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.     

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.     

Human thymine DNA glycosylase (TDG) and methyl-CpG-binding protein 4 (MBD4) excise thymine glycol (Tg) from a Tg:G mispair

The repair enzymes thymine DNA glycosylase (TDG) and methyl-CpG-binding protein 4 (MBD4) remove thymines from T:G mismatches resulting from deamination of 5-methylcytosine. Thymine glycol, a common DNA lesion produced by oxidative stress, can arise from oxidation of thymine or from oxidative deamination of 5-methylcytosine, and is then present opposite adenine or opposite guanine, respectively. Here we have used oligonucleotides with thymine glycol incorporated into different sequence contexts and paired with adenine or guanine. We show that TDG and MBD4 can remove thymine glycol when present opposite guanine but not when paired with adenine. The efficiency of these enzymes for removal of thymine glycol is about half of that for removal of thymine in the same sequence context. The two proteins may have evolved to act specifically on DNA mismatches produced by deamination and by oxidation-coupled deamination of 5-methylcytosine. This repair pathway contributes to mutation avoidance at methylated CpG dinucleotides.     

Measurement of deaminated cytosine adducts in DNA using a novel hybrid thymine DNA glycosylase

The hydrolytic deamination of cytosine and 5-methylcytosine drives many of the transition mutations observed in human cancer. The deamination-induced mutagenic intermediates include either uracil or thymine adducts mispaired with guanine. While a substantial array of methods exist to measure other types of DNA adducts, the cytosine deamination adducts pose unusual analytical problems, and adequate methods to measure them have not yet been developed. We describe here a novel hybrid thymine DNA glycosylase (TDG) that is comprised of a 29-amino acid sequence from human TDG linked to the catalytic domain of a thymine glycosylase found in an archaeal thermophilic bacterium. Using defined-sequence oligonucleotides, we show that hybrid TDG has robust mispair-selective activity against deaminated U:G and T:G mispairs. We have further developed a method for separating glycosylase-released free bases from oligonucleotides and DNA followed by GC–MS/MS quantification. Using this approach, we have measured for the first time the levels of total uracil, U:G, and T:G pairs in calf thymus DNA. The method presented here will allow the measurement of the formation, persistence, and repair of a biologically important class of deaminated cytosine adducts.     

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.     

Role of base excision repair in maintaining the genetic and epigenetic integrity of CpG sites

Cytosine methylation at CpG dinucleotides is a central component of epigenetic regulation in vertebrates, and the base excision repair (BER) pathway is important for maintaining both the genetic stability and the methylation status of CpG sites. This perspective focuses on two enzymes that are of particular importance for the genetic and epigenetic integrity of CpG sites, methyl binding domain 4 (MBD4) and thymine DNA glycosylase (TDG). We discuss their capacity for countering C to T mutations at CpG sites, by initiating base excision repair of G·T mismatches generated by deamination of 5-methylcytosine (5mC). We also consider their role in active DNA demethylation, including pathways that are initiated by oxidation and/or deamination of 5mC.     

Dependence of substrate binding and catalysis on pH, ionic strength, and temperature for thymine DNA glycosylase: Insights into recognition and processing of G·T mispairs

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.     

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.     

A DNA repair process in Escherichia coli corrects U:G and T:G mismatches to C:G at sites of cytosine methylation

Escherichia coli contains a base mismatch correction system called VSP repair that is known to correct T:G mismatches to C:G when they occur in certain sequence contexts. The preferred sequence context for this process is the site for methylation by the E. coli DNA cytosine methylase (Dcm). For this reason, VSP repair is thought to counteract potential mutagenic effects of deamination of 5-methylcytosine to thymine. We have developed a genetic reversion assay that quantitates the frequency of C to T mutations at Dcm sites and the removal of such mutations by DNA repair processes. Using this assay, we have studied the repair of U: G mismatches in DNA to C: G and have found that VSP repair is capable of correcting these mismatches. Although VSP repair substantially affects the reversion frequency, it may not be as efficient at correcting U: G mismatches as the uracil DNA glycosylase-mediated repair process.     

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.     

A DNA repair process in Escherichia coli corrects U:G and T:G mismatches to C:G at sites of cytosine methylation

Escherichia coli contains a base mismatch correction system called VSP repair that is known to correct T:G mismatches to C:G when they occur in certain sequence contexts. The preferred sequence context for this process is the site for methylation by the E. coli DNA cytosine methylase (Dcm). For this reason, VSP repair is thought to counteract potential mutagenic effects of deamination of 5-methylcytosine to thymine. We have developed a genetic reversion assay that quantitates the frequency of C to T mutations at Dcm sites and the removal of such mutations by DNA repair processes. Using this assay, we have studied the repair of U: G mismatches in DNA to C: G and have found that VSP repair is capable of correcting these mismatches. Although VSP repair substantially affects the reversion frequency, it may not be as efficient at correcting U: G mismatches as the uracil DNA glycosylase-mediated repair process.     

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.     

Role of MED1 (MBD4) Gene in DNA repair and human cancer

The human protein MED1, also known as MBD4, was isolated in a yeast two-hybrid screening as an interactor of the mismatch repair protein MLH1. MED1 contains an N-terminal 5-methylcytosine binding domain (MBD), which allows binding to methylated DNA, and a C-terminal catalytic domain with homology to bacterial DNA damage-specific glycosylases/lyases. This suggests that DNA methylation may play a role in human DNA repair. MED1 acts as a mismatch-specific DNA N-glycosylase active on thymine, uracil, 5-fluorouracil and, weakly, 3,N(4)-ethenocytosine paired with guanine. The glycosylase activity of MED1 prefers substrates in which the G:T mismatch is present in the context of methylated or unmethylated CpG sites. Since G:T mismatches can originate via spontaneous deamination of 5-methylcytosine to thymine, MED1 appears to act as a caretaker of genomic fidelity at CpG sites. Mutagenesis caused by these deamination events is a frequent mechanism of genetic instability in cancer; thus, based on the biochemical activity of its gene product, MED1 is a candidate tumor suppressor gene. Indeed, frameshift mutations of the MED1 gene have been reported in human colorectal, gastric, endometrial, and pancreatic cancer. In the future, efforts should be directed toward investigations of the functional role of the MED1 gene in the pathogenesis, prevention, and treatment of human cancer.     

Spontaneous Mutation at a 5-Methylcytosine Hotspot Is Prevented by Very Short Patch (Vsp) Mismatch Repair

In many strains of Escherichia coli, the product of gene dcm methylates the internal cytosines in the sequence 5'CC(A or T)GG. Spontaneous deamination of 5-methylcytosine produces thymine which, if not corrected, can result in a transition mutation. 5-Methylcytosines in the lacI gene are hotspots for spontaneous C to T mutations. dcm is linked to vsr, a gene required for very short patch (VSP) repair. VSP repair corrects T.G mispairs in the following contexts:CTAAGGGGTCC, CTTGGGGACC, TAGGGTCC and CTAGGGTC. I have investigated the relationships between cytosine methylation, mutation, and VSP repair. Spontaneous mutations in the repressor (cI) gene of lambda prophage were isolated in wild-type and mutant lysogens. A hotspot for spontaneous mutation that corresponds with a 5-methylcytosine was observed in wild-type lysogens but was not present in bacteria lacking both methylase and VSP repair activity. Introduction of a plasmid containing dcm+ and vsr+ restored the mutation hotspot. If the added plasmid carried only dcm+, the frequency of spontaneous mutations at the 5-methylcytosine was over 10-fold higher than in Dcm+Vsr+ lysogens. The addition of vsr on a plasmid to a wild-type lysogen resulted in a 4-fold reduction in mutation at the hotspot. These findings support the previously untested hypothesis that VSP repair prevents mutations resulting from deamination of 5-methylcytosine.     

HhaI and HpaII DNA methyltransferases bind DNA mismatches, methylate uracil and block DNA repair.

The hydrolytic deamination of 5-methylcytosine (5-mC) to thymine (T) is believed to be responsible for the high mutability of the CpG dinucleotide in DNA. We have shown a possible alternate mechanism for mutagenesis at CpG in which HpaII DNA-(cytosine-5) methyltransferase (M.HpaII) can enzymatically deaminate cytosine (C) to uracil (U) in DNA [Shen, J.-C., Rideout, W.M., III and Jones, P.A., Cell, 71, 1073-1080, (1992)]. Both the hydrolytic deamination of 5-mC and enzymatic deamination of C create premutagenic DNA mismatches (G:U and G:T) with the guanine (G) originally paired to the normal C. Surprisingly, we found that DNA-(cytosine-5) methyltransferases have higher affinities for these DNA mismatches than for their normal G:C targets and are capable of transferring a methyl group to the 5-position of U, creating T at low efficiencies. This binding by methyltransferase to mismatches at the recognition site prevented repair of G:U mismatches by uracil DNA glycosylase in vitro.     

DNA mismatch correction by Very Short Patch repair may have altered the abundance of oligonucleotides in the E. coli genome.

A base mismatch correction process in E. coli K-12 called Very Short Patch (VSP) repair corrects T:G mismatches to C:G when found in certain sequence contexts. Two of the substrate mismatches (5'-CTWGG/3'-GGW'CC; W = A or T) occur in the context of cytosine methylation in DNA and reduce the mutagenic effects of 5-methylcytosine deamination to thymine. However, VSP repair is also known to repair T:G mismatches that are not expected to arise from 5-methylcytosine deamination (example--CTAG/GGT-C). In these cases, if the original base pair were a T:A, VSP repair would cause a T to C transition. We have carried out Markov chain analysis of an E. coli sequence database to determine if repair at the latter class of sites has altered the abundance of the relevant tetranucleotides. The results are consistent with the prediction that VSP repair would tend to deplete the genome of the 'T' containing sequences (example--CTAG), while enriching it for the corresponding 'C' containing sequences (CCAG). Further, they provide an explanation for the known scarcity of CTAG containing restriction enzyme sites among the genomes of enteric bacteria and identify VSP repair as a force in shaping the sequence composition of bacterial genomes.     

miR-21 Gene expression triggered by AP-1 is sustained through a double-negative feedback mechanism

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.     

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.