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INTRODUCTION |
It is well established that the presence of DNA adducts in the
template strand can impede or block DNA synthesis at a replication fork. Although most bulky adducts inhibit DNA synthesis strongly, some
can be bypassed readily in vitro. The well studied
carcinogen N-acetyl-2-aminofluorene
(AAF)1 can form both types of
adducts in DNA; N-(deoxyguanosin-8-yl)-2-acetylaminofluorene adducts (dG-C8-AAF) are known to be strong blocks to DNA synthesis, whereas N-(deoxyguanosin-8-yl)-2-aminofluorene adducts
(dG-C8-AF) can be bypassed by all polymerases tested (1). The mutagenic consequences of each adduct are also quite distinct. The dG-C8-AAF adduct results in mostly frameshift mutations in bacteria, whereas the
dG-C8-AF adduct produces predominantly base substitution mutations (2-4). These different properties are likely to be the result of
differences in the structures that these adducts assume in DNA in the
active site of the DNA polymerase. Structural and enzymatic studies on
duplex DNA molecules have demonstrated that the AF adduct produces less
distortion in the DNA helix than the AAF adduct (2, 5).
Multidimensional NMR experiments show that the guanine bearing the
C8-AAF adduct rotates from anti to syn conformation in the double-stranded DNA helix with the fluorene ring
inserted into the helix (base displacement model (6)). This contrasts
with the AF adduct, which can adopt interchangeable conformations: in
one the fluorene remains outside the helix (outside binding model),
whereas the other has the fluorene ring stacked within the helix (5).
The ratio of these conformations seems to be dependent on the sequence
within which the adduct lies (7). Although it has been assumed that the
structural differences between the AAF and AF adducts are responsible
for the observed in the biological effects, the molecular mechanism
that is operating is not known in any detail.
The Klenow fragment of Escherichia coli DNA polymerase I is
a 68-kDa protein that carries a polymerase and 3'-5'-exonuclease activities on a single polypeptide chain. Because of its simple structure, this enzyme has served as a model enzyme for studying the
mechanism of DNA synthesis for three decades. In efforts to understand
this process better, several crystal structures of the Klenow fragment
and other polymerases have been solved (8-13), and mutations in
conserved positions have indicated sites that are responsible for
polymerization, proofreading, and DNA binding (for a review, see Ref.
8).
In addition, pre-steady-state kinetic experiments have provided a
quantitative description of each step in the nucleotide insertion
pathway by Klenow fragment (14-16). It is widely accepted that the
process of incorporation of a dNTP into the nascent DNA chain involves
a number of sequential steps that are generally described in terms of
two different conformations of the DNA polymerase. It is thought that
in the open conformation the polymerase can bind to the primer terminus
after which it is converted to the closed conformation by the incoming
dNTP (17, 18). Upon formation of a new phosphodiester bond,
pyrophosphate is released, and the DNA polymerase returns to the open
conformation, allowing for translocation of the enzyme to the new
primer terminus. The nonchemical event that occurs between nucleotide
binding and phosphodiester bond formation (presumably the
conformational change) has been shown to limit the rate of
incorporation of a correct nucleotide (14), whereas the chemical step
itself limits the rate of a nucleotide misincorporation (15). These
results, together with the fact that the energy difference between
right (Watson-Crick) versus wrong (non-Watson-Crick) base
pairing cannot account for the extraordinary fidelity of DNA
replication (10
6-10
7 even in the absence
of proofreading), imply a selection mechanism based on the geometry of
the base pair and the protein active site. In this model, a wrong
nucleotide cannot be positioned properly for the nucleophilic attack in
the active site of the polymerase, and this slows down the rate of the
bond formation significantly. The major role of the base pair geometry
in replication has been confirmed recently by Kool and co-workers (19,
20), who showed that a thimidine triphosphate shape analog lacking
Watson-Crick pairing ability is replicated with high sequence selectivity.
In the present study we have determined the equilibrium dissociation
constant for the interaction of Klenow fragment with AF- and
AAF-modified primer-templates and compared them with the values for
unmodified primer-templates in the presence or absence of dNTPs. Our
results demonstrate that the enzyme-DNA complex is stabilized by the
presence of the next correct nucleotide, but binding is weaker when a
noncomplementary nucleotide is present. Binding of the polymerase to
AAF-modified DNA was stronger and almost independent of the nature of
the nucleotide present. These data provide additional insight into the
mechanisms of replication and mutagenesis.
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EXPERIMENTAL PROCEDURES |
Materials--
The Klenow fragment of E. coli DNA
polymerase I (exonuclease-free) was purchased from Amersham Pharmacia
Biotech. The protein had been overexpressed and purified from a strain
carrying a double mutation D355A,E357A, which results in about a
105-fold reduction of endogenous 3'-5'-exonuclease
activity (11). T7 DNA polymerase (exo
), T4 polynucleotide
kinase, and T4 DNA ligase were also purchased from Amersham Pharmacia Biotech.
Oligonucleotides were obtained from Midland Certified, Inc.
Site-specifically modified 12-mer (GTGATG(C8-AAF)ATAAGT)
was synthesized and purified as described previously (21). All dNTPs
and dideoxy-NTPs were ordered from Amersham Pharmacia Biotech.
[
-32P]ATP was from ICN Biomedicals.
Methods--
The sequences of oligonucleotides that were used in
this study to create model primer-templates are shown in Fig.
1. The 28-mer template oligonucleotide
was modified with an AF or AAF adduct at G6, which positions the adduct
on the junction of the single strand and double strand when the 22-mer
is used as a primer and leaves it in the single-stranded region when
the 20-mer or the 21-mer is annealed to the template. The 21-mer and
22-mer either had the normal 3'-OH or lacked the 3'-hydroxyl to prevent
a nucleotide incorporation. Construction and purification of all
oligonucleotides are described below.

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Fig. 1.
Oligonucleotide primers and templates.
Primers shown contained either a 3'-OH or were terminated with a
dideoxy sugar at the 3'-end. The 28-mer that was used as a template was
either unmodified or modified at the C8 position of G6 by an AF or AAF
adduct as indicated.
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Construction and Purification of the 28-Mer Oligonucleotides with
a Single AAF or AF Adduct--
All synthetic oligonucleotides used in
this study were first purified by denaturing polyacrylamide gel
electrophoresis. To construct the 28-mer with a single AAF adduct at
position G6 (Fig. 1), the site-specifically modified 12-mer was ligated
to a 5'-phosphorylated 16-mer, using a 21-mer as a template. The
ligated 28-mer containing the adduct and the complementary 21-mer were
denatured and separated on a 20% polyacrylamide gel in the presence of
8 M urea. The AAF-modified 28-mer was then recovered from
the gel and desalted with Sep-Pak C18 cartridges (Waters
Chromatography) according to the protocol supplied by the manufacturer.
The 28-mer containing the AF adduct was obtained by treatment of the
AAF-modified 28-mer with 0.1 M NaOH in the presence of 0.2 mM 2-mercaptoethanol as described (22) followed by high
performance liquid chromatography (HPLC) purification. The HPLC
separation was carried out on a Hamilton PRP-1 350 × 7-mm column.
The running buffer contained 0.5-15% acetonitrile in 20 mM sodium phosphate, pH 7, over 30 min with a flow rate of
1.5 ml/min.
Synthesis of the Primers with a 3'-Dideoxynucleotide--
A
21-mer and 22-mer lacking a 3'-OH were obtained by extension of the
corresponding 20-mer and 21-mer according to the following procedure.
First, the primers was labeled at the 5'-end by
[
-32P]ATP using T4 polynucleotide kinase. The
32P-labeled 20-mer and 21-mer were then annealed to the
template 28-mer in 40 mM Tris-HCl, pH 7.5, 20 mM MgCl2, and 50 mM NaCl. The
corresponding dideoxy-NTP (0.4 mM) and Sequanase 2.0 (2 units) were added to the reaction mixture. The resulting 21-mer and
22-mer were purified by electrophoresis in 20% polyacrylamide gel with 8 M urea.
Primer Extension Reactions--
To show the absence of a 3'-OH
on the primer terminus of the oligonucleotides, primer extension
reactions were carried out using these oligonucleotides as primers.
Thus, primer-templates consisting of an unmodified 28-mer and the
32P-labeled primer (approximately 1 nM each)
were incubated with a 0.4 mM concentration of each of the
four dNTPs and 2 units of the Klenow fragment in 20 µl of 50 mM Tris-HCl, pH 7, containing 10 mM
MgCl2, 1 mM dithiothreitol, and 0.05 mg/ml
bovine serum albumin. 2-µl aliquots of the reaction mixture were
taken, and the reaction was stopped with 10 µl of gel loading buffer
containing 90% formamide and 5 mg/ml bromphenol blue and xylene
cyanol. The samples were analyzed on 20% denaturing polyacrylamide gel
electrophoresis (Fig. 2). An identical
procedure was used for the primer extension studies on the AAF- and
AF-modified templates (Fig. 3).

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Fig. 2.
Characterization of the dideoxy-terminated
primers. The absence of a 3'-OH group was confirmed by primer
extension of each primer on the unmodified 28-mer template followed by
polyacrylamide gel electrophoresis. Extension of the primers shown in
Fig. 1 was carried out using Klenow fragment (exo ) and
all four dNTPs as described under "Experimental Procedures."
Lanes 1-3 are 20-, 21-, and 22-mers, respectively, having a
3'-OH. Lanes 4-6 are the same oligonucleotides after primer
extension. Lanes 7 and 8 are 21- and 22-mers with
3'H. Lanes 9 and 10 are the same oligonucleotides
after primer extension.
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Gel Retardation Assay--
Determination of the equilibrium
dissociation constants for the polymerase-primer templates was carried
out by a method similar to that described by Astatke et al.
(23). This method has been employed successfully to measure the binding
constants for DNA polymerase I and polymerase I mutants (23). The DNA
binding reactions were performed in 50 mM Tris-HCl, pH 7, containing 10 mM MgCl2, 1 mM
dithiothreitol, 0.05 mg/ml bovine serum albumin, and 4% glycerol. The
binding was carried out at 25 °C for 30 min in a 10-µl reaction
containing 10-100 pM labeled duplex DNA, increasing amounts of Klenow exo
(typically 0.05-81
nM), and 36 mM dNTP (if present). The reaction mixtures were loaded onto a native 7% polyacrylamide gel
preequilibrated with 0.4 × TB buffer (0.04 M Tris
borate, pH 8.3). Gels were fixed with 7% acetic acid, dried, and
scanned using a PhosphorImager. The amount of complex formed at
equilibrium was estimated as the difference in the band intensities of
free primer-template and the intensity of this band without the
polymerase addition.
Data Analysis--
The dissociation constant
(Kd) of a protein-DNA complex measured by gel
retardation assay is equal to the protein concentration at which half
of the DNA is bound to the protein, providing that protein
concentration used is much higher than the DNA concentration. The
protein-bound and free oligonucleotides separated by gel
electrophoresis were quantified by scanning the gels in a Molecular
Dynamics PhosphorImager SF. To obtain the Kd,
the fraction of the DNA bound to the protein was plotted against the
initial protein concentrations, and the data were analyzed using the
program Ultrafit (Biosoft, Cambridge, U. K.) and fitted to the
equation for single-site ligand binding. The values for the
Kd ± S.E. were obtained from the resulting fit
of this equation.
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RESULTS |
Primer Extensions Using AAF- and AF-modified Templates--
The
dG-C8-AAF adduct is generally known to block DNA replication much more
strongly than the dG-C8-AF adduct, but this effect depends upon the
sequence context and the polymerase used. To test the effect of these
adducts in the particular system used in this study, primer extension
analysis was carried out using the Klenow fragment of DNA polymerase I,
the 21-mer as a primer, and the AAF- or AF-modified 28-mer template. In
agreement with previous studies, we find here that the AAF adduct is
much more inhibitory of DNA synthesis than the AF adduct (Fig.
3). In fact the results obtained for the
unmodified and AF-modified templates are almost identical, whereas the
AAF-modified template gave less than 3% bypass at the experimental
conditions used. Note also that the presence of some unextended primer
is presumably the result of a slight molecular excess of the primer and
a small fraction of the duplex which denatures during the
incubation.

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Fig. 3.
Primer extension analysis of AF- and
AAF-modified primer-templates. The 21-mer primer was annealed to
either the unmodified 28-mer or the AAF- or AF-containing template.
Primer extension was carried out using the Klenow fragment
(exo ), and the reaction was terminated after the period
of time indicated under each lane as described under
"Experimental Procedures."
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Determination of the Equilibrium Dissociation Constants--
To
determine the dissociation constants for the binding of the Klenow
fragment to DNA, 32P-labeled primers were annealed to the
templates, and these duplexes were incubated with increasing amounts of
polymerase. These mixtures were then immediately loaded and run on a
nondenaturing polyacrylamide gel producing a band that was the result
of the formation of the polymerase-DNA complex (Fig.
4A). The small amount of free
primer is caused by the dissociation of the primer-template which is unavoidable at the low DNA concentrations used for these experiments. The polymerase does not form any detectable complexes with the single-stranded primer even at protein concentrations much higher than
that used in this study (Fig. 4A, lane 9), nor
does it bind to the modified or unmodified templates (not shown). The
amount of complex formed at equilibrium was estimated from the ratio of
band intensities of free primer-template to the intensity of this band
without the addition of polymerase. This analysis allows measurement of
the amount of complex formed in solution before it was loaded on the
gel and the neglect of the slight amount of dissociation of DNA-protein
complexes which occurs in the gel. The data obtained this way were then
fitted to an equation for single-site ligand binding (Fig.
4B), and apparent Kd values were
calculated. A slight variation of this method was used successfully in
prior studies to determine the dissociation constants for polymerase I
binding to primer-templates (23).

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Fig. 4.
Determination of the equilibrium dissociation
constants (Kd) of the Klenow fragment-DNA
complex. Panel A, typical gel shift assay used to
determine the Kd of Klenow fragment-DNA
complexes. 32P-Labeled primer-templates were incubated with
increasing amounts of Klenow fragment (exo ) and then
loaded on a 7% nondenaturing polyacrylamide gel as described under
"Experimental Procedures." Lanes 1-8, binding of
increasing concentrations of polymerase to the primer-template. In
lane 9, the polymerase (600 nM) was mixed with
the primer (100 pM) under conditions otherwise identical to
those used for binding to the primer-template. Panel B,
binding curves for the interaction of the Klenow fragment with the
unmodified primer-template in the presence of dCTP (+), dTTP ( ),
dATP (×), or dGTP ( ). The fraction of bound DNA determined as
described under "Experimental Procedures" was plotted
versus the initial Klenow concentration. Each data point
represents the average of at least three different experiments. Each of
the values shown in Tables I-III was determined using this
method.
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Absence of a 3'-OH on the Primer Does Not Change the Dissociation
Constant--
To determine the effect of the presence of dNTPs on the
dissociation constant, the binding experiments need to be carried out
under the conditions where nucleotides cannot be incorporated. To
prevent this reaction, a primer-template having a dideoxynucleotide on
the 3'- end of the primer was prepared and used to form the primer-template. In the absence of a dNTP, the dissociation constant determined for interaction of the protein with the 3'-dideoxy terminated primer-template was found to be identical to that for interactions with normal primer-templates (Table
I). Consistent with the available
structural information (8), this result implies that there is no direct
interaction of the DNA polymerase active site carboxylates with the
3'-OH of the primer terminus in the ground state. Also, as shown below,
binding was the same in the presence and absence of a 3'-OH in the
primer for the modified templates.
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Table I
Dissociation constants of the Klenow fragment-DNA complexes in the
absence of dNTPs in which the adducts are positioned in the polymerase
active site
The primer-templates used in the binding studies are described in Fig.
1. The Kd values were determined as described under
"Experimental Procedures" by measuring the difference in the band
intensities of the free primer-template and the intensity of this band
before the addition of the polymerase and plotting the fraction of
bound DNA versus the initial protein concentration (Fig. 4).
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Binding to AAF- and AF-modified Templates--
To gain an
understanding of which step of a nucleotide incorporation is influenced
by the DNA adduct, we first determined the dissociation constants in
the absence of dNTPs for the Klenow fragment on primer-templates
containing AAF and AF adducts (Table I). These data indicate that when
the AAF adduct is positioned so that it is the next position for
incorporation, the polymerase binding is about 10-fold stronger than to
the unmodified primer template. Surprisingly, binding to an identical
template but containing an AF adduct gave dissociation constants
identical to the unmodified template. The absence of a 3'-OH had no
effect on these results (Table I). If the adduct is positioned at the
+1 or +2 position in line for incorporation, placing them in the
single-stranded region of the template, then no differences were
observed between the unmodified or modified templates (Table
II), suggesting that the interaction with
the AAF adduct was highly dependent on the position in which it resided
within the active site.
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Table II
Dissociation constants of the Klenow fragment-DNA complexes in which
the adducts are not in the active site
The primer-templates used in the binding studies are described in Fig.
1. The Kd values were determined as described under
"Experimental Procedures" by measuring the difference in the band
intensities of the free primer-template and the intensity of this band
before the addition of the polymerase and plotting the fraction of
bound DNA versus the initial protein concentration (Fig. 4).
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Klenow Fragment Binding in the Presence of dNTPs--
It has been
shown that E. coli DNA polymerase I shows little
discrimination against nonpairing dNTPs at the stage of nucleotide binding (24). Thus the dissociation constants for the four nucleotides from the polymerase bound to the primer-template junction are approximately the same. However, the dissociation constants for the
polymerase in the presence of each nucleotide have never been measured.
To make these measurements, we prepared primer-templates where the
primer contained a 3'-dideoxy-terminated nucleotide and measured the
dissociation constants of the polymerase in the presence of each dNTP.
Interestingly, unlike the data measured for dNTP dissociation, the
binding of the polymerase to DNA is much stronger in the presence of
the correctly paired nucleotide than in the presence of an incorrect
nucleotide (Table III). Indeed, the
complex is about 100 times more stable when the next correct nucleotide
(dCTP in this case) is present than in the presence of a dATP or dGTP
and about 15 times more stable in the presence of another pyrimidine,
dTTP. Virtually identical results were obtained for an unmodified
template using a 3'-dideoxy-terminated 21-mer primer; in this case the
next correct nucleotide (dTTP) caused the strongest binding, dATP and
dGTP caused 100-fold higher dissociation constants, and dCTP was about
10-fold higher (data not shown). Thus the differences in binding are
not the result of a special interaction with one of the nucleotides but
are clearly dependent on having the correct versus incorrect
nucleotide bound in the active site.
When the binding studies were carried out on the primer-template that
positions an AAF adduct in the active site of the polymerase, the
presence or identity of the dNTP had no effect on the dissociation constants; in all cases the Kd was about 0.1 nM (Table III), very close to the value in the absence of
the nucleotide for the AAF-modified primer-template (Table I). When an
AF adduct was present in the active site of the protein, the
Kd in each case was intermediate between the
value determined for binding to the unmodified and AAF-modified
primer-template (Table III). The only exception was dCTP, the next
correct nucleotide, where the binding strength was lower than that
determined for the unmodified and AAF-modified templates.
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DISCUSSION |
Acetylaminofluorene adducts have been studied intensively as model
DNA lesions to understand the mechanism of mutagenesis and
carcinogenesis. Despite a great deal of structural, mutational, and
kinetic data available regarding these DNA lesions, the exact mechanism
of the mutagenicity and the reason for the different behavior of AF and
AAF adducts during DNA replication are not clear. We have begun to
address these questions by attempting to understand the molecular
interactions that occur between DNA polymerases and primer templates
that position these adducts within the enzyme active site. In the
experiments described here, we have measured the dissociation constants
of the Klenow fragment from AAF- and AF-modified primer-template
complexes where the adducts are positioned in or near the active site
and in the presence or absence of each of the dNTPs.
Measurement of equilibrium binding constants of DNA polymerases
with DNA has been used extensively to study the mechanism of DNA
polymerase action and the function of polymerase mutants. These
constants are typically measured using single-turnover kinetics experiments (14, 25-27), DNase I footprinting methods (28), or gel
mobility shift assays (23). The values that have been measured for
wild-type Klenow fragment range from 0.2 to 8 nM, and prior
measurements using the gel mobility assay in the absence of dNTP gave a
value of 0.3 nM (23). The present study, which used a
slightly different DNA construct, gave a comparable value of 0.4 nM under otherwise similar conditions.
No prior studies have measured dissociation constants in the presence
of incorrect dNTPs because the kinetic method normally used to measure
dissociation constants needs to have the correct nucleotide present. In
the present study, measurements in the presence of each of the dNTPs
was accomplished by the use of a primer-template containing a
nonreactive 3'-dideoxynucleotide primer terminus. Under these
experimental conditions, it was impossible for the dNTP to be
incorporated, and the only possible fate of the complex was
dissociation presumably because the rate of dissociation of the
Klenow-DNA complex is much faster than the rate of shifting of the
primer terminus to the exonuclease domain (15, 29, 30). Also the
absence of a 3'-OH did not effect the binding strength; in the absence
of a dNTP the dissociation constants were identical for templates
containing or lacking a 3'-OH (Table I). This result provides further
evidence that the 3'-OH is not involved directly in the ground state
DNA binding of the polymerase, consistent with the published crystal
structures of polymerase-DNA complexes (8), (10). Although
Mg2+- mediated interactions between one of the
active site carboxylates and the 3'-OH of the primer have been
proposed, these must be too weak to be detected in this system or might
be formed after the conversion to the closed conformation (see below).
A great deal is known regarding the interactions of the polymerase with
the primer-template (17). It is fairly well established that most DNA
polymerases can adopt two different conformations during the process of
nucleotide incorporation. These enzymes apparently bind to DNA in a
so-called "open" conformation and then undergo a conformational
change upon binding of a nucleotide (closed complex). This
conformational rearrangement is thought to help bring the incoming
nucleotide to the polymerase active site and align it properly for the
nucleophilic attack by the 3'-hydroxyl of the primer. Unlike many other
polymerases, DNA polymerase I apparently does not discriminate between
the correct and incorrect dNTP at the time of nucleotide binding (24).
Instead, checking for proper pairing is thought to take place during or after the conformational change that occurs when the polymerase adopts
a closed structure. However, apparently all polymerases are able to
reach a fully active catalytic configuration if the nucleotide can
adopt a Watson-Crick geometry, and it is thought that this so-called
induced fit mechanism provides the selectivity during nucleotide
incorporation. Moreover, it has been shown for both the HIV reverse
transcriptase (26) and the T7 DNA polymerase (17) that the rate of
dissociation of the polymerase decreases when the polymerase is bound
to a nucleotide and in the closed conformation and increases when the
polymerase is bound to a nucleotide in the open conformation.
Consistent with these, we find that positioning of a nonpairing
nucleotide in the Klenow active site decreases the stability of the
complex with DNA, whereas a correct nucleotide increases the stability
(Table III). It is possible that the geometry of an incorrect dNTP
bound to the active site does not allow the protein to complete the
conformational change to the closed structure or that if the
conformational change occurs, the structure is perturbed in the
presence of a noncomplementary base. In either case the interactions
between protein and the DNA are disturbed thereby decreasing the
stability of the complex.
The dissociation constants obtained when an AAF or AF adduct is
positioned in the polymerase active site clearly indicate that their
presence affect the interaction of the polymerase with both the
template and incoming nucleotide. In the absence of a dNTP, the
dissociation constant is about 5-fold lower when an AAF adduct is
positioned in the active site, whereas an AF adduct has no effect on
the binding (Table I). The enhanced binding was somewhat unexpected
because the AAF adduct is known to cause a substantial distortion in
the structure in the DNA helix (2) and has been shown to inhibit
strongly the incorporation across from the adduct by most polymerases
(1). Thus, it is possible that the AAF adduct may drastically alter the
structure of the primer-template within the active site which results
in a complex having vastly different binding properties possibly
involving specific interactions between the AAF moiety and amino acid
side chains in the active site. In agreement with this model, a
recently published footprinting analysis of the complex of T7
polymerase with AAF-modified primer-template (31) demonstrates that the presence of an AAF adduct reduces the hypersensitive sites seen in the
footprint of unmodified DNA. It is important to note that a kinetic
analysis has shown that the presence of an AAF adduct does not affect
the dissociation rate for the T7 DNA polymerase (32), which may mean
that our results are specific to polymerase I.
A second important observation from this study is that this enhanced
AAF-induced binding is not affected by the presence of a complementary
or noncomplementary dNTP, whereas on an unmodified template the
Watson-Crick nucleotide enhances binding, and the other nucleotides
greatly reduce it (Table III). A recent molecular modeling study of DNA
polymerase
bound to a primer-template modified with the bulky
polycyclic aromatic hydrocarbon, benzo[a]pyrene, showed that the
adduct may interact with the same amino acids that are involved into
the nucleotide binding (33). It is tempting to propose that the AAF
adduct can also interfere with the nucleotide binding either sterically
or by hydrogen bonding with one of the active site amino acids.
Although no structural information has yet been published for an AAF
adduct positioned at a primer-template junction, it has been shown that
in some sequence contexts the dG-C8-AF adduct structure can resemble
that determined for a dG-benzo[a]pyrene adduct (34). Thus, it is
possible that the AAF adduct can resemble a benzo[a]pyrene adduct at
a primer-template junction where the benzo[a]pyrene adduct has been
suggested to displace the modified guanine into the major groove and
can stack over the junctional base pair (35). Alternatively, it may be
possible that the presence of the AAF adduct inhibits the
conformational change that occurs upon nucleotide binding and removes
any possible contribution that a nucleotide would have on the
dissociation constants.
Consistent with its ability to adopt two different conformations, the
AF adduct produced a set of dissociation constants that had
intermediate values between the unmodified and AAF-modified primer-templates when a noncomplementary nucleotide was present (Table
III). One possible explanation for this observation is that what was
being measured is the average of two sets of data, one with the
adducted guanine in the normal anti conformation and the
other having the more distorted AAF-like syn conformation. We also note that the ternary complex is most stable in the presence of
dCTP, which is in agreement with incorporation data showing that dCTP
is most often incorporated across from an AF adduct (36).
Finally, no differences in the dissociation constants compared with the
unmodified case are observed when either adduct is positioned at the +1
or +2 position on the primer-template (Table II). This result is
consistent with the data from a recent study by Miller and Grollman
(37) who demonstrated that kinetic parameters for nucleotide
incorporation are not influenced by an adduct positioned away from the
primer-template junction (37). Thus, whatever interactions are causing
the AAF-induced enhanced binding they are not simply a nonspecific
interaction of the adduct with the hydrophobic amino acids and are most
probably related to the mechanism of polymerization in the active site.
This is supported further by the differences observed between the AAF
and AF adduct, which are chemically very similar but lead to very
different behaviors when positioned in the active site. A full
understanding of these interactions awaits a crystal structure of a DNA
polymerase complexed with a primer-template containing an AAF and AF adduct.