From the Departments of Biochemistry and Molecular
Biology and § Cellular and Molecular Physiology, The
Pennsylvania State University College of Medicine,
Hershey, Pennsylvania 17033
Received for publication, November 20, 2002
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The mutagenic and cytotoxic effects of many
endogenous and exogenous alkylating agents are mitigated by the actions
of O6-alkylguanine-DNA alkyltransferase (AGT).
In humans this protein protects the integrity of the genome, but it
also contributes to the resistance of tumors to DNA-alkylating
chemotherapeutic agents. Here we report properties of the interaction
between AGT and short DNA oligonucleotides. We show that although AGT
sediments as a monomer in the absence of DNA, it binds cooperatively to both single-stranded and double-stranded deoxyribonucleotides. This
strong cooperative interaction is only slightly perturbed by active
site mutation of AGT or by alkylation of either AGT or DNA. The
stoichiometry of complex formation with 16-mer oligonucleotides, assessed by analytical ultracentrifugation and electrophoretic mobility
shift assays, is 4:1 on single-stranded and duplex DNA and is unchanged
by several active site mutations or by protein or DNA alkylation. These
results have significant implications for the mechanisms by which AGT
locates and interacts with repairable alkyl lesions to effect DNA repair.
O6-Alkylguanine-DNA alkyltransferase is a
ubiquitous repair protein that plays a vital role in minimizing the
mutagenic effects of alkylating agents (1-4). It catalyzes the
stoichiometric transfer of a variety of alkyl substituents from the
O6-position of guanine to an active site
cysteine, preventing incorrect base pairing caused by these adducts.
More than 100 alkyltransferases are now known, and the crystal
structures are available for three family members: the Ada-C protein
from Escherichia coli (5), the human alkyltransferase
(hAGT)1 (6), and the protein
from the thermophilic archaeon, Pyrococcus kodakaraensis (7). All of the known alkyltransferases lack the
ability to dealkylate themselves, and no dealkylation activity has been
found in cell extracts to date. On this basis, it is widely thought
that alkyltransferase participates in a single reaction and is then
irreversibly inactivated. Given the apparently nonenzymatic nature of
the protein, the protection afforded by alkyltransferase is likely to
depend on the regulation of its synthesis and degradation and on its
ability to efficiently locate repairable lesions throughout the genome.
The mechanisms by which AGT interacts with adduct-containing and
adduct-free DNAs are poorly understood. Two contrasting mechanisms have
been proposed to date. In the first, single AGT proteins bind normal
and lesion-containing DNA, and the distribution of AGT between normal
and lesion sites depends on a difference in binding affinity. This
model is consistent with the observation that a single AGT monomer is
necessary and sufficient to dealkylate a single
O6-alkyl guanine adduct within a DNA duplex (3).
It is supported by the observation that single AGT-DNA complexes are
detected by gel shift assay when AGT binds short DNA molecules. These
complexes have been interpreted as having a 1:1 AGT:DNA stoichiometry,
and the binding affinities have been calculated based on that
assumption (8). The second mechanism was proposed when it was found
that some AGT-DNA complexes have stoichiometries greater than 1:1 and form without the accumulation of detectable binding intermediates (9).
This pattern strongly suggests a cooperative binding mechanism for
AGT.
Here we more thoroughly characterize the cooperative binding mechanism.
We show that it functions on both single-stranded and duplex 16-mer
DNAs and with unmodified and alkylated hAGTs. In all cases the
stoichiometries of hAGT:16-mer complexes were 4:1. In this binding
mode, hAGT discriminates poorly between lesion-containing and
lesion-free DNA. Together these results support a novel model of
binding site search and recognition that involves the cooperative formation and processive movement of multi-protein complexes.
Reagents--
T4 polynucleotide kinase was purchased
from New England Biolabs, and [ AGT Protein--
Recombinant human AGT (wild type and C145A
mutant proteins) were prepared as previously described (10). Both of
the proteins were homogeneous as judged by electrophoresis (not shown).
The wild type protein was 100% active in debenzoylating
O6-benzylguanine. The C145A mutant lacks the
active site cysteine and is not active against alkyl-DNA or
alkyl-guanine substrates (11). The construction of modified pQE-30
vectors encoding C-terminally His6-tagged wild type and
C145A and C145S mutant AGT proteins for expression in E. coli was accomplished as described (12). His-tagged proteins were
purified from cell lysates using TALON® affinity resin
(Clontech), according to the manufacturer's
instructions. The samples were dialyzed against 50 mM Tris
buffer (pH 7.6) containing 5 mM DTT and stored frozen at
Human AGT concentrations were measured both with the BCA dye binding
assay (15) and spectrophotometrically using a molar extinction
coefficient, Nucleic Acids--
A 16-residue oligodeoxyribonucleotide
(sequence 5'-GAC TGA CTG ACT GAC T-3') and its complement
were purchased from Invitrogen. A substrate oligonucleotide with the
same sequence and a methyl substitution at the
O6-position of the 3'-most guanine (shown above
in bold type) was purchased from Synthegen LLC (Houston, TX). When
duplex DNA was required, the oligonucleotide samples were combined and
annealed as described (17). The DNA samples were labeled at the 5'
termini with 32P as described by Maxam and Gilbert (18) and
transferred into 10 mM Tris (pH 8.0 at 21 °C) using
Sephadex G-25 centrifuge columns (Amersham Biosciences). Stock DNA
concentrations were measured spectrophotometrically, using
Electrophoretic Mobility Shift Assays--
The
binding reactions were carried out at 20 ± 1 °C in 10 mM Tris (pH 7.6), 1 mM dithiothreitol, and 10 µg/ml bovine serum albumin, supplemented with NaCl as indicated.
Protein-DNA complexes were formed by adding appropriate amounts of hAGT
to solutions containing 32P-labeled
oligodeoxyribonucleotides. The mixtures were equilibrated at 20 ± 1 °C for 30 min. Duplicate samples incubated for longer periods gave
identical results, indicating that equilibrium had been attained.
Electrophoresis was performed using 10% polyacrylamide gels
(acrylamide:N,N'-methylene bisacrylamide = 75:1), cast, and run at 8 V/cm in buffer consisting of 10 mM Tris acetate (pH 7.6) supplemented with NaCl to match
the conductivity of the protein-DNA samples. Autoradiograms were
obtained with Kodak X-Omat Blue XB-1 film exposed at 4 °C. Gel
segments containing individual electrophoretic species were excised
using the developed film as a guide and counted in a scintillation
counter by the Cerenkov method (20). Similar results were obtained
using scanning densitometry.
The serial dilution method (19) was used to obtain self-consistent
estimates of the binding stoichiometry (n) and the
association constant (Ka). For a binding mechanism
of the type nP + D
In many cases the association constant was also evaluated by direct
titration. hAGT protein was directly added to 32P-DNA
solutions (typically ~5 × 10 Analytical Ultracentrifugation--
hAGT protein and
oligodeoxyribonucleotides were dialyzed against 10 mM Tris
(pH 7.6), 1 mM DTT, 1 mM EDTA, 100 mM NaCl. Analytical ultracentrifugation was performed at
20 ± 0.1 °C in a Beckman XL-A centrifuge using an AN 60 Ti
rotor. Scans were obtained at 260 and 280 nm with a step size of 0.001 cm. Equilibrium was considered to be attained when scans made 6 h
apart were indistinguishable. Typically, equilibration times
For a system in which hAGT protein is in binding equilibrium with DNA
according to the mechanism nP + D Wild Type and Representative Mutant hAGT Proteins Are
Monomeric--
Solutions containing hAGT protein at two nominal
concentrations (3.6 and 13.7 µM) were brought to
sedimentation equilibrium at three different centrifuge speeds (22,000, 31,000, and 43,000 rpm). A representative data set for a 13.7 µM sample of His6-tagged C145S hAGT taken at
43,000 rpm and 20 °C is shown in Fig.
1 (curve A). The solid
line is the result of fitting the single-species version of
Equation 3 (n = 1) to the data. This fit returned a value of Mr = 21,800 ± 400, which is in
excellent agreement with the monomer molecular weight derived from the
protein sequence (Mr = 21,860). The small,
uniformly distributed residuals indicate that the monomer model is
consistent with the data over the entire concentration range present in
the centrifuge cell. Extension of the model to include oligomers of
hAGT (Equation 3 with n > 1 and Mn = nM1) did not improve the quality of the fit as
judged by the correlation coefficient or by the magnitude of the
residuals (result not shown). Similar results were obtained for wild
type hAGT (Mr = 21,500 ± 200), C145A hAGT
(Mr = 21,400 ± 200),
His6-tagged wild type hAGT (Mr = 22,000 ± 500), and His6-tagged C145A hAGT
(Mr = 21,800 ± 200). These molecular
weights agree well with values predicted from protein sequence,
consistent with the interpretation that all of the preparations were
monomeric within the concentration range tested. Importantly, neither
the His6 tag nor the active site mutation C145A changed the
monomeric state of free hAGT. As discussed below, hAGT forms oligomeric complexes with DNA. The absence of detectable hAGT oligomers in the
absence of DNA demonstrated here argues against models in which protein
association precedes DNA binding.
AGT Forms 4:1 Complexes with a Single-stranded
Oligodeoxyribonucleotide 16-mer and Its Cognate Duplex--
Mixtures
containing hAGT and single-stranded DNA were brought to sedimentation
equilibrium at four different centrifuge speeds (11,000, 15,000, 20,500, and 27,000 rpm). Representative data are shown in Fig. 1
(curve B); the solid curve represents the global
fit of Equation 4 to the data ensemble. The small, uniformly distributed residuals indicate that the simple mechanism nP + D
Similar experiments were carried out with hAGT and a 16-bp duplex DNA.
Samples were brought to sedimentation equilibrium at 11,000, 15,000, 20,500, and 27,000 rpm. Representative data are shown in Fig. 1
(curve C); the solid curve represents the global fit of Equation 4 to the data ensemble. As before, the high quality of
the fit indicates that the simple mechanism nP + D
Electrophoretic mobility shift assays (25) were performed
to explore a range of hAGT and DNA concentrations below those accessible in the analytical ultracentrifuge. The binding of hAGT to
DNA produced a single mobility-shifted complex at all protein and DNA
concentrations that gave detectable binding (Fig.
2A). This binding pattern is
consistent with a mechanism of the type nP + D
Binding stoichiometries were also measured by the
continuous variation (Job plot) method (26). With the input
concentrations of protein and DNA ([P]o + [D]o) held constant, the ratio of
[P]o:[D]o that yields the greatest
concentration of complex (the optimal combining ratio) is a measure of
the association stoichiometry. As shown in Fig.
3 and summarized in Table I, this method
returns hAGT-DNA stoichiometries close to 4:1, in good agreement with values obtained by the serial dilution and analytical ultracentrifuge methods. Taken with the fact that higher stoichiometry complexes are
readily observed with larger DNAs
(9),2 the absence of
detectable complexes with stoichiometries greater than 4:1, even at
high [hAGT], suggests that this stoichiometry represents protein
saturation for both single-stranded and duplex 16-mer DNAs. Together,
the presence of free DNA in equilibrium with the 4:1 complex and the
absence of complexes with protein:DNA ratios <4:1, suggest that hAGT
binds cooperatively to both single-stranded and double-stranded DNAs.
Because free hAGT is monomeric (Fig. 1), this pattern suggests that the
protein assembly forms on DNA and not in free solution prior to DNA
binding.
Equilibrium Constants Depend Only Weakly on DNA Secondary
Structure, Sequence Changes at the Active Site, or the Presence of a
C-terminal His6 Tag--
Association constants for the
interaction of wild type and C145A, His6-wild type,
His6 C145A, and His6-C145S hAGTs with
single-stranded and duplex 16-mer DNAs were calculated from serial
dilution data as described above and were also determined by direct
titration of DNA by hAGT (Fig. 4; data
summarized in Table I). Measured in these ways, the formation constant
(Ka) for the complex of wild type protein with
single-stranded 16-mer is ~1.5 × 1023
M DNA Interactions of Alkylated hAGT--
Proteolysis is the
ultimate fate of alkyl-AGT (29), but it is not known whether the
alkyl-protein plays a role in DNA repair prior to degradation. Studies
were undertaken to examine the DNA binding properties of hAGT protein
modified by reaction with substrate analogues methylguanine and
benzylguanine. Although these alkyl-proteins were too unstable to
analyze by analytical ultracentrifugation, the more rapid
electrophoretic mobility shift assay allowed assessment of
their interactions with small DNA molecules. The binding affinities of
methylated and benzylated hAGT for 16-mer DNAs were determined by
direct titration (Fig. 5A) and
by serial dilution methods (not shown). Stoichiometries of binding were
also determined using by serial dilution and continuous variation
methods (Fig. 5C). As summarized in Table
III, alkylated hAGT proteins bind DNA,
with somewhat lower affinity than nonalkylated proteins. The larger benzyl adduct causes a greater decrease in affinity than the methyl adduct, but neither modification has a profound effect on binding. Alkylated hAGT proteins bind
O6-alkylguanine-containing DNA; the affinities
of these proteins for lesion-containing oligonucleotides are slightly
but significantly elevated over that for lesion-free molecules (Table
IV). Together with the observation that
alkyl and native hAGT proteins form similar 4:1 complexes with 16-mer
DNAs, the elevated affinity for
O6-alkylguanine-containing DNA suggests that the
mechanism of binding is not greatly perturbed by protein
alkylation.
O6-Alkylguanine-DNA
alkyltransferases reduce the mutagenicity of DNA-alkylating agents and
enhance the resistance of tumor cells to chemotherapeutic agents (30).
Despite these important functions, little is known of the mechanisms by
which the human protein interacts with alkylated and nonalkylated DNAs.
Most models of these interactions are based on results obtained with
Ada, a two-domain bacterial protein that shares sequence similarity with human AGT (31). However, Ada has a simple, noncooperative DNA
binding mechanism (32) quite distinct from the cooperative one that we
have found for human AGT. This disparity may reflect a functional
divergence of the eukaryotic and bacterial proteins (33). The
differences in the binding mechanisms of hAGT and Ada suggest that
caution should be used in modeling functions of the human protein on
the basis of its well studied Ada homologue.
We have shown that hAGT is rigorously monomeric in free solution and
that it forms multiprotein complexes with short DNA molecules (Ref. 9
and this work). Human AGT binds with a 4:1 stoichiometry to
16-nucleotide single strands and 16-bp duplexes, regardless of the DNA
association state, the presence of repairable
O6-alkylguanine lesions, active site mutations
(C154A and C154S), or the C-terminal His6 affinity tag.
This binding mechanism is robust. Its qualitative features are not
affected by changes in [NaCl] (Figs. 1, 2, and 4), temperatures
between 4 and 30 °C, or the presence of divalent
cations.2 The absence of detectable complexes with
stoichiometries greater than 4 suggests that the 4:1 ratio
represents saturation for 16-mer DNA. The presence of the 4:1 complex
in equilibrium with free DNA and the absence of detectable complexes of
lower stoichiometry indicate that the binding is highly cooperative.
These results are particularly significant in view of previous studies
in which association constants for AGT-DNA complexes were derived for
assumed 1:1 binding models (8). For a cooperative binding mechanism, differences in the stability of the protein-DNA assembly may be due to
differences in the intrinsic affinity of AGT for a given DNA or to
differences in the stability of the AGT-AGT interaction. Our results
are consistent with binding mechanisms in which the aggregate stability
of the 4:1 complex is large enough to effectively mask any difference
in affinity for single-stranded and duplex DNAs or any difference in
affinity for alkylated and nonalkylated DNAs.
As shown in Tables III and IV, mutation of the active site cysteine to
alanine or serine does not eliminate DNA binding cooperativity. This
supports the conclusion (28) that conservative mutation of the active
site cysteine has little effect on hAGT structure. Because such mutants
lack alkyltransferase activity, they are useful for the study of
interactions with O6-alkylguanine-containing
DNAs (see below). Analogous results were obtained with proteins in
which residues 202-207 of hAGT were converted to histidines, forming a
C-terminal His6 affinity tag. Although the DNA affinities
of His6-AGT proteins were slightly elevated when compared
with proteins without the tag, the His6 modification did
not alter the binding stoichiometry or the cooperative nature of the
interaction. This outcome suggests that the wild type sequence of the
C-terminal residues (202-207) may not be a determinant of hAGT
self-association or binding cooperativity. The elevated affinity
observed with His6-modified proteins is consistent with the
notion that these residues might interact electrostatically with
DNA.3
Experiments designed to probe the effect of the DNA
association state on binding affinity returned the striking result that hAGT displays little preference for duplex 16-mers over single-stranded 16-mers (summarized in Tables I and II). This accords well with results
obtained by competition assay using high molecular weight DNAs (9) but
is a smaller difference than that found by Bender et al. (8)
using 29-mer oligonucleotides. Because the apparent binding site size
varies from a 4-nucleotide/hAGT monomer on a 16-nucleotide
single-stranded DNA to an ~9-nucleotide/hAGT monomer on an
80-nucleotide DNA (9), this contrast might reflect stoichiometric or
geometric differences between our complexes and those of Bender et al. (8).4
Alkylation of the active site cysteine has been shown to
destabilize the native fold of hAGT (13). Recently, it has been proposed that an alkylation-mediated conformation change promotes DNA
release from the active site (34). However, results shown above
indicate that although alkylation is associated with a mild reduction
in DNA affinity, methylated and benzoylated proteins retain significant
binding activity.5 In
addition, the cooperative binding mechanism appears to be unchanged by
alkylation. Together, these observations raise the unexpected
possibility that alkylated protein molecules participate (through
cooperative DNA binding) in the DNA binding and repair activities of
other hAGT molecules.
It was not possible to measure the affinity of wild type
hAGT for O6-methylguanine-containing DNAs,
because of the rapid alkylation of the protein and dealkylation of the
DNA, under native binding conditions. However, the modestly elevated
affinities of C145A hAGT, His6-C145A hAGT, and
His6 C145S hAGT for DNAs containing O6-methyl guanine relative to nonalkylated DNAs
(Tables I and II) are indications that hAGT possesses some specificity
that may enhance its binding to lesion-containing
sites.6 In view of the
relatively small preference for
O6-methylguanine-containing DNA (a factor of
3-4/monomer in our 4:1
complexes),7 it seems
possible that cooperative interactions may provide an alternative
mechanism for "scanning" segments of DNA that does not require much
preferential binding to lesion sites. Processive binding of large
cooperative units of hAGT molecules to long stretches of DNA may
provide a mechanism by which alkyl adducts are located and repaired in
an efficient manner, despite the low specificity of hAGT monomers for
lesion sites. hAGT-containing repair complexes may potentially form
wherever alkylation damage occurs, overcoming differences in intrinsic
affinity based upon DNA sequences (9). Cooperativity may represent an
essential feature of hAGT-DNA binding that enables efficient repair of
a wide variety of lesions contained in any genomic sequence.
By the same token, the cooperative binding has the potential to mask
differences in the intrinsic affinity of hAGT for competing binding
sites on a DNA molecule. For example, in the binding of His6-C145A hAGT to
O6-methylguanine-containing single stranded
16-mer, our data do not distinguish between a single specific high
affinity interaction at the lesion site averaged with three nonspecific
interactions and four identical interactions of modestly elevated
affinity. In this example, binding to the lesion-containing
oligonucleotide is characterized by n = 3.89 ± 0.2, Ka = 37.3 ± 22 × 1024
M
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]ATP was purchased
from PerkinElmer Life Sciences. Acrylamide and
N,N'-methylene bisacrylamide were purchased from
Aldrich. O6-Methylguanine and
O6-benzylguanine were generously provided by Dr.
R. C. Moschel (ABL-Basic Research Program, NCI-Frederick Cancer
Research and Development Center, Frederick, MD).
80 °C until needed. Wild type hAGT proteins were alkylated at the
active site cysteine by incubation (at 37 °C for 30 min) with either
1 mM O6-methylguanine or 1 mM O6-benzylguanine according to
Kanugula et al. (13). Alkylation was detected by
matrix-assisted laser desorption ionization time-of-flight mass
spectrometric analysis of trypsin-digested protein, in which the
conversion of the fragment containing the active site cysteine (Gly136-Arg147,
Mpredicted = 1314.72, m/zobs = 1315.82) to its methylated
derivative (Mpredicted = 1328.73, m/zobs = 1328.81) or benzoylated
derivative (Mpredicted = 1391.80, m/zobs = 1393.66) was observed.
Complete conversion of hAGT to the alkyl form (as monitored by mass
spectrometry) eliminated detectable alkyl transferase activity assayed
with [3H]methyl calf thymus DNA as substrate (results not
shown) (14). Human AGT undergoes a conformational change upon
alkylation that reduces its in vivo and in vitro
half-life (13). Accordingly, the samples were used immediately
following alkylation to avoid problems of instability.
280 = 3.93 × 104
M
1 cm
1, calculated from data of
Roy et al. (16). The values of
215/
280 = 8.2 and
260/
280 = 0.63 were obtained from UV
spectra of the purified protein dissolved in 10 mM Tris
buffer (pH 7.6) at 21 °C.
260 = 1.3 × 104
M
1 cm
1 (per base pair) for
duplex samples and
260 = 1.04 × 104
M
1 cm
1 (per base) for
single-stranded samples.
PnD, the association constant is
Kn = [PnD]/[P]n[D]. Separating
variables and taking logarithms gives the following equation.
Dilution of an AGT-DNA mixture changes the binding ratio
[PnD]/[D] by mass action. A recursive method (14) was used
to evaluate n and Ka, starting with an
initial value of n = 4 deduced from the value of
Mr (complex) measured by sedimentation equilibrium.
(Eq. 1)
7 M), and
the samples were analyzed by native gel electrophoresis. The free
protein concentration [P] was estimated using the conservation relation [P] = [P]o
n[PnD] in
which [P]o is the total hAGT concentration in the
reaction mixture, and an initial value of n = 4 was
assumed on the basis of our sedimentation equilibrium results (see Fig.
1). For the highly cooperative formation of a 4:1 complex under
conditions of large protein excess, the fractional saturation
Y is given by (20) the following.
Estimates of Ka were obtained by fitting
Equation 2 to the experimentally determined dependence of Y
on [P].
(Eq. 2)
24 h met
this criterion for AGT-DNA mixtures. Five scans were averaged for each
sample at each wavelength and rotor speed. For analysis of hAGT protein
alone, models incorporating different assembly stoichiometries were
based on the following general equation.
Here A(r) is the absorbance at radial position
r, and
(Eq. 3)
n is the absorbance of the nth
species at the reference radius (ro). The
parameter
n is the reduced molecular weight [
n = Mn(1
)
2/(2RT)],
Mn is the molecular weight of the nth
species,
its partial specific volume,
is the
solvent density,
is the rotor angular velocity, R is the
gas constant, T is the absolute temperature, and
is the
base-line offset. Solvent density (1.004 g/ml) was measured using a
Mettler density meter. The partial specific volume of hAGT (0.744 ml/g)
was calculated by the method of Cohn and Edsall (21), using partial
specific volumes of amino acids tabulated by Laue et al.
(22).
PnD, Equation 3 becomes
(Eq. 4)
Here, most terms are defined as for Equation 3;
D
and
PnD are absorbances of DNA and
protein-DNA complex at ro, the reduced molecular
weights of DNA and protein-DNA complex are given by
D = MD(1
D
)
2/(2RT)
and
PnD = (nMP + MD)(1
PnD
)
2/(2RT),
and n is the protein:DNA ratio of the complex. In this analysis, the known molecular weights of recombinant hAGT proteins (21,614
Mr
21,876) and DNA
(Mr = 4,881 for single-stranded DNA and
Mr = 9,762 for double-stranded DNA) were used as
constants. The partial specific volume of NaDNA at 0.1 M
NaCl (0.502 ml/g) was estimated by interpolation of the data of Cohen
and Eisenberg (23). Partial specific volumes of each of protein-DNA
complexes were estimated using Equation
Here n is the stoichiometric ratio of protein to DNA
in the complex. Equation 5 is based on the assumption that there is no
significant change in partial specific volumes of the components upon
association. Although we do not know whether such a volume change
occurs, it seems reasonable that values of
(Eq. 5)
PnD for complexes containing
a large mass proportion of protein (like those analyzed here) should
reflect that proportion. Equation 4 was used in global analysis of
multiple data sets obtained at different macromolecular concentrations
and/or rotor speeds (24). In this method, the values of
P,
D,
PnD,
and
are unique to each sample, but the value of n must
be common to all of the data sets. Nonideality was not considered,
because there was no evidence of nonideal effects.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (45K):
[in a new window]
Fig. 1.
Sedimentation equilibrium analyses of hAGT
protein and solutions containing hAGT and DNA. Curve A,
sedimentation profile for His6-tagged C145S hAGT. Protein
(nominal concentration, 13.7 µM) was brought to
equilibrium at 43,000 rpm and 20 ± 0.1 °C. The absorbance
measurements were made at 280 nm. The smooth curve
represents the global fit of Equation 3 to data sets obtained at two
protein concentrations (13.7 and 3.6 µM) and three rotor
speeds (43,000, 31,000, and 22,000 rpm). This analysis returned
Mr = 21,800 ± 400 in good agreement with
the monomer molecular weight derived from sequence data (21,860).
Curve B, sedimentation profile of a mixture containing
His6-tagged C145S hAGT (10 µM) and
single-stranded 16-mer DNA (0.5 µM strands) brought to
equilibrium at 15,000 rpm and 20 ± 0.1 °C. The absorbance
measurements were made at 260 nm. The smooth curve
represents the global fit of Equation 3 to data sets obtained at
11,000, 15,000, 20,500, and 27,000 rpm. This analysis returned
Mr = 91,900 ± 1,500, consistent with a
binding stoichiometry of 3.98 ± 0.07. The small, symmetrical
residuals demonstrate the compatibility of this model to the data.
Curve C, sedimentation profile of a mixture containing
His6-tagged C145S hAGT (10 µM) and
double-stranded 16-mer DNA (0.5 µM duplex) brought to
equilibrium at 15,000 rpm and 20 ± 0.1 °C. The absorbance
measurements were made at 260 nm. The smooth curve
represents the global fit of Equation 3 to data sets obtained at
11,000, 15,000, 20,500, and 27,000 rpm. This analysis returned
Mr = 95,900 ± 1,700, consistent with a
binding stoichiometry of 3.94 ± 0.08.
PnD, with
n = 3.98 ± 0.07 is consistent with the data.
Sedimentation models with additional species did not fit the data
significantly better than Equation 4 (results not shown). This outcome is intriguing because it suggests that the
n = 4 complex forms without significant accumulation of
intermediates of lower stoichiometry. This interpretation is supported
by the gel mobility shift experiments described below. Parallel
experiments carried out with wild type and C145A, His6
wild type, His6-C145A, and His6-C145S
hAGTs returned closely similar protein-DNA stoichiometries (Table
I), indicating that neither
modification of the active site cysteine nor presence of a C-terminal
His6 tag alters the stoichiometry of association.
Association constants and binding stoichiometries for the interaction
of 16-mer oligodeoxyribonucleotides with recombinant hAGT proteins
PnD, with
n = 3.94 ± 0.08 is consistent with the data.
Inclusion of additional species in the sedimentation model did not
improve the quality of the fit (results not shown), suggesting that, as with single-stranded DNA, stoichiometric intermediates are not present
in significant concentrations. This suggestion is supported by gel
shift results (discussed below). The fact that the hAGT stoichiometry
is the same, within error, for both single-stranded and duplex 16-mers
is intriguing, because it suggests that factors determining
stoichiometry may not be sensitive to the association state of the DNA.
PnD in which the maximum
stoichiometry (n) is reached without accumulation of
significant concentrations of intermediates (19). The dependence of
ln([PD]/[D]) on ln[P] is shown in Fig. 2B. The values
of stoichiometry and Ka were calculated from these
data as described under "Experimental Procedures." The
stoichiometry values most consistent with the data for
His6-tagged C145S hAGT binding to 16-mer are 3.84 ± 0.2 for single-stranded DNA and 3.99 ± 0.1 for double-stranded
DNA. These stoichiometry values (summarized in Table I) agree well with
ones obtained by analytical ultracentrifugation, despite a difference
in the salt concentration of the buffers used in the two techniques
(serial dilution assays, ~10 mM; sedimentation equilibrium assays, ~110 mM). Closely similar
stoichiometry values were obtained by the electrophoretic mobility
shift assay method for the active site mutant C145A hAGT as well as
His6-wild type hAGT and His6-C145A hAGT (Table
I), supporting the conclusion that neither the C145A modification of
the active site nor the presence of a C-terminal His6 tag
has a significant effect on the stoichiometry of these
interactions.
View larger version (57K):
[in a new window]
Fig. 2.
Serial dilution analysis of the interaction
between hAGT and 16-mer oligonucleotides. A,
electrophoretic mobility shift assay. The initial mixture contained
His6-tagged C145S hAGT (3.5 µM) and
double-stranded 16-mer (0.8 µM duplex) in 10 mM Tris (pH 7.6), 1 mM DTT, and 10 µg/ml
bovine serum albumin (lane a). Serial dilutions were
performed using the same buffer (dilution factor 0.85/step; lanes
b-m), giving decreasing concentrations of both protein and DNA.
Electrophoresis was carried out as described under "Experimental
Procedures." Band B, bound DNA; band F, free
DNA. B, graph of the dependence of ln([PD]/[D]) on
ln[P]. The error bars indicate the range of values
obtained in three parallel experiments. The solid lines
represent linear least squares fits of Equation 1 to data obtained for
wild type hAGT with single-stranded DNA ( ) and double-stranded DNA
(
). For single-stranded 16-mer, this analysis returned
n = 3.84 ± 0.2 and KPD = 6.9 ± 1.0 × 1023 M
4.
For double-stranded 16-mer, n = 3.96 ± 0.1 and
KPD = 4.3 ± 0.9 × 1023
M
4.
View larger version (52K):
[in a new window]
Fig. 3.
Continuous variation analysis of hAGT-DNA
complexes. A, electrophoretic mobility shift assay of
the binding of wild type hAGT to single-stranded 16-mer DNA. The total
macromolecular concentration was fixed ([hAGT] + [DNA] = 6 × 10 6 M) with the mole fraction varied
regularly in increasing increments of protein across the gel
lanes a-n. Binding was carried out at 20 ± 1 °C in
10 mM Tris (pH 7.6), 1 mM DTT, and 10 µg/ml
bovine serum albumin. Electrophoresis was performed as described under
"Experimental Procedures." Band B, bound DNA; band
F, free DNA. B, job plot of wild type hAGT binding
single-stranded 16-mer DNA. The data were from A and
replicate experiments. The error bars (most obscured by data
symbols) indicate the range of values obtained in three parallel
experiments. The solid lines are least squares fits to
rising and falling subsets of the data. Their intersection yields a
binding stoichiometry of 3.9.
4. Assuming equipartition of the binding
free energies among the four hAGT monomers, this corresponds to a
monomer association constant of ~6.2 × 105
M
1, which is in reasonable agreement with
values reported for small (9) and large (27) DNA molecules. The very
narrow range of association constants for wild type, active site
mutant, and His6-tagged hAGT proteins (best seen by
comparison of the effective monomer association constants given in
Table II) is an especially interesting result. It indicates that neither the C145A mutation of the active site
nor the presence of a C-terminal His6 affinity
tag significantly alters DNA binding affinity under our assay
conditions. A similar result with active site cysteine mutants was
previously obtained for untagged C145A and C145S proteins under
different binding conditions, by Hazra et al. (28). Under
our experimental conditions, hAGT appears to bind double-stranded DNA
with slightly higher affinity than single-stranded DNA (best seen by
comparison of the effective monomer association constants given in
Table II). In addition, the presence of a methylated guanine nucleotide
also enhances hAGT binding, and that enhancement is slightly more
pronounced in single-stranded DNA than in double-stranded DNA (Table
II). However, the small differences in affinity and the closely similar stoichiometries suggest that the overall mechanism of hAGT interaction is altered little by changes in DNA secondary structure or alkylation, hAGT active site mutation, or the presence of a C-terminal
His6 affinity tag.
View larger version (49K):
[in a new window]
Fig. 4.
Measurement of binding affinities by direct
titration. Interaction of single-stranded 16-mer DNA with hAGT.
A, binding carried out at 20 ± 1 °C in 10 mM Tris (pH 7.6), 1 mM DTT, 10 µg/ml bovine
serum albumin. B, binding carried out at 20 ± 1 °C
in the same buffer as that shown in A, adjusted to contain
70 mM NaCl. All of the samples contained 8.75 × 10 7 M DNA; the samples in lanes
b-l of each gel contained increasing concentrations of
His6-tagged C145A hAGT. Electrophoresis was performed as
described under "Experimental Procedures." Band B, bound
DNA; band F, free DNA. C, binding isotherms for
hAGT. The measurements were performed in triplicate. The error
bars indicating the ranges of values are obscured by the data
symbols. The smooth curves are fits of Equation 2 to the
data, returning association constants (KPD) of
2.7 ± 0.6 × 1023 M
4
in 10 mM Tris buffer without added KCl and 1.7 ± 0.5 × 1023 M
4 in 10 mM Tris, 70 mM KCl buffer, respectively.
Association constants and binding stoichiometries for the interaction
of methylated 16-mer oligodeoxyribonucleotides with active site
mutant human AGT proteins
View larger version (36K):
[in a new window]
Fig. 5.
Analysis of the interaction of alkylated hAGT
with and double-stranded DNA. A, titration of the
16-mer duplex with Cys145 methyl hAGT (Me AGT). A 1 µg/µl stock of wild type hAGT was alkylated at the active site
cysteine as described under "Experimental Procedures." Binding was
carried out at 20 ± 1 °C in 10 mM Tris (pH 7.6), 1 mM DTT, and 10 µg/ml bovine serum albumin. All of the
samples contained 8.75 × 10 7 M DNA; the
samples in lanes b-l contained increasing concentrations of
Me AGT. Electrophoresis was carried out as described under
"Experimental Procedures." Band B, bound DNA; band
F, free DNA. B, binding isotherms; dependence of the
fractional saturation of DNA (Y) on free [hAGT]; data from the
experiment shown in A and from parallel titrations of
double-stranded DNA with wild type hAGT and benzoylated hAGT (Bz AGT;
not shown). The solid lines are fits of Equation 2 to the
data. Apparent monomer-equivalent dissociation constants
(KD) were 1.05 ± 0.05 × 10
6 M for wild type hAGT, 3.50 ± 0.08 × 10
6 M for Me AGT, and 4.40 ± 0.05 × 10
6 M for Bz AGT.
C, job plots for Me AGT and Bz AGT binding to
double-stranded 16-mer DNA.
, Me AGT;
, Bz AGT. Experiments like
that shown in Fig. 3A were performed in triplicate using
double-stranded 16-mer DNA as the substrate. The concentration ranges
of AGT-DNA complexes are given by the error bars. The
solid lines are least squares fits to rising and falling
subsets of the data for Me AGT. The optimal combining ratio for Me AGT
and Bz AGT with DNA are 4.1 and 4.2, respectively.
Association constants and binding stoichiometries for the interaction
of 16-mer oligodeoxyribonucleotides with alkylated human AGT proteins
Association constants and binding stoichiometries for the interaction
of substrate 16-mer oligodeoxyribonucleotides with alkylated human
AGT proteins
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
4 (Table II), whereas the binding of the
same protein to the homologous nonalkylated 16-mer is characterized by
n = 3.92 ± 0.1, Ka = 0.269 ± 0.059 × 1024
M
4 (Table I). Assuming equipartition of
binding free energy, the ~138-fold difference in the aggregate
stabilities of these complexes corresponds to a difference of ~3.4 in
the average, monomer-scale association constants. However, if the 4:1
complex contains one protein bound with high affinity to the lesion
site and three others bound nonspecifically (with Ka
equal to that of the protein for nonalkylated DNA), the entire 138-fold
affinity difference between complexes on alkyl and nonalkyl DNAs might be a measure of the binding specificity of His6-C145A hAGT
for an O6-methylguanine-containing site in a
single-stranded DNA. Similarly large affinity differences are found for
other mutant or alkylated hAGT proteins and double-stranded
oligonucleotide (Table V).
Alterations in binding free energy as a consequence of DNA alkylation
Alone, the observed level of preferential binding cannot drive an
efficient lesion search in a genome containing ~109 bp,
like those present in many eukaryotes. Although it is possible that
other (currently unknown) interactions provide the necessary specificity in vivo, we propose an alternative surveillance
mechanism that requires no additional components. High binding
cooperativity may allow AGT to occupy any exposed DNA region, including
those near replication forks. The movement of this available DNA with replication would produce a processive search for alkylated sites that
does not require high lesion binding specificity. A greater understanding of the mechanism by which AGT interacts with target and
nonspecific DNAs may ultimately lead to novel methods to control its
activities for therapeutic purposes.
![]() |
FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grants GM-48517 (to M. G. F.) and CA-18137 (to A. E. P.) and Medical Scientist Training Program Grant 5 T32 GM-08601-05.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed. Tel.: 717-531-5250; Fax: 717-531-7072; E-mail: mfried@psu.edu.
Published, JBC Papers in Press, December 20, 2002, DOI 10.1074/jbc.M211854200
2 J. J. Rasimas, unpublished results.
3 Also consistent with this idea is the finding that the DNA affinities of His6 AGT proteins are more sensitive to changes in [salt] than those of nontagged homologues (J. J. Rasimas, unpublished results).
4 In previous analytical ultracentrifugation experiments, five or six hAGT molecules were found to bind a single-stranded DNA 30-mer (9). It seems likely that the complex described by Bender et al. (8) had a similar stoichiometry.
5 This observation is consistent with a previous indication that the binding of hAGT to DNA is not grossly affected by the methylation status of the active site cysteine (35).
6 Wild type hAGT may lack the specificity demonstrated by the mutants. However, because C145A and C145S mutants and methylated and benzylated forms of wild type protein demonstrate specificity (Tables III-V), we regard this possibility as unlikely.
7 A similar enhancement of the apparent, monomer scale association constant has been reported (3, 26), but both studies employed repair-competent AGT, so the results represent average affinities for native- and alkyl-AGT with native- and alkyl-DNA. As a result, the agreement of these results with ours may be coincidental.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: hAGT, human AGT; AGT, O6-alkylguanine-DNA alkyltransferase; DTT, dithiothreitol.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Samson, L. (1992) Mol. Microbiol. 6, 825-831[Medline] [Order article via Infotrieve] |
2. | Sekiguchi, M., Nakabeppu, Y., Sakumi, K., and Tuzuki, T. (1996) J. Cancer Res. Clin. Oncol. 122, 199-206[Medline] [Order article via Infotrieve] |
3. | Pegg, A. E., Dolan, M. E., and Moschel, R. C. (1995) Prog. Nucleic Acids Res. Mol. Biol. 51, 167-223[Medline] [Order article via Infotrieve] |
4. | Pegg, A. E. (2000) Mutat. Res. 462, 83-100[CrossRef][Medline] [Order article via Infotrieve] |
5. | Moore, M. H., Gulbus, J. M., Dodson, E. J., Demple, B., and Moody, P. C. E. (1994) EMBO J. 13, 1495-1501[Abstract] |
6. | Daniels, D. S., and Tainer, J. A. (2000) Mutat. Res. 460, 151-163[Medline] [Order article via Infotrieve] |
7. | Hashimoto, H., Inoue, T., Nishioka, M., Fujiwara, S., Takagi, M., Imanaka, T., and Kai, Y. (1999) J. Mol. Biol. 292, 707-716[CrossRef][Medline] [Order article via Infotrieve] |
8. |
Bender, K. B.,
Federwisch, M.,
Loggen, U.,
Nehls, P.,
and Rajewsky, M. F.
(1996)
Nucleic Acids Res.
24,
2087-2094 |
9. | Fried, M. G., Kanugula, S., Bromberg, J. L., and Pegg, A. E. (1996) Biochemistry 35, 15295-15301[CrossRef][Medline] [Order article via Infotrieve] |
10. | Pegg, A. E., Boosalis, M., Samson, L., Moschel, R. C., Byers, T. L., Swenn, K., and Dolan, M. E. (1993) Biochemistry 32, 11998-12006[Medline] [Order article via Infotrieve] |
11. | Crone, T. M., and Pegg, A. E. (1993) Cancer Res. 53, 4750-4753[Abstract] |
12. | Liu, H., Xu-Welliver, M., and Pegg, A. E. (2000) Mutat. Res. 452, 1-10[Medline] [Order article via Infotrieve] |
13. | Kanugula, S., Goodtzova, K., and Pegg, A. E. (1998) Biochem. J. 329, 545-550[Medline] [Order article via Infotrieve] |
14. | Xu-Welliver, M., Kanugula, S., and Pegg, A. E. (1998) Cancer Res. 58, 1936-1945[Abstract] |
15. | Walker, J. M. (1994) Methods Mol. Biol. 32, 5-8[Medline] [Order article via Infotrieve] |
16. | Roy, R., Shiota, S., Kennel, S. J., Raha, R., von Wronski, M., Brent, T. P., and Mitra, S. (1995) Carcinogenesis 16, 405-411[Abstract] |
17. | Kanugula, S., Goodtzova, K., Edara, S., and Pegg, A. E. (1995) Biochemistry 34, 7113-7119[Medline] [Order article via Infotrieve] |
18. | Maxam, A. M., and Gilbert, W. (1977) Proc. Natl. Acad. Sci. U. S. A. 74, 560-565[Abstract] |
19. | Fried, M. G., and Crothers, D. M. (1984) J. Mol. Biol. 172, 241-262[Medline] [Order article via Infotrieve] |
20. | van Holde, K. E. (1985) Physical Biochemistry , pp. 51-92, Prentice Hall, Englewood Cliffs, NJ |
21. | Cohn, E. J., and Edsall, J. T. (1943) Proteins, Amino Acids and Peptides as Ions and Dipolar Ions , pp. 140-154, Reinhold, New York |
22. | Laue, T. M., Shah, B. D., Ridgeway, T. M., and Pelletier, S. L. (1992) in Analytical Ultracentrifugation in Biochemistry and Polymer Science (Harding, S. E. , Rowe, A. J. , and Harding, J. C., eds) , pp. 90-125, The Royal Society of Chemistry, Cambridge, UK |
23. | Cohen, G., and Eisenberg, H. (1968) Biopolymers 6, 1077-1100[Medline] [Order article via Infotrieve] |
24. | Johnson, M. L., Correia, J. J., Yphantis, D. A., and Halvorson, H. R. (1981) Biophys. J. 36, 575-588[Abstract] |
25. | Fried, M. G., and Crothers, D. M. (1981) Nucleic Acids Res. 9, 6505-6525[Abstract] |
26. | Huang, C. Y. (1982) Methods Enzymol. 87, 509-525[Medline] [Order article via Infotrieve] |
27. | Chan, C., Z., W., Ciardelli, T., Eastman, A., and Bresnick, E. (1993) Arch. Biochem. Biophys. 300, 193-200[CrossRef][Medline] [Order article via Infotrieve] |
28. | Hazra, T. K., Roy, R., Biswas, T., Grabowski, D. T., Pegg, A. E., and Mitra, S. (1997) Biochemistry 36, 5769-5776[CrossRef][Medline] [Order article via Infotrieve] |
29. | Pegg, A. E., Wiest, L., Mummert, C., Stine, L., Moschel, R. C., and Dolan, M. E. (1991) Carcinogenesis 12, 1679-1683[Abstract] |
30. | Becker, K., Dosch, J., Gregel, C. M., Martin, B. A., and Kaina, B. (1996) Cancer Res. 56, 3244-3249[Abstract] |
31. | Clubb, R. T., Omichinski, J. G., Savilahti, H., Mizuuchi, K., Gronenborn, A. M., and Clore, G. M. (1994) Structure 2, 1041-1048[Medline] [Order article via Infotrieve] |
32. | Takahashi, M., Sakumi, K., and Sekiguchi, M. (1990) Biochemistry 29, 3431-3436[Medline] [Order article via Infotrieve] |
33. |
Wibley, J. E. A.,
Pegg, A. E.,
and Moody, P. C. E.
(2000)
Nucleic Acids Res.
28,
393-401 |
34. |
Daniels, D. S.,
Mol, C. D.,
Arval, A. S.,
Kanugula, S.,
Pegg, A. E.,
and Tainer, J. A.
(2000)
EMBO J.
19,
1719-1730 |
35. | Bhattacharyya, D., Foote, R. S., Boulden, A. M., and Mitra, S. (1990) Eur. J. Biochem 193, 337-343[Abstract] |