From the Department of Biochemistry and Molecular Biology, University College London, Gower Street, London WC1E 6BT, United Kingdom
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ABSTRACT |
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The time course of removal of thymine by thymine
DNA glycosylase has been measured in vitro. Each molecule
of thymine DNA glycosylase removes only one molecule of thymine from
DNA containing a G·T mismatch because it binds tightly to the
apurinic DNA site left after removal of thymine. The 5'-flanking base
pair to G·T mismatches influences the rate of removal of thymine:
kcat values with C·G, T·A, G·C, and A·T
as the 5'-base pair were 0.91, 0.023, 0.0046, and 0.0013 min1, respectively. Thymine DNA glycosylase can also
remove thymine from mismatches with
S6-methylthioguanine, but, unlike G·T
mismatches, a 5'-C·G does not have a striking effect on the rate:
kcat values for removal of thymine from
SMeG·T with C·G, T·A, G·C, and A·T as the 5'-base
pair were 0.026, 0.018, 0.0017, and 0.0010 min
1,
respectively. Thymine removal is fastest when it is from a G·T mismatch with a 5'-flanking C·G pair, suggesting that the rapid reaction of this substrate involves contacts between the enzyme and
oxygen 6 or the N-1 hydrogen of the mismatched guanine as well as the
5'-flanking C·G pair. Disrupting either of these sets of contacts
(i.e. replacing the 5'-flanking C·G base pair with a
T·A or replacing the G·T mismatch with SMeG·T) has
essentially the same effect on rate as disrupting both sets
(i.e. replacing CpG·T with TpSMeG·T), and
so these contacts are probably cooperative.
The glycosylase removes uracil from G·U, C·U, and T·U base pairs faster than it removes thymine from G·T. It can even remove uracil from A·U base pairs, although at a very much lower rate. Thus, thymine DNA glycosylase may play a backup role to the more efficient general uracil DNA glycosylase.
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INTRODUCTION |
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G·T mismatches are produced in DNA by replication errors and by the deamination of 5-methylcytosine. In human cells, errors from these two sources are probably repaired in different ways. The G·T mismatches from replication errors are thought to be repaired by the postreplicative mismatch repair pathway (1, 2), but the G·T mismatches from the deamination of 5-methylcytosine are repaired by base excision repair (reviewed in Refs. 3 and 4) initiated by excision of the thymine by thymine DNA glycosylase (5, 6). The strong preference of thymine DNA glycosylase for G·T mismatches in the sequence CpG·T (7-10) is consistent with the view that the role of thymine DNA glycosylase is to remove thymine produced through deamination of 5-methylcytosine, because cytosine is methylated almost exclusively in CpG sequences. Although thymine DNA glycosylase can remove uracil as well as thymine (11), cloning of the human enzyme (12) showed that it has no sequence homology to the general uracil DNA glycosylase (EC 3.2.2.3). However, it is homologous to a class of uracil glycosylases specific for G·U mismatches (13). Because of their specificity for uracil in G·U mismatches, and to distinguish them from the general uracil DNA glycosylases, these mismatch-specific uracil glycosylases have been called mismatch-specific uracil DNA glycosylases (MUGs)1 (14). Like thymine DNA glycosylase, they will act only on double-stranded DNA, in contrast to uracil DNA glycosylase, which will act on both single- and double-stranded DNA. Up to now, there have been no published structural studies on thymine DNA glycosylase, but a crystal structure of the Escherichia coli mismatch-specific uracil glycosylase has recently been published (14) that has allowed comparisons to be made with the previously published structures of uracil DNA glycosylases (15, 16). This comparison has shown that despite the lack of any sequence homology between mismatch-specific glycosylases and the general uracil glycosylases, there are similarities in the crystal secondary structures of the two classes of enzymes, and perhaps more important, some of the amino acids in the uracil DNA glycosylase catalytic site are conserved in the proposed catalytic site of E. coli MUG (14-16).
The work on thymine DNA glycosylase reported now began as an extension of an interest in the cytotoxicity of thioguanine. It has been proposed that a crucial step in the mechanism of the delayed cytotoxicity is the formation S6-methylthioguanine·thymine (SMeG·T) base pairs in DNA and the recognition of these mispairs by the proteins of the postreplicative DNA repair system (17, 18). This binding leads, by a process that is not yet understood, to chromosomal damage and cell death (19, 20). If this proposal were correct, one would expect that any other enzyme system that could repair the SMeG·T mispairs would affect the cytotoxicity of thioguanine. In the closely analogous case of N-methyl-N-nitrosourea poisoning, in which the toxicity depends on recognition of O6-methylguanine·thymine (OMeG·T) pairs by the proteins of the postreplicative mismatch repair system (19, 21-23), it is known that two other DNA repair systems can intervene. The methyl group can be removed from the O6-methylguanine by O6-methylguanine-DNA-alkyltransferase (reviewed in Ref. 24), and the thymine can be removed from these mismatches by thymine DNA glycosylase (9). It has been shown that O6-methylguanine-DNA-alkyltransferase removes the methyl group from S6-methylthioguanine residues in DNA, although 106 times slower than it removes the methyl from O6-methylguanine (17), but it was not known whether thymine DNA glycosylase can remove the thymine from SMeG·T mispairs. We now report that thymine DNA glycosylase can remove thymine from SMeG·T mispairs, and we also report a quantitative comparison of the effect of different base pairs on the 5'-side of the mismatched guanine on the rate of removal of thymine from both G·T and SMeG·T mismatches. It was found that the glycosylase can remove thymine from mispairs with all the natural bases and that all mismatches with uracil are better substrates for the enzyme than the analogous mispairs with thymine. The rate of action of the glycosylase on all these substrates was measured and compared. We also report that the enzyme will remove only a stoichiometric amount of thymine or uracil from mismatches because it binds very strongly to the apurinic site generated by its action as a glycosylase. These results, particularly the tight binding to the apurinic site, suggest that the role of the enzyme is not confined to the repair of G·T mismatches in the sequence CpG.
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EXPERIMENTAL PROCEDURES |
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Expression and Purification of Thymine DNA Glycosylase
Thymine DNA glycosylase was expressed in E. coli as described previously (12). After lysis of the cells and removal of cell debris by centrifugation, the soluble protein fraction was loaded onto a 5-ml Hi-Trap Heparin column (Amersham Pharmacia Biotech) equilibrated at 4 °C with 100 mM NaCl, 10% glycerol, and 0.5 mM phenylmethylsulfonyl fluoride in Buffer A (25 mM Hepes (pH 7.6), 1 mM EDTA, and 2 mM dithiothreitol). After washing with 25 ml of the buffer, the column was eluted with three 8.5-ml steps of 350 mM NaCl, 600 mM NaCl, and 800 mM NaCl. Thymine DNA glycosylase elution was monitored by its reaction with a DNA duplex containing a G·T mismatch, by a band shift assay of binding to the same DNA, and by SDS-polyacrylamide gel electrophoresis. The glycosylase eluted at the beginning of the 600 mM NaCl step. The pooled thymine DNA glycosylase fractions were diluted with buffer to 80 mM NaCl and loaded onto a Mono Q (5/5) anion exchange column (Amersham Pharmacia Biotech) that had been equilibrated at 4 °C with 60 mM NaCl and 10% glycerol in Buffer A at a flow rate of 0.5 ml/min. After washing with 7.5 ml of buffer, a gradient of 60-360 mM NaCl was run over 24 ml. Collected fractions were monitored as described above, and thymine DNA glycosylase was found to elute at around 150 mM NaCl. At this stage, two main protein bands were visible by SDS-polyacrylamide gel electrophoresis: one at 60 kDa, corresponding to full-length thymine DNA glycosylase, and one at 55 kDa that is probably derived from full-length thymine DNA glycosylase through proteolytic degradation (12). The thymine DNA glycosylase preparation was further purified on a Mono S (5/5) cation exchange column (Amersham Pharmacia Biotech) at room temperature. The glycosylase was loaded in Buffer A containing 95 mM NaCl and 10% glycerol and then eluted using a salt gradient of 240-360 mM NaCl at a flow rate of 0.5 ml/min. In this way, the full-length thymine DNA glycosylase could be separated from most of the 55-kDa species. The thymine DNA glycosylase was then finally purified by gel filtration using a Superdex-200 (10/30) column (Amersham Pharmacia Biotech) eluted with 25 mM Hepes (pH 7.6), 0.5 mM KCl, 0.1 mM EDTA, 2 mM dithiothreitol, and 15% glycerol at a flow rate of 0.6 ml/min. The final thymine DNA glycosylase preparation was judged to be >95% pure by SDS-polyacrylamide gel electrophoresis, with the main contaminant being the 55-kDa species.
Synthesis and Purification of Oligodeoxynucleotides
34-base pair DNA duplexes of the general sequence AGC TTG GCT GCA GGN XGA CGG ATC CCC GGG AAT T (where N is A, C, G, or T and X is G or SMeG) were synthesized and purified as described previously (18). Oligodeoxynucleotides with a uracil opposite the X position were purchased from Amersham Pharmacia Biotech. They were purified by ion exchange chromatography at pH 12 and desalted (25).
DNA containing an apurinic site opposite a guanine in position X was prepared by reacting a single-stranded oligodeoxynucleotide containing a single uracil with E. coli uracil DNA glycosylase at a molar ratio of 100:1. It was then purified by chromatography as described above and annealed with the complementary strand containing a guanine in position X. Ion exchange chromatography showed that less than 5% of the apurinic sites had been cleaved spontaneously. Radioactively labeled DNA duplexes containing an apurinic site were prepared by reacting 32P-labeled, double-stranded DNA containing a G·U or an SMeG·U base pair with uracil DNA glycosylase at a molar ratio of 50:1. These 32P-labeled duplexes were used in band shift experiments without further purification.
Glycosylase Assays
34-Base pair DNA duplexes, in which the strand containing the mismatched thymine or uracil was 5'-labeled with 32P, were reacted at room temperature with thymine DNA glycosylase in reaction buffer (25 mM Hepes (pH 7.6), 50 mM KCl, 1 mM EDTA, 0.01 mM ZnSO4, 2 mM dithiothreitol, and 0.5 mg/ml bovine serum albumin). 10-µl samples were removed at various times and quenched with 5 µl of 0.3 M NaOH/30 mM EDTA. Apurinic sites produced by the glycosylase reaction were cleaved by heating the samples at 90 °C for 30 min, and the amount of DNA cleaved was measured by gel electrophoresis or by chromatography.
For the electrophoretic assay, an equal volume of formamide containing 10 mM EDTA was added to the samples before loading 4 µl onto a 7 M urea, 20% polyacrylamide sequencing gel. Electrophoresis was at 2000 V for 55 min. The gel was then fixed in aqueous 10% methanol/10% acetic acid, dried, and autoradiographed. The amount of cleaved and intact DNA was quantified by cutting out the bands and scintillation counting.
For the chromatographic assay (which was used in all cases except that shown in Fig. 1A), an equal volume of saturated urea containing 10 mM EDTA and 0.5% Triton X-100 (Sigma) was added to the samples. Cleaved product DNA was separated from full-length unreacted DNA using a 2.1 × 30 mm Poros HQ anion exchange column (PerSeptive Biosystems). A 4-min gradient of 360-500 mM NaCl in 10 mM NaOH, 1 mM EDTA, and 0.05% Triton X-100 at a flow rate of 0.8 ml/min was generally used for separating cleaved from uncleaved DNA, although slight variations in the gradient were needed from column to column. The eluent was monitored using a Berthold LB 506 C-1 radioactivity detector, and radiolabeled DNA was quantified by integration of the peaks.
Band Shift Assays
DNA Containing an Apurinic Site Opposite Guanine-- 0.5 nM 32P-labeled DNA duplex containing an apurinic site in the sequence CpG·AP was incubated for 30 min at room temperature with 1.3 nM thymine DNA glycosylase in binding buffer (25 mM Hepes (pH 7.6), 50 mM KCl, 1 mM EDTA, 0.01 mM ZnSO4, 5 mM dithiothreitol, 0.5 mg/ml bovine serum albumin, and 4% Ficoll 400). Competitor oligodeoxynucleotides in 40-fold excess were premixed with the radiolabeled DNA before adding the thymine DNA glycosylase. After the incubation, samples were loaded onto a nondenaturing 6% polyacrylamide gel for electrophoresis (2 h at 8 V/cm and 10-15 °C). The gel was then dried, and DNA bands were visualized by autoradiography.
DNA Containing an Apurinic Site Opposite S6-Methylthioguanine-- The band shift experiment was carried out in the same way as for CpG·AP DNA except that 1 nM 32P-labeled DNA duplex containing an apurinic site in the sequence CpSMeG·AP was incubated with 1.5 nM thymine DNA glycosylase.
DNA Containing a G·T Mismatch-- 10 nM 32P-labeled DNA containing a G·T mismatch in the sequence CpG·T was incubated in binding buffer with five different concentrations of thymine DNA glycosylase (as indicated in the figure legend) for 1 h at room temperature. After incubation, part of each sample was analyzed for thymine removal using the chromatography assay, and another portion was electrophoresed on a nondenaturing gel as described above. Free DNA and protein-DNA complexes were quantified by cutting the bands from the dried gel and scintillation counting.
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RESULTS |
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Thymine DNA Glycosylase Is a Single Turnover Enzyme-- DNA glycosylases remove their target base by cleavage of the glycosidic bond. This leaves behind an apurinic site that can be cleaved by treatment of the DNA at high temperature and high pH (26). Usually, the extent of base removal is assayed by gel electrophoresis on a denaturing sequencing gel that separates the hydroxide cleaved DNA from uncleaved DNA, as shown in Fig. 1A. The bands are then cut out and quantified by scintillation counting. However, we separated cleaved DNA from uncleaved DNA using perfusion chromatography (27). Fig. 1B, which presents a few example chromatograms, shows that this technique takes only a few minutes. The extent of product formation can be determined from the chromatogram by integration of the substrate and product peaks. Data derived from the gel and the chromatography methods are plotted in Fig. 1C, which shows that they give broadly the same results, although chromatography gives fewer erratic points.
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Thymine DNA Glycosylase Removes Thymine from Base Pairs with
S6-Methylthioguanine--
Early (0-1.6 min) data points
for the removal of thymine from DNA containing a G·T mismatch are
shown in Fig. 2A. With an enzyme concentration of 10 nM, a somewhat slow initial
reaction rate of 9.1 nM min1 was calculated
from the curve. When DNA of the same sequence containing an
SMeG·T base pair was tested under the same conditions,
thymine was removed, but at an initial reaction rate of 0.26 nM min
1, 35 times slower than for the G·T
mismatch (Fig. 2B). Experiments similar to that shown in
Fig. 1 showed that in this case also the final amount of thymine
removed from SMeG·T base pairs did not go beyond the
amount of glycosylase present.
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Rate of Thymine Removal by Thymine DNA Glycosylase Is Dependent
Upon the Base 5' to the Mismatched Guanine or
S6-Methylthioguanine--
The rate of removal of thymine
from a G·T mismatch was measured for DNA duplexes of the same
sequence but with different bases 5' to the mismatched guanine (Fig. 2,
C-E). The initial single turnover rates, listed
in Table I, show that the rate of thymine removal depends upon the 5'-base in the order C T > G > A. This order is in agreement with previously published results,
which have shown a strong preference for a 5'-cytosine (7, 9, 10). Our
results show that thymine is removed from the second best substrate, a
G·T mismatch with thymine 5' to the mismatched guanine (TpG·T),
almost 40 times more slowly than from CpG·T. G·T mismatches with a
5'-guanine or adenine are very poor substrates, with rates of thymine
removal approximately 200 and 700 times slower than CpG·T,
respectively.
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The Slower Reaction Rates Are Not Due to Reduced Substrate
Binding--
Because the measured reaction rate is influenced by the
binding constant of the glycosylase to the DNA mismatch as well as by
the absolute rate of scission of the glycosidic bond
(Kd and kcat, respectively,
in Scheme I), one possible reason for the difference in reaction rates
between the DNA sequences is that they are bound by thymine DNA
glycosylase to different extents. If substrate binding were a limiting
factor in the measured reaction rate, then doubling the concentrations
of DNA and thymine DNA glycosylase should result in a greater than
2-fold increase in the reaction rate. Fig.
3A shows the results of the
reaction between equimolar thymine DNA glycosylase and CpG·T at
concentrations of 2.5, 5, and 10 nM. In order to compare
the relative rates of the three concentrations on the same graph, they
have been plotted as mol of thymine removed/mol of glycosylase. The
results show that, within experimental error, the relative rate of
reaction is independent of concentration. This means that at these
concentrations essentially all of the CpG·T substrate is bound by the
glycosylase. Therefore, the initial reaction rate measured for 10 nM thymine DNA glycosylase acting on CpG·T of 9.1 nM min1 is the maximum rate for 10 nM glycosylase, giving a kcat value of 0.91 min
1 (= 0.015 s
1).
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Thymine DNA Glycosylase Binds to Apurinic DNA Sites Opposite Either Guanine or S6-Methylthioguanine-- The fact that each thymine DNA glycosylase could remove only one thymine suggested either that the enzyme becomes inactivated or that it binds so tightly to the product that it is not released and is therefore prevented from reacting further. To see whether thymine DNA glycosylase bound to the product of its reaction with G·T mismatches, we incubated it with a 32P-labeled DNA duplex containing an apurinic site opposite a guanine (CpG·AP). In a band shift assay, a retarded band was observed that is due to binding of the DNA by the glycosylase (Fig. 4, left panel). This complex is not covalent because it does not persist under conditions of SDS-polyacrylamide gel electrophoresis. The glycosylase also bound to a DNA duplex containing an apurinic site opposite S6-methylthioguanine (Fig. 4, right panel). When the 32P-labeled DNA containing CpG·AP was mixed with a 40-fold excess of unlabeled CpG·AP DNA before addition of the glycosylase, the amount of radioactive complex was greatly reduced. However, excess unlabeled CpG·C DNA could not compete for the complex. This indicates that formation of the complex requires the apurinic site. The amount of complex was also greatly reduced by competition with excess unlabeled CpG·T DNA. Because thymine DNA glycosylase has been reported to bind to DNA containing a G·T base pair (6), this competition could be due to the enzyme binding to the unlabeled CpG·T DNA. However, the complex between thymine DNA glycosylase and a 32P-labeled DNA duplex containing a G·T base pair migrated at the same position as the complex seen with CpG·AP DNA, and when samples of the same incubation mixture were treated with hydroxide at 90 °C and analyzed by chromatography to measure the amount of apurinic DNA, it was found that the amount of DNA bound was exactly equal to the amount of apurinic DNA (Fig. 5). This was true for a range of thymine DNA glycosylase concentrations and demonstrates that the complex seen when thymine DNA glycosylase is incubated with CpG·T DNA is in fact the binding of the enzyme to the DNA containing an apurinic site left after removal of thymine, and not to the original G·T DNA substrate.
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Thymine DNA Glycosylase Removes Uracil from DNA-- Thymine DNA glycosylase is known to be able to remove uracil from base pairs with guanine at a faster rate than it removes thymine (11). The reaction of thymine DNA glycosylase with CpG·U shown in Fig. 7A confirms this finding. The initial rate is approximately 10 times faster than the removal of thymine from CpG·T under the same conditions (Table II). We found that uracil is also removed quite efficiently from CpC·U and CpT·U base pairs (Fig. 7, B and C; Table II). In fact, the rate of removal of uracil from both CpC·U and CpT·U base pairs is faster than the removal of thymine from CpG·T DNA. Somewhat surprisingly, the glycosylase also removes uracil from base pairs with adenine (Fig. 7D; Table II). This is in contrast to a previous report that found that thymine DNA glycosylase did not remove uracil from an A·U base pair in a different DNA duplex (11).
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DISCUSSION |
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This paper reports some general aspects of the kinetics of thymine DNA glycosylase and shows that this enzyme can remove thymine from base pairs with S6-methylthioguanine. To help in these studies, we developed a new method for measuring the action of DNA glycosylases. When thymine DNA glycosylase is reacted with an oligodeoxynucleotide duplex containing a single G·T mismatch an apurinic site is produced that can be cleaved by heat treatment under basic conditions (26). In previous reports, this cleaved product DNA has been separated from the longer, intact substrate DNA by electrophoresis on a sequencing gel, as shown in Fig. 1A, but here we used perfusion chromatography to separate the cleaved and intact DNA (Fig. 1B). This is faster than the gel method; one gets an immediate view of the progress of the reaction, which allows failed reactions to be abandoned immediately; and it gives less erratic results (see Fig. 1C). This method was used to follow the removal by thymine DNA glycosylase of thymine from mismatched base pairs with guanine, S6-methylthioguanine, cytosine, and thymine, and also the removal of uracil from various mismatches.
Fig. 1 shows that in vitro thymine DNA glycosylase removes an approximately equimolar amount of thymine and the reaction then stops. Sibghat-Ullah et al. (9) have also reported a limiting level of reaction for thymine DNA glycosylase and have ascribed it to inactivation of the enzyme. However, the results reported here show that the enzyme is not inactivated, but that it binds so tightly to the apurinic site it produces that it is unable to process more than one mismatch. The evidence for binding to the apurinic site is, first, thymine DNA glycosylase forms a complex with DNA containing an apurinic site opposite either guanine or S6-methylthioguanine (Fig. 4); second, preincubation of thymine DNA glycosylase with DNA containing an apurinic site opposite guanine almost completely prevented it from reacting with DNA containing a G·T mismatch (Fig. 6). Interestingly, the E. coli MUG, a homologue of thymine DNA glycosylase (13), also binds tightly (Kd = 6 nM) to an apurinic site opposite a guanine (14). It is not yet clear whether this strong binding to the apurinic site has any physiological significance. It is possible that, in vivo, the bound glycosylase might recruit other proteins involved in the repair of G·T mismatches, such as an apurinic endonuclease, DNA polymerase, and a DNA ligase. One advantage of such a system would be that complete repair of the G·T mismatch could occur in a concerted fashion. This would minimize the length of time that unstable intermediate states of the repair pathway are exposed and thus reduce the likelihood of a DNA strand break. Alternatively, it is possible that the binding of the glycosylase to the apurinic site acts to block replication of the potentially mutagenic apurinic site.
In many previous reports, the reaction rate of thymine DNA glycosylase
has been measured by determining the level of thymine removal after a
set time, and not as a function of time (6-8). The reaction plateau
seen in Fig. 1 shows that such "single point" measurements do not
necessarily give an accurate determination of the rate of reaction.
Consequently, we have compared the initial rates of a single turnover
reaction of thymine DNA glycosylase with various substrates. When
equimolar amounts of thymine DNA glycosylase and DNA containing a G·T
mismatch in the sequence CpG·T were reacted together at three
different concentrations (2.5, 5, and 10 nM), the initial
rate of reaction, expressed as mol of thymine removed/mol of
glycosylase/min, was independent of reactant concentration (Fig.
3A), implying that at these concentrations, essentially all
of the CpG·T DNA and thymine DNA glycosylase are bound together and
that Kd in Scheme I must be below 2.5 nM. This is consistent with the results of Schärer
et al. (10), who report a Kd of 60 pM for the binding of thymine DNA glycosylase to DNA
containing a nonhydrolyzable uracil analogue opposite guanine in a CpG
sequence. In addition, because all of the DNA was bound to the
glycosylase, the initial rate (0.91 min1; Table I) must
be close to the actual value of kcat. The rate of similar reactions of thymine DNA glycosylase with DNA containing CpSMeG·T or ApG·T were also independent of
concentration (Fig. 3, B and C). Therefore, the
differences between the rates of removal of thymine from CpG·T, from
CpSMeG·T, and from ApG·T (Table I) are not due to
differences in binding of the substrates, but are due to differences in
kcat. It should be pointed out that
kcat measured here may not represent a single step, but may include structural rearrangement as well as the actual
cleavage of the glycosidic bond. Such rearrangement could conceivably
include flipping the thymine base out of the DNA helix, as happens with
uracil in the uracil DNA glycosylase reaction (29) and has been
suggested to happen when the E. coli homologue of thymine
DNA glycosylase, MUG, removes uracil from G.U mismatches (14).
The kcat (0.91 min1) for the
removal of thymine from CpG·T base pairs by thymine DNA glycosylase
is much lower than the kcat of uracil DNA
glycosylase (2500 min
1) (30), but it is of a magnitude
similar to the kcat of other DNA glycosylases.
For example, the kcat of human 3-methyladenine DNA glycosylase is 10 min
1 for removal of
3-methyladenine and 0.38 min
1 for removal of
7-methylguanine (31), and murine N-methylpurine DNA
glycosylase has kcat values of 0.8 and 0.2 min
1 for removal of 3-methyladenine and 7-methylguanine,
respectively (32).
The quantitative comparisons showing the effect on kcat of replacing the mismatched guanine with S6-methylthioguanine or changing the 5'-flanking base pair (Table I) give some possible insight into the structural interactions between enzyme and substrate. The rate of reaction of thymine DNA glycosylase with G·T and SMeG·T mismatches is greatly influenced by the base pair 5' to the mismatched guanine or S6-methylthioguanine (Fig. 2 and Table I). These substrates fall into three groups. The first group consists of the CpG·T DNA in which the G·T mismatch is flanked on the 5'-side by a C·G base pair. This was by far the best substrate. The second group includes the TpG·T DNA, the CpSMeG·T DNA, and the TpSMeG·T DNA. Thymine was removed from all these substrates at a similar rate, which was between 30- and 50-fold slower than the rate of removal of thymine from the CpG·T DNA. Finally, there is a third group, consisting of the GpG·T DNA, the ApG·T DNA, the GpSMeG·T DNA, and the ApSMeG·T DNA, which all have very slow reaction rates.
The most striking observation is the strong preference for a G·T mismatch in a CpG sequence: changing the base 5' to the G·T from cytosine to thymine (i.e. from CpG·T to TpG·T) reduces the rate by 40-fold. The preference for G·T mismatches in this sequence has been reported previously and is consistent with the view that the primary role of thymine DNA glycosylase is to repair G·T mismatches that arise through deamination of 5-methylcytosine (7-10). The much higher rate of removal of thymine from G·T mismatch in a CpG·T sequence than from a G·T mismatch in any other sequence suggests that the enzyme makes a contact to the 5'-C·G base pair during the reaction. This has been confirmed by recent methylation interference studies that have shown that when the glycosylase is bound to DNA with a CpG·U mismatch containing a uracil with a noncleavable glycosidic link, the enzyme makes a strong contact to the 7-nitrogen of the guanine in the 5'-C·G base pair (10). A similar contact to the 7-nitrogen of adenine in the 5'-T·A base pair of TpG·T DNA may explain why thymine is removed 5-20-fold faster from G·T mismatches with a 5'-T·A base pair than from mismatches with a 5'-G·C or A·T (Table I). However, the fact that thymine is removed 40-fold slower from TpG·T than from CpG·T, despite both substrates having this potential contact to the 7-nitrogen of the purine in the 5'-flanking base pair, suggests that the enzyme makes other contacts to the 5'-C·G base pair of CpG·T, in addition to the contact to the 7-nitrogen of guanine.
Another striking observation is that the quantitative reduction in the rate of removal of thymine when the mismatched guanine in CpG·T is changed to S6-methylthioguanine (i.e. CpSMeG·T), is almost identical to the quantitative reduction in rate observed when the 5'-flanking base pair is changed from C·G to T·A (Table I). Because in S6-methylthioguanine, oxygen 6 of guanine has been replaced and the hydrogen in the N-1 position has been lost, the simplest interpretation of the reduction in rate when the mismatched guanine is replaced with an S6-methylthioguanine is that the enzyme makes a contact to oxygen 6 or the N-1 hydrogen of the mismatched guanine of CpG·T during the reaction. The possibility of a contact between the enzyme and the N-1 hydrogen of the mismatched guanine is supported by the recent crystal structure of the homologous E. coli MUG bound to a G·AP site (14). This shows that MUG inserts a "wedge" involving Gly143, Leu144, Ser145, and Arg146 into the apurinic site and that the main chain carbonyl oxygen of Gly143 forms a bifurcated hydrogen bond to the N-1 hydrogen and the 2-amino group of the mismatched guanine. It is possible that thymine DNA glycosylase could make a similar hydrogen bond from a peptide carbonyl to the mismatched guanine. However, before drawing inferences about the structure of thymine DNA glycosylase from the structure of this particular uracil glycosylase, it should be remembered that although the enzymes are homologous, they differ quite considerably in their amino acid sequence and enzymic specificity. Only one of the four amino acids of this uracil glycosylase wedge is identical in the analogous sequence (Ser272, Ser273, Ala274, and Arg275) of thymine DNA glycosylase. Also, in thymine DNA glycosylase, there are 14 amino acids immediately on the carboxyl side of Arg275 that are absent from MUG. Furthermore, under the normal conditions of enzyme assay, MUG will not remove thymine from G·T mismatches (13). One very important feature of the results is that if both the mismatched guanine is changed to S6-methylthioguanine and the 5'-base pair is changed to T·A then the reaction rate is reduced by about the same amount as if either change is made on its own (i.e. thymine is removed from TpG·T, CpSMeG·T and TpSMeG·T at similar rates; Table I). Therefore, these changes do not have independent effects, suggesting that the selectivity for G·T mismatches with a 5'-flanking C·G is the result of cooperativity between the contacts that the enzyme makes to the 5'-C·G base pair and those made to the mismatched guanine in CpG·T. Unfortunately, the homologous mismatch-specific uracil glycosylase does not have a strong preference for mismatches with a 5'-flanking C·G2, so its crystal structure (14) does not suggest a way in which the cooperative contacts of thymine DNA glycosylase might be made.
We also found that thymine DNA glycosylase can remove thymine from
CpC·T and CpT·T base pairs but not from CpA·T base pairs. The
results in Table II show that the order of preference of the glycosylase for mismatches involving thymine is G·T C·T > T·T (with no reaction at A·T), which is in agreement with a
previous report (6). Thymine DNA glycosylase also removes uracil from base pairs with any of the four DNA bases, including a slow removal of
uracil from base pairs with adenine. The rate of removal of uracil is
much faster than the removal of thymine from equivalent mismatches, and
whereas removal of thymine from C·T and T·T mismatches is 25 and
200 times slower, respectively, than from G·T, the removal of uracil
from C·U and T·U mismatches is only 2 and 9 times slower, respectively, than from G·U. The removal of uracil by thymine DNA
glycosylase is not harmful to the cell (and may act as a useful backup
to the general uracil DNA glycosylase) because the base being removed
(uracil) is not a normal component of DNA. However, removal of thymine
from T·T and C·T base pairs has the potential of fixing a DNA error
into a permanent mutation because thymine DNA glycosylase cannot
distinguish which is the "correct" base in the pair. Presumably,
the extremely slow rate of reaction with T·T and C·T reduces the
probability of thymine DNA glycosylase incorrectly removing a thymine
from such sites in vivo.
In addition, thymine DNA glycosylase can remove thymine from SMeG·T base pairs. The reaction is 10-20-fold faster if the base 5' to the S6-methylthioguanine is a pyrimidine rather than a purine (Table I). This sequence dependence is similar to that for the structurally related O6-methylguanine·thymine base pairs (OMeG·T) because Sibghat-Ullah et al. (9) reported that significant amounts of thymine were removed after 90 min of incubation of the glycosylase with CpOMeG·T DNA, and with TpOMeG·T DNA, but that no reaction could be detected after incubation with GpOMeG·T or ApOMeG·T DNA. The removal of thymine from SMeG·T base pairs suggests that some SMeG·T base pairs may be processed by this base excision pathway and thus avoid recognition by the proteins of the postreplicative mismatch repair pathway. Although it has been suggested that, in the closely analogous case of OMeG·T base pairs, base excision repair might itself contribute to toxicity (33), it is clear that postreplicative mismatch repair, and not base excision repair, is the predominant factor in the cytotoxicity of thioguanine, because a cell line that has lost postreplicative mismatch repair is resistant to 6-thioguanine, despite retaining thymine DNA glycosylase activity (7).
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ACKNOWLEDGEMENTS |
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We are extremely grateful to Josef Jiricny (Institute for Medical Radiobiology, Switzerland) for providing the thymine DNA glycosylase-overproducing strain and for useful discussions. We also thank Renos Savva and Laurence Pearl (University College London, United Kingdom) for the kind gifts of E. coli uracil DNA glycosylase and UGI.
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FOOTNOTES |
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* This work was supported by the Medical Research Council, United Kingdom.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.
To whom correspondence should be addressed. Tel.: 44-171-3807117;
Fax: 44-171-3807193; E-mail: t.waters{at}biochem.ucl.ac.uk.
The abbreviations used are: MUG, mismatch-specific uracil DNA glycosylase; SMeG, S6-methylthioguanineOMeG, O6-methylguanineAP, apurinic/apyrimidinic siteUGI, uracil DNA glycosylase inhibitor.
2 T. R. Waters and P. F. Swann, unpublished observations.
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REFERENCES |
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