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J. Biol. Chem., Vol. 275, Issue 43, 33449-33456, October 27, 2000
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From the Institute of Medical Radiobiology, University of
Zürich and the Paul Scherrer Institute, August Forel Strasse 7, Zürich 8008, Switzerland
Received for publication, June 13, 2000, and in revised form, July 20, 2000
Human thymine DNA glycosylase (TDG) was
discovered as an enzyme that can initiate base excision repair at sites
of 5-methylcytosine- or cytosine deamination in DNA by its ability to
release thymine or uracil from G·T and G·U mismatches. Crystal
structure analysis of an Escherichia coli homologue
identified conserved amino acid residues that are critical for its
substrate recognition/interaction and base hydrolysis functions. Guided
by this revelation, we performed a mutational study of structure
function relationships with the human TDG. Substitution of the
postulated catalytic site asparagine with alanine (N140A) resulted in
an enzyme that bound mismatched substrates but was unable to catalyze
base removal. Mutation of Met-269 in a motif with a postulated role in
protein-substrate interaction selectively inactivated stable binding of
the enzyme to mismatched substrates but not so its glycosylase
activity. These results establish that the structure function model
postulated for the E. coli enzyme is largely applicable to
the human TDG. We further provide evidence for G·U being the
preferred substrate of TDG, not only at the mismatch recognition step
of the reaction but also in base hydrolysis, and for the importance of
stable complementary strand interactions by TDG to compensate for its comparably poor hydrolytic potential.
DNA of all organisms is susceptible to modification and damage
through the action of a variety of exogenous and endogenous reagents. A
prominent form of spontaneous damage arises through hydrolytic
deamination of bases carrying exo-cyclic amino groups such as cytosine
and 5-methylcytosine. Deamination of cytosine in double-stranded DNA
(dsDNA)1 generates a
uracil·guanine mispair and, similarly, deamination of 5-methycytosine
generates a thymine·guanine mispair. In vitro, both events
occur at appreciable rates, with 5-methylcytosine deamination being
slightly faster than that of cytosine (1) and, in vivo, both
deamination products are mutagenic and will produce C A G·T mismatch-specific thymine glycosylase activity was discovered
in HeLa cell extracts (3). It was purified to apparent homogeneity (4),
and the encoding cDNA was cloned (5). The biochemical properties of
this thymine DNA glycosylase (TDG) are compatible with a function of
the enzyme in cellular defense against mutagenesis by cytosine and
5-methylcytosine deamination. It is capable of recognizing G·T and
G·U mismatches in DNA and initiating their restoration to G·C base
pairs through a base excision repair process involving DNA polymerase
More recent evidence suggested a wider range of possible substrates and
functions for TDG homologues. Both the E. coli and the human
enzymes were found to efficiently process a mutagenic cyclic adduct of
cytosine, 3,N4-ethenocytosine
( The three-dimensional structure of E. coli Mug was analyzed
by x-ray crystallography and resolved at a resolution of 1.8 Å (14).
This revealed striking structural similarities between Mug and the
functionally related uracil DNA glycosylases, despite very limited
conservation at the amino acid sequence level. Similar to uracil DNA
glycosylase, Mug forms an active site pocket, which penetrates into the
core of the enzyme. The inferred catalytic mechanism for Mug suggests
that the mispaired base to be released is flipped out of the DNA double
helix and accommodated within the active site pocket in a manner that
allows the N-glycosidic bond to be hydrolytically attacked
by an activated water molecule. Positioning of the water molecule is
coordinated by the Asn-40 of the highly conserved putative active site
motif GINPGL. Base flipping by Mug is accompanied by intercalation of a
3-amino acid wedge into the DNA double helix. The less-conserved
intercalating residues around Gly-143 occupy the abandoned space
opposite the guanine and maintain the base-stacking interactions to
avoid the bases flanking the flipped-out residue collapsing on each
other. A notable difference to the mechanism employed by uracil DNA
glycosylase (15) is that the Mug residues involved in helix
intercalation engage in specific complementary DNA strand interactions
in a way that mimics hydrogen bonding to the widowed guanine (14, 16).
To test the functional predictions from the Mug crystal structure on
the human TDG and to better understand the structural relationship
between different TDG homologues, we performed site-directed mutagenesis of critical residues and examined the biochemical activities of the mutant proteins. We were able to separate the DNA
glycosylase and the substrate interaction functions in human TDG by
mutating the implicated residues Asn-140 and Met-269. By studying the
differential effects of the amino acid substitutions on binding and
excision of thymine, uracil, and 5-fluorouracil opposite guanine, we
found the relative inefficiency of TDG in G·T versus G·U
processing to be a consequence of both a lower G·T binding affinity
and a lower catalytic efficiency in thymine release. We further
established experimental evidence for the importance of stable
complementary strand interactions by human TDG as compensation for its
comparably poor hydrolytic potential.
Reagents and Oligonucleotides--
All oligonucleotides were
synthesized by Microsynth (Switzerland). The substrate oligonucleotides
were polyacrylamide gel electrophoresis-purified after synthesis.
Restriction enzymes and uracil DNA glycosylase inhibitor were supplied
by New England BioLabs (Beverly, MA), and the UDG was supplied by Roche
Diagnostics (Switzerland). All other chemicals and reagents were
purchased from Sigma (Switzerland).
Plasmids and Expression Vectors--
pPRS202b: pQE30 (Qiagen,
Germany), containing a BamHI and SalI polymerase
chain reaction fragment of full-length cDNA encoding human TDG, was
cloned into the respective restriction sites (polymerase chain reaction
primers were: BamATG,
5'-GCACGTGGATCCATGGAAGCGGAGAACGCG-3'; TAASal,
5'-CCGTGTCGACTTAAGCATGGCTTTCTTCTTCCTG-3'). This cloning strategy generates a 6-histidine-TDG fusion open reading frame downstream of a promoter-operator element consisting of phage T5
promoter and two lac operator sequences. The plasmids
encoding mutant proteins were as follows. pPRS203b: N140A
mutant; pPRS204b: N140D mutant; pPRS205: M269H mutant; pPRS209: A145S mutant.
Site-directed Mutagenesis--
In vitro mutagenesis
of human TDG was performed using the QuikChange site-directed
mutagenesis kit from Stratagene (San Diego, CA) according to the
manufacturer's instructions. pPRS202b served as template for
mutagenesis, and the oligonucleotide primers used to generate the
individual mutations were as follows (sense strand sequences shown
only): hsTDG-N140D:
5'-GTCATTATTGGCATAGACCCGGGACTAATGGC-3'; hsTDG-A145S:
5'-CCCGGGACTAATGTCTGCTTACAAAGGGC-3'; hsTDG-N140A: 5'-GTCATTATTGGCATAGCCCCGGGACTAATGGC-3'; hsTDG-M269H-u:
5'-GAAACTCTCTGCTATGTTCATCCATCATCCAGTGC-3'.
Purification of Recombinant TDG Proteins--
Expression
constructs for the individual TDG variants were cotransformed with the
lacI-repressor encoded on plasmid pREP4 (Qiagen) into
E. coli BL21(DE3) cells by electroporation. Transformants were selected on LB plates containing 100 µg/ml ampicillin, 50 µg/ml kanamycin, and 2% glucose after incubation at 30 °C. TDG expression cultures of 1 liter of LB medium containing the same ingredients were inoculated with 25 ml of an overnight culture and
incubated at 30 °C until an A600 of
0.8 was reached. After lowering the temperature of the culture to
20 °C over a period of 30 min, TDG expression was induced by the
addition of 200 µM isopropyl-1-thio- Glycosylase Activity Assay--
The enzymatic activity of the
recombinant wild-type and mutant proteins was monitored by means of a
standardized nicking assay. 60-mer double-stranded oligonucleotide
substrates containing different mismatches were prepared by annealing
of an unlabeled upper strand oligonucleotide
5'-TAGACATTGCCCTCGAGGTACCATGGATCCGATGTCXACCTCAAACCTAGACGAATTCCG-3' to a 5'-fluorescein-labeled lower oligonucleotide strand
5'-F-CGGAATTCGTCTAGGTTTGAGGTYGACATCGGATCCATGGTACCTCGAGGGCAATGTCTA-3', where X = G or A and Y = C, T, U,
or 5-fluorouracil (FU). Strand annealing was carried out in 10 mM Tris-HCl, pH 8.0, 50 mM NaCl with 0.5 µM labeled and 1 µM unlabeled
oligonucleotide by heating to 95 °C for 5 min and gradual cooling to
25 °C over 30 min.
Standardized nicking reactions were carried out in a 20-µl total
volume in 1× nicking buffer (50 mM Tris-HCl, pH 8.0, 1 mM DTT, 0.1 mg/ml bovine serum albumin, 1 mM
EDTA) containing 1 pmol of substrate DNA, 1 pmol of TDG protein, and
0.5 unit of uracil DNA glycosylase inhibitor. The reactions were
incubated for 15 min at 37 °C. Deviating assay conditions are
indicated where appropriate. The reactions were then stopped, and the
generated AP-sites were cleaved by the addition of 1 N NaOH
to a final concentration of 90 mM and heating to 99 °C
for 10 min. Subsequently, the DNA was ethanol-precipitated at
Electrophoretic Mobility Shift Assays--
EMSAs were performed
to measure the DNA-binding ability of wild-type and mutant TDG
proteins, using the double-stranded oligonucleotide substrates
described above. Substrate containing an AP-site was generated as
follows: 10 pmol of duplex DNA containing a uracil opposite G or A at
position Y in the labeled oligonucleotide (see above) was incubated
with 1 unit of uracil-DNA-glycosylase (Roche Diagnostics, Switzerland)
in 1× UDG buffer (50 mM Tris-HCl, pH 8.0, 1 mM
DTT) in a total volume of 20 µl for 2 h at 37 °C. Accuracy and completion of AP-site formation was tested by NaOH treatment and
denaturing gel-electrophoresis as described above for the nicking
assay. Fully processed substrate DNA was then stored at
In standard EMSA, 4 pmol of TDG protein were incubated in a 10-µl
reaction mixture containing 1 pmol of labeled oligonucleotide substrate, 10 pmol of unlabeled homoduplex competitor DNA, 50 mM Tris-HCl, pH 8.0, 1 mM DTT, 5% glycerol,
and 1 mM EDTA. After 15 min at 37 °C the reactions were
loaded immediately onto 6% native polyacrylamide gels in 0.5 × TBE (Bio-Rad, Mini Protean II cell), and electrophoresis was carried
out in 0.5 × TBE for 50 min at 100 V at room temperature. The
fluorescent probes were visualized using a Storm 860 (Molecular
Dynamics) in the blue fluorescence mode, and the ImageQuaNT software
(version 1.2) was used for the subsequent quantitative analysis of the
fluorescent signals.
In substrate dissociation assays, 2 pmol of TDG protein was
preincubated with 1 pmol of labeled substrate in 1× binding buffer and
a total volume of 10 µl. After 10 min at 37 °C, different molar
excesses (0, 10-fold, and 20-fold) of unlabeled unspecific competitor
(homoduplex) or specific, mismatch-containing competitor substrate was
added and the reaction was left to proceed for another 10 min at
37 °C. Substrate binding was then analyzed on 6% native polyacrylamide gels as described above.
Rationale of Site-directed Mutagenesis--
The existence of TDG
homologues in organisms ranging from bacteria to humans documents that
the enzyme is of ancient origin and has a highly conserved structure
and function. The degree of evolutionary relationship between the
catalytic core domains of TDG homologues across the species E. coli, Schizosaccharomyces pombe, Drosophila
melanogaster, and Homo sapiens is illustrated in Fig.
1. Highlighted are two conserved sequence
motifs that build critical parts of the enzyme's active site, as
deduced from the crystal structure of E. coli Mug. These are
the N-terminal GINPGL and the C-terminal NPSGLSR sequences. The amino
acid Asn-18 in the N-terminal motif is the predicted catalytic residue
in Mug (14) and is present in all TDG homologues identified thus far.
The structural model suggests a role for this asparagine in activation
of a water molecule for the hydrolytic attack of the
N-glycosidic bond of the flipped-out base to be released. Residues of the less conserved C-terminal motif NPSGLSR form a structural interface for the protein-DNA interactions involved in the
base-flipping/helix-intercalation mechanism proposed for Mug (14).
These functions are comparable to those assigned to equivalent motifs
in the well-characterized uracil DNA glycosylase enzyme (15, 17) (Fig.
1). Yet, the helix-intercalating residues of Mug, unlike those of
uracil DNA glycosylase enzyme, establish and maintain specific contacts
to the Watson-Crick face of the widowed G in the complementary DNA
strand (14, 16). Ser-23 of Mug is part of a small, flexible helix
contributing to the fold of the active site and appears to interfere
with the accommodation of the hydrophobic thymine within the catalytic
pocket. The equivalent residue in the human enzyme is the smaller and
less polar Ala-145, a substitution that was proposed to account for the
human enzyme's ability to process thymine-containing substrates
(16).
To test whether the functional predictions from the Mug crystal
structure are applicable to the human TDG enzyme, we mutagenized the
putative active site residue Asn-140 to Ala (N140A) or Asp (N140D), the
presumed DNA binding site residue Met-269 to His (M269H), as well as
Ala-145 to Ser (A145S) according to standard site-directed mutagenesis procedures.
Expression and Purification of Recombinant TDG Variants--
To
facilitate purification, we expressed the wild-type and mutant variants
of human TDG in E. coli as N-terminal fusions with a
6-histidine-tag. Using a simple two-step purification scheme involving
metal affinity chromatography (nickel-nitrilotriacetic acid) and fast
protein liquid chromatography (Resource Q), we were able to produce
5-10 mg of pure protein from 1 liter of culture. Consistent with
previous observations (5), we noticed that moderate expression at low
temperature (20 °C) was required to avoid the formation of inclusion
bodies with insoluble protein. Regarding solubility, the proteins
carrying mutations at position Asn-140 and Ala-145 behaved as wild-type
TDG, whereas the M269H mutant gave rise to a lower amount of soluble
protein. All TDG variants showed fractionation properties identical to
the wild-type TDG.
Enzymatic Properties of the TDG Variants--
Human TDG releases
thymine and uracil from G·T and G·U mismatches to generate AP-sites
opposite G (7). We examined the ability of the mutant TDG proteins to
catalyze this reaction. As substrate we used fluorescein-labeled,
double-stranded and single-stranded oligonucleotides of 60-base pair
length carrying a target lesion 24 base pairs away from the labeled
5'-end. Upon incubation with TDG, we monitored the generation of
alkaline-sensitive AP-sites by treatment of the reactions with NaOH and
separation of the cleaved products from the alkaline-resistant
substrate fragments in denaturing gels. To analyze the DNA-binding
capacity of the mutant proteins, we performed gel retardation assays
with fluorescently labeled oligonucleotide substrates in the presence or absence of specific and unspecific competitor DNA. Because TDG is
very strongly product-inhibited under in vitro assay
conditions, we based our comparisons on single turnover kinetics of
AP-site formation under standard reaction conditions. The specific
conditions applied were equimolar enzyme substrate ratios and
incubation at 37 °C, the parameters for comparison were total amount
of substrate processed (Pmax) and the time
requirement for processing 50% of the plateau levels for each
substrate (T50). These values are summarized for
each TDG variant in Table I.
Enzyme-substrate interactions were assessed as equilibrium binding
capacity under standardized EMSA conditions described under
"Experimental Procedures."
Consistent with previously reported observations (8), we found that
purified recombinant wild-type TGD efficiently processed G·U and
G·T substrates and very weakly also uracil in A·U base pairs,
whereas uracil in ssDNA was resistant to cleavage by the enzyme (Fig.
2A). In the absence of
competitor DNA, the wild-type enzyme bound homoduplex DNA nearly as
well as the G·T and G·U heteroduplex substrates or an AP-site
opposite G (G·AP) (Fig. 4A). Only when substrate binding
was assayed in the presence of an excess of homoduplex competitor DNA
did differences in affinities become more evident (Fig.
3A). Under these conditions,
substrate binding efficiencies followed the order G·AP = G·U > G·T
The N140A mutation abolished the catalytic potential of the enzyme. No
G·T or G·U mismatch processing was detectable under standard assay
conditions, neither in the presence of a 20-fold excess of enzyme over
substrate as shown in Fig. 2B, nor after prolonged
incubation times (not shown). Yet, this hydrolytic mutation was still
proficient in substrate binding (Fig. 3B). Although, in
comparison with the wild-type protein, G·C and G·AP binding by the
mutant was nearly unaffected, G·U and G·T binding efficiency was
decreased, with G·T binding being only marginally stronger than
binding to the homoduplex substrate. This mutant thus displayed the
following order of decreasing binding affinity: G·AP > G·U > G·T > G·C. The apparently stronger affinity of
wild-type TDG for the G·U and G·T substrates is explained by the
fact that, under standard EMSA conditions, the wild-type enzyme
releases the mismatched uracil and thymine bases from these substrates and remains bound to the G·AP sites (our data not shown (8)).
Mutation of the same site to Asp (N140D) produced an enzyme with
reduced catalytic activity. Whereas G·U was still processed (T50 = 13 min), the mutant has lost the ability
to measurably act on the G·T and A·U substrates (Fig.
2C, Table I). Even in the presence of a 20-fold excess of
enzyme over substrate and after prolonged incubation, no processing of
the latter substrates was detectable (data not shown). Substrate
binding by the N140D mutant appeared to be slightly destabilized
compared with the wild-type protein or the N140A variant, but overall,
it showed the same order of binding preference as did the N140A mutant, namely G·AP > G·U > G·T = G·C (Fig.
3C). Binding to G·C and G·T was more affected than
binding to G·U and G·AP substrates, which is consistent with the
enzyme converting G·U but not G·T substrates to G·AP products
that are bound with the highest affinity.
Mutation of Met-269 to His resulted in an enzyme with reduced
glycosylase activity on both G·T and G·U substrates. Fig.
2D shows that a higher than 10-fold excess of the mutant
protein over substrate is needed to yield near wild-type amounts of
product under otherwise standard conditions. The substrate specificity as expressed by the ratio between G·U (T50 = 1.0 min) and G·T (T50 = 3.25 min) processing
efficiency remained unaffected in this TDG variant (Table I). A more
dramatic effect appeared in the DNA binding assays. No mobility shift
was detectable with either substrate indicated in Fig. 3D.
Even in the presence of a large molar excess (30-fold) of enzyme over
substrate, no interaction was evident under EMSA conditions (not
shown). This documents an inability of this mutant protein to stably
interact with the DNA substrate, which may account for the general
reduction in its uracil and thymine glycosylase activity.
The double mutant N140D,M269H was unable to process or bind any of the
substrates used (not shown), indicating that weakening both the
catalytic activity and substrate binding capacity of the enzyme has a
synergistic effect on its biochemical function.
Mutating Ala-145 to Ser (A145S) did not notably change the enzymatic
properties of TDG. In particular, we did not observe an effect on the
enzyme's ability to process the G·T mismatched substrate (data not
shown). Thus, contrary to predictions based on the Mug crystal
structure (16), conversion of the equivalent of Ser-23 to Ala in the
human enzyme cannot adequately account for its acquired ability to
process G·T mismatches.
Insights into DNA Interaction Modes of Human TDG--
TDG has
properties of a general DNA-binding protein, it efficiently binds
different dsDNA substrates ranging from homoduplex DNA to mismatch- and
AP-site-containing duplexes. The molecular nature of these protein-DNA
interactions is largely unknown. While homoduplex binding was not
investigated, mismatch binding studies have been complicated by the
fact that, under EMSA conditions, TDG readily converts the mismatched
substrates to AP-sites to which it remains bound (8, 18). Having
generated a non-hydrolytic mutant, we were interested to explore
substrate recognition and binding by TDG under conditions where no
processing occurs. To assess the stability of TDG-substrate
interactions, we preincubated the wild-type and the mutant proteins
with labeled G·C, G·T, G·U, and G·AP substrates under
conditions where the binding and nicking reactions were completed. We
then added nonspecific or specific competitor DNA in 10- and 20-fold
molar excess and allowed the reaction to proceed. Fig.
4A shows that binding of
wild-type TDG to G·C homoduplex DNA was significantly reduced when
G·C or G·AP competitor DNA was added. We therefore consider this
mode of substrate interaction reversible. In contrast, no reduction in
the amounts of protein-DNA complexes was seen when the wild-type enzyme
was preincubated with either G·T, G·U, or G·AP, even after
addition of a 20-fold or higher molar excess of specific competitor DNA (Fig. 4A) or after prolonged incubation (not shown). This
implies a different mode of DNA interaction, where TDG remains tightly bound to its substrate and cannot be turned over in our experimental system. Taking into account that wild-type TDG immediately processes G·T and G·U mispairs under EMSA conditions, these results document the ability of TDG to stably interact with AP-sites opposite G but do
not reflect the substrate binding preference of the enzyme. Therefore,
we performed the same experiments with the hydrolysis-deficient N140A
mutant. This TDG variant was indistinguishable from the wild-type
enzyme with respect to G·C binding, but its interaction with G·T
substrate was significantly more labile and reduced to undetectable
levels when challenged with a 20-fold excess of specific competitor DNA
(Fig. 4B). In contrast, G·U binding was only marginally affected by the addition of specific competitor DNA, and the complex with G·AP totally resisted the presence of excess amounts of specific competitor DNA. Therefore, we conclude that interaction of human TDG
with a substrate containing a G·T mismatch is reversible and, thus,
similar to its mode of homoduplex DNA interaction, whereas G·U and
G·AP site binding is more specific and highly stable.
Processing of 5-Fluorouracil and Its Implications on Mechanistic
Properties of TDG--
Human TDG processes G·T mismatches with a
10-fold lower rate than G·U (8). This can be explained by a lower
affinity of the enzyme for the G·T mismatch and/or by a higher
chemical stability of the N-glycosidic bond connecting the
thymine with the deoxyribose. Although our substrate dissociation
experiments indeed document a weaker interaction of TDG with the G·T
mispair, the latter possibility gains some support from the fact that
the 5-methyl group distinguishing thymine from uracil can act as an
electron donor and, thus, may exert a stabilizing effect on the
glycosidic bond. If so, the contrary might be expected for a uracil
carrying a strong electron-withdrawing group at the same position. We
were, therefore, interested to test the efficiency of base hydrolysis
with the cytotoxic anticancer drug 5-fluorouracil (FU). We found that
the wild-type enzyme processed substrates containing FU very
efficiently, and, interestingly, it did so not only when FU was
mismatched with G but also when it was opposite A, or present in a
single-stranded oligonucleotide (Fig.
5A, Table I). The non-binding
mutant M269H behaved as the wild-type protein, but was generally less
efficient (Fig. 5B, Table I). Surprisingly, our
non-hydrolytic mutant N140A was also able to process the G·FU
substrate, but not A·FU and FU in ssDNA (Fig. 5C, Table
I). The fact that this catalytic mutant processed G·FU, whereas no
cleavage was detectable with G·U and G·T (Fig. 2B),
supports the view that the 5-fluoro substitution affects the stability
of uridine such that a lower activation energy is required to hydrolyze
the N-glycosidic bond. This is also in agreement with the
effect of the N140D mutation, which has a less dramatically reduced
catalytic potential. This protein failed to process the G·T
substrate, showed intermediate activity on G·U, and displayed highest
activity with the G·FU substrate (Figs. 2C, 5D,
Table I).
Given these results, we examined whether transient interactions of the
fully active wild-type enzyme with an energetically favorable substrate
such as FU could be sufficient for base hydrolysis. This would predict
that TDG is able to process FU substrate without engaging into stable
complementary strand interactions and, therefore, in a reaction showing
enzymatic turnover. To investigate this hypothesis, we performed
comparative kinetic experiments with wild-type TDG protein and
different ssDNA and dsDNA substrates. Initial rate comparisons of
single turnover reactions revealed that TDG released FU faster than U
and U faster than T in dsDNA substrates, whereas FU excision from ssDNA
proceeded with a lower initial rate (Fig.
6A). However, in contrast to
the reactions with double-stranded substrate, TDG appeared to
dissociate more easily from the single-stranded AP-sites and showed
steady-state kinetics of FU processing at higher substrate
concentrations (Fig. 6B). Therefore, we examined the kinetic
properties of this reaction and determined its Michaelis-Menten
parameters (Fig. 6C). The results substantiated that TDG was
able to process the single-stranded FU substrate with a slow but steady
turnover (kcat = 0.041 min We generated and characterized mutant variants of human TDG
protein that explored two structural motifs critical for its catalytic activity and its ability to interact with DNA. The crystal structure of
E. coli Mug predicts the Asn in the highly conserved motif GINPGL to be essential for the hydrolytic activity of the protein. Accordingly, we found that mutation of the equivalent Asn of TDG to Ala
reduced the enzyme's catalytic potential to undetectable levels. By
analogy to the postulated catalytic mechanism for Mug, we conclude that
this mutation abolished the activation of a water molecule in the
putative active site pocket, which was then not available for efficient
hydrolysis of the N-glycosidic bond. Interestingly, G·U as
well as G·AP-site binding was largely unaffected in this mutant, but
its interaction with the G·T mismatched substrate was clearly
destabilized. Because in gel retardation experiments with wild-type
TDG, AP-site binding rather than G·T or G·U mismatch binding is
measured, and assuming that the N140A mutation does not alter the
substrate recognition properties of the enzyme, we propose that the
mismatch binding capacities of this mutant reflect the genuine
substrate preferences of wild-type TDG. This view is consistent with
the repeated observation that the wild-type enzyme processes G·T
mismatches with a lower efficiency than G·U mismatches (Table
I (8, 9)).
The substrate dissociation characteristics of the N140A mutant revealed
that TDG engages in at least two qualitatively different modes of DNA
interaction, one being loose and reversible as observed with homoduplex
and G·T mismatched DNA, and another being more rigid and resistant to
competition as observed with G·U and G·AP substrates. Thus, the
thymine of a G·T mismatch might be less well accommodated within the
catalytic pocket of the enzyme than the uracil residue of a G·U
mismatch. In consequence, the specific contacts to the Watson-Crick
face of the mispaired G as evident in the structure of substrate-bound
Mug (14, 16) could not be properly established with a G·T substrate
and, thus, G·T interaction would resemble homoduplex binding rather
than G·U and G·AP binding. The structural analysis of Mug suggested
that the side chain of Ser-23 would clash with the 5'-methyl group of a
thymine inside the active site pocket and therefore may account for the
G·T discrimination of the bacterial enzyme (16). The equivalent
position in the human TDG is occupied by a more hydrophobic alanine
(Ala-145), which may more easily facilitate the accommodation of a
thymine, although crowding around the 5'-methyl group is still expected to occur. Therefore, the active site geometry of TDG could indeed explain a tolerated but disfavored interaction with G·T mispairs, but
our finding that the critical A145S mutation in the human enzyme had no
effect on its ability to process G·T substrate is inconsistent with
such a mode for thymine discrimination. Also, it should be remembered
at this point that the G·T processing activity of TDG was lost
through the deletion of 112 N-terminal amino acids (9) without any
changes occurring in the active site. This implies that G·T
recognition and processing involves regions of TDG other than the
immediate proximity of the substrate binding pocket. Because the
N-terminal extension is absent from Mug, the crystal structure of the
bacterial TDG homologue can be of little help here. This phenomenon
thus warrants further study.
Mutation of the methionine to histidine in the proposed DNA-interaction
motif MPSSSAR generated a protein that failed to detectably bind to its
substrate but was still able to hydrolytically process the mismatched
substrates. Although a more than 10-fold higher enzyme concentration
was needed to achieve wild-type levels of base removal, the substrate
specificity of the mutant remained unchanged as compared with that of
the wild-type enzyme. This suggests that a rate-limiting step in
reactions with the M269H mutant is substrate recognition/interaction
rather than base hydrolysis and that the loss of affinity for the
substrate can be compensated for by increasing the enzyme
concentration. However, the precise role of the mutated methionine in
DNA interaction cannot be assigned solely on the basis of our
biochemical data. By analogy to the situation in Mug, where the
equivalent Asn is an N-terminal coordinate of the helix-intercalating
and complementary strand-interacting residues, we propose a similar
role for Met-269 in the human enzyme.
We found that the human wild-type TDG processes the artificial base FU
more efficiently than any other substrate tested. In vivo,
FU is known to inhibit the thymidylate synthase with the cytotoxic
consequence of a reduction in the dTTP pool and an increased incorporation of uridine into DNA. The molecular basis of the toxicity
of FU is complex, but increased base excision repair activity due to
misincorporation of uracil and possibly also FU has been proposed to
lead to an accumulation of DNA strand breaks that can trigger cell
death (19). Some tumors acquire resistance to FU by an unknown
mechanism, but it appears likely that changes in relevant DNA repair
functions may be involved (20). Although it is uncertain to what extent
FU is incorporated into the DNA of proliferating cells (21), TDG could
conceivably contribute to the toxicity of the drug through its ability
to efficiently process FU in a mismatch-independent manner. Thus,
inactivation of TDG by mutation could well provide one route for
acquiring tolerance to FU.
FU is distinct from uracil or thymine only at the 5 position, where it
carries a fluoro substituent instead of a hydrogen or a methyl group,
respectively. Whereas uracil DNA glycosylase actively excludes thymine
from its catalytic pocket by interference mediated by a strategically
positioned tyrosine residue, no such function is apparent in the Mug
structure (14). Although we have demonstrated that TDG has a lower
affinity for a G·T substrate than for a G·U substrate, our results
on FU processing show that active site exclusion cannot fully account
for its substrate specificity. In particular, it fails to explain why
G·FU is more efficiently processed than G·U and why the enzyme
excises FU but not U from ssDNA. If the chemical stability of the
N-glycosidic bond rather than active site geometry
discriminates between the substrates in question, FU would be expected
to be released very easily, uracil with intermediate, and thymine with
lowest efficiency, and this is consistent with our experimental
evidence. The fact that wild-type TDG releases FU even from ssDNA
supports the view that transient substrate interactions are sufficient
for hydrolysis of the N-glycosidic bond if only a low
activation energy is required, and this is in agreement with the
observation that reducing the catalytic potential of TDG by the
mutation (N140A) limits its activity to the energetically most
favorable G·FU substrate and brings back the need for a stable
interaction with this substrate. It seems a reasonable strategy for an
enzyme that hydrolyzes normal bases from DNA to make its action
dependent on specific complementary strand interactions. In the case of
thymine release by TDG, a sufficiently stable DNA interaction is
established only if the opposite base is a guanine, thus providing the
substrate discrimination needed to avoid inappropriate and nonspecific
base hydrolysis. Thus, evolution may have balanced the catalytic power
of TDG with its substrate interaction properties to achieve the
required substrate specificity. Such requirement may be less important
for glycosylases that release damaged bases from DNA, because in such
cases, the specificity is achieved through physical recognition of
structural irregularities of the inappropriate bases.
Our data establish that the E. coli Mug structure is a valid
model for human TDG as far as the overall three-dimensional fold of its
core domain and, thus, its basic reaction mechanism is concerned.
Obviously, the bacterial model is inaccurate when it comes to
explaining properties specific for the human enzyme, e.g.
the wider substrate spectrum and the activity modulating role of its
extra N- and C-terminal domains. Therefore, the ultimate understanding
of the molecular mechanisms involved in substrate recognition, binding,
and hydrolysis by human thymine DNA glycosylase will have to await
structural analysis of this enzyme in interaction with its substrates.
This is where the separation of function mutants characterized
in this paper will be instrumental.
We express our gratitude to Teresa Lettieri,
Orlando Schärer, Laurence Pearl, Giancarlo Marra, and all our
colleagues at the Institute of Medical Radiobiology for stimulating
discussions. We also thank Margret Fäsi and Daniel Kokot for
excellent technical assistance.
*
This study was supported by grants from the Schweizeriche
Krebsliga (to U. H., J. J., P. S.) and the Swiss National Science Foundation (to M. B., P. S.).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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AJ277958 and AJ277789.
Published, JBC Papers in Press, August 10, 2000, DOI 10.1074/jbc.M005095200
The abbreviations used are:
dsDNA, double-stranded DNA;
ssDNA single-stranded DNA, TDG, thymine DNA
glycosylase;
UDG, uracil DNA glycosylase;
FU, 5-fluorouracil;
AP-site, apurinic/apyrimidinic site;
DTT, dithiothreitol;
EMSA, electrophoretic
mobility shift assay;
Mug, mismatch-specific uracil DNA-glycosylase;
cv, column-volume;
T50, time requirement for
processing 50% of the plateau levels for each substrate.
Separating Substrate Recognition from Base Hydrolysis in Human
Thymine DNA Glycosylase by Mutational Analysis*
ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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INTRODUCTION
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T transitions
upon DNA replication, if left unrepaired. Whereas accurate repair of
G·U mispairs is mediated by enzymes that specifically recognize and
process uracil in DNA, e.g. uracil DNA glycosylase (UDG)
(2), correction of G·T mispairs to G·C base pairs requires a repair
function that is able to discriminate between a mutagenic thymine in a
G·T mismatch and a normal thymine base-paired with adenine.
(6-8). Two bacterial open reading frames with significant homology
to the central part of human TDG were discovered, and expression of the
Escherichia coli homologue produced an enzyme with G·U
mismatch-dependent uracil DNA glycosylase activity but no
G·T mismatch processing activity (9). This protein was, therefore,
named Mug for mismatch-specific uracil DNA-glycosylase. Deletion
analysis of the human enzyme revealed that its conserved central domain
is sufficient for G·U but not for G·T processing activity and that
additional non-conserved amino acid residues of the N terminus are
required for G·T processing (9).
C), which arises in DNA as a consequence of lipid
peroxidation or the exposure to chemical carcinogens such as vinyl
chloride (10, 11). A vertebrate homologue of TDG was reported to
copurify with a 5-methylcyotsine DNA glycosylase activity from extracts
of chicken embryos and to have weak 5-methylcytosine glycosylase
activity when purified as a recombinant protein from overexpressing
bacteria (12). Furthermore, a different line of investigation revealed
physical and functional interactions of mammalian TDG with retinoid
receptors and, therefore, implicated a role in nuclear
receptor-mediated control of transcription (13).
EXPERIMENTAL PROCEDURES
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EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-D-galactopyranoside, and incubation
was allowed to proceed at 20 °C for a further 4 h. The cells
were harvested by centrifugation (Sorvall SLA-3000, 5000 rpm, 4 °C,
30 min), and the pellets were stored at 
80 °C. The thawed cell
pellets were resuspended in 3 ml/g sonication buffer (50 mM
sodium phosphate, pH 8.0, 300 mM NaCl, 10% glycerol, 1 mM imidazole 10 mM -mercaptoethanol, 1 mM phenylmethylsulfonyl fluoride) and lysed by sonication
on ice (25 × 10-s bursts with intermittent chilling for 10 s). After removal of cell debris by centrifugation (Sorvall SS34,
15,000 rpm, 4 °C, 30 min), 1 ml of sonication-buffer-equilibrated
nickel-nitrilotriacetic acid-agarose (Qiagen) was added to the crude
lysate and incubated for 1 h at 4 °C with gentle shaking. The
suspension was then packed into a disposable column from which unbound
protein was washed out with sonication buffer containing stepwise
increasing concentrations of imidazole: 1 × 15 column-volumes
(cv) 1 mM imidazole; 5 × 5 cv 20 mM
imidazole; 1 × 5 cv 60 mM imidazole. Finally, bound
histidine-tagged TDG protein was eluted with 5 × 1 cv sonication
buffer containing 300 mM imidazole. The 300 mM
imidazole fractions were pooled and dialyzed overnight at 4 °C
against binding buffer (50 mM Tris-HCl, pH 8,0, 10%
glycerol, 5 mM -mercaptoethanol). After loading the dialyzed fraction onto a 1-ml Resource Q fast protein liquid
chromatography column (Amersham Pharmacia Biotech) and washing with 10 ml of binding buffer, bound protein was eluted with a linear gradient of 0-500 mM NaCl in 30 ml. The nearly homogeneous TDG
protein (>98% pure) eluted as a major protein peak in fractions
containing approximately 150 mM NaCl. After a last dialysis
step against storage buffer (50 mM Tris-HCl, pH 8.0, 50 mM NaCl, 10% glycerol, 5 mM
-mercaptoethanol), the pure TDG protein was stored in aliquots at
80 °C.
20 °C for 1 h, following the addition of tRNA to a final
concentration of 0.4 mg/ml and of sodium acetate (pH 5.2) to 0.3 M. It was collected by centrifugation (Eppendorf, 14,000 rpm, 4 °C, 20 min) and washed in 80% ethanol (20 °C). The
dried pellets were resuspended in 10 µl of formamide gel loading buffer (90% formamide, 1× TBE), heated for 5 min at 99 °C, and chilled on ice immediately. The samples were then loaded onto a 15%
denaturing polyacrylamide gel (acrylamide:bisacrylamide = 19:1,
1× TBE, 8 M urea) (Bio-Rad, Mini Protean II cell), pre-run for 15 min at 450 V. The gels were run for 5 min at 450 V and subsequently at 250 V until the bromphenol blue dye reached the bottom
of the gel. Fluorescein-labeled DNA was visualized using the blue
fluorescence mode of the Storm 860 (Molecular Dynamics) and analyzed by
ImageQuaNT software (Molecular Dynamics). For kinetic assays, the
reactions were performed in larger volumes with an enzyme concentration
of 50 nM and substrate concentrations ranging from 50 nM to 25 µM. After different time points,
samples were withdrawn, stopped by the addition of NaOH, and further
treated as described above.
20 °C and
used for EMSA.
RESULTS
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EXPERIMENTAL PROCEDURES
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DISCUSSION
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View larger version (68K):
[in a new window]
Fig. 1.
Evolutionary conservation of the catalytic
domain in TDG homologues. Shown is a partial amino acid sequence
alignment spanning the core domains of the TDG homologues of human
(hsTDG, EMBL accession number U51166), D. melanogaster (dmThd1, EMBL accession number AJ277789), S. pombe (spThp1, EMBL accession number AJ277958), and E. coli (ecMug; Swissprot accession number P43342). Identical
residues are shaded and the amino acid motifs forming the
essential parts of the proposed active site pocket are
framed. Residues in italics indicate the
structural equivalent motifs present in the herpes simplex virus uracil
DNA glycosylase (hsvUDG; Swissprot accession number P10186).
Asterisks indicate the sites mutagenized in the human TDG
(N140A, N140D, M269H, or A145S).
Quantitative assessment of DNA glycosylase activities of wild-type and
mutant variants of human thymine DNA glycosylase
G·C.
View larger version (23K):
[in a new window]
Fig. 2.
Catalytic activity of wild-type and mutant
TDG proteins. The ability to generate alkaline sensitive sites in
standard substrates was assayed for wild-type TDG (A) and the mutant
variants N140A (B), N140D (C) and M269H (D) as described in
`Experimental Procedures'. Shown are the results obtained with dsDNA
substrates containing either G·C, G·T, G·U or A·U base pairs or
ssDNA containing a single uracil (U) at an identical position. The
reactions were done in 20 µl volume and in the presence of 1 pmol of
substrate DNA. The amounts of enzyme were 1 pmol in (A) and (C), 20 pmol in (B) and 10 pmol in (D). Reaction products were separated from
substrates on 15% denaturing polyacrylamide gels. The positions of the
60-mer substrate DNA and 23-mer product fragment are indicated. The
slightly shorter migrating fragment in the reaction with ssDNA reflects
processing of a G·T mismatch generated in the secondary structure of
the single-stranded substrate oligonucleotide.
View larger version (16K):
[in a new window]
Fig. 3.
Binding of wild-type and mutant TDG proteins
to substrate and product DNA. Gel retardation assays were
performed with wild-type TDG (A) and the mutant variants
N140A (B), N140D (C), and M269H (D) as
described under EMSA under "Experimental Procedures." The 10-µl
reactions contained 1 pmol of labeled substrate DNA as indicated, 10 pmol of unlabeled homoduplex competitor DNA, and 4 pmol of TDG. Bound
fluorescein-labeled DNA was separated from free substrate DNA in 6%
native polyacrylamide gels. A representative experiment is shown, with
numbers at the bottom of the lanes representing the amounts of bound
substrate (%) under steady-state conditions.
View larger version (31K):
[in a new window]
Fig. 4.
Substrate dissociation of wild-type TDG and
the N140A mutant. In this substrate dissociation assay, 2 pmol of
wild-type or mutant TDG enzyme was preincubated at 37 °C for 10 min
with 1 pmol of labeled substrate DNA under EMSA conditions. The
preformed protein-DNA complexes were then challenged by the addition of
a 0, 10-fold, or 20-fold molar excess of unlabeled nonspecific or
specific competitor DNA as indicated, and incubation was continued for
a further 10 min at 37 °C. The reaction products were separated in
6% native polyacrylamide gels. The positions of the free and bound
substrate DNA are indicated, and numbers below the lanes represent the
amounts of bound substrate (%) in each reaction. Shown is the result
of a representative experiment.
View larger version (22K):
[in a new window]
Fig. 5.
Processing of 5-fluorouracil and uracil
substrates by wild-type and mutant TDG variants. Generation of
alkaline-sensitive sites was assayed with the dsDNA substrates G·U,
A·U, G·FU, A·FU, and with the ssDNA substrates containing U or FU
as indicated. All reactions contained 1 pmol of substrate DNA. The
amounts of protein were 1 pmol for the wild-type TDG (A),
the N140A (C), and the N140D mutant (D) and 10 pmol for the M269H mutant (B). Reactions were set up in
20-µl volumes, incubated at 37 °C for 15 min, and the products
were separated on 15% denaturing polyacrylamide gels.
1) and a
Km of 114 nM. Under standard reaction
conditions with an excess of dsDNA substrates, TDG processed less than
one molar equivalent of G·U and G·T mispairs. The reactions with
the G·5FU substrate also reached plateaus but at higher and substrate concentration-dependent product levels corresponding to
1-5 molar equivalents of enzyme (e.g. Fig. 6B).
The lack of turnover with the G·U and G·T substrates is explained
by the fact that TDG needs to establish specific complementary strand
contacts to be able to hydrolyze the mismatched uracil and thymine and
then remains bound to the G·AP-site product after base release (Fig.
4A (14, 18)). The limited turnover with the G·FU substrate
most likely reflects the ability of TDG to hydrolyze FU from G·FU
without engaging in stable complementary strand interactions. This
results in an enzymatic turnover until the concentration of generated G·AP-site products is high enough to inhibit TDG through competitive binding. Taken together, these observations document that
destabilization of the N-glycosidic bond can enhance the
catalytic activity of TDG and obviate its need for stable interactions
with the substrate.
View larger version (11K):
[in a new window]
Fig. 6.
Kinetic properties of 5-fluorouracil
processing by human TDG. The time-dependent generation
of alkaline-sensitive sites was assayed by incubation of human TDG with
double-stranded 60-mer substrate containing a single G·U ( 
), G·T
(
), or G·FU (
) mismatch or ssDNA containing FU (
).
A, equimolar concentrations (50 nM) of substrate
and TDG protein; or B, a 5-fold molar excess of substrate
over TDG protein were incubated at 37 °C, and the reactions were
stopped after the indicated times by the addition of NaOH (see
"Experimental Procedures"). Product formation was monitored and
quantified after denaturing gel electrophoresis and fluorescent
scanning. C, Lineweaver-Burk plot derived from a series of
time course experiments with TDG protein and single-stranded FU
substrate. The concentrations were 50 nM for TDG protein
and 250 nM to 25 µM for the ssDNA substrate.
The resulting kinetic parameters are: Km = 114 nM; Vmax = 2.05 nM
min1; kcat = 0.041 min1.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
ACKNOWLEDGEMENTS
FOOTNOTES
To whom correspondence should be addressed: Tel: 41-1-634-8926;
Fax: 41-1-634-8904; E-mail: schaer@imr.unizh.ch.
ABBREVIATIONS
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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