Differential and Simultaneous Adenosine Di- and Triphosphate Binding by MutS*

Keith P. Bjornson and Paul ModrichDagger §

From the Department of Biochemistry and Dagger  Howard Hughes Medical Institute, Duke University Medical Center, Durham, North Carolina 27710

Received for publication, January 31, 2003, and in revised form, March 3, 2003

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The roles of ATP binding and hydrolysis in the function of MutS in mismatch repair are poorly understood. As one means of addressing this question, we have determined the affinities and number of adenosine di- and triphosphate binding sites within MutS. Nitrocellulose filter binding assay and equilibrium fluorescence anisotropy measurements have demonstrated that MutS has one high affinity binding site for ADP and one high affinity site for nonhydrolyzable ATP analogues per dimer equivalent. Low concentrations of 5'-adenylylimidodiphosphate (AMPPNP) promote ADP binding and a large excess of AMPPNP is required to displace ADP from the protein. Fluorescence energy transfer and filter binding assays indicate that ADP and nonhydrolyzable ATP analogues can bind simultaneously to adjacent subunits within the MutS oligomer with affinities in the low micromolar range. These findings suggest that the protein exists primarily as the ATP·MutS·ADP ternary complex in solution and that this may be the form of the protein that is involved in DNA encounters in vivo.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 MutSalpha 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 MutSalpha 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.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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]ATPgamma S (adenosine 5' (gamma -thio) triphosphate, 1250 Ci/mmol) were obtained from PerkinElmer Life Sciences. [alpha -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: ATPgamma 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]ATPgamma 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.
[SA]=<FR><NU>K<SUB>d</SUB>+A<SUB>t</SUB>+S<SUB>t</SUB>−<RAD><RCD>(<UP>−</UP>K<SUB>d</SUB>−A<SUB>t</SUB>−S<SUB>t</SUB>)<SUP>2</SUP>−4S<SUB>t</SUB>A<SUB>t</SUB></RCD></RAD></NU><DE>2</DE></FR> (Eq. 1)
[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).
R<SUB>0</SUB><SUP>6</SUP>=<FENCE><FR><NU>9000 · (<UP>ln10</UP>) · &kgr;<SUP>2</SUP> · Q<SUB>D</SUB></NU><DE>128 · <UP>&pgr;</UP><SUP>5</SUP> · N · n<SUP>4</SUP></DE></FR></FENCE>×<FR><NU><LIM><OP>∫</OP><LL>0</LL><UL>∞</UL></LIM> F<SUB>D</SUB>(&lgr;) · ϵ<SUB>A</SUB>(&lgr;) · &lgr;<SUP>4</SUP>d&lgr;</NU><DE><LIM><OP>∫</OP><LL>0</LL><UL>∞</UL></LIM> F<SUB>D</SUB>(&lgr;)d&lgr;</DE></FR> (Eq. 2)
QD is the quantum yield of the donor, BODIPY FL (2' or 3')-AMPPNP for which a value of 0.9 was used. The kappa 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 kappa 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.
<UP>Ratio </UP>A=E · d<SUP>+</SUP> · <FR><NU>ϵ<SUP>FL</SUP>(490<UP> nm</UP>)</NU><DE>ϵ<SUP>TR</SUP>(590 <UP>nm</UP>)</DE></FR>+<FR><NU>ϵ<SUP>TR</SUP>(490 <UP>nm</UP>)</NU><DE><UP>ϵ<SUP>TR</SUP></UP>(<UP>590 nm</UP>)</DE></FR> (Eq. 3)
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 epsilon FL(490 nm)/epsilon 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).
R=<FENCE><RAD><RCD><FR><NU>1−E</NU><DE>E</DE></FR></RCD><RDX>6</RDX></RAD></FENCE> · R<SUB>0</SUB> (Eq. 4)


    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 (open circle , "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 [alpha -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.

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 (open circle ) 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.

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 (open circle ) 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.

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.

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).

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 ATPgamma S. Complexes between MutS (10 µM) and [35S]ATPgamma S (5 µM) were prepared (Fig. 6). Since this [35S]ATPgamma 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]ATPgamma S·MutS complexes with [3H]ADP led to retention of both nucleotides on the filter, and the yield of MutS-bound [35S]ATPgamma S was unaffected by [3H]ADP addition until the diphosphate concentration equaled that of [35S]ATPgamma S. Further [3H]ADP addition resulted in a modest reduction of [35S]ATPgamma 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 ATPgamma S when protein and nucleotide concentrations are in the low micromolar range.


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Fig. 6.   ADP binds to MutS·ATPgamma S complex with half-site reactivity. 10 µM MutS (monomer equivalent) and 5 µM [35S]ATPgamma S was titrated with [3H]ADP. The concentration of [35S]ATPgamma S retained on nitrocellulose is plotted on the right axis, (black-square), and retained [3H]ADP is plotted on the left axis, (open circle ). 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.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 ATPgamma 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 ATPgamma 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 (approx 46 Å, Fig. 5) in the absence of DNA is greater than that in the MutS·ADP·heteroduplex crystalline complex (approx 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.

    FOOTNOTES

* This work was supported in part by Grant GM23719 from the NIGMS, National Institutes of Health.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.

§ Investigator of the Howard Hughes Medical Institute. To whom correspondence should be addressed: Howard Hughes Medical Institute, Box 3711, Duke University Medical Center, Durham, NC 27710. Tel.: 919-684-2775; Fax: 919-681-7874; E-mail: modrich@biochem.duke.edu.

Published, JBC Papers in Press, March 6, 2003, DOI 10.1074/jbc.M301101200

2 K. P. Bjornson, L. J. Blackwell, H. Sage, C. Baitinger, D. Allen, and P. Modrich, submitted for publication.

3 K. P. Bjornson, T. Polhaus, J. Warren, J. Genschel, L. Beese, and P. Modrich, unpublished observations.

    ABBREVIATIONS

The abbreviations used are: ABC, adenine nucleotide binding cassette; AMPPNP, 5'-adenylylimidodiphosphate; FRET, fluorescence resonance energy transfer.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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