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INTRODUCTION |
The base excision repair pathway provides the cell with a major
line of defense against damage to DNA bases by excising damaged bases
and resynthesizing DNA. Base excision repair is initiated by the
activity of DNA glycosylases, which function to identify and excise
damaged bases. Because these enzymes recognize DNA base damage, they
are key to the overall effectiveness of the pathway. Monofunctional DNA
glycosylases such as human alkyladenine DNA glycosylase
(hAAG)1 excise damaged DNA
bases by hydrolysis of the C1'-N glycosylic bond, forming a free DNA
base and an abasic sugar residue. Once the damaged base is removed,
other enzymes in the pathway remove the remaining sugar residue and
resynthesize DNA to fill in the gap.
DNA glycosylases are damage-specific; different enzymes are responsible
for excising different types of damaged DNA bases. Some glycosylases
such as uracil DNA glycosylase are very specific and excise only a
single damaged base, uracil, in this case. Other DNA glycosylases have
broader substrate specificities. For example, formamidopyrimidine DNA
glycosylase (FaPy) recognizes oxidative damage to DNA bases and excises
7,8-dihydro-8-oxoguanine (8-oxoG), FaPy, and 5-hydroxycytosine. Based
on both structural (for recent reviews, Refs. 1-3) and spectroscopic
(4) data, DNA glycosylases are believed to "flip" damaged
nucleotides out of the DNA helix and into an enzyme active site, where
catalysis takes place. Given this type of flipping mechanism, it is
easy to imagine how a DNA glycosylase may recognize a specific damaged
DNA base through interactions in the active site that provide a
"tight" fit and align the glycosylic bond for chemistry. For DNA
glycosylases with broader substrate specificities, the nature of the
structural interactions and mechanisms that provide specificity are
less clear. We are interested in the mechanisms by which DNA
glycosylases are able to efficiently identify and excise damaged DNA bases.
Alkyladenine DNA glycosylase (also referred to as 3-methyladenine
DNA glycosylase and N-methylpurine DNA
glycosylase) is the only glycosylase identified to date in human
cells that excises alkylation-damaged bases. This glycosylase has been
shown to have a broad substrate specificity and has been reported to
excise at least 12 different damaged bases including 3-methyladenine (5-10), 7-methylguanine (5-7, 10, 11),
1,N6-ethenoadenine (8, 9, 11, 12),
etheno adducts of guanine (12), 7,8-dihydro-8-oxoguanine (13),
hypoxanthine (11, 14, 15), and undamaged purines (16, 17). These
damaged purine bases are structurally diverse and contain modifications
to both the major and minor groove sides of base pairs as well as to
groups involved in base pairing. For example, the methyl group of
3-methyladenine projects into the minor groove, whereas that of
7-methylguanine projects into the major groove, but neither of these
methyl groups interrupts hydrogen bonding interactions with its base
pairing partner. On the other hand, hydrogen-bonding interactions are disrupted for ethenoadenine and the etheno adducts of guanine. Deamination of adenine to form hypoxanthine alters base pairing and
converts a Watson-Crick base pair with T to a wobble base pair, whereas
8-oxoG is still capable of forming a Watson-Crick base pair with C. Given these different base and base pair structures, it is difficult to
formulate one model for the structural and mechanistic bases of
recognition and excision of these chemically diverse substrates.
To begin to define the mechanistic basis for substrate recognition and
excision by hAAG, kinetics of excision and DNA binding affinities were
measured for DNA containing different damaged DNA bases within the same
sequence context. In addition, the effects on excision rates and
binding constants of varying the base paired with a damaged DNA base
were examined. Although the alkylated DNAs processed by hAAG appear to
have few characteristics in common, the goal of our experiments is to
determine whether there are common underlying structural features that
are recognized by hAAG. Base excision and DNA binding by hAAG were
measured for more than 20 different base pair combinations. Fig.
1 illustrates structures of some of the
base pairs that were incorporated into DNA substrates. We found
ethenoadenine (
A) and hypoxanthine (Hx) to be the most efficiently
excised; however, excision of Hx was affected dramatically by its
base-pairing partner.

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Fig. 1.
Structures for some of the DNA base pairs
tested as substrates for hAAG. When paired opposite T, A and Hx
were excised most rapidly. Changing the base opposite A to C and U
had only modest effects on base excision rates and DNA binding. In
contrast, changing the base opposite Hx had a dramatic effect on base
excision and DNA binding. Replacing T with U opposite Hx resulted in a
decrease in excision rate and an increase in DNA binding affinity,
whereas replacing T opposite Hx with either C or 5-MeC reduced both the
excision rate and binding affinity. The effects of the base opposing a
lesion on DNA binding and base excision activity of hAAG demonstrate
that recognition and excision of a damaged base is not simply a
function of the structure of the damaged base alone but is also a
function of the structure of a damaged base pair.
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EXPERIMENTAL PROCEDURES |
Oligonucleotides--
Synthetic oligonucleotides were either
purchased from Fisher-Genosys or made on an Applied Biosystems, Inc.
392 DNA synthesizer using standard
-cyanoethylphosphoramidite
chemistry and reagents from Glen Research. Oligonucleotides were
purified by denaturing polyacrylamide gel electrophoresis.
Concentrations of purified single-stranded oligonucleotides were
determined from absorbances measured at 260 nm using extinction
coefficients calculated for each oligonucleotide at 260 nm (18). The
extinction coefficient used for
A was 5000 M
1 cm
1
(extinction coefficient for
1,N6-ethenoadenosine (19)), and
extinction coefficients for
A dinucleotides were estimated to be the
average of mononucleotide extinction coefficients. For Hx, extinction
coefficients for A and A dinucleotides were used, and for
O6-methylguanine (O6-MeG) and
7,8-dihydro-8-oxoguanine (8-oxoG), extinction coefficients for G and G
dinucleotides were used. The overall error in oligonucleotide extinction coefficients contributed by using estimated values for Hx,
O6-MeG, and 8-oxoG is small because the damaged base is
only 1 out of 25 total nucleotides. All oligonucleotides were 25 nucleotides in length and of identical sequence
(5'-GCGTCAAAATGTDGGTATTTCCATG-3') except for the central
damaged base (D). Duplex DNA substrates were made by annealing labeled
oligonucleotides to an equal concentration of unlabeled complementary
oligonucleotides. Annealed duplexes were typically prepared at
20 times greater concentrations than used in excision or binding assays
and then diluted directly into assay mixtures without further purification.
Human 3-Methyladenine DNA Glycosylase (hAAG)--
A deletion
mutant of hAAG that is missing the first 79 amino acids from the N
terminus (hAAG
79) was used in all assays. Deletion of this
unconserved N-terminal region has been shown to have no effect on
either base excision or DNA binding activities of the enzyme (7, 20,
21), but the truncated enzyme is more soluble at low ionic strength. A
catalytically inactive mutant, hAAG
79E125Q, containing a single
point mutation, Glu-125
Gln, was used in DNA binding assays. Both
hAAG
79 and hAAG
79E125Q were overexpressed in Escherichia
coli and purified as previously described (21).
Excision Assays--
Base excision was measured using a chemical
cleavage/gel assay. DNA strands containing a damaged DNA base were
5'-end-labeled with 32P and annealed to a complementary
strand. Excision reactions were performed by incubating hAAG
79 with
a DNA substrate at 37 °C in 50 mM HEPES, pH 8.0, 100 mM NaCl, 10 mM EDTA, 1 mM DTT, and 9.5% v/v glycerol. Typical reaction mixtures contained 400 nM hAAG
79 and 50 nM duplex DNA. At several
time points during the course of excision reactions, an aliquot of the
reaction mixture was quenched in 0.2 M NaOH (final
concentration) and heated at 90 °C for 5 min to cleave DNA products
containing apurinic sites. After heating, samples were diluted with 2 volumes of loading buffer consisting of 95% formamide and 20 mM EDTA. Unreacted substrates were separated from cleaved
products by electrophoresis on 16% denaturing polyacrylamide gels and
quantitated using a Molecular Dynamics Storm PhosphorImager and
ImageQuant software.
DNA Binding Assays--
DNA binding was measured in
electrophoretic mobility shift assays (EMSAs). The DNA strand
containing the damaged base was 5'-end-labeled with 32P and
annealed to a complementary strand containing either T, C, or U
opposite the damaged base. Labeled oligos (50 nM) were incubated with increasing concentrations of hAAG for 10 min at 4 °C,
diluted with loading buffer, and loaded directly onto a 6%
nondenaturing polyacrylamide gel. Polyacrylamide gel electrophoresis was performed at 4 °C for 180 min at 8 V/cm. The EMSA assay buffer was identical to the buffer used in excision assays and contained 50 mM HEPES, pH 8.0, 100 mM NaCl, 10 mM EDTA, 1 mM DTT, and 9.5% v/v glycerol. The
fraction of DNA bound by hAAG was quantitated using a Molecular
Dynamics Storm PhosphorImager and ImageQuant software.
Apparent binding constants (Kd,app) for
hAAG binding to DNA substrates were calculated using Equation 1 for a
simple two-state binding model where Eo is the total
enzyme concentration, Do is the total DNA
concentration, and EDtotal is the total
concentration of all enzyme-bound species (see Equation 3 under
"Results").
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(Eq. 1)
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RESULTS |
Excision of Damaged and Undamaged DNA Bases by hAAG--
A survey
was done of the excision activity of hAAG on several different damaged
and undamaged DNA bases to determine which of these bases were most
efficiently excised by hAAG. Damaged DNA bases were incorporated at a
single site in the center of synthetic oligonucleotides 25 nucleotides
in length using standard
-cyanoethylphosphoramidite chemistry.
Duplex DNA substrates were made by annealing to the complementary
oligonucleotide and were identical in sequence except for the central
"damaged" base pair. Excision activity was measured in time course
assays by incubating these 32P-labeled DNA substrates (50 nM) with hAAG
79 (400 nM) at 37 °C for
periods up to 160 min. A chemical cleavage/gel assay was used to
measure the amount of excision of each damaged base at several times
during the course of the excision reaction. In this assay, DNA products
containing apurinic sites were chemically cleaved by heating in 0.2 M NaOH at 90 °C for 5 min. Cleaved DNA products were
then separated from uncleaved substrates by denaturing polyacrylamide gel electrophoresis and quantitated by phosphorimaging. Time courses for excision of several of the bases that were tested as substrates are
shown in Fig. 2 (see Fig. 1 for
structures of base pairs). Of the damaged DNA bases tested, Hx and
A
were excised most efficiently; excision of Hx was nearly complete in 10 min, and excision of
A was nearly complete in 80 min. In contrast,
significant excision of 8-oxoG and O6-MeG did not occur
over a period of 160 min. Changing the base opposite 8-oxoG and
O6-MeG from C to T did not affect excision rates (data not
shown). It has been reported that hAAG is capable of excising undamaged purines (16, 17); however, under our assay conditions, significant excision of A and G were not observed when present as correct pairs or
when present as G·T and A·C mispairs.

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Fig. 2.
Excision of different damaged and undamaged
DNA bases by hAAG. DNA duplexes (50 nM), 25 nucleotides in length and of identical sequence except for the central
base pair, were incubated with hAAG (400 nM) at 37 °C
for periods up to 160 min. At several time points, an aliquot of each
reaction mixture was quenched in 0.2 M NaOH and heated at
90 °C for 5 min to cleave DNA products containing apurinic sites.
Unreacted substrates were separated from cleaved products by denaturing
polyacrylamide gel electrophoresis and imaged and quantitated by
phosphorimaging. Assay buffer consisted of 50 mM HEPES, pH
8.0, 100 mM NaCl, 10 mM EDTA, 9.5% glycerol,
and 1 mM DTT.
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Single Turnover Kinetics of Excision of
1,N6-Ethenoadenine and Hypoxanthine--
The kinetics of
excision of
A and Hx were examined in more detail. Because excision
kinetics were extremely slow under steady-state kinetic conditions and
the enzyme loses activity with prolonged incubation at 37 °C, single
turnover kinetics of excision were measured (note: when hAAG is
incubated with DNA under conditions where DNA binding occurs, the
enzyme is protected from inactivation at
37 °C2). For these assays,
the 25-nucleotide DNA duplex substrates above containing a central
A·T or Hx·T base pair were used. In these experiments, 50 nM DNA was incubated with increasing concentrations of
hAAG
79 up to 800 nM in separate reactions. Aliquots were
withdrawn at several time points during each reaction, quenched with
0.2 M NaOH, and analyzed by the chemical cleavage/gel assay
method described above. For each concentration of hAAG
79, two or
three separate time course reactions were performed. The averages and S.D. for time courses at 400, 600, and 800 nM
concentrations of hAAG
79 are plotted in Fig.
3. For both damaged base pairs,
A·T and Hx·T, reaction time courses are essentially the same at these three enzyme concentrations, demonstrating that the concentration of
enzyme is saturating, and reaction kinetics are not a function of
enzyme-substrate binding rates.

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Fig. 3.
Single-turnover kinetics of excision of
1,N6-ethenoadenine and hypoxanthine when
paired opposite thymine. DNA duplexes (50 nM)
containing either a central A·T (left panel) or Hx·T
(right panel) base pair were incubated with 400 (circles), 600 (squares), and 800 (triangles) nM hAAG 79 in separate reactions.
Assay buffer contained 50 mM HEPES, pH 8.0, 100 mM NaCl, 10 mM EDTA, 9.5% glycerol, and 1 mM DTT. Excision products were quantitated using a chemical
cleavage/gel assay as shown in Fig. 2 and described under
"Experimental Procedures." Two or three separate time course
reactions were done at each enzyme concentration, and the average
values and S.D. are plotted. The solid lines are exponential
fits to these time course data.
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Individual time course reactions at each enzyme concentration were fit
to an exponential rise (Equation 2) to determine values for
kobs.
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(Eq. 2)
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For
A, kobs values were 0.080 ± 0.003, 0.075 ± 0.001, and 0.076 ± 0.001 min
1 for duplicate measurements at
concentrations of 400, 600, and 800 nM enzyme,
respectively. Calculated values of kobs were
4-5 times greater for Hx and were 0.31 ± 0.01, 0.32 ± 0.01, 0.37 ± 0.01 min
1 for triplicate
measurements at 400, 600, and 800 nM enzyme, respectively.
Effects of Base-pairing Partners on Excision of
A and
Hx--
To determine whether the base paired opposite
A or Hx had
any effect on the efficiency of hAAG-catalyzed excision, thymidine (T)
was replaced with both 2'-deoxycytidine (C) and 2'-deoxyuridine (U).
The pyrimidine base paired opposite the damaged base had a larger
effect on excision of Hx than on excision of
A. Time course assays
(Fig. 4) were done in duplicate using 400 nM hAAG and 50 nM "damaged" DNA as above.
Replacing T with a C resulted in little if any effect on the observed
rate (kobs) of excision of
A, which was 0.066 min
1, but decreased
kobs for Hx by a factor of about 5 to 0.062 min
1. Surprisingly, replacing T with U
resulted in a decrease in the rates of excision of both
A and Hx.
Again, the effect on the rate of excision of
A was smaller and was
reduced by a factor of 1.7 to 0.045 min
1.
Excision of Hx was reduced by a factor of about 15 to 0.022 min
1 when T was replaced with U. As a
control, the DNA strand that contained U was labeled, and excision was
measured in assays using both hAAG
79 and E. coli uracil
DNA glycosylase. No uracil DNA glycosylase activity was observed in
reactions with hAAG
79, whereas quantitative excision of U was seen
in reactions with uracil DNA glycosylase (data not shown). The effect
of replacing T with U is striking because U differs from T in that it
simply lacks the 5-methyl group, which extends into the major groove.
To determine whether a 5-methyl group affects base excision by hAAG, C
was replaced with 5-methylcytosine (5-MeC) in base pairs with
A and Hx. In this case, the 5-methyl group had no effect, and excision was
the same for base pairs with C and 5-MeC (data not shown).

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Fig. 4.
Excision of
1,N6-ethenoadenine and hypoxanthine when
paired opposite cytosine and uracil. Time course assays for
excision of A and Hx paired opposite C and U were performed in
reactions containing 50 nM DNA and 400 nM
hAAG 79 at 37 °C as in Fig. 2.
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Binding of hAAG
79 and a Catalytically Inactive Mutant to
DNA Containing an
A·T Base Pair--
To better define the
interactions between hAAG and different damaged DNA bases, the binding
affinity of hAAG to DNA duplexes containing different damaged DNA bases
was measured. For these experiments, a catalytically inactive mutant of
hAAG, hAAG
79E125Q, was used so that binding to DNA substrates could
be measured in the absence of excision. In this mutant, glutamic acid
125 was replaced by glutamine. This Glu residue has been proposed to
act as a general base to activate water for hydrolysis of the
glycosylic bond (21). In excision assays with
A and Hx paired
opposite T, hAAG
79E125Q was unable to excise either
A or Hx over
a period of 80 min under conditions as in Fig. 2 (data not shown).
To determine whether the mutation of Glu-125 to Gln affected binding
activity, binding of "wild type" hAAG
79 and hAAG
79E125Q to
DNA duplexes containing an
A·T base pair was measured. Incubation of hAAG
79 with
A-containing DNA at 4 °C significantly reduces the rate of excision of
A, so that binding to this substrate can be
measured in the absence of significant product formation. The same
25-nucleotide duplex DNA substrates used in excision assays were used
in binding assays, and the DNA strand containing the damaged base was
5'-end-labeled with 32P. Binding experiments were done by
incubating the
A·T-containing DNA duplex with different
concentrations of enzyme at 4 °C for 10 min. After 10 min, an
aliquot of these reaction mixtures was removed and analyzed using an
EMSA. Two additional aliquots of each reaction mixture were removed:
the first, when the EMSA gel was loaded, and the second, after the gel
was completed. These additional aliquots were immediately quenched with
0.2 M NaOH and analyzed to determine the amount of excision
of
A that occurred during the time course of the EMSA.
Results from EMSAs are shown in Fig.
5A for hAAG
79 and Fig.
6A (upper panel)
for hAAG
79E125Q. Binding isotherms for the wild type and
catalytically inactive mutant are virtually identical (Fig.
5B), indicating that the point mutation reduces excision activity but has little if any effect on DNA substrate binding. For
wild type hAAG
79, only about 16% of the substrates were converted to abasic DNA products during the time course of the EMSA at the highest enzyme concentration.

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Fig. 5.
Binding of hAAG 79
and hAAG 79E125Q to DNA containing a
1,N6-ethenoadenine·thymine base
pair. A, an EMSA gel is shown for binding of
catalytically active hAAG 79. An EMSA gel for hAAG 79E125Q is shown
in Fig. 6A, for comparison. Assay conditions were identical
to those of excision assays containing 50 nM DNA, 50 mM HEPES, pH 8.0, 100 mM NaCl, 10 mM EDTA, 9.5% glycerol, and 1 mM DTT.
B, the fraction of A excised during the EMSA time course
(open circles) was quantitated and is plotted along with the
fraction of A·T DNA bound by hAAG 79 (filled circles)
and hAAG 79E125Q (filled triangles).
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Fig. 6.
Binding of
hAAG 79E125Q to DNA containing
1,N6-ethenoadenine and hypoxanthine base
pairs. DNA duplexes containing A or Hx paired with T, C, or U
were incubated with increasing concentrations of hAAG 79E125Q.
Assays contained 50 nM DNA, 50 mM HEPES, pH
8.0, 100 mM NaCl, 10 mM EDTA, 9.5% glycerol,
and 1 mM DTT. A, EMSAs were used to quantitate
the fraction of DNA bound by protein. B, binding isotherms
are shown for DNA containing A·T (circles), A·C
(squares), and A·U (triangles) pairs
(left panel) as well as Hx·T (circles), Hx·C
(squares), and Hx·U (triangles) pairs
(right panel). Each EMSA experiment was performed in
triplicate, and average values and S.D. for the different
concentrations of enzyme-bound DNA are shown. Solid curves
through the data points are the results of fits to calculate
dissociation constants (Kd,app) assuming
a simple two-state binding mechanism (see "Experimental
Procedures").
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Binding of hAAG
79E125Q to DNA Substrates Containing Different
A and Hx Base Pairs--
The catalytically inactive mutant,
hAAG
79E125Q, was used to measure the binding affinity of the enzyme
to DNA substrates containing different damaged DNA bases.
Electrophoretic mobility shift assays were done as above using the same
damaged duplex DNA substrates used in excision assays. Representative
phosphorimager scans of binding data are shown in Fig.
6A for DNA duplexes containing
A and Hx base pairs with
T, C, and U. In general, hAAG binds with greater affinity to DNA
containing
A base pairs than Hx base pairs. The base-pairing partner
has a significant effect on the enzyme affinity for DNA substrates
containing Hx base pairs. For each DNA substrate, three separate EMSA
experiments were performed and quantitated. Binding isotherms showing
the average and S.D. of these three independent experiments are shown
in Fig. 6B. Data were fit to a simple two-state binding
model shown in Equation 3 using a quadratic equation (see
"Experimental Procedures," Equation 1) to determine an apparent
dissociation constant (Kd,app) where
EDtotal represents all species of ED
complexes formed (i.e. both complexes where the damaged
nucleotide are flipped (EDflip) and not flipped
(EDun)).
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(Eq. 3)
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Apparent dissociation constants calculated for
A base pairs
were 20 ± 2, 23 ± 2, and 6.3 ± 1.0 nM for
base pairs with T, C, and U, respectively. Dissociation constants for
DNA duplexes containing Hx base pairs were affected to a much greater
extent by changing the base-pairing partner. Apparent dissociation
constants were 92 ± 2 and 12 ± 2 nM for Hx·T
and Hx·U base pairs, respectively. For the duplex containing Hx·C
base pairs, Kd,app is ~600
nM and is too great to accurately determine because at high enzyme concentrations, bands "smear" on EMSA gels, probably due to
nonspecific binding of the enzyme to undamaged DNA.
In addition to measuring binding to DNA containing
A and Hx lesions,
hAAG binding was measured to DNA containing each of the base pairs that
were used in excision assays. These base pairs included
O6-MeG opposite C and T, 8-oxoG opposite C and T, G
opposite T, C, and U, and A opposite T, C, U, and 5-MeC. Significant
binding to these DNA substrates was not observed (data not shown).
Although U opposite a lesion increased binding to DNA containing
A
and Hx, it had no effect when placed opposite G or A.
Binding of hAAG
79 to DNA Duplexes Containing Abasic
Sites--
Several DNA glycosylases including E. coli MutY
(22), human thymine DNA glycosylase (23), and human
methyl-CpG-binding endonuclease 1 (24) have been shown to bind very
tightly to the products of their excision reactions. To determine
whether hAAG has a high affinity for apurinic DNA products, binding of hAAG
79 to duplex DNA substrates containing a synthetic abasic site
was measured. A synthetic "reduced" abasic site was used in place
of the natural abasic site because this substrate is more stable and
can be incorporated at a specific site using standard synthetic
chemistry. As a control, binding of hAAG
79 to DNA containing a
natural abasic site was measured (data not shown) and found to be
similar to binding to a reduced abasic site, as has been observed by
others (15). Results from EMSA experiments with DNA substrates
containing abasic sites are shown in Fig.
7. As with damaged bases, the affinity of
the enzyme for abasic sites is affected by the base opposite the abasic
site. The enzyme only binds duplexes that contain pyrimidines opposite
the abasic site. Apparent binding constants calculated for substrates
with pyrimidines opposite the abasic site were 140, 300, and 56 nM for T, C, and U, respectively. Interestingly, when
either
A or Hx was placed opposite the abasic site, the enzyme did
not bind DNA duplexes, suggesting that the enzyme cannot recognize
these damaged bases unless a base is paired opposite them.

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Fig. 7.
Binding of hAAG 79 to
DNA containing abasic sites. Binding of hAAG 79 to product
analogs was measured by EMSA under conditions identical to excision
assays and binding assays with damaged bases (Figs. 2 and 5). A reduced
abasic site analog was used in place of the natural abasic site in
these assays. A, hAAG 79 binds DNA containing an abasic
site opposite a pyrimidine, T, C, or U. B, no binding of
hAAG 79 was observed for DNAs containing an abasic site opposite a
purine base, even if the purine was A or Hx.
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DISCUSSION |
The human alkyladenine DNA glycosylase has been shown to have a
broad substrate specificity excising damaged purines, particularly alkylated purines. Various studies have shown that hAAG is capable of
excising 3-methyladenine (5-10), 7-methylguanine (5-7, 10, 11),
1,N6-ethenoadenine (8, 9, 11, 12),
etheno adducts of guanine (12), 7,8-dihydro-8-oxoguanine (13),
hypoxanthine (11, 14, 15), and undamaged purines (16, 17). However,
the relative efficiencies of excision of all of these damaged bases
have not been firmly established by direct comparison of excision
kinetics for each within the same DNA sequence context. This study
examines the structural and mechanistic principles for recognition and excision of damaged DNA bases by hAAG. In essence, our approach was to
perform "site-directed mutagenesis" on damaged base pairs to
determine which structural features of a base pair were important in
binding and excision. Much of the previous work in the field has
focussed largely on the damaged base alone, but more recent evidence
suggests that its base-pairing partner plays a role (9, 11, 14, 25).
Our results demonstrate that for some damaged bases, the opposing base
can have a dramatic effect on binding and excision. This result is
surprising based on the structural data available for the enzyme-DNA
complex, which shows no specific contacts between the enzyme and
opposing base.
The crystal structure of hAAG complexed with DNA containing a
pyrrolidine abasic site analog (21) and a more recent structure of hAAG
bound to
A-containing DNA (26) have revealed that this enzyme, like
other DNA glycosylases, flips a damaged nucleotide out of the DNA helix
and into an enzyme binding pocket where hydrolysis takes place. A
hairpin projects into the minor groove and widens the minor groove at
the site of damage and at base pairs immediately 3' to the pyrrolidine,
suggesting that the enzyme may scan DNA from the minor groove to detect
damage. A tyrosine residue (Tyr-162) projects from this
hairpin and
intercalates in the DNA helix in the "hole" where the damaged base
would have been. In contrast to cocrystal structures of uracil DNA
glycosylase with DNA, little compression of the sugar-phosphate
backbone is seen in the hAAG-DNA complexes (27, 28). For hAAG,
"pushing" the damaged nucleotide out of the helix may be
accomplished by the action of Tyr-162 along with other residues of the
-hairpin without the assistance of "pinching" due to backbone
compression that is seen for uracil DNA glycosylase. Although the
binding site of uracil DNA glycosylase has a geometry that provides a
"tight fit" for uracil through specific amino acid-uracil
interactions (29, 30), the binding site of hAAG must be able to
accommodate a structurally diverse group of damaged bases. Aromatic
amino acid side chains, including Tyr-127, are present in the active
site of hAAG that stack with the damaged base. It has been proposed
that base-stacking interactions between electron-deficient damaged
bases and aromatic side chains may provide the basis for recognition
and excision by hAAG (21, 26) and E. coli 3-methyladenine
DNA glycosylase II (21, 31, 32). In addition, interactions between a
hydrogen bond donor on hAAG and a hydrogen bond acceptor at position 6 of a damaged purine may play a role in recognition (26). Although these
structures have provided significant insights into the mechanism of
recognition and excision by hAAG, questions about substrate specificity
remain to be answered.
To gain further insight into the structural and mechanistic principles
for excision of damaged DNA bases, excision and binding activities of
hAAG were measured for different damaged substrates within an identical
DNA sequence context. Initial assays were done to qualitatively compare
the excision of four damaged bases, 1,N6-ethenoadenine, hypoxanthine,
7,8-dihydro-8-oxoguanine, and O6-methylguanine,
as well as undamaged purines both correctly paired and mispaired with
pyrimidines. Hypoxanthine and
1,N6-ethenoadenine, both paired
opposite T, were the only bases excised during the 160-min time courses
of these assays. Another study using full-length His-tagged hAAG also
found that 8-oxoG was not excised (11). It is possible that undamaged
purines, 8-oxoG, and O6-MeG may be excised after much
longer times, but since excision of these bases was so inefficient,
further characterization was not done. Neither 3-methyladenine nor
7-methylguanine were examined in this study because 3-MeA cannot be
incorporated site-specifically into DNA, and 7-MeG is relatively labile.
A comparison of the structures of these base pairs, shown in Fig. 1,
highlights structural similarities and differences that may be
important in the excision reaction. Both
A and Hx have two hydrogen
bond acceptors that project into the major groove, N7 for both and an exocyclic nitrogen at the 6 position for
A and an exocyclic oxygen at the 6 position for Hx.
However, the base pair that each forms with T is different. The
exocyclic etheno group of
A creates a more bulky base and prevents
hydrogen-bonding interactions with T. NMR studies show that to
accommodate the larger size of
A, an
A·T pair adopts a
conformation where both bases are stacked in the helix but skewed
relative to one another so that they do not form a planar base pair
(33). Hypoxanthine hydrogen bonds with T but forms a wobble pair rather
than a Watson-Crick-type pair. This wobble pair differs from a
Watson-Crick pair in that the purine is shifted into the minor groove,
and the pyrimidine is shifted into the major groove. If hAAG scans the
minor groove, it may detect either of these distortions. The fact that
Hx forms hydrogen bonds with T and
A does not may make
A easier
to flip, whereas the smaller size of Hx may increase the rate of
excision by a better fit in the binding pocket.
It is interesting that excision of G was not observed when placed
opposite T because a G·T pair forms a wobble base pair very similar
in structure to Hx·T, the major difference being the 2-amino group
that is present on G but not on Hx. Perhaps the 2-amino group is not
accommodated within the enzyme active site or it misaligns the
nucleotide in the active site so that hydrolysis of the glycosylic bond
is not efficient (26). In contrast, 7-MeG also has a 2-amino group but
is excised by hAAG (5-7, 10, 11). The 7-methyl group acts to increase
the lability of the purine base and may also serve to enhance the
efficiency of hydrolysis of the glycosylic bond in the enzyme active
site even though alignment of the nucleotide may not be optimal.
To further characterize the excision of
A and Hx, single turnover
kinetics were performed to establish the maximal rate for excision of
each base. When paired opposite T, the observed rate constant
(kobs) for excision of Hx (0.33 min
1) was about 4-fold greater than that for
A (0.077 min
1). Since observed rates in
these experiments are not limited by the rate of enzyme-DNA binding and
the assay measures both enzyme-bound products and free products,
kobs values reflect the rate of conversion of an
enzyme-substrate complex to an enzyme-product complex. Depending on the
kinetic mechanism, this rate could be limited by the rate of nucleotide
flipping or the actual rate of hydrolysis of the glycosylic bond but in
any case reflects the rate of conversion of enzyme-bound substrates to products.
The pyrimidine base opposing the lesion has a much greater affect on
excision of Hx than
A. For both lesions, excision rates decreased in
the order T > C > U. For Hx, changing from T to C and T to
U reduced the observed rates by factors of 5 and 15, respectively.
Replacing T with U resulted in a more modest 1.7-fold decrease in
excision of
A. A similar study done by Asaeda et al. (11)
using full-length His-tagged hAAG also found that excision of Hx was
affected to a much greater extent by its base-pairing partner than
excision of
A. The fact that the base-pairing partner has a much
larger effect on excision of Hx than
A may be due to differences in
hydrogen-bonding interactions in the base pairs. Because the etheno
group bridges N1 and the exocyclic amino group
of adenine,
A is prevented from making hydrogen-bonding interactions
with T, C, and U (Fig. 1). If nucleotide flipping is important in the
mechanism of excision, the lack of hydrogen-bonding interactions may
simply make
A relatively easy to flip regardless of which base
opposes it. It is important to note that although excision of
A by
hAAG is relatively insensitive to the base opposite
A, a base is
required. The fact that hAAG does not excise either
A or Hx when
placed opposite an abasic site further suggests that hAAG does not
simply capture damaged bases that transiently assume extrahelical
positions but instead actively finds and flips damaged bases. The lack
of base-pairing interactions at an abasic site is likely to increase
the frequency of transient spontaneous flipping of a damaged DNA base.
The effects that the pyrimidine base-pairing partner has on excision
rates for Hx is somewhat surprising, particularly when a T is replaced
by a U. The major difference between a T and U base-pairing partner is
the presence or absence of a 5-methyl group that extends into the major
groove (Fig. 1). The fact that the enzyme discriminates between and
Hx·T and Hx·U suggests that the structure of the base pair rather
than simply the damaged DNA base plays a role in the mechanism of
recognition and excision by hAAG. Two possible explanations are that
the enzyme either initially interacts with both the damaged base and
its partner or that the base-pairing partner affects the interaction of
the enzyme with the damaged base in some way. Although it is possible that the enzyme could interact with both the damaged base and its
partner by binding DNA at the major groove before flipping the damaged
base from the minor groove, it seems unlikely. Instead, the crystal
structure suggests that the presence of uracil opposite the lesion
could affect the alignment of the flipped base in the enzyme active
site. Intercalation of Tyr-162 into the space formerly occupied by the
damaged base may help push the nucleotide into the enzyme active site
so that the glycosylic bond is aligned properly for hydrolysis. Uracil
opposite hypoxanthine may reduce the rate of catalysis by
preventing Tyr-162 from intercalating into the DNA far enough to push
the damaged nucleotide into the enzyme active site in the proper
alignment. The unpaired uracil may shift back into the helix toward the
minor groove to maximize base stacking interactions, and this shift may
prevent Tyr-162 from intercalating into the DNA far enough to push the
damaged base into its proper orientation. The 5-methyl group on T may prevent T from shifting as far back into the helix due to its bulkiness
and unfavorable steric interactions with the bases above and below T. In the crystal structure, the T opposite the damaged base is also
pushed into the major groove by about 1.5 Å (21).
The effect of the base-pairing partner on excision efficiency may have
an important biological role in helping to ensure that the damage is
repaired correctly. Hypoxanthine is excised more efficiently when
placed opposite T than opposite C. Initially, Hx would be formed in DNA
from deamination of A opposite T. Once in DNA, Hx is mutagenic,
miscoding for C so that if replication occurs before repair, then an
Hx·C pair may be formed. Once an Hx·C pair is formed, excision of
Hx and repair by base excision repair would create a G·C mutation.
The structural basis for this difference may be due to the fact that
Hx·C forms a Watson-Crick-like base pair, whereas Hx·T or Hx·U
form wobble base pairs (Fig. 1). The addition of a 5-methyl group to C
does not enhance the excision of Hx as does replacing U with T. Perhaps, the normal Watson-Crick-type structure of the base pair masks
the presence of the Hx.
To further characterize the interaction of hAAG with damaged DNA bases,
binding to DNA containing damaged bases was also measured with a
catalytically inactive mutant, hAAG
79E125Q. In general, DNA binding
and base excision activities were correlated. For damaged bases that
were poorly excised by hAAG such as 8-oxoG, O6-MeG, and
undamaged G and A, significant binding was not observed. There was one
exception to this rule; binding to
A·U and Hx·U pairs was
relatively strong. Overall, hAAG had a greater affinity for DNA
containing
A (Kd = 20 nM for
A·T, as also reported by Kartalou et al. (34)) than for
DNA containing Hx (Kd = 92 nM for
Hx·T). Binding affinities decreased with the base opposing the lesion
in the order U > T > C, and the effect was much greater for
Hx than
A. Binding to DNA containing Hx pairs was 7.6-fold greater
for Hx·U than Hx·T and at least 50-fold greater for Hx·U than
Hx·C, whereas the affinity of hAAG for DNA containing
A·U was
3.2- and 3.6-fold greater than for DNA containing
A·T and
A·C, respectively. The same trend was seen for hAAG binding to DNA
containing abasic sites. hAAG does not bind to abasic site DNA
containing purine residues opposite the abasic site even if these
purine residues are damaged
A and Hx. The magnitude of the binding
interactions to DNA products containing an abasic site is on the same
order of magnitude as binding to a DNA substrate containing an Hx·T
base pair and about 7-fold weaker than binding to DNA containing an
A·T base pair. This result seems to imply that hAAG may not remain
tightly bound to DNA products after excision, as has been found for
some other DNA glycosylases including E. coli MutY (22),
human thymine DNA glycosylase (23), and human methyl-CpG-binding
endonuclease 1 (24).
Which is the better substrate for hAAG,
A or Hx? To some extent this
depends on the base opposite the lesion, because for Hx both DNA
binding and excision are affected significantly by the opposing base.
Both damaged bases are likely to be initially formed opposite a T since
they arise from damage to A. Hx opposite T is excised about 4 times
more rapidly than
A opposite T; however, this rapid excision rate is
balanced by a greater binding affinity of hAAG for DNA containing
A.
hAAG binds DNA containing an
A·T pair about five times better than
DNA containing an Hx·T. So when
A and Hx are paired with T, they
are about equally good substrates for hAAG.
Based on these initial experiments, we have developed a working model
for damaged base recognition and excision by hAAG. In this model, there
are two important criteria for efficient base excision, initial
identification of the damaged DNA base and proper alignment of this
damaged nucleotide in the enzyme active site for cleavage of the
glycosylic bond. Initially, the enzyme must find the damaged base amid
the vast excess of undamaged DNA bases. Initial recognition of damage
may depend on recognition of structural distortions in DNA induced by
the damage followed by nucleotide flipping, which checks for fit of the
damaged base in the enzyme active site. Alternatively, damaged base
recognition may occur solely through flipping the damaged base into the
active site. If the damaged base does not fit properly into the enzyme
active site then it will not attain the proper geometry for hydrolysis to take place and excision will be inefficient. The base opposite a
damaged base might affect excision by influencing either the initial
recognition of damage and/or substrate alignment in the enzyme active
site. For example, a C opposite Hx may mask Hx from efficient
recognition because it forms a Watson-Crick-type base pair, whereas U
opposite Hx may affect how Hx is aligned in the enzyme active site.
This model will be tested further with more extensive kinetic and
mechanistic experiments.