colon tumor origin. Together these results suggest that derivative A1235-MR4 cells are defective in m6G:T incision activity and that the efficiency of this activity in the parental (A1235) cells may depend on the presence of several ill-defined mismatch repair recognition proteins along with TDG and ATP.     

Base-flipping mechanism in postmismatch recognition by MutS

DNA mismatch recognition and repair is vital for preserving the fidelity of the genome. Conserved across prokaryotes and eukaryotes, MutS is the primary protein that is responsible for recognizing a variety of DNA mismatches. From molecular dynamics simulations of the Escherichia coli MutS-DNA complex, we describe significant conformational dynamics in the DNA surrounding a G·T mismatch that involves weakening of the basepair hydrogen bonding in the basepair adjacent to the mismatch and, in one simulation, complete base opening via the major groove. The energetics of base flipping was further examined with Hamiltonian replica exchange free energy calculations revealing a stable flipped-out state with an initial barrier of ∼2 kcal/mol. Furthermore, we observe changes in the local DNA structure as well as in the MutS structure that appear to be correlated with base flipping. Our results suggest a role of base flipping as part of the repair initiation mechanism most likely leading to sliding-clamp formation.     

Unique mutational profile associated with a loss of TDG expression in the rectal cancer of a patient with a constitutional PMS2 deficiency

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.     

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.     

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.     

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.     

Intrinsic conformational properties of deoxyribonucleosides: implicated role for cytosine in the equilibrium among the A, B, and Z forms of DNA.

Structural properties of biomolecules are dictated by their intrinsic conformational energetics in combination with environmental contributions. Calculations using high-level ab initio methods on the deoxyribonucleosides have been performed to investigate the influence of base on the intrinsic conformational energetics of nucleosides. Energy minima in the north and south ranges of the deoxyribose pseudorotation surfaces have been located, allowing characterization of the influence of base on the structures and energy differences between those minima. With all bases, chi values associated with the south energy minimum are lower than in canonical B-DNA, while chi values associated with the north energy minimum are close to those in canonical A-DNA. In deoxycytidine, chi adopts an A-DNA conformation in both the north and south energy minima. Energy differences between the A and B conformations of the nucleosides are <0.5 kcal/mol in the present calculations, except with deoxycytidine, where the A form is favored by 2.3 kcal/mol, leading the intrinsic conformational energetics of GC basepairs to favor the A form of DNA by 1.5 kcal/mol as compared with AT pairs. This indicates that the intrinsic conformational properties of cytosine at the nucleoside level contribute to the A form of DNA containing predominately GC-rich sequences. In the context of a B versus Z DNA equilibrium, deoxycytidine favors the Z form over the B form by 1.6 kcal/mol as compared with deoxythymidine, suggesting that the intrinsic conformational properties of cytosine also contribute to GC-rich sequences occurring in Z DNA with a higher frequency than AT-rich sequences. Results show that the east pseudorotation energy barrier involves a decrease in the furanose amplitude and is systematically lower than the inversion barrier, with the energy differences influenced by the base. Energy barriers going from the south (B form) sugar pucker to the east pseudorotation barrier are lower in pyrimidines as compared with purines, indicating that the intrinsic conformational properties associated with base may also influence the sugar pseudorotational population distribution seen in DNA crystal structures and the kinetics of B to A transitions. The present work provides evidence that base composition, in addition to base sequence, can influence DNA conformation.     

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.     

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.     

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.     

Role of Two Strictly Conserved Residues in Nucleotide Flipping and N-Glycosylic Bond Cleavage by Human Thymine DNA Glycosylase

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; Asn¹⁴⁰, implicated in the chemical step, and Arg²⁷⁵, 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 ∼10^4.4-fold) for G·U, G·FU, and G·BrU substrates. Thus, Asn¹⁴⁰ does not contribute substantially to substrate binding but is essential for the chemical step, where it stabilizes the transition state by ∼6 kcal/mol (compared with 11.6 kcal/mol stabilization provided by TDG overall). Our recent crystal structure revealed that Arg²⁷⁵ 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 Arg²⁷⁵ 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). Arg²⁷⁵ 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.     

The Effect of a G:T Mispair on the Dynamics of DNA

Distortions in the DNA sequence such as damages or mispairs are specifically recognized and processed by DNA repair enzymes. A particular challenge for the enzymatic specificity is the recognition of a wrongly-placed native nucleotide such as thymine in T:G mispairs. An important step of substrate binding which is observed in many repair proteins is the flipping of the target base out of the DNA helix into the enzyme’s active site. In this work we investigate how much the intrinsic dynamics of mispaired DNA is changed compared to canonical DNA. Our molecular dynamics simulations of DNA with and without T:G mispairs show significant differences in the conformation of paired and mispaired DNA. The wobble pair T:G shows local distortions such as twist, shear and stretch which deviate from canonical B form values. Moreover, the T:G mispair is found to be kinetically less stable, exhibiting two states with respect to base opening: a closed state comparable to the canonical base pairs, and a more open state, indicating a proneness for base flip. In addition, we observe that the thymine base in a T:G mispair is significantly more probable to be flipped than thymine in a T:A pair or cytosine in a C:G pair. Such local deformations and in particular the existence of a second, more-open state can be speculated to help the target-site recognition by repair enzymes.     

GATC sequence and mismatch repair in Escherichia coli.

The Escherichia coli mismatch repair system greatly improves DNA replication fidelity by repairing single mispaired and unpaired bases in newly synthesized DNA strands. Transient undermethylation of the GATC sequences makes the newly synthesized strands susceptible to mismatch repair enzymes. The role of unmethylated GATC sequences in mismatch repair was tested in transfection experiments with heteroduplex DNA of phage phi 174 without any GATC sequence or with two GATC sequences, containing in addition either a G:T mismatch (Eam+/Eam3) or a G:A mismatch (Bam+/Bam16). It appears that only DNA containing GATC sequences is subject to efficient mismatch repair dependent on E. coli mutH, mutL, mutS and mutU genes; however, also in the absence of GATC sequence some mut-dependent mismatch repair can be observed. These observations suggest that the mismatch repair enzymes recognize both the mismatch and the unmethylated GATC sequence in DNA over long distances. The presence of GATC sequence(s) in the substrate appears to be required for full mismatch repair activity and not only for its strand specificity according to the GATC methylation state.     

DNA-substrate sequence specificity of human G:T mismatch repair activity.

G:T mispairs in DNA originate spontaneously via deamination of 5-methylcytosine. Such mispairs are restored to normal G:C pairs by both E. coli K strains and human cells. In this study we have analyzed the repair by human cell extracts of G:T mismatches in various DNA contexts. We performed two sets of experiments. In the first, repair was sequence specific in that G:T mispairs at CpG sites at four different CpG sites were repaired, but a G:T mismatch at a GpG site was not. Cytosine hemimethylation did not block repair of a substrate containing a CpG/GpT mismatch. In the second set of experiments, substrates with a G:T mismatch at a fixed position were constructed with an A, T, G, or C 5' to the mismatched G, and alterations in the complementary strand to allow otherwise perfect Watson-Crick pairing. All were incised just 5' to the mismatched T and competed for repair incision with a G:T substrate in which a C was 5' to the mismatched G. Thus human G:T mismatch activity shows sequence specificity, incising G:T mismatched pairs at some DNA sites, but not at others. At an incisable site, however, incision is little influenced by the base 5' to the mismatched G.     

Multiplicity of strand incision at G:T base mismatches in DNA by human cell extracts

Cell extract from the HT29 human colon carcinoma cell line (lacking mutator phenotype) was used to study the ATP-dependent G:T mismatch repair. We found that when a 45-bp (model) DNA with a single CpG/TpG mispair was incubated with the cell extract and ATP, it was incised immediately 5' and 3' to the mismatched T, and we noted that the actual 5'- and 3'-labeled fragments were similar to the cleaved products of thymine DNA glycosylase (TDG). This TDG-like cleavage product was enhanced (5-fold) with stimulation of several novel fragments, as inferred from the effect on incision at CpG/TpG site of the addition of G:U competitor DNA and ATP to the HT29 extract. The novel fragments were compatible with a strand incision on both sides of the mismatch (the third phosphodiester bond 5' and the second phosphodiester bond 3' to the mismatched T) and an incision 3' to the mismatched T, respectively. This suggests that while the ATP-dependent (TDG-like) incision activity, contrary to expectation, shows a lack of substrate competition, its catalytic property is likely modified by an interaction with G:U mispair. These multiple ATP-dependent incision events were not detected when extracts of the mismatch repair (MMR) defective HCT15 or HCT116 cell line were augmented with ATP and G:U. We postulate that these multiple ATP-dependent incision events possibly require the same MMR factors, and together they constitute a modified single ATP-dependent G:T incision activity. This activity toward the CpG/TpG was competitively inhibited by a 45-bp DNA with an ApG/TpT mispair; incision at a single site 5' to the latter mismatch compares with one of the multiple sites incised 5' to the former mismatch. These results suggest that one of several mismatch-incision factors is required by the human ATP-dependent G:T incision activity, in addition to MMR factors and ATP.     

Detection of single DNA base mutations with mismatch repair enzymes.

A novel method for identifying DNA point mutations has been developed by using mismatch repair enzymes. The high specificity of the Escherichia coli MutY protein has permitted the development of a reliable and sensitive method for the detection and characterization of point mutations in the human genome. The MutY protein is involved in a repair pathway that can convert A/G or A/C mismatches to C/G or G/C basepairs, respectively. A/G or A/C mismatches formed by hybridization between two amplified genomic DNA samples or between specific DNA probes and target DNA are nicked at the mispaired adenine strand by MutY protein. As little as 1% of the mutant sequence can be detected by the mismatch repair enzyme cleavage (MREC) method in a mixture of normal and mutated DNAs (e.g., mutant cells are only present in 1% of the normal cell background). By using different probes, the assay also can determine the nucleotide sequence of the mutation. We have applied this method to detect single-base substitutions in human oncogenes.     

HhaI DNA methyltransferase uses the protruding Gln237 for active flipping of its target cytosine

Access to a nucleotide by its rotation out of the DNA helix (base flipping) is used by numerous DNA modification and repair enzymes. Despite extensive studies of the paradigm HhaI methyltransferase, initial events leading to base flipping remained elusive. Here we demonstrate that the replacement of the target C:G pair with the 2-aminopurine:T pair in the DNA or shortening of the side chain of Gln237 in the protein severely perturb base flipping, but retain specific DNA binding. Kinetic analyses and molecular modeling suggest that a steric interaction between the protruding side chain of Gln237 and the target cytosine in B-DNA reduces the energy barrier for flipping by 3 kcal/mol. Subsequent stabilization of an open state by further 4 kcal/mol is achieved through specific hydrogen bonding of the side chain to the orphan guanine. Gln237 thus plays a key role in actively opening the target C:G pair by a "push-and-bind" mechanism.