The Main Role of Human Thymine-DNA Glycosylase Is Removal of Thymine Produced by Deamination of 5-Methylcytosine and Not Removal of Ethenocytosine*

Mika Abu and Timothy R. WatersDagger

From the Department of Biochemistry and Molecular Biology, University College London, Gower Street, London WC1E 6BT, United Kingdom

Received for publication, October 30, 2002, and in revised form, December 11, 2002

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Metabolites of vinyl chloride react with cytosine in DNA to form 3,N4-ethenocytosine. Recent studies suggest that ethenocytosine is repaired by the base excision repair pathway with the ethenobase being removed by thymine-DNA glycosylase. Here single turnover kinetics have been used to compare the excision of ethenocytosine by thymine-DNA glycosylase with the excision of thymine. The effect of flanking DNA sequence on the excision of ethenocytosine was also investigated. The 34-bp duplexes studied here fall into three categories. Ethenocytosine base-paired with guanine within a CpG site (i.e. CpG·epsilon C-DNA) was by far the best substrate having a specificity constant (k2/Kd) of 25.1 × 106 M-1 s-1. The next best substrates were DNA duplexes containing TpG·epsilon C, GpG·epsilon C, and CpG·T. These had specificity constants 45-130 times smaller than CpG·epsilon C-DNA. The worst substrates were DNA duplexes containing ApG·epsilon C and TpG·T, which had specificity constants, respectively, 1,600 and 7,400 times lower than CpG·epsilon C-DNA. DNA containing ethenocytosine was bound much more tightly than DNA containing a G·T mismatch. This is probably because thymine-DNA glycosylase can flip out ethenocytosine from a G·epsilon C base pair more easily than it can flip out thymine from a G·T mismatch. Because thymine-DNA glycosylase has a larger specificity constant for the removal of ethenocytosine, it has been suggested its primary purpose is to deal with ethenocytosine. However, these results showing that thymine-DNA glycosylase has a strong sequence preference for CpG sites in the excision of both thymine and ethenocytosine suggest that the main role of thymine-DNA glycosylase in vivo is the removal of thymine produced by deamination of 5-methylcytosine at CpG sites.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

It has been known for nearly 30 years that exposure to vinyl chloride can cause cancer in humans (1). Vinyl chloride is metabolized by cytochrome P450 2E1 to form chloroethylene oxide (2) which can rearrange spontaneously to give chloroacetaldehyde (3). Both these metabolites react in vitro with DNA to form ethenoadducts of adenine, guanine, and cytosine (Fig. 1). Three of these four possible ethenobases have been detected in animals exposed to vinyl chloride (reviewed in Ref. 4). Ethenobases have also been found in the DNA of rats and humans not exposed to vinyl chloride. These are probably formed endogenously by the reaction of lipid peroxidation products with DNA (5). The ethenobases cause mutations by misincorporating during DNA replication, and there is evidence that these mutations are responsible for the carcinogenicity of vinyl chloride and related chemicals (6).


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Fig. 1.   Formation of 3,N4-ethenocytosine. The vinyl chloride metabolites chloroethylene oxide (i) and chloroacetaldehyde (ii) react with cytosine bases in DNA to form 3,N4-ethenocytosine. Similar reactions occur at the 1,N2- and N2,3-positions of guanine and at the 1,N6-position of adenine (4).

Extracts from human cells remove all four ethenoadducts from DNA (7). Because they are released from the DNA as the ethenobases, it is likely that they are repaired by the base excision repair pathway. The base excision repair pathway (reviewed in Refs. 8 and 9) involves initial removal of the damaged base by a DNA glycosylase. In the short-patch repair pathway the resultant abasic site is cut by an apurinic endonuclease, probably human apurinic endonuclease 1 (APEX1; also known as HAP1, APE1, or Ref-1). The single nucleotide gap is filled by DNA polymerase beta  which also removes the abasic sugar-phosphate. Finally, the phosphate backbone is restored by a DNA ligase. Thymine-DNA glycosylase (TDG) is the enzyme believed to repair G·T mismatches arising from spontaneous deamination of 5-methylcytosine (10). Support for this comes from the observation that TDG excises thymine from G·T mismatches at sites of cytosine methylation (i.e. CpG) much more efficiently than from other DNA sequences (11-14). Recently, two groups (15, 16) independently found that TDG can also remove ethenocytosine from DNA.

A common feature of many DNA glycosylases is their tight binding to the abasic sites that they produce (14, 17-21). Failure to consider this product inhibition led to the so-called single strand-selective monofunctional uracil-DNA glycosylase (SMUG1) originally being incorrectly designated as a single-stranded DNA glycosylase (18, 22). Product binding is particularly strong for TDG and is so tight that in vitro each glycosylase molecule can only process one G·T mismatch (14). The next enzyme in the repair pathway, APEX, can relieve this product inhibition and increase the turnover of TDG by releasing the glycosylase from the abasic site (23). The mechanism of this release is not yet known. The initial experiments showing that TDG can remove ethenocytosine from DNA did not consider product inhibition of the glycosylase (15, 16), and so we decided to study TDG excision of ethenocytosine using single turnover experiments. Because the base pair flanking the mismatched guanine has a remarkably strong effect on the rate of thymine excision from G·T mismatches, the effect of the flanking base pair on the excision of ethenocytosine was also measured.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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Synthesis and Purification of Oligodeoxynucleotides-- 34-Base deoxynucleotides of the general sequence AGC TTG GCT GCA GGX GGA CGG ATC CCC GGG AAT T (where X is A, C, G, or T) were annealed with the complementary strand that had either thymine or ethenocytosine opposite the underlined G. The nomenclature used in the following text is XpG·T-DNA for the guanine·thymine mismatch containing duplexes and XpG·epsilon C-DNA for the guanine·ethenocytosine containing duplexes. X is either A, C, G, or T and refers to the base 5' to the mismatched guanine. Normal base-containing oligodeoxynucleotides were synthesized on an Applied Biosystems 391 DNA synthesizer and purified as described previously (24). Deoxyethenocytidine was synthesized from deoxycytidine using the protocol of Zhang et al. (25). The correct structure of the synthesized deoxyethenocytidine was confirmed by UV and 1H NMR spectroscopy, which gave data agreeing with that published previously (25). The 5'-dimethoxytrityl-protected deoxyethenocytidine phosphoramidite was prepared by standard procedures (26). Oligodeoxynucleotides containing ethenocytosine opposite the underlined G were synthesized using the standard DNA synthesis protocol except that the coupling time for the ethenocytidine phosphoramidite was increased to 2 min. The coupling yield, as judged by trityl cation release, was the same for the ethenocytidine phosphoramidite as for the unmodified phosphoramidites. Full-length oligodeoxynucleotides were separated from failure sequences using Nensorb columns (DuPont), further purified by ion exchange chromatography at pH 12 (27) using a Mono-Q column (Amersham Biosciences), and finally desalted. Oligodeoxynucleotides prepared in this way were >95% pure as judged by 260 nm absorbance of their ion exchange chromatography traces. Samples of the oligodeoxynucleotides were digested with nuclease P1 plus alkaline phosphatase and analyzed by reverse-phase high pressure liquid chromatography. A fifth peak eluted after the four natural deoxynucleosides. This had the absorbance expected for a single deoxyethenocytidine and co-eluted with standard deoxyethenocytidine, thus confirming the presence of ethenocytosine in the oligodeoxynucleotides.

Enzymes-- Thymine-DNA glycosylase was expressed in Escherichia coli from the pT7-hTDG plasmid as described previously (28) and was purified in three chromatographic steps (14). The concentrations of the dilute TDG samples used for the kinetic experiments were determined accurately using a bandshift assay. For this, five different amounts of TDG were incubated for 30 min with 32P-labeled CpG·T-DNA in binding buffer (25 mM Hepes (pH 7.6), 50 mM KCl, 1 mM EDTA, 2 mM dithiothreitol, 0.5 mg/ml bovine serum albumin, and 4% Ficoll 400). Electrophoresis was performed as described (14). The amount of DNA bound was plotted against the volume of TDG added, and the concentration of the glycosylase was determined from linear regression analysis of this plot.

Human APEX was a gift from Dr. I. Hickson and Dr. D. Rothwell (Oxford University, UK).

Glycosylase Assays-- 34-Base pair DNA duplexes were 5'-labeled with 32P in the strand containing the mismatched thymine or ethenocytosine. They were reacted at room temperature with TDG in reaction buffer (25 mM Hepes (pH 7.6), 2.5 mM MgCl2, 2 mM dithiothreitol, 0.2 mM EDTA, 0.5 mg/ml bovine serum albumin) containing either 50 or 140 mM KCl. Because of the sensitivity of the TDG reaction to salt, consistent results can only be attained if extra care is taken to ensure that the concentrations of MgCl2 and KCl are kept constant (i.e. salt present in the protein and DNA stock solutions must be allowed for). For the inhibition experiments, labeled DNA and inhibitor were mixed first, and TDG was then added to start the reaction. Samples from the G·T reactions were removed at various times and quenched by addition of NaOH and EDTA to a concentration of 0.1 M and 10 mM, respectively. Abasic sites produced by the glycosylase were cleaved by heating at 90 °C for 30 min. Because of the greater lability of deoxyethenocytidine to alkali, milder conditions must be used for the ethenocytosine oligonucleotides. Ethenocytosine samples were heated at 90 °C for 30 min in 30 mM piperidine, 10 mM EDTA. This completely cleaved abasic sites, whereas cleavage of the parent DNA containing ethenocytosine was kept to less than 2%. The cleaved DNA was separated from full-length, unreacted DNA by perfusion chromatography using a 2.1 × 30 mm Q HyperD (Biosepra) anion exchange column as described (14). Radiolabeled DNA was detected by Cerenkov counting using a Berthold LB 506 C-1 monitor and was quantified by integration of the peaks.

For the determination of kinetic constants, reactions were carried out using equimolar concentrations of TDG and DNA at seven different concentrations as follows: 0.1, 0.2, 0.5, 1, 2, 5, and 10 times the approximate value of Kd. The reaction was analyzed using the reaction model given in Scheme I that assumes no product is released by the glycosylase during the time that the reaction is monitored. Data for all seven concentrations were fitted simultaneously using the differential equation solving program Berkeley Madonna (version 8.0.1; www.berkeleymadonna.com). The program was used to obtain the best fit of the theoretical lines to all of the experimental data by varying the values of k1, k-1, and k2.


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Scheme I.  


    RESULTS
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ABSTRACT
INTRODUCTION
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Single Turnover Excision of Ethenocytosine by TDG Is Slower Than the Excision of Thymine-- The single turnover excision of ethenocytosine from CpG·epsilon C-DNA was compared with the excision of thymine from CpG·T-DNA (Fig. 2). Under the conditions used for this experiment (10 nM DNA + 10 nM TDG in buffer containing 2.5 mM MgCl2 and 50 mM KCl), the initial rate of excision for ethenocytosine (0.72 nM min-1) was more than four times slower than the initial rate of excision for thymine (3.2 nM min-1). Decreasing the concentration of enzyme and DNA had no effect upon the rate of excision of ethenocytosine showing that in Fig. 2 all the enzyme is bound to the CpG·epsilon C-DNA. The value of Kd must therefore be much less than 10 nM. Also, the initial rate for CpG·epsilon C-DNA in Fig. 2 must be Vmax, giving a value of 0.0012 s-1 for k2. In contrast, reducing the concentration of TDG and CpG·T-DNA significantly reduced the rate of thymine excision (data not shown). Thus, under the conditions of Fig. 2, not all the glycosylase is bound to the CpG·T-DNA. Therefore, the initial rate for CpG·T-DNA in Fig. 2 is less than Vmax, and so k2 for CpG·T-DNA must be greater than 0.0053 s-1.


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Fig. 2.   Rate of excision by TDG of ethenocytosine from CpG·epsilon C-DNA is slower than the excision of thymine from CpG·T-DNA. 10 nM TDG was reacted with 10 nM of 32P-labeled CpG·epsilon C-DNA () or CpG·T-DNA () in reaction buffer containing 50 mM KCl. Samples were removed at various times, and the extent of base excision was measured as described under "Experimental Procedures."

Determination of Kd and k2 for TDG Excision of Ethenocytosine and Thymine-- An attempt to get accurate values for Kd and k2 in the buffer conditions used in Fig. 2, by measuring the rates of excision at different concentrations of glycosylase and substrate, was unsuccessful when CpG·epsilon C-DNA was used, because there was no change in the rate of excision even at the lowest concentration detectable by our assay (0.01 nM). Thus, Kd values for the ethenocytosine oligonucleotide must be <0.01 nM under these buffer conditions. Increasing the concentration of salt decreases the DNA binding affinity of most proteins, mainly because the protein displaces cations from the DNA phosphates when it binds to the DNA (29, 30). By increasing KCl to 140 mM (which is isotonic to mammalian tissue), Kd and k2 could be measured for the action of TDG on both CpG·T-DNA and CpG·epsilon C-DNA (Fig. 3 and Table I). Data for the base excision at seven different equimolar concentrations of DNA and TDG were fitted simultaneously to the reaction model in Scheme I. When fitting was performed allowing all three rate constants to vary, k1 reached the maximum value expected for a diffusion-controlled reaction (109 M-1 s-1). However, equally good fits to the experimental data were obtained with k1 fixed at values up to 2 orders of magnitude lower and allowing just k-1 and k2 to vary. These fits gave essentially identical values for k2 and k-1/k1 (i.e. Kd). Although the absolute values of k1 and k-1 were poorly defined by the experimental data, the results do show that k-1 k2, and so the reaction is not limited by association of TDG with the substrates. In close agreement with the results in 50 mM KCl, the maximum rate of excision (k2) for ethenocytosine is six times slower than the excision of thymine (Table I). Most strikingly Kd for the CpG·epsilon C-DNA substrate is nearly 800 times smaller than the Kd of CpG·T-DNA.


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Fig. 3.   Determination of Kd and k2 for excision by TDG of thymine from CpG·T-DNA and of ethenocytosine from CpG·epsilon C-DNA. Equimolar concentrations of TDG and DNA containing either a G·T (A) or a G·epsilon C (B) mismatch were incubated together in reaction buffer containing 140 mM KCl. Base excision was measured as described under "Experimental Procedures." For each experiment, all data points were simultaneously fitted to Scheme I to determine values of Kd and k2. A, excision of thymine from CpG·T-DNA. Concentrations of TDG and CpG·T-DNA were 2.5 (black-diamond ), 5 (), 12.5 (open circle ), 25 (+), 50 (triangle ), 125 (×), and 250 nM (). B, excision of ethenocytosine from CpG·epsilon C-DNA. Concentrations of TDG and CpG·epsilon C-DNA were 0.01 (black-diamond ), 0.02 (), 0.05 (open circle ), 0.1 (+), 0.2 (triangle ), 0.5 (×), and 1 nM (). Note that the concentrations used in the CpG·T experiment (A) were 250 times greater than those used in the CpG·epsilon C experiment (B) and that the time scale for the CpG·T experiment is approximately half that for the CpG·epsilon C experiment.

                              
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Table I
Kinetic constants k2 and Kd for TDG excision of ethenocytosine and thymine from mismatches with different flanking DNA sequences
The rate of base excision of seven different equimolar concentrations of TDG and mismatched DNA was measured in 140 mM KCl as described under "Experimental Procedures." Values for Kd and k2 were determined by fitting data for all seven concentrations simultaneously to the reaction model given in Scheme I, as shown for the examples of CpG·T-DNA and CpG·epsilon C-DNA in Fig. 3. Errors in parentheses are S.D. for at least three different experiments.

TDG Flips Ethenocytosine Out of DNA More Easily Than It Flips Thymine-- The binding of DNA, represented by Kd in Scheme I, involves flipping the mismatched base out of the DNA helix into a pocket of TDG (31). There are two possible explanations of why CpG·epsilon C-DNA has a much smaller Kd than CpG·T. 1) TDG binds CpG·epsilon C-DNA more tightly by making more and/or better contacts to the DNA substrate, or 2) less energy is required to flip out the ethenocytosine from a G·epsilon C base pair than to flip out a thymine from a G·T mismatch. In terms of potential contacts, the only difference between the two substrates is the base to be excised. The first explanation therefore implies that TDG binds ethenocytosine more tightly than thymine. This was tested by investigating whether free ethenocytosine base inhibits the TDG reaction more than free thymine base. Addition of either thymine or ethenocytosine up to a concentration of 5 mM had no effect upon the reaction of TDG (data not shown). Higher concentrations of free base were not practical because of their poor solubility. TDG therefore has little affinity for either thymine or ethenocytosine. This is in contrast to uracil-DNA glycosylase where 2 mM uracil inhibited the reaction by 58% (32).

Inhibition of the TDG reaction by single-stranded oligonucleotides containing either ethenocytosine or thymine was also investigated. TDG does not excise thymine (23) or ethenocytosine (16) from single-stranded DNA, and so these oligonucleotides would act as reversible inhibitors. The results in Fig. 4 show that the rate of excision of thymine from 25 nM CpG·T-DNA can be reduced to ~50% by inhibition with 250 nM single-stranded thymine containing oligonucleotide. A similar but slightly lower level of inhibition was given by 60 nM single-stranded ethenocytosine oligonucleotide. The results in Fig. 4 suggest that TDG binds single-stranded DNA containing ethenocytosine approximately three times more tightly than single-stranded DNA containing thymine.


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Fig. 4.   TDG excision of thymine from CpG·T-DNA is inhibited by single-stranded DNA containing either thymine or ethenocytosine. Rate of removal of thymine from 25 nM CpG·T-DNA by 25 nM TDG in reaction buffer containing 140 mM KCl in the presence of no inhibitor (), 250 nM single-stranded oligonucleotide containing thymine (open circle ), or 60 nM single-stranded oligonucleotide containing ethenocytosine (×). Base excision was measured as described under "Experimental Procedures."

Excision of Ethenocytosine Is Very Dependent Upon the Flanking DNA Sequence-- The rate of excision of thymine from G·T mismatches is dependent upon the base pair 5' to the mismatched guanine (11-14). To see whether the excision of ethenocytosine exhibited a similar dependence upon flanking sequence, Kd and k2 values for the excision of ethenocytosine from TpG·epsilon C-DNA, GpG·epsilon C-DNA, and ApG·epsilon C-DNA were determined. For comparison, Kd and k2 were also measured for the excision of thymine from TpG·T-DNA under identical buffer conditions (the excision of thymine from other sequence contexts was too slow to allow accurate determination of Kd). The results in Table I show that excision of ethenocytosine also is very dependent upon the 5' base pair. In terms of the specificity constant, k2/Kd, an ethenocytosine in a CpG·epsilon C site is 45 times more efficiently removed than the next best ethenocytosine substrate, TpG·epsilon C-DNA. This is very similar to the difference in specificity constant between CpG·T-DNA and TpG·T-DNA. However, although with the G·T substrates this drop in specificity is entirely because of a reduced k2, the drop in specificity for ethenocytosine is the result of TpG·epsilon C-DNA having both reduced k2 and decreased binding to TDG (larger Kd).

The other ethenocytosine oligonucleotides are even worse substrates; GpG·epsilon C-DNA and ApG·epsilon C-DNA had 77- and 1630-fold, respectively, lower specificity constants than CpG·epsilon C-DNA. Again these reduced specificities are because of both decreased k2 and increased Kd. An earlier study using slightly different buffer conditions found that k2 for the excision of thymine from G·T mismatches depended upon the base 5' to the mismatched guanine in the order C T > G > A (14). Here, excision of ethenocytosine from G·epsilon C base pairs follows a similar trend except that k2 is faster with a 5'-guanine than with a 5'-thymine (i.e. C G > T > A).

Effect of APEX on Product Release by TDG-- In vitro, the reaction of TDG with G·T mismatches is limited by extremely slow release of the abasic DNA product (14, 33, 34). This dissociation is so slow that each TDG molecule removes only one thymine. The apurinic endonuclease, APEX, increases the turnover number of TDG by displacing the glycosylase from the abasic site. The effect of APEX on the turnover of TDG with CpG·T-DNA was compared with its effect on the turnover of TDG with CpG·epsilon C-DNA (Fig. 5). With both substrates in the absence of APEX, the reaction stops after a stoichiometric amount of base has been removed. In the presence of APEX, the turnover of both substrates is increased. This increase in turnover is dependent upon the concentration of APEX and is essentially the same for CpG·T-DNA and for CpG·epsilon C-DNA.


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Fig. 5.   The effect of the apurinic endonuclease APEX upon the removal of ethenocytosine and thymine by TDG. 10 nM TDG was reacted with 100 nM of either CpG·T-DNA (A) or CpG·epsilon C-DNA (B) in reaction buffer containing 50 mM KCl in the absence of (triangle ) or the presence of 20 (), 40 (open circle ), or 80 nM (+) APEX.


    DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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In this paper the effect of flanking DNA sequence on the excision of ethenocytosine by TDG has been examined. To avoid the problem of product inhibition often encountered with DNA glycosylases and which is particularly strong for TDG, single turnover kinetics were used to determine the kinetic constants k2 and Kd (Scheme I). Saparbaev et al. (16) previously calculated kcat and Km values for the reaction of TDG with oligonucleotides containing GpG·T or GpG·epsilon C mismatches assuming that the reaction followed Michaelis-Menten kinetics. Their conclusion that G·epsilon C is a much better substrate for TDG than G·T broadly agrees with our results for the mismatches in this sequence context, but, although the conclusion is correct, since product release by TDG is so slow a Michaelis-Menten analysis is not appropriate. It was originally believed that the physiological purpose of TDG was to remove thymine from the G·T mismatches produced by deamination of 5-methylcytosine (10), but the observation that TDG removes ethenocytosine faster than thymine led to the suggestion that the real in vivo role of TDG is to remove ethenocytosine and that the removal of thymine from G·T mismatches was a fortuitous accident (15, 16). However, from Table I it is clear that TDG has evolved to have a strong sequence preference for base excision at CpG sites. Because G·T mismatches produced by the deamination of 5-methylcytosine occur exclusively in the sequence context CpG·T, the main role of TDG in cells must therefore be to remove deaminated 5-methylcytosine from CpG sites. In E. coli the mismatch-specific uracil-DNA glycosylase, which is a homologue of TDG, appears to be the only glycosylase that removes ethenocytosine efficiently (35). In humans, besides TDG there are two other enzymes known to remove ethenocytosine. These are SMUG1 (36),2 and the methyl-CpG binding domain protein 4 (MBD4, also known as MED1) which has a weak ethenocytosine glycosylase activity (20). It is not yet known which (if any) of these enzymes is responsible for removing ethenocytosine in vivo.

The specificity constant for CpG·T-DNA mismatches is 56 times higher than for the next best G·T substrate, TpG·T-DNA (Table I). This difference in specificity is almost entirely because of a drop in k2 with virtually no change in Kd. In an earlier study we deduced that when it binds to a G·T mismatch in the sequence CpG·T, the glycosylase makes cooperative contacts to the mismatched guanine and to the C·G base pair on the 5' side of the mismatched guanine (14). The fact that Kd does not change when the 5'-flanking base pair is changed shows that the binding energy of the contacts to the mismatched guanine and to the 5'-C·G base pair is used to stabilize the transition state (to lower k2) and not to stabilize the enzyme-substrate complex. There is a similar drop in specificity (45-fold) on going from CpG·epsilon C-DNA to TpG·epsilon C-DNA suggesting that TDG makes the same contacts to the mismatched guanine and the 5'-C·G base pair of the CpG·epsilon C-DNA substrate. However, in this case the drop in specificity is due both to a lower k2 and to an increase in Kd, and so the binding energy of these contacts is used to stabilize both the transition state and the enzyme-substrate complex.

TDG excised ethenocytosine from three of the four DNA sequences containing G·epsilon C tested with specificity constants (k2/Kd) that are at least as good as the physiologically relevant G·T oligonucleotide, CpG·T-DNA. However, in all of the G·epsilon C substrates ethenocytosine was excised more slowly than thymine, and the increased specificity constants result from much lower Kd values. TDG binds CpG·epsilon C-DNA nearly 800 times more tightly than CpG·T-DNA, corresponding to a difference in binding energy of ~4 kcal/mol. In the enzyme-substrate complex the base to be excised would be flipped out of the DNA helix into a binding pocket of the glycosylase (31, 33). In this complex the enzyme makes contacts to the DNA backbone, to the mismatched guanine, and to the flipped out base itself. Because the contacts to the mismatched guanine and to the DNA backbone would be the same for CpG·T-DNA and for CpG·epsilon C-DNA, the 800-fold lower Kd of CpG·epsilon C-DNA may result from TDG binding the flipped out ethenocytosine more tightly than the flipped out thymine. If ethenocytosine was bound very strongly one might expect free ethenocytosine to inhibit the TDG reaction, but up to 5 mM ethenocytosine had no effect. Sub-micromolar concentrations of single-stranded DNA containing either an ethenocytosine or a thymine did inhibit the TDG reaction (Fig. 4), but the single-stranded DNA containing an ethenocytosine was only three times more effective than the single-stranded DNA containing a thymine. This suggests that the majority of the 800-fold tighter binding of CpG·epsilon C-DNA compared with CpG·T-DNA is not because of TDG binding the flipped out ethenocytosine more tightly than thymine. An alternative explanation for the lower Kd of CpG·epsilon C-DNA is that less energy is needed to flip out the ethenocytosine from a G·epsilon C base pair than is needed to flip out thymine from a G·T mismatch. Melting temperature studies show that a G·epsilon C base pair is less stable than a G·T base pair, although the magnitude of this difference in stability varies considerably between different authors. One melting temperature study found that on average a G·T mismatch contributes 3.5 kcal/mol less energy than a G·C base pair to the stability of a DNA duplex (37). In another study, 13 bp DNA duplexes containing a G·epsilon C base pair were 13.4-15.3 kcal/mol less stable than the parental G·C-containing duplexes (38). A further study directly comparing the melting temperature of 15-bp DNA duplexes containing either a G·epsilon C base pair or a G·T mismatch found that the G·epsilon C containing duplexes were 0.43-1.63 kcal/mol less stable than the corresponding G·T-containing duplex (39). Interestingly, the difference in stability between G·epsilon C and G·T was greatest when the mismatch was in a CpG site. Structural studies also indicate that a G·epsilon C base pair is considerably less stable than a G·T mismatch. Both crystal (40) and NMR (41) structures show that a G·T mismatch forms a stable "wobble" base pair that involves two good hydrogen bonds (Fig. 6). Also, the guanine and thymine are stacked well with the adjacent bases in the DNA helix. Although the NMR structure of a G·epsilon C base pair reveals a similar wobble geometry, this base pair is much more distorted, and there is only one hydrogen bond between the ethenocytosine and the guanine (42). In addition to this weaker hydrogen bonding, the ethenocytosine base is very poorly stacked with the adjacent bases, suggesting that the G·epsilon C base pair is considerably less stable than a G·T mismatch.


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Fig. 6.   Schematic diagram of the normal Watson-Crick A·T base pair and the two mismatches, G·T and G·epsilon C. The G·T mismatch is based on the structures found by NMR (41) and crystallographic (40) studies. The G·epsilon C base pair is based on a structure determined by NMR (42). Note that in the A·T and G·T structures the two bases of each base pair are in the same plane, whereas in the G·epsilon C structure the ethenocytosine is distorted out of the plane preventing proper stacking with the flanking bases.

An investigation of the reaction of chloroacetaldehyde with DNA found that, although the distribution of ethenoadducts was not random, there was no obvious DNA sequence preference for the formation of ethenoadducts (43). Ethenocytosine is therefore expected to occur in all sequence contexts. If TDG were the sole enzyme responsible for repairing ethenocytosine, the results in Table I suggest that ethenocytosine formed at ApG·epsilon C sites would be very poorly repaired. Thus one might expect a predominance of mutations of G·C base pairs at ApG sites in vinyl chloride-treated animals. Analysis of liver tumors from rats exposed to vinyl chloride found that 3 of 25 angiosarcomas had a mutation of a G·C base pair in their p53 gene (44). In another study, analysis of hepatacellular carcinomas taken from workers exposed to vinyl chloride found that 5 of 18 had a mutated G·C base pair in their p53 gene (45). Although the number of mutations studied so far is very small, it is perhaps significant that none of these G·C mutations occur at ApG sequences. This suggests that poor repair of ethenocytosine at ApG·epsilon C sites by TDG is not an important factor in vinyl chloride-induced carcinogenesis.

Acting alone, TDG removes a stoichiometric amount of mismatched base because the glycosylase remains bound to the abasic site product, but the next enzyme in the base excision repair pathway, the apurinic endonuclease APEX, releases the TDG from the abasic site (Fig. 5 and Refs. 23 and 46). Tight binding of abasic DNA and the stimulatory effect of apurinic endonucleases has now been found for several other DNA glycosylases (21, 47-49). The mechanism of the release of the glycosylase by APEX is unknown and the subject of some controversy. One theory that has been proposed is that the apurinic endonuclease acts passively by simply "mopping up" free abasic DNA to prevent re-binding of the glycosylase to the abasic DNA, thus allowing it to react with more substrate DNA (21, 50). This is probably true for some glycosylases but not for TDG. The fact that APEX increases the turnover of TDG to more than 40 times the observed rate of TDG dissociation from abasic DNA suggests that APEX actively displaces the glycosylase from the abasic site (23, 46), either by interacting directly with the bound TDG to displace the glycosylase or by binding to the DNA and distorting the DNA structure in such a way that disrupts the TDG-DNA complex (51, 52). The recent discovery that mouse TDG interacts, albeit weakly, with mouse apurinic endonuclease 1 also supports an active displacement mechanism (53). As shown in Fig. 5, APEX increases the turnover of TDG with CpG·epsilon C-DNA to the same extent as with CpG·T-DNA. Because the reaction of TDG with CpG·epsilon C-DNA gives the same glycosylase-abasic DNA complex as the reaction of TDG with CpG·T-DNA, this is consistent with a model where APEX actively displaces the TDG from the abasic site.

    ACKNOWLEDGEMENTS

We thank Professor Peter Swann (University College London, UK) for helpful discussions and for critical reading of the manuscript.

    FOOTNOTES

* This work was supported by the Wellcome Trust, UK.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed. Tel.: 44-20-7679 2323; Fax: 44-20-7679 7193; E-mail: t.waters@biochem.ucl.ac.uk.

Published, JBC Papers in Press, December 18, 2002, DOI 10.1074/jbc.M211084200

2 J. E. A. Wibley, T. R. Waters, K. Haushalter, G. L. Verdine, and L. H. Pearl, manuscript in preparation.

    ABBREVIATIONS

The abbreviations used are: APEX, apurinic endonuclease 1; TDG, thymine-DNA glycosylase; SMUG1, single strand-selective monofunctional uracil-DNA glycosylase.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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