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INTRODUCTION |
In mammalian cells, 2-7% of the total cytosine is methylated.
Spontaneous deamination of 5-methylcytosine, which is somewhat faster
than cytosine (1), generates G·T mispairs in DNA. The repair of these
G·T mismatches is initiated by thymine DNA glycosylase which excises
the mismatched thymine (2, 3). 5-Methylcytosine occurs almost
exclusively in the sequence MeCpG, and in keeping with its
proposed role in the repair of G·T mispairs resulting from the
deamination of 5-methylcytosine, thymine DNA glycosylase shows a strong
preference for removal of thymine from CpG·T sequences (4-7). The
human enzyme has been cloned and overexpressed (8) and has been shown
to belong to a family of uracil DNA glycosylases that remove uracil
from G·U base pairs but that are distinct from the general uracil DNA
glycosylase enzyme (9). Thymine DNA glycosylase removes uracil from
G·U base pairs more rapidly than it removes thymine from G·T base pairs (10) and can also remove uracil from C·U, T·U, and A·U base
pairs (7) and may therefore provide a backup function to the general
uracil DNA glycosylase. The glycosylase also removes thymine from base
pairs with S6-methylthioguanine
(SMeG)1 that are
thought to occur in the DNA of cells treated with the drug
6-thioguanine (7, 11). Kinetic studies in our laboratory showed that
each molecule of thymine DNA glycosylase can remove only one thymine
molecule because the glycosylase remains bound to the apurinic site it
produces (7). Since this strong binding may have physiological
significance, it has been studied in more detail.
Removal of an incorrect base by a DNA glycosylase is the first step of
base excision repair (reviewed in Refs. 12 and 13), and one possible
role of the bound thymine DNA glycosylase might be to recruit the
enzymes needed to complete the repair process. Removal of the base is
followed by cleavage of the phosphodiester bond 5' to the abasic sugar
(Fig. 1). In humans, this reaction is
most probably carried out by the apurinic endonuclease, HAP1 (also
known as APE, APEX, and Ref-1), as this enzyme is responsible for
around 95% of all incisions at apurinic sites in HeLa cell extracts
(14) and can function satisfactorily in reconstituted in
vitro base excision repair systems (15, 16). Following cleavage of
the sugar-phosphate backbone, DNA polymerase
then removes the
deoxyribose 5'-phosphate and fills the single base gap (17-19).
Ligation of the nick completes repair and is believed to be carried out
by DNA ligase III (15), which is present in cells as a heterodimer with
XRCC1 (20). The XRCC1 may act to ensure that polymerase
adds only
one nucleotide (15). The strong binding of thymine DNA glycosylase to
apurinic sites suggested that it might recruit the other proteins
involved in base excision repair. Consequently we investigated possible
interactions between the thymine DNA glycosylase bound to the apurinic
site and the other proteins involved in base excision repair. We found
that repair cannot take place while the thymine DNA glycosylase is bound to the apurinic site, and so the extremely slow dissociation of
thymine DNA glycosylase from these sites would greatly inhibit repair.
However, the apurinic endonuclease HAP1, which catalyzes the second
step in base excision repair, can displace the bound thymine DNA
glycosylase. This effect, which couples the first and second steps in
base excision repair, is concentration-dependent and would
be expected to be significant at the concentrations of HAP1 reported in
mammalian cells.

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Fig. 1.
Schematic of the steps involved in the base
excision repair of G·T mismatches. Thymine is removed from G·T
mismatches through cleavage of the glycosidic bond by thymine DNA
glycosylase to produce an apurinic site. HAP1 then cuts the phosphate
backbone on the 5'-side of the apurinic site. The abasic
sugar-phosphate moiety is then removed by a deoxyribophosphodiesterase
activity, possibly polymerase , leaving a gap opposite the
mismatched guanine. Polymerase then fills the gap using dCTP, and
finally the nick in the phosphate backbone is sealed by DNA ligase to
give fully repaired G·C DNA.
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EXPERIMENTAL PROCEDURES |
Enzymes--
Thymine DNA glycosylase was expressed in
Escherichia coli as described previously (8) and was
purified in four chromatographic steps (7). HAP1 was a gift from Dr. I. Hickson and Dr. D. Rothwell; DNA polymerase
, XRCC1, and DNA ligase
I were gifts from Dr. G. Daly and Dr. T. Lindahl; and E. coli Endonuclease IV was provided by Dr. T. Barrett and Dr. L. Pearl.
Synthesis and Purification of Oligodeoxynucleotides--
34-Base
pair DNA duplexes of the general sequence AGC TTG GCT GCA GGC
XGA CGG ATC CCC GGG AAT T (where X is A, C, G, T,
or S6-methylthioguanine, SMeG) were
synthesized and purified as described previously (21). Oligodeoxynucleotides with a uracil opposite position X were
purchased from Amersham Pharmacia Biotech. They were purified by ion
exchange chromatography at pH 12 and desalted (22). DNA duplexes
containing an apurinic site were prepared by removing the uracil from
DNA containing a single uracil opposite position X with
E. coli uracil DNA glycosylase as described previously
(7).
Glycosylase Assays--
34-Base pair DNA duplexes were
5'-labeled with 32P in the strand containing the mismatched
thymine or uracil. They were reacted at room temperature with thymine
DNA glycosylase in either EDTA buffer (1 mM EDTA, 25 mM Hepes (pH 7.6), 50 mM KCl, 0.01 mM ZnSO4, 2 mM dithiothreitol, 0.5 mg/ml bovine serum albumin) or magnesium buffer (2 mM
MgCl2, 25 mM Hepes (pH 7.6), 50 mM
KCl, 0.2 mM EDTA, 0.01 mM ZnSO4, 2 mM dithiothreitol, 0.5 mg/ml bovine serum albumin). When
measuring the total amount of base removed, samples were taken at
various times, quenched with a half-volume of 0.3 M NaOH, 30 mM EDTA, and the apurinic sites cleaved by heating at
90 °C for 30 min. After cooling, an equal volume of saturated urea
containing 10 mM EDTA and 0.5% Triton X-100 (Sigma) was
added. To measure the enzymatic cleavage at apurinic sites by HAP1 or
Endonuclease IV, the hydroxide heat treatment was omitted, and the
samples were quenched with an equal volume of saturated urea containing 10 mM EDTA and 0.5% Triton X-100 and frozen in liquid
nitrogen until chromatographic analysis. The concentrations of proteins and DNA substrate in each reaction are given in the figure legends.
The cleaved DNA was separated from full-length, unreacted DNA by
perfusion chromatography using a 2.1 × 30-mm Poros HQ (PerSeptive Biosystems) anion exchange column as described (7). Radiolabeled DNA
was detected by Cerenkov counting using a Berthold LB 506 C-1 monitor
and was quantified by integration of the peaks.
Experiments involving polymerase
, XRCC1, and DNA ligase III were
carried out in magnesium reaction buffer containing 20 µM
dCTP. In these experiments, all proteins (including thymine DNA
glycosylase and HAP1) were preincubated for 12 min at room temperature
before adding the DNA containing a G·T mismatch. When used, ATP was
included at 2 mM concentration.
Band Shift Assay--
The complexes between the proteins and the
DNA containing a mismatch or an apurinic site were formed in either
EDTA buffer or magnesium buffer (see above) containing 4% Ficoll 400. Complexes with DNA containing an apurinic site were formed by
incubating thymine DNA glycosylase (or HAP1) with
32P-labeled apurinic DNA duplex for 30 min at room
temperature. When used, competitor oligodeoxynucleotides were pre-mixed
with the radiolabeled DNA at a 40-fold excess, before adding the
thymine DNA glycosylase. After incubation, samples were loaded onto a non-denaturing 6% polyacrylamide gel, and electrophoresis was carried
out for 2 h (8 V/cm at 10-15 °C). The gel was then dried and
the position of the DNA visualized by autoradiography.
The band shift experiment in Fig. 6 was carried out as above except
that DNA containing a G·T mismatch was incubated with thymine DNA
glycosylase and HAP1 (amount as shown in the legend) for 2.5 h at
room temperature before loading onto the gel. Also, part of each sample
was assayed by chromatography, as described above, to measure the
extent of removal of thymine by thymine DNA glycosylase, and the extent
of scission of the resultant apurinic site by HAP1.
Measurement of the Rate of Dissociation of the Glycosylase from
an Apurinic Site in DNA--
Complexes of thymine DNA glycosylase and
DNA containing an apurinic site opposite guanine (G·AP) were
preformed by incubating 1.4 nM thymine DNA glycosylase with
1 nM 32P-labeled G·AP DNA in the Ficoll
containing buffer described above for 20 min at room temperature.
Samples were removed from this master mix at various times and added to
100-fold excess of unlabeled G·AP DNA, and incubation was continued
at room temperature. These samples were all loaded at the same time
onto a non-denaturing polyacrylamide gel, and electrophoresis was
carried out as described for the band shift experiments. After
electrophoresis the gel was dried, the position of bound and free
32P-labeled DNA was located by autoradiography, and then
cut out. The amount of bound 32P-labeled DNA was quantified
by scintillation counting and was plotted against the time between
addition of the sample to the unlabeled competitor G·AP DNA and
loading it onto the gel.
The dissociation of thymine DNA glycosylase complexes with
32P-labeled DNA containing an apurinic site opposite
S6-methylthioguanine (SMeG·AP) or
opposite cytosine (C·AP) was measured slightly differently. The
complexes were preformed as above, but after the initial incubation, 100-fold excess of unlabeled G·AP DNA was added to the master mix. At
various times after adding the G·AP DNA, samples of this mix were
loaded directly onto the electrophoresis gel with the current running.
Electrophoresis was carried out as above, and the gels were dried and
autoradiographed. The amount of bound 32P-labeled DNA was
plotted against the time between adding the unlabeled competitor G·AP
DNA and loading the sample onto the gel.
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RESULTS |
Thymine DNA Glycosylase Binds to DNA Containing an Apurinic Site
Opposite All Four DNA Bases--
In a previous report (7) we showed
that thymine DNA glycosylase forms a complex with DNA containing an
apurinic site opposite guanine (Fig.
2A). Tight binding of the
glycosylase requires the apurinic site because 40-fold excess unlabeled
DNA containing a G·AP site virtually eliminates formation of the
radioactive complex, whereas excess perfectly matched DNA only reduces
the complex by about 60%. Formation of the complex does not depend upon the apurinic site being opposite a guanine, since the glycosylase also bound to DNA containing an apurinic site opposite
S6-methylthioguanine (7), thymine (Fig.
2B), cytosine (Fig. 2C), or adenine (Fig.
2D). Again, these complexes could be competed out by excess
G·AP DNA but were much less affected by perfectly matched competitor
DNA.

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Fig. 2.
Thymine DNA glycosylase can bind to an
apurinic site opposite any of the four natural DNA bases. 1.3 nM thymine DNA glycosylase was incubated in EDTA binding
buffer with 0.5 nM 32P-labeled DNA containing
an apurinic site opposite either guanine (A), thymine
(B), cytosine (C), or adenine (D) for
30 min, and then the samples were run on a non-denaturing gel as
described under "Experimental Procedures." Competitor
oligodeoxynucleotides, as indicated above the lanes, were
mixed with the 32P-DNA at a 40-fold excess before addition
of the glycosylase. The positions of bound and free DNA are shown by
arrows.
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The significant competition by perfectly matched duplex DNA (Fig.
2A) suggests that thymine DNA glycosylase binds to it,
although with much less affinity than it binds DNA containing an
apurinic site. The previous observation (7) that addition of a
perfectly matched DNA duplex reduces the rate that thymine DNA
glycosylase removes thymine from a G·T mismatch in DNA also suggests
that the glycosylase binds nonspecifically to DNA.
The Rate of Dissociation of Thymine DNA Glycosylase from Apurinic
Sites Limits the Glycosylase Reaction--
Because thymine DNA
glycosylase binds tightly to an apurinic site in DNA, the rate of
dissociation of the complex could be measured using electrophoresis
(23). Complexes between thymine DNA glycosylase and
32P-labeled apurinic DNA were preformed, and then a
100-fold excess of unlabeled DNA containing a G·AP site was added to
trap the glycosylase as it dissociated from the labeled apurinic site. The mixtures were incubated and samples were loaded at intervals onto a
non-denaturing gel for electrophoresis to separate and quantitate the
radioactive DNA-glycosylase complex remaining in each sample. The rates
of dissociation were measured in the absence and the presence of 2 mM magnesium. The results for the dissociation of complexes
between thymine DNA glycosylase and DNA with a G·AP, a
SMeG·AP, or a C·AP site are shown in Fig.
3, A-C, and were fitted to an
exponential curve to obtain the rate constants shown in Table
I.

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Fig. 3.
A-C, dissociation rates of complexes
between thymine DNA glycosylase and DNA containing an apurinic site and
the effect of magnesium. Complexes between 1.4 nM thymine
DNA glycosylase and 1 nM 32P-labeled apurinic
DNA containing an apurinic site opposite guanine (A),
S6-methylthioguanine (B), and
cytosine (C) were preformed in binding buffer containing
either EDTA ( ) or 2 mM magnesium ( ). The dissociation
rates were determined using the band shift assay described under
"Experimental Procedures." The dissociation rates determined from
the exponential curves are given in Table I. D-G, effect of
magnesium on the action of thymine DNA glycosylase. In buffer
containing EDTA ( ), thymine DNA glycosylase could only remove a
stoichiometric amount of thymine or uracil from mismatches. In the
presence of 2 mM magnesium ( ), the increased
dissociation rates of complexes between thymine DNA glycosylase and
SMeG·AP or C·AP allowed the glycosylase to remove more
than an equimolar amount of mismatched base from SMeG·T
or C·U substrates. Magnesium failed to increase the amount of
mismatched base removed from G·T or G·U mismatches. D,
removal of thymine by thymine DNA glycosylase (30 nM) from
DNA containing a G·T mismatch (100 nM); E-G,
removal by thymine DNA glycosylase (3 nM) of uracil or
thymine from DNA (10 nM) containing either a G·U mismatch
(E), an SMeG·T mismatch (F), or a
C·U mismatch (G). The amount of mismatched uracil or
thymine removed was monitored by chromatographic assay.
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Table I
Rates of dissociation for the complex between thymine DNA glycosylase
and DNA containing an apurinic site in the presence and absence of 2 mM magnesium
Apparent dissociation rates were calculated by fitting exponential
curves to the data shown in Fig. 3, A-C.
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Fig. 3A shows that the complex formed between thymine DNA
glycosylase and the G·AP site it produces when it removes thymine from a G·T mismatch dissociates very slowly (half-life ~10 h). Fig.
3, D and E, shows the glycosylase action of
thymine DNA glycosylase on a G·T and a G·U mismatch. Thymine DNA
glycosylase rapidly removed a stoichiometric amount of thymine from a
G·T mismatch, or uracil from a G·U mismatch, before the reaction
stopped. Magnesium had little effect on this. The fact that thymine DNA
glycosylase could remove only 1 eq of the mismatched base reflects the
fact that thymine DNA glycosylase binds strongly to the apurinic site
produced by its glycosylase action. The lack of influence of added
magnesium on the progress of the glycosylase reaction is consistent
with the observation that magnesium does not affect the rate of
dissociation of the enzyme from the G·AP site (Fig. 3A).
In contrast magnesium had a significant effect on the dissociation of
thymine DNA glycosylase from an SMeG·AP site. In the
absence of magnesium thymine DNA glycosylase dissociated only slightly
more rapidly from an SMeG·AP site than from a G·AP
site, but magnesium increased the dissociation of the glycosylase from
an SMeG·AP site by about 20-fold (Fig. 3B and
Table I). This affects the glycosylase activity of the enzyme on an
SMeG·T mismatch. In the absence of magnesium, thymine DNA
glycosylase could only remove a stoichiometric amount of thymine from
an SMeG·T mismatch because it bound to the
SMeG·AP site that it produced, but in the presence of
magnesium its more rapid dissociation from the SMeG·AP
site allowed turnover of SMeG·T mismatches (Fig.
3F).
Magnesium has an even greater effect on the dissociation of thymine DNA
glycosylase from C·AP sites (Fig. 3C and Table I). As one
would expect, this faster dissociation from the C·AP site affected
the glycosylase action of thymine DNA glycosylase on C·U mismatches
to an even greater extent than on SMeG·T mismatches (Fig.
3G).
In the experiments shown in Fig. 3, E-G, the concentration
of DNA was 10 nM, and the concentration of thymine DNA
glycosylase was 3 nM, whereas in Fig. 3D the
concentration of G·T DNA was 100 nM and glycosylase 30 nM. These higher concentrations were used because when 10 nM G·T DNA was incubated with 3 nM enzyme, it
was found that the initial reaction rate was lower in 2 mM magnesium than in EDTA (Fig.
4A). Although magnesium
increased the amount of thymine finally removed from
SMeG·T DNA by thymine DNA glycosylase, it did not affect
the initial rate of removal of the thymine (Fig. 3F). The
reactions with G·U DNA (Fig. 3E) and C·U DNA (Fig.
3G) were too fast to be able to see whether the initial
rates were affected by magnesium.

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Fig. 4.
The effect of magnesium upon the initial rate
of reaction of thymine DNA glycosylase with G·T DNA.
A, reaction of thymine DNA glycosylase (3 nM)
with 32P-labeled DNA (10 nM) containing a G·T
mismatch in buffer containing either EDTA ( ) or 2 mM
magnesium ( ). The amount of thymine removed was measured by
chromatographic assay. B, reaction between equal amounts of
thymine DNA glycosylase and DNA containing a G·T mismatch in 2 mM magnesium. The concentrations of DNA and enzyme were 20 nM ( ), 10 nM ( ), 5 nM (×),
and 2 nM ( ). For easier comparison of the relative
rates, the reactions have been plotted as moles of mismatched thymine
removed per mol of glycosylase.
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The slower initial rate in the presence of magnesium for the removal of
thymine from a G·T mismatch suggested that magnesium might lower the
affinity of the glycosylase for the G·T mismatch. To investigate the
effect of magnesium on the initial rate of thymine removal from G·T
DNA, rates were determined at four different concentrations of DNA and
enzyme (Fig. 4B). The results are plotted as moles of
thymine removed per mol of enzyme to facilitate comparison between the
different concentrations. As the concentration increased, so did the
relative rate of reaction, implying that at these concentrations not
all of the substrate G·T DNA was bound to the enzyme (i.e. Kd in Scheme I must be greater than 2 nM). This is in contrast to the reaction in EDTA in which
the Kd has been reported to be considerably less
than 2.5 nM (7, 24). The data in Fig. 4B could
not be used to calculate a Kd because the rate of
reaction at the lowest concentrations was less than one would expect
from a single reaction with a single Kd and because
the reaction appeared to be biphasic (see Fig. 4A). Taken
together, these suggest that at the lowest concentrations some of the
enzyme was inactivated or bound in a non-productive complex.
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The Action of the Apurinic Endonuclease HAP1 on Apurinic Sites
Complexed with Thymine DNA Glycosylase--
The accessibility of the
apurinic site in the thymine DNA glycosylase-apurinic DNA complex was
measured. Naked apurinic sites in DNA are cleaved rapidly by the human
type II apurinic endonuclease, HAP1. The reported Kd
for the binding of HAP1 to an apurinic site in a 49-base pair
oligodeoxynucleotide is 0.8 nM and the kcat 10 s
1 (25). As would be
expected from these values, we found that when 20 nM DNA
containing a G·AP site was treated with 6 nM HAP1 the DNA
was entirely cleaved in less than 1 min. The effect of two unrelated
type II apurinic endonucleases, HAP1 from human cells and Endonuclease
IV from E. coli, on the apurinic site in the complex with
thymine DNA glycosylase is shown in Fig.
5. In this experiment thymine DNA
glycosylase and DNA containing a G·T mismatch were incubated together
with one of the apurinic endonucleases. The effect of adding
Endonuclease IV was very different from the effect of adding HAP1.
Endonuclease IV had no discernable effect on the progress of the
reaction (Fig. 5A), even when it was present in a
considerable excess (60 nM) over the amount of thymine DNA glycosylase (6 nM) or DNA (20 nM). By contrast
in the presence of HAP1 the reaction of thymine DNA glycosylase with
DNA containing a G·T mismatch (Fig. 5B) continued after
reaching the stoichiometric limit seen in Figs. 3D and
5A. Although this second phase was slow, it was much faster
than in the absence of HAP1. The samples from the incubation were
analyzed to measure the approximate amount of intact DNA still
containing the G·T mismatch, DNA containing an apurinic site, and DNA
containing a strand break where the apurinic site had been cleaved by
HAP1. The amount of DNA cleaved by HAP1 was measured by adding an equal
amount of saturated urea containing 10 mM EDTA and 0.5%
Triton X-100 before chromatography to separate cleaved from intact DNA.
The total amount of DNA from which thymine had been removed
(i.e. the combined amount of DNA cleaved by HAP1 and DNA
still containing an apurinic site) was measured by treating the samples
with NaOH to cleave the apurinic sites before chromatography. Fig.
5B shows the rate of thymine removal and the rate of
production of DNA cleaved by HAP1. It can be seen that thymine DNA
glycosylase rapidly produced an approximately stoichiometric amount of
apurinic DNA. After this initial burst, the glycosylase removed thymine
at a much slower rate. The amount of DNA cleaved by HAP1 accumulated at
approximately the same rate as the slower phase of the thymine DNA
glycosylase reaction so that there was always an approximately constant
amount of intact apurinic DNA complexed with the glycosylase. The
amount of this intact apurinic DNA, estimated by subtracting the amount
of DNA cleaved by HAP1 from the total cleaved by HAP1 and by NaOH, was, on average, only about 4 nM (i.e. two-thirds of
the glycosylase present). If thymine DNA glycosylase bound only to DNA
containing an apurinic site one would expect that there would be 6 nM bound apurinic DNA. This discrepancy is probably an
artifact arising from the method of analysis. In this analysis the
reaction was stopped, the DNA and protein dissociated, and the DNA
strands separated, by addition of urea and EDTA. It is probable that
this treatment exposed some of the apurinic sites in the thymine DNA glycosylase-apurinic DNA complex before all the HAP1 activity had been
lost. This explanation is supported by the observation (Fig. 7,
discussed below) that an exactly stoichiometric amount of apurinic DNA
and glycosylase is not repaired by a complete base excision repair
system and also by analysis of the products of reaction with
Endonuclease IV using the same method as used for the study of HAP1.
Endonuclease IV does not cut the apurinic sites in the thymine DNA
glycosylase-apurinic DNA complex, and no cleaved DNA should have been
obtained, but when the reaction mixture at the end of the experiments
shown in Fig. 5A was analyzed by the same method as used for
the HAP1 experiment shown in Fig. 5B, it was found that
25-39% of the apurinic sites had been cleaved.

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Fig. 5.
The effect of the apurinic endonucleases HAP1
and Endonuclease IV upon the reaction of thymine DNA glycosylase.
A, E. coli. Endonuclease IV does not cut the
apurinic site in the glycosylase-G·AP complex and does not affect the
progress of the glycosylase reaction with a G·T mismatch. 20 nM G·T DNA was incubated with 6 nM thymine
DNA glycosylase in the absence ( ) or the presence of 6 nM ( ) and 60 nM (×) Endonuclease IV.
B, the apurinic site in the complex of thymine DNA
glycosylase and apurinic DNA is also inaccessible to HAP1, but HAP1
increases the rate of reaction by increasing the dissociation of
thymine DNA glycosylase from the apurinic site. DNA containing a G·T
mismatch (20 nM) was incubated with 6 nM
thymine DNA glycosylase and 6 nM HAP1 in 2 mM
magnesium buffer. At intervals samples were taken and were either
analyzed by chromatography to measure the amount of DNA cleaved by HAP1
( ) or treated with NaOH to cleave any remaining apurinic DNA and
then analyzed by chromatography to measure the total amount of thymine
removed ( ). C, concentration dependence of the effect of
HAP1 on the glycosylase reaction with a G·T mismatch. G·T DNA (20 nM) was incubated with thymine DNA glycosylase (6 nM) in 2 mM magnesium in the absence of ( )
or the presence of 6 nM ( ), 12 nM (×), 24 nM ( ), and 600 nM ( ) HAP1. D,
effect of HAP1 on the glycosylase reaction with a C·U mismatch. C·U
DNA (10 nM) was incubated in 2 mM magnesium
with thymine DNA glycosylase (3 nM) in the presence of 10 nM HAP1 ( ). The first four data points from Fig.
3G for the reaction in magnesium buffer have also been
plotted to show the rate of reaction in the absence of HAP1
( ).
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The results in Fig. 5B suggest that HAP1 can displace
thymine DNA glycosylase from an apurinic site in DNA. The dependence of
the rate of this displacement on the concentration of HAP1 is shown in
Fig. 5C. From 6 to 24 nM HAP1 the rate of
removal of thymine after the stoichiometric limit had been reached was roughly proportional to the amount of HAP1 present. In the presence of
600 nM HAP1, almost all of the 20 nM G·T DNA
had reacted after 1 h.
HAP1 had a similar, but greater, effect on the removal of uracil from a
C·U mismatch. Despite the fact that thymine DNA glycosylase dissociates more rapidly from a C·AP site than a G·AP site (Table I), the removal of uracil from C·U DNA by thymine DNA glycosylase is
still limited by slow product release (Fig. 3G). Addition of 10 nM HAP1 increased the rate of turnover of C·U DNA by
more than 150-fold so that the reaction was complete in around 15 min
(Fig. 5D). The increased rate of uracil removal seen when
HAP1 was added was not due to contamination of the HAP1 preparation by
uracil DNA glycosylase because addition of the uracil glycosylase
inhibitor did not affect the rate. In addition, no uracil was removed
when C·U DNA was incubated with HAP1 alone.
The results in Fig. 5 suggested that HAP1 and the glycosylase interact.
This interaction is probably with the complex of glycosylase and the
apurinic site and not with the free glycosylase, because essentially
the same results were obtained when HAP1 was mixed with the DNA before
the glycosylase was added, as were obtained when thymine DNA
glycosylase was preincubated with HAP1 before addition of the DNA. The
possibility that this interaction might involve the formation of a
ternary complex of apurinic DNA, glycosylase, and HAP1 was investigated
by a band shift experiment (Fig. 6). 32P-Labeled DNA containing a G·T mismatch was incubated
for 2.5 h with thymine DNA glycosylase and various concentrations
of HAP1 and then subjected to electrophoresis on a non-denaturing
polyacrylamide gel. When only thymine DNA glycosylase was present, a
band corresponding to the complex between the glycosylase and DNA
containing a G·AP site was observed. The mobility of this complex of
thymine DNA glycosylase with G·AP DNA was not altered in the presence
of up to 24 nM HAP1, indicating that HAP1 does not bind
tightly to the glycosylase-apurinic DNA complex. However, a new band
appeared that became more intense as the concentration of HAP1
increased. This complex had the same mobility as a complex seen when
G·AP DNA was incubated with HAP1 (Fig. 6B,
+HAP1). Chromatographic analysis of the DNA in Fig.
6B showed that in the presence of thymine DNA glycosylase
alone (+TDG) the G·AP DNA was intact, whereas in the
presence of HAP1 only (+HAP1) the DNA was entirely cleaved,
and thus HAP1 was bound to DNA that it had already cleaved. Complexes
between HAP1 and DNA containing an uncleaved tetrahydrofuran analogue
of an apurinic site (26) to intact apurinic DNA that could not be
cleaved because of the absence of magnesium (25) and to a DNA fragment
with a 3'-phosphoribose produced by heat-induced cleavage of an
apurinic DNA (25) have been detected previously in band shift
experiments, but this seems to be the first report of a complex between
HAP1 and apurinic DNA that it had itself cleaved.

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Fig. 6.
Non-denaturing gel of the reaction shown in
Fig. 5C. A,
32P-labeled DNA containing a G·T mismatch was incubated
with thymine DNA glycosylase in the presence of four different
concentrations of HAP1 for 2.5 h under the same conditions as Fig.
5C and then run on a non-denaturing gel as described under
"Experimental Procedures." B, 10 nM
32P-labeled DNA containing a G·AP site was incubated with
either 5 nM thymine DNA glycosylase (+TDG) or 5 nM HAP1 (+HAP1) for 30 min at room temperature
and then analyzed on a non-denaturing gel. The positions of free DNA,
HAP1-DNA complexes, and thymine DNA glycosylase-DNA complexes are shown
by arrows. Note that the free DNA migrates more slowly in
the presence of HAP1 than in the presence of thymine DNA glycosylase.
This is presumably because the nicked DNA produced by HAP1 migrates
differently to the intact G·AP DNA in the 1st lane
(+TDG).
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Complete In Vitro Repair of G·T Mismatches--
The experiments
reported above show that thymine DNA glycosylase binds particularly
strongly to the apurinic sites produced from its supposed physiological
substrate, a G·T mismatch in the sequence CpG·T, but that the
thymine DNA glycosylase can be displaced from this complex by HAP1, the
enzyme that catalyzes the second step in base excision repair. This
suggested the possibility that the physiological function of the bound
thymine DNA glycosylase might be to recruit proteins involved in base
excision repair, i.e. HAP1, DNA polymerase
, XRCC1, and
DNA ligase III (15), to the site of DNA damage. The influence of these
proteins on the repair of a G·T mismatch is shown in Fig.
7. The DNA containing the G·T mismatch
was incubated with either (i) thymine DNA glycosylase; (ii) thymine DNA
glycosylase and HAP1; (iii) thymine DNA glycosylase, HAP1, DNA
polymerase
, XRCC1, and DNA ligase III without ATP; or
(iv) with thymine DNA glycosylase, HAP1, DNA polymerase
, XRCC1, and
DNA ligase III and ATP. At intervals samples were removed, treated with hydroxide, and the amount of cleaved DNA measured. With
thymine DNA glycosylase alone the cleavable DNA that is measured represents the total DNA containing an apurinic site, either as free
apurinic DNA or as the thymine DNA glycosylase-apurinic site complex.
When both thymine DNA glycosylase and HAP1 are present in the
incubation, the cleaved DNA that is measured represents the sum of the
apurinic DNA cleaved by HAP1 and the apurinic DNA in the thymine DNA
glycosylase-apurinic site complex that has been cleaved by the
hydroxide. When all the proteins are present, but without ATP
(i.e. in the absence of ligase action), the cleaved DNA
measured represents the sum of three different species: (i) apurinic
DNA that has been cut by HAP1; (ii) apurinic DNA that has been cut by
HAP1 and a deoxycytidine nucleotide added to the 3'-end by polymerase
; and (iii) the apurinic DNA in the thymine DNA glycosylase-apurinic
site complex that has been cleaved by the hydroxide. The final mixture
in which there are all the proteins and ATP represents a positive
control because if all the proteins are active, and present in the
required amounts, then all the apurinic DNA, except that bound in the
thymine DNA glycosylase complex, will be repaired and the two fragments
religated. The results showed that, in the presence of all the
proteins, but in the absence of ATP, the cleaved DNA accumulated at a
similar rate to that seen with thymine DNA glycosylase and HAP1 alone. This shows that, unlike HAP1, these other repair enzymes do not enhance
the rate of dissociation of thymine DNA glycosylase from the apurinic
site. When all these proteins and ATP were present the amount of
cleavable DNA remained at a level similar to the amount of thymine DNA
glycosylase. This is most likely because any apurinic DNA that was
released from the glycosylase-G·AP complex was immediately repaired
and ligated to form G·C DNA. This repair of apurinic DNA shows that
the enzymes used were active and present in sufficient quantity to
carry out complete repair. The observation that the glycosylase-G·AP
complex persisted even in the presence of all these proteins confirms
the conclusion drawn from the experiments shown in Fig. 5B
that the apurinic site in the complex cannot be repaired until the
glycosylase is displaced from it. Although the glycosylase-apurinic DNA
complex slows down the process, the DNA in the complex was repairable
since overnight incubation of the reaction shown in Fig. 7, in which
all of the repair components were present, resulted in more than 90%
repair.

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Fig. 7.
The effect of polymerase , XRCC1, and DNA
ligase III upon the reaction of thymine DNA glycosylase with G·T
mismatches. DNA containing a G·T mismatch (20 nM) in
2 mM magnesium buffer containing 20 µM dCTP
was incubated with either 6 nM thymine DNA glycosylase
alone (×); 6 nM thymine DNA glycosylase, and 11 nM HAP1 ( ); 6 nM thymine DNA glycosylase, 11 nM HAP1, 10 nM polymerase , 10 nM XRCC1, and 10 nM DNA ligase III without ATP
( ); or 6 nM thymine DNA glycosylase, 11 nM
HAP1, 10 nM polymerase , 10 nM XRCC1, and 10 nM DNA ligase III in the presence of 2 mM ATP
( ). The results have been plotted as hydroxide-cleaved DNA. This
includes DNA containing an apurinic site that is cleaved by the
hydroxide treatment as well as all of the intermediates in base
excision repair that contain a nick in the phosphate backbone (see Fig.
1). Remaining DNA containing the G·T mismatch and the final ligated
DNA in which a G·C pair has replaced the mismatch are not cleaved
under these conditions.
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DISCUSSION |
Having recently found (7) that each molecule of thymine DNA
glycosylase can only remove one thymine because the glycosylase binds
so tightly to the G·AP product of the reaction that it is unable to
react with another G·T mismatch, we investigated the interaction of
thymine DNA glycosylase with apurinic sites in DNA in more detail. We
found that the glycosylase can bind to DNA containing an apurinic site
opposite guanine, S6-methylthioguanine (7),
cytosine, thymine, and adenine (Fig. 2). Thus thymine DNA glycosylase
shows no absolute preference for the base opposite the apurinic site
but seems only to require the apurinic site itself. Furthermore,
thymine DNA glycosylase binds to all apurinic sites with a relatively
low Kd. These findings suggest that thymine DNA
glycosylase may bind to apurinic sites in the cell produced by the
following: (i) spontaneous depurination; (ii) the action of alkylating
agents and other chemicals that react with the bases in DNA to produce
adducts with unstable glycosidic bonds, such as 3-alkyladenine or
7-methylguanine (27); and, (iii) the action of other DNA glycosylases
such as uracil DNA glycosylase and the glycosylases that remove
modified bases (28).
We have measured the dissociation rates for glycosylase-apurinic DNA
complexes using a band shift assay that has been used previously for
measuring dissociation rates of protein-nucleic acid complexes (23, 29,
30). The absolute koff values obtained here need
to be confirmed using other techniques and should be treated with
caution. However, the dissociation rates are consistent with the
results for the reaction of thymine DNA glycosylase (see Fig. 3 and
discussion below). In EDTA buffer, the dissociation rates of complexes
between thymine DNA glycosylase and all three apurinic containing DNA
duplexes tested in Fig. 3 are extremely slow (1.8-3.6 × 10
5 s
1; see Table I) and correspond to
half-lives for the complexes of between 5 and 10 h. Assuming a
typical second-order association rate (kon) of
107 M
1 s
1, these
results imply that the binding constants for thymine DNA glycosylase to
these apurinic sites in DNA are between 2 and 4 pM. These
results confirm our previous finding that the thymine DNA glycosylase
action is limited by extremely slow product release (7). The E. coli mismatch-specific uracil glycosylase, MUG, which is a
homologue of thymine DNA glycosylase (9), also binds to apurinic sites
in DNA (31), although the reported Kd (6 nM) for the binding of MUG to an apurinic site opposite
guanine is a thousand times larger than the value obtained here for
human thymine DNA glycosylase.
Thymine DNA glycosylase does not require magnesium, and initially we
followed the practice of previous authors (2, 4, 6, 8) and carried out
the thymine DNA glycosylase experiments in buffer containing EDTA. As
the following step in base excision repair, cutting of the apurinic
site by the apurinic endonuclease HAP1 requires at least 0.1 mM magnesium for maximum activity (32), the dissociation
rates were also measured in the presence of 2 mM magnesium.
Magnesium had little effect upon the rate of dissociation of thymine
DNA glycosylase from G·AP DNA (only a 60% increase in
koff), but it increased the rate of dissociation
of thymine DNA glycosylase from SMeG·AP and C·AP sites
in DNA by 20- and 28-fold, respectively (Table I). This increase in the
dissociation rate produced by magnesium has a great influence on the
reaction of thymine DNA glycosylase with DNA containing a
SMeG·T or a C·U mismatch allowing it to remove more
than a stoichiometric amount of thymine from SMeG·T and
uracil from C·U mismatches (Fig. 3, F and G).
In contrast, magnesium had little effect on the dissociation of thymine
DNA glycosylase from a G·AP site in DNA, so even in the presence of magnesium the glycosylase could remove only a stoichiometric amount of
the mismatched thymine or uracil from DNA containing a G·T or a G·U
mismatch. These results suggest that in the presence of magnesium and
with limiting thymine DNA glycosylase, both C·U mismatches and
SMeG·T mismatches would be better substrates for the
glycosylase than G·T DNA. This is probably irrelevant to the repair
of C·U base pairs because they would occur very rarely in cells, and it is almost certain that in vivo uracil would be removed
from C·U by the more efficient and abundant uracil DNA glycosylase. However, thymine DNA glycosylase is the only glycosylase known to be
able to remove thymine from SMeG·T base pairs so the
reasonably rapid attack of thymine DNA glycosylase on
SMeG·T mismatches may be important in regard to the
cytotoxicity of 6-thioguanine (11).
Glycosylases mediate the first step in the base excision repair
pathway. The apurinic sites produced are then cut at the apurinic site
by an apurinic endonuclease, probably HAP1 (reviewed in Ref. 33). We
had expected that the bound thymine DNA glycosylase might recruit HAP1
and thus facilitate the cleavage of the apurinic site, but unexpectedly
it was found that neither HAP1 nor Endonuclease IV can cut at the
apurinic site while thymine DNA glycosylase is bound to it (Fig. 5,
A and B). Thus dissociation of the thymine DNA
glycosylase from the apurinic site is a prerequisite for complete repair to take place (Fig. 7). HAP1 increases the rate of this dissociation. This increases the removal of thymine from G·T
mismatches, because the displacement allows turnover of the glycosylase
(Fig. 5) and accelerates the repair because it allows HAP1 to cut the apurinic site, which in turn allows the other enzymes involved in base
excision repair to refill the gap in the DNA. The displacement of
thymine DNA glycosylase from the apurinic site appears to be specific
to HAP1 since Endonuclease IV, a type II endonuclease from E. coli, has no effect on the glycosylase reaction (Fig. 5A). No similar interaction between a DNA glycosylase and
HAP1 has been reported before. The increase induced by 6 nM
HAP1 in the rate of turnover of 6 nM thymine DNA
glycosylase was quite small, but the increase is
concentration-dependent and at 600 nM HAP1 a much
larger increase in turnover was induced. From the purification of HAP1
from HeLa cells (14), we estimate that the concentration of HAP1 is
about 0.1-1 mM (a similar figure is given in Ref. 34), and
at this concentration HAP1 should produce a very substantial increase
in the rate of dissociation of thymine DNA glycosylase from apurinic
sites. The rate of removal of thymine from G·T mismatches by thymine
DNA glycosylase in the presence of such a large concentration of HAP1
should be more than adequate to cope with the rate of deamination of
5-methylcytosine in cells (35). The high concentration of HAP1 in cells
may, in part, reflect its role in maintaining the redox state of some transcription factors (33), but it may also reflect the necessity for a
large concentration of HAP1 to dissociate the thymine DNA glycosylase,
and conceivably other DNA glycosylases, from apurinic sites.
A multiprotein complex that fully repairs uracil sites in DNA has been
purified suggesting that components of base excision repair may exist
as a "repairosome" that can carry out complete repair in a
concerted manner (36). Kubota et al. (15) reconstituted in vitro the repair of uracil in DNA using the human
proteins uracil DNA glycosylase, HAP1, polymerase
, XRCC1, and DNA
ligase III. In an analogous experiment, we reconstituted in
vitro repair of DNA containing a G·T mismatch using thymine DNA
glycosylase, HAP1, polymerase
, XRCC1, and DNA ligase III. Our
present data do not support the view that these proteins act as a
repair complex analogous to that seen in nucleotide excision repair
(reviewed in Ref. 37). Although this combination of proteins could
completely repair the G·T mismatch, the rate of the glycosylase
reaction was no greater than that seen with thymine DNA glycosylase and HAP1 alone (Fig. 7) showing that these other repair proteins are not
able to cooperate with HAP1 in the displacement of the glycosylase from
the apurinic site. Furthermore, there was no evidence from band shift
assays that HAP1, which is the only one of these proteins that had any
effect on the dissociation of the thymine DNA glycosylase from the
apurinic site, formed a complex with the thymine DNA glycosylase bound
to the apurinic site (Fig. 6).
The interaction between thymine DNA glycosylase and HAP1 would loosely
coordinate the first and second step of base excision repair and
appears similar to the recently reported interaction between HAP1 and
DNA polymerase
(34) that would loosely coordinate the second and
third step. An interaction between polymerase
and a complex of DNA
ligase III and XRCC1 has also been described (15). The overall effect
of these interactions might be to link all the steps in the pathway.
However, this coordination does not entirely explain why the
glycosylase binds so strongly to apurinic sites. If the site were not
protected by the bound glycosylase it would be very rapidly cut by
HAP1, which is a relatively fast enzyme (25), and so the binding of the
glycosylase to apurinic sites slows, rather than accelerates, their
repair. One possibility comes from the observation that in human
tissue, the amount of HAP1 measured in some cells types is quite low,
and in some cells HAP1 is predominantly cytoplasmic and thus may not be
available for DNA repair (38). One could foresee that in cells where
there was a lack of HAP1, the binding of thymine DNA glycosylase to apurinic sites might prevent oxidative damage to the apurinic site or
might act as a physical block to DNA replication or transcription and
prevent misincorporation opposite the apurinic site. Alternatively, it
might act as a signal to halt the cell cycle until DNA repair can be
completed or, in extreme cases, might act as the signal for apoptosis.