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INTRODUCTION |
Mismatch repair contributes genetic stability by correcting DNA
biosynthetic errors and preventing recombination between
quasi-homologous DNA sequences (1-5). The Escherichia coli
reaction responsible for correction of replication errors has been
reconstituted in a pure system (6-8). The key protein responsible for
initiation of this reaction is MutS, which recognizes mismatched base
pairs (9, 10). In addition to its mismatch recognition function, MutS
has a carboxyl-terminal ATPase that is required for function of the
protein in mismatch repair (11, 12). Structural studies have shown
bacterial MutS (13-15) and the DNA repair protein RAD50 (16) to be
members of the adenine nucleotide binding cassette (ABC)1 family of proteins,
which is largely composed of proteins that couple the energy of ATP
hydrolysis to transport of molecules across biological membranes (17,
18).
For members of the ABC family, shared motifs around the nucleotide
binding domain comprise an integral part of a subunit-subunit interface
(17). As a consequence, nucleotide binding and hydrolysis within one
subunit can induce conformational changes across this interface to
cooperatively influence nucleotide binding and hydrolysis by the other
(19, 20). Stimulation of ATPase by the cognate ligand implies
interaction of nucleotide binding centers and ligand binding sites of
ABC transporters (17). In the case of MutS homologs and RAD50, this is
manifested as ATPase activation in the presence of DNA (16,
21-24).
While the MutS ATPase center is required for function of the protein in
mismatch repair, the functional roles of nucleotide binding and
hydrolysis in modulating interaction of the protein with DNA is
controversial. Several possible functions for the ATPase have been
suggested in this regard. One class of model is based on the
observation that ATP promotes release of bacterial MutS and human
MutS
from the mismatch, resulting in movement of the proteins along
the helix contour (25-29). This movement has been postulated to play a
role in signaling between the mismatch and the secondary DNA site that
serves as the strand signal that directs repair. This sort of model is
appealing because it accounts for the preferential utilization of
proximal strand signals (7, 30) and for the fact that the excision
system is loaded at the strand signal with an orientation bias
(31).
Two distinct mechanisms for MutS homolog movement along the helix have
been suggested. One model posits directional movement along the helix
that is coupled to ATP binding and hydrolysis by the DNA-bound protein
(25). The problem with this proposal is that it is difficult to
reconcile the rate of MutS movement along the helix (several hundred to
several thousand base pairs per minute) with the modest turnover rate
of its ATPase (about 4 min
1 per monomer equivalent in the
presence of heteroduplex) (24, 25). However, a mechanism that supports
directional movement along the helix with limited energy input has been
suggested (26).
Study of human MutS
has led to proposal of a molecular switch model
for movement of MutS homologs along the helix. This mechanism postulates mismatch recognition by the MutS·ADP complex, with the
mispair promoting exchange of ADP for ATP (27, 32). Since the resulting
MutS·ATP complex is envisioned to freely diffuse along the helix, the
modest turnover number of the MutS ATPase is readily accommodated
within this model. On the other hand, several lines of evidence
indicate that movement of MutS homologs from the mismatch is dependent
on ATP hydrolysis by the heteroduplex-bound protein (25, 26, 28).
A distinct function for the MutS ATPase that does not invoke movement
of the protein along the helix has also been described (15, 33). This
model invokes ATPase function in a kinetic proofreading process that
enhances mismatch specificity of MutS-DNA interaction. In this
proposal, ATP binding by DNA-bound MutS preferentially reduces the
affinity of the protein for homoduplex sequences, leading to abortion
of an incorrect recognition complex that might occur at a canonical
Watson-Crick base pair. The model also suggests that ATP binding by
heteroduplex-bound MutS serves to verify mismatch recognition. The
finding that ATP promotes release of MutS homologs from a mismatch (22,
25-28, 34-36) and the observation that mismatch specificity of
bacterial MutS is abolished in the presence of a non-hydrolyzable ATP
analogue (29) are seemingly incompatible with these ideas.
To some extent, these disparate models reflect the lack of information
concerning the effects of ATP binding and hydrolysis and, hence,
potential states of nucleotide occupancy on MutS-DNA interaction.
Structural studies of carboxyl-terminal MutS truncations have shown
that the MutS dimer is capable of mismatch recognition and that the two
subunits within dimer·heteroduplex complex are not equivalent (13,
14). Consequently, the two nucleotide binding sites within the MutS
dimer can potentially be filled in nine different ways (37).
Furthermore, recent work indicates that MutS dimers undergo a
concentration-dependent assembly reaction that is promoted
by binding of nucleotide or heteroduplex DNA, probably to a tetramer
end state (14, 24).2,3 This
dramatically increases the complexity of
the system. To clarify potential nucleotide occupancy states of the
protein, we have determined the number and the affinities of di- and
triphosphate binding sites within MutS. We show that MutS binds ADP and
non-hydrolyzable ATP analogues with similar affinities, that the
protein has one high affinity ADP site and one high affinity
triphosphate site per dimer equivalent, and that di- and triphosphate
binding sites can be simultaneously occupied.
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EXPERIMENTAL PROCEDURES |
MutS and Nucleotides--
MutS preparations were those described
previously (29). Concentrations of the protein are expressed in terms
of monomer equivalents. [2,8-3H]ADP (33.9 Ci/mmol) and
[35S]ATP
S (adenosine 5' (
-thio) triphosphate, 1250 Ci/mmol) were obtained from PerkinElmer Life Sciences.
[
-32P]AMPPNP (5'-adenylylimidodiphosphate, 50 Ci/mmol)
was from ICN. BODIPY FL (2' or 3')-AMPPNP and BODIPY TR (2' or 3')-ADP
(custom synthesis) were purchased from Molecular Probes.
Nucleotide Filter Binding Assays--
Nucleotide binding assays
were performed using a modified Schleicher and Schuell 96-well filter
apparatus (38), which was stored at 4 °C prior to use.
Nitrocellulose membranes (PROTRAN BA85, Schleicher and Schuell) were
briefly soaked in 0.4 M KOH, extensively rinsed with
distilled water until the pH was neutral, and then equilibrated with
binding buffer (25 mM Tris-HCl, pH 7.6 (determined at
4.0 °C), 100 mM KCl, 5 mM MgCl2,
1 mM dithiothreitol, 10% (v/v) glycerol). Binding
reactions (100 µl) containing nucleotides and MutS as indicated were
allowed to equilibrate on ice for 10 min. Thirty-µl samples of each
binding reaction were applied to three different wells, filtered
(filtration time less than 10 s), and then rinsed with 100 µl of
binding buffer. Affinity measurements for [3H]ADP and
[32P]AMPPNP were performed using only radiolabeled
nucleotides, whereas the stoichiometric binding experiments contained a
fixed amount of radiolabeled nucleotide (<0.5 µM), which
was isotopically diluted with unlabeled nucleotide (ADP, USB Corp.;
AMPPNP, ICN: ATP
S, Calbiochem-Novabiochem) to yield the desired
concentration. After filtration, the nitrocellulose membrane was
blotted dry and further dried under a heat lamp. For single label
experiments, [3H]ADP and [32P]AMPPNP
binding was quantitated using a Molecular Dynamics PhosphorImager and
ImageQuant software with a low energy PhosphorImager screen used for
the former isotope. For dual label experiments using [35S]ATP
S and [3H]ADP, filter-retained
radioactivity was quantitated by liquid scintillation counting, and
counts in the tritium channel were corrected for a 5% spillover from
the 35S channel.
Nucleotide-MutS interaction under stoichiometric binding conditions was
analyzed by nonlinear regression fit (39) to Equation 1.
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(Eq. 1)
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[SA] is the concentration of bound nucleotide,
At is the total added nucleotide concentration,
Kd is the dissociation constant for nucleotide
binding, and St is the total concentration of
available nucleotide binding sites that was determined in this analysis.
Fluorescence Nucleotide Binding Assay--
Binding of BODIPY FL
(2' or 3')-AMPPNP and BODIPY TR (2' or 3')-ADP to MutS was monitored by
equilibrium fluorescence anisotropy determination using an SLM 8100 fluorometer (Jobin Yvon, Inc.) equipped with Glan-Thomson
calcite polarizers. Reactions contained binding buffer (above), 100 nM fluorescent nucleotide, and MutS as indicated. BODIPY FL
(2' or 3')-AMPPNP was excited at 490 nm, and fluorescence emission
determined at 514 nm, whereas the BODIPY TR (2' or 3')-ADP fluorophore
was excited at 590 nm, and emission measured at 620 nm. Anisotropy
measurements were taken in the L-format using binding buffer as blank
and correcting for the instrument G-factor as described (40).
Fluorescence Energy Transfer--
The theoretical value for
R0, the distance between the energy transfer
pair which gives a 50% efficiency, was calculated from overlap of the
emission spectra of the donor, BODIPY FL (2' or 3')-AMPPNP (arbitrary
units versus wavelength) with the excitation spectra of the
acceptor, BODIPY TR (2' or 3')-ADP (extinction coefficient
versus wavelength). R0 was calculated
from physical constants and the overlap integral using Equation 2
(40).
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(Eq. 2)
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QD is the quantum yield of the donor,
BODIPY FL (2' or 3')-AMPPNP for which a value of 0.9 was used. The
2 parameter is a factor relating the relative
orientation of the transition dipoles of the donor and acceptor. A
value of 2/3 was used, assuming random dynamic averaging of donor and
acceptor dipoles. Although this assumption may not be valid in all
cases, the sixth root dependence R0 on
2 limits the potential error in the calculated distance
to less than 36% (40). N is Avogadro's number and
n is the refractive index of the medium, for which a value
of 1.33 was used. The overlap integral was calculated numerically using
the program Kaleidagraph and normalized by dividing by the total
integrated area under the emission spectra. Substitution of these
values into Equation 2 yielded an R0 distance of
38.1 Å for this donor-acceptor pair.
The efficiency, E, of fluorescence energy transfer (FRET)
between BODIPY FL (2' or 3')-AMPPNP (donor) and BODIPY TR (2' or 3')-ADP (acceptor) was obtained using the Ratio A procedure
as described (41). Using this method the efficiency, E, of
fluorescence energy transfer can be determined in a single cuvette by
measuring the ratio of the fluorescence intensity of the acceptor when
the donor is excited (490 nm) to the fluorescence intensity observed upon direct excitation of the acceptor (590 nm) as shown in Equation 3.
|
(Eq. 3)
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The fluorescence intensity at 620 nm contains emission intensity
due to energy transfer as well as some spillover fluorescence intensity
from the emission of the donor. To correct for this non-FRET
contribution, the fluorescence spectra of the donor was subtracted from
the FRET emission spectra using the SLM 8100 software as described
(41). Ratio A is the ratio of the corrected fluoresce intensity of the acceptor, BODIPY TR (2' or 3')-ADP at 620 nm upon
donor excitation at 490 nm divided by the fluorescence intensity of the
acceptor, BODIPY TR (2' or 3')-ADP upon direct excitation (no FRET) at
590 nm. The term d+ is the efficiency of donor
labeling for which a value of unity was used. This term will be less
than unity if less than 100% of donor and acceptor-labeled nucleotides
are bound by MutS (see "Results"). The factor
FL(490 nm)/
TR(590
nm) is the ratio of the extinction coefficients of the donor at 490 nm
and the acceptor at 590 nm and was experimentally determined as 0.51. The last term in Equation 3 is the ratio of the fluorescence intensity
of the acceptor upon excitation at 490 nm to that observed when
excitation was at 590 nm, for which a value of 0.036 was experimentally
determined. Using Equation 3, the efficiency, E, can be
calculated upon the determination of Ratio A. The efficiency of energy transfer was converted into the molecular distance
R, separating the donor and acceptor pair using Equation 4
(40).
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(Eq. 4)
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 |
RESULTS |
MutS Binds ADP and AMPPNP with Similar Affinities--
Nucleotide
binding by MutS was examined by filter assay using radiolabeled ADP and
nonhydrolyzable AMPPNP and by fluorescent anisotropy determination
using nucleotides chemically modified with the BODIPY (FL or TR)
fluorophore on the 2' or 3' positions of the ribose moiety (see
"Experimental Procedures"). The latter method is based on the
observation that whereas free nucleotides have a low anisotropy in
solution, binding to MutS leads to an approximate 8-fold increase in
this parameter. As shown in Fig. 1,
similar binding profiles were obtained with radiolabeled and fluorophore-tagged nucleotides, indicating that the derivatization of
ADP and AMPPNP with the BODIPY fluorophore has little effect on
nucleotide binding by MutS.

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Fig. 1.
Binding of radiolabeled and fluorescent
derivatives of ADP and AMPPNP. Binding of 100 nM
BODIPY TR (2' or 3')-ADP (Panel A) and BODIPY FL (2' or
3')-AMPPNP (Panel B) was determined as a function of MutS
concentration (monomer equivalents) by anisotropy measurement ( ,
"Experimental Procedures"). At each MutS concentration,
fluorescence was determined (vertical and horizontal emission). MutS
binding to 310 nM [3H]ADP (A) and
6.2 nM [ -32P]AMPPNP (B) was
determined by nitrocellulose filter assay ( , "Experimental
Procedures"). Filter retention efficiency was determined to be unity
for AMPPNP and 0.8 for ADP at high concentrations of MutS. Error
bars correspond to one standard deviation for triplicate
determinations.
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As judged by either assay method, the MutS concentration where half of
the nucleotide is bound is ~1 µM for both ADP and
AMPPNP. Because these experiments were performed by titrating a fixed nucleotide concentration with a multivalent protein, this analysis addresses only the highest affinity nucleotide binding site(s) within
the MutS oligomer. Inspection of ADP and AMPPNP binding profiles at low
MutS concentrations (0-0.2 µM) reveals that the curves
are sigmoidal in shape, indicating an apparent increase in nucleotide
affinity with increasing MutS concentration. This observation is
consistent with the finding that MutS dimer oligomerization in this
concentration range is promoted by the presence of ATP (24).
MutS Binds Nucleotides with Half-site Specificity--
The
stoichiometry of ADP and AMPPNP binding was determined by nucleotide
titration of a high fixed concentration of the protein (10 µM monomer, Fig. 2). Under
these conditions binding is expected to be essentially stoichiometric.
This analysis yielded stoichiometries of 2.1 ± 0.2 MutS
monomers/ADP and 2.4 ± 0.3 MutS monomers/AMPPNP, suggesting that
only half of the nucleotide binding sites within the protein are
available for binding of ADP or the triphosphate.

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Fig. 2.
Stoichiometries of ADP and AMPPNP
binding. Stoichiometries of [3H]ADP (A)
and [32P]AMPPNP (B) binding were determined by
filter assay at a fixed high concentration of MutS and MutS800 (10 µM as monomer) by titration with radioactive nucleotide.
The concentration of nucleotide retained by MutS ( ) and MutS800
( ) was calculated from the fraction of radioactive counts retained
on the filter divided by the retention efficiency times the total
nucleotide concentration. Data were fit by nonlinear regression to
Equation 1 ("Experimental Procedures"), yielding a concentration of
available ADP binding sites of 6.5 ± 0.2 µM for
MutS and 5.2 ± 0.2 µM for MutS800. The
concentration of AMPPNP binding sites was determined 5.8 ± 0.3 µM for MutS and 4.9 ± 0.2 µM for
MutS800.
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This finding is in agreement with structural analysis of
heteroduplex-protein co-crystals obtained in the presence of ADP with
E. coli MutS800, a carboxyl-terminal truncation that
oligomerizes to yield dimers only (14). ADP occupancy is restricted to
one MutS subunit within the DNA·(MutS800)2 complex (14,
37). Using titration assay, we have confirmed half-site occupancy for
ADP binding by MutS800 (2.9 ± 0.3 MutS800 monomers/ADP) in the
absence of DNA and have obtained similar results with AMPPNP (2.2 ± 0.3 MutS800 monomers/AMPPNP, Fig. 2). These observations suggest
that half-site nucleotide specificity is determined at the dimer level.
AMPPNP Is a Weak Competitor for High Affinity ADP Binding--
The
experiments described above indicate one high affinity ADP binding site
and one high affinity triphosphate binding site per MutS dimer
equivalent. Since ADP and AMPPNP bind to MutS with similar affinities
(Fig. 1), each nucleotide should be a highly effective competitor of
the other if both bind to a common site. Fig.
3 shows a competition binding study in
which MutS-bound [3H]ADP was examined as a function of
increasing concentrations of unlabeled ADP or AMPPNP. Under initial
conditions (4 µM MutS and 2 µM
[3H]ADP), the [3H]ADP was only partially
bound, and as expected, addition of unlabeled ADP competed for binding
of the radiolabeled diphosphate. By contrast, [3H]ADP
binding by MutS was potentiated by low concentrations of AMPPNP (< 5 µM). Significant AMPPNP competition was evident only when
the triphosphate analogue was present at 10-fold molar excess over
[3H]ADP, and near complete competition was observed at
100-fold excess. In view of the similar affinities of MutS for the ADP and AMPPNP (Fig. 1), the pattern of AMPPNP competition is inconsistent with the possibility that the two nucleotides share a common high affinity site. Indeed, the potentiation of [3H]ADP
binding observed at low AMPPNP concentrations indicates that the two nucleotides bind simultaneously to distinct sites with
apparent positive cooperativity. A similar cooperative interaction has
been demonstrated for the nucleotide binding centers of the multidrug
resistance protein MRP1, another ABC superfamily member (19, 20). While
these observations indicate the presence of distinct di- and
triphosphate binding sites within MutS, the specificity of these sites
is not absolute as judged by the ability of high concentrations of
AMPPNP to compete with ADP binding.

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Fig. 3.
Competition for
[3H]ADP binding by ADP and AMPPNP.
Complex formation between MutS (4 µM) and
[3H]ADP (2 µM) was determined by
nitrocellulose filter assay (see "Experimental Procedures") as a
function of nonradioactive ADP ( ) or AMPPNP ( ) competitors. The
dashed line corresponds to the yield of
[3H]ADP·MutS complexes observed in the absence of
competitor. Note that the x-axis is discontinuous above 25 µM.
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MutS Can Bind ADP and AMPPNP Simultaneously--
To
further address the possibility that di- and triphosphate binding
involves distinct, high affinity sites within the MutS oligomer, we
asked whether the protein is capable of binding ADP and AMPPNP
simultaneously. These experiments exploited the fact that BODIPY FL (2'
or 3')-AMPPNP and BODIPY TR (2' or 3')-ADP form a fluorescence energy
transfer pair. As shown in Fig. 4, the
emission spectrum of the donor BODIPY FL (2' or 3')-AMPPNP overlaps the
absorption spectrum of the BODIPY TR (2' or 3')-ADP acceptor. The
overlap integral shown in Fig. 4 permits calculation of
R0, the distance between the donor and acceptor
that yields 50% energy transfer efficiency (40), for which a value of
38.1 Å was obtained (see "Experimental Procedures").

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Fig. 4.
BODIPY FL (2' or 3')-AMPPNP and BODIPY
TR (2' or 3')-ADP form a fluorescence energy transfer pair. The
fluorescence emission spectrum for BODIPY FL (2' or 3')-AMPPNP
(red line, arbitrary units) was determined upon excitation
at 490 nm. The absorption spectrum of BODIPY TR (2' or 3')-ADP is shown
in units of extinction coefficient (left axis, blue
line). The overlap integral (right axis, dashed
black line) was calculated from the spectra shown (Equation 1,
"Experimental Procedures") to yield a theoretical
R0 value for this donor-acceptor pair of 38.1 Å.
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Fig. 5 shows the fluorescence emission
spectrum of an equimolar (4 µM) mixture of the BODIPY FL
(2' or 3')-AMPPNP and BODIPY TR (2' or 3')-ADP upon excitation at 490 nm (AMPPNP donor excitation). As can be seen, the emission intensity is
almost exclusively that of the donor at 514 nm, and there is no
discernable peak at the wavelength (620 nm) expected for emission of
the BODIPY TR (2' or 3')-ADP acceptor. However, addition of 8 µM (monomer) MutS or MutS800, resulted in a decrease in
donor emission at 514 nm and a concomitant appearance of fluorescence
intensity at 620 nm as expected for acceptor emission. Furthermore, the
sensitized emission at 620 nm observed in the presence of MutS and
MutS800 was largely abolished by addition of excess ATP, and this was accompanied by partial restoration of donor emission at 514 nm. These
observations are consistent with MutS bringing ADP and AMPPNP into
proximity by virtue of simultaneous binding of the two nucleotides.

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Fig. 5.
MutS-dependent
fluorescence energy transfer between BODIPY FL (2' or 3')-AMPPNP and
BODIPY TR (2' or 3')-ADP. The fluorescence emission spectrum was
determined for a mixture of 4 µM BODIPY FL (2' or
3')-AMPPNP (donor) and 4 µM BODIPY TR (2' or 3')-ADP
(acceptor) using the excitation wavelength of the donor (490 nm) in the
absence of MutS (red line), and in the presence of 8 µM MutS (solid blue line) or MutS800
(dashed blue line). MutS- and MutS800-dependent
sensitized emission was largely abolished by subsequent addition of an
ATP competitor to 200 µM (solid and
dashed green lines). Residual donor quenching and sensitized
emission of the acceptor in the presence of ATP represents a
kinetically trapped fraction of fluorescent-tagged ADP and AMPPNP
because addition of the ATP competitor prior to MutS completely
abolished donor quenching and energy transfer (data not shown). The
inset plot shows an expanded view of the fluorescence
emission in the region of acceptor emission (620 nm).
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By taking the ratio of the sensitized emission intensity of the
acceptor (Fig. 5, inset) to that obtained by directly
exciting the acceptor molecule bound to MutS or MutS800 (data not
shown), the efficiency of energy transfer was determined (Equation 3, "Experimental Procedures" (41)). The efficiency calculated in this
manner is 25% for both MutS and MutS800, corresponding to a 46-Å
separation between the fluorophores on the BODIPY FL (2' or 3')-AMPPNP
donor and BODIPY TR (2' or 3')-ADP acceptor (Equation 4,
"Experimental Procedures"). This distance estimate represents an
upper limit because it assumes 100% binding of both nucleotides as
well as 100% labeling efficiency of the AMPPNP donor molecule (that
d+ = 1 in Equation 4). If these assumptions are
not valid, then the actual distance between the nucleotides would be
less than 46 Å. Since assembly of MutS800 is restricted to dimer
formation (13, 14), the similar energy transfer efficiencies for native MutS and the MutS800 indicate that simultaneous ADP and AMPPNP binding
occurs at the dimer level and does not depend on oligomerization of
this species.
The possibility that MutS di- and triphosphate binding sites can be
simultaneously occupied was confirmed by filter binding assay using
differentially radiolabeled ADP and ATP
S. Complexes between MutS (10 µM) and [35S]ATP
S (5 µM)
were prepared (Fig. 6). Since this
[35S]ATP
S concentration is 25 times the
Kd (0.2 µM, data not shown), binding
was near stoichiometric, and essentially all the input label was
recovered as filter-retained complexes in the presence of MutS. As
shown in Fig. 6, titration of [35S]ATP
S·MutS
complexes with [3H]ADP led to retention of both
nucleotides on the filter, and the yield of MutS-bound
[35S]ATP
S was unaffected by [3H]ADP
addition until the diphosphate concentration equaled that of
[35S]ATP
S. Further [3H]ADP addition
resulted in a modest reduction of [35S]ATP
S binding,
consistent with the competition results shown in Fig. 3. The apparent
stoichiometry of [3H]ADP·MutS complex formation
determined from this analysis is 2.3 ± 0.2 MutS monomers per ADP
bound, in good agreement with that observed in the absence of the ATP
analogue (2.1 ± 0.2 monomers per ADP, Fig. 2). These results
demonstrate that MutS can simultaneously bind ADP and ATP
S when
protein and nucleotide concentrations are in the low micromolar
range.

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Fig. 6.
ADP binds to
MutS·ATP S complex with half-site
reactivity. 10 µM MutS (monomer equivalent) and 5 µM [35S]ATP S was titrated with
[3H]ADP. The concentration of [35S]ATP S
retained on nitrocellulose is plotted on the right axis,
( ), and retained [3H]ADP is plotted on the left
axis, ( ). Fitting of the [3H]ADP data to Equation 1 ("Experimental Procedures") yielded a value of 4.4 ± 0.4 µM for the concentration of available ADP binding sites,
corresponding to a stoichiometry of 2.3 ± 0.2 MutS monomers per
ADP bound under these conditions.
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 |
DISCUSSION |
Structural analysis of the E. coli MutS800 complex with
heteroduplex DNA and ADP has demonstrated asymmetry with respect to interaction of the two subunits with DNA and with nucleotide (14, 37).
Whereas the nucleotide binding site is occupied within the MutS800
subunit responsible for mismatch recognition, the nucleotide binding
center within the other subunit is empty. This ADP binding
stoichiometry is recapitulated in the solution studies described here.
Since our experiments were performed in the absence of DNA, it is clear
that half-site ADP occupancy does not depend on the presence of DNA.
Binding of nonhydrolyzable AMPPNP and ATP
S (data not shown) also
displays a similar half-site stoichiometry, and the diphosphate and
triphosphate sites can be simultaneously and differentially occupied at
nucleotide concentrations in the low micromolar range. The specificity
of the sites is not absolute, however, because high concentrations of
AMPPNP can displace ADP from MutS (Fig. 3) and excess ADP competes
weakly for ATP
S binding (Fig. 6).
Differential and simultaneous occupancy of nucleotide binding sites
have been reported for other members of the ABC superfamily. For
example, cross-linking of azido-ATP to MRP1 is stimulated by ADP up to
equimolar amounts, although excess ADP inhibits the cross-linking
reaction (19, 20). These results are similar to our finding that AMPPNP
increases ADP binding but competes for ADP binding when present in
excess (Fig. 3). Further studies will be required to determine whether
the observed heterogeneity in MutS binding site specificities reflects
a stable asymmetry in the oligomer that is maintained during
steady-state ATP hydrolysis or whether the individual subunits
alternate specificity in an ordered fashion. Precedent for both
possibilities has been documented with other ABC transporters. For
example, nucleotide hydrolysis by P-glycoprotein has been attributed to
a catalytic cycle in which the two subunits alternately bind and
hydrolyze ATP (42). By contrast, the nucleotide centers of the cystic
fibrosis transmembrane conductance regulator function in a different
manner, with one of the two distinct domains supporting more rapid
hydrolytic turnover than the other (43).
We presume that the MutS·adenine nucleotide complexes described here
reflect occupancy states that occur during the course of ATP hydrolysis
by the protein. Pre-steady-state analysis of ATP hydrolysis by MutS in
the absence of DNA has demonstrated a burst of ADP production during
the first hydrolytic turnover that is substoichiometric with respect to
the available number of MutS monomer equivalents (24). Although the
basis of this effect has not been established, this observation
indicates that only a subset of MutS protomers is capable of ATP
hydrolysis at any given instant. Because the ADP burst indicates that
the rate-limiting step for turnover occurs after hydrolysis, it is also
clear that a hydrolytically active protomer spends most of its lifetime
as the ADP complex in the absence of mismatched DNA. Given the
millimolar concentrations of ATP in vivo, available
triphosphate binding sites would certainly be filled under these
conditions. Since we have shown that MutS supports simultaneous binding
of di- and triphosphate, these arguments strongly suggest that the
protein exists predominantly as an ATP·MutS·ADP ternary complex in
solution. This suggestion is in contrast to the molecular switch model
that restricts nucleotide occupancy to either the MutS·ADP or
MutS·ATP complex (27).
Recognition of the multivalent nature of MutS with regard
to nucleotide occupancy allows for the possibility of multiple roles for ATP binding and hydrolysis to coordinate the intricate functions attributed to MutS in the mismatch repair pathway. Indeed, distinct roles for ATP binding and hydrolysis have already been proposed. ATP
and ADP binding by MutS is coupled to tetramer formation (Ref. 24 and
Fig. 1), whereas ATP hydrolysis has been postulated to be involved in
the production of mobile complexes on DNA (25, 26, 28). ATP binding is
also postulated to function in a signaling manner to promote the
recruitment of downstream repair activities like MutL (34, 44, 45).
As noted above, the complex with bound diphosphate and triphosphate may
represent the form of the enzyme that encounters DNA. In the crystal
structure of the MutS·ADP· heteroduplex complex the protein
envelops the DNA in a clamp-like channel (14, 37). However, the
distance between the nucleotide binding sites in the ATP·MutS·ADP
complex measured by FRET (
46 Å, Fig. 5) in the absence of DNA is
greater than that in the MutS·ADP·heteroduplex crystalline complex
(
20 Å, (14, 37)). Although the separation distance estimated by
FRET is a maximum value, an attractive hypothesis is that the
ATP·MutS·ADP complex represents an open form of the protein that
allows DNA to enter the heteroduplex binding site. Further studies will
be required to test this idea. However, proposals concerning the role
of ATP binding and hydrolysis by MutS in mismatch repair must consider
the complexity inherent in the possibility of differential
occupancy of multiple nucleotide binding sites.