Crystal Structure and Biochemical Analysis of the MutS·ADP·Beryllium Fluoride Complex Suggests a Conserved Mechanism for ATP Interactions in Mismatch Repair*,

Eric AlaniDagger §||, Jae Young Lee§, Mark J. Schofield**, Amanda W. KijasDagger , Peggy Hsieh**, and Wei Yang§DaggerDagger

From the Dagger  Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York 14853-2703, the § Laboratory of Molecular Biology, NIDDK, National Institutes of Health, Bethesda, Maryland 20892, and the ** Genetics and Biochemistry Branch, NIDDKD, National Institutes of Health, Bethesda, Maryland 20892

Received for publication, December 26, 2002, and in revised form, January 14, 2003

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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During mismatch repair ATP binding and hydrolysis activities by the MutS family proteins are important for both mismatch recognition and for transducing mismatch recognition signals to downstream repair factors. Despite intensive efforts, a MutS·ATP·DNA complex has eluded crystallographic analysis. Searching for ATP analogs that strongly bound to Thermus aquaticus (Taq) MutS, we found that ADP·beryllium fluoride (ABF), acted as a strong inhibitor of several MutS family ATPases. Furthermore, ABF promoted the formation of a ternary complex containing the Saccharomyces cerevisiae MSH2·MSH6 and MLH1·PMS1 proteins bound to mismatch DNA but did not promote dissociation of MSH2·MSH6 from mismatch DNA. Crystallographic analysis of the Taq MutS·DNA·ABF complex indicated that although this complex was very similar to that of MutS·DNA·ADP, both ADP·Mg2+ moieties in the MutS· DNA·ADP structure were replaced by ABF. Furthermore, a disordered region near the ATP-binding pocket in the MutS B subunit became traceable, whereas the equivalent region in the A subunit that interacts with the mismatched nucleotide remained disordered. Finally, the DNA binding domains of MutS together with the mismatched DNA were shifted upon binding of ABF. We hypothesize that the presence of ABF is communicated between the two MutS subunits through the contact between the ordered loop and Domain III in addition to the intra-subunit helical lever arm that links the ATPase and DNA binding domains.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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The mismatch repair (MMR)1 system dramatically improves the fidelity of DNA replication by excising DNA mismatches that result from misincorporation errors. In Escherichia coli, MutS, MutL, and MutH play critical roles in initiating the MMR process (1). MutS protein displays both mismatch DNA binding and ATPase activities that are essential for its function. These activities enable recruitment of MutL, a matchmaker protein that interacts with MutH endonuclease and UvrD helicase, two proteins that act in conjunction with Dam methylase to impose strand specificity during MMR (2-7). During DNA replication, interactions between MMR components result in the removal of DNA mismatches via excision and resynthesis steps that occur on the newly replicated DNA strand. ATP binding and hydrolysis by both MutS and MutL appear to be essential for the mismatch recognition and for the formation of a MutS·MutL complex that recruits and activates MutH-directed incision of the newly replicated DNA strand.

Homologs of MutS and MutL have been identified in lower and higher eukaryotes; MutH homologs, however, have not been found (reviewed in Refs. 8 and 9). The yeast Saccharomyces cerevisiae, for example, contains six MutS (MSH) and four MutL (MLH) homolog proteins. In yeast DNA mismatches are recognized primarily by MSH2·MSH6 and MSH2·MSH3 complexes that display distinct mismatch binding specificities. Both of these complexes primarily interact with a single MLH1·PMS1 complex in steps that are ATP-dependent (10, 11). The mechanism of strand discrimination is unclear in eukaryotes. Recent in vitro and in vivo studies (12-19) in yeast and mammalian cells, however, have suggested that MMR proteins interact with the replication fork through an association with the DNA polymerase processivity factor proliferating cell nuclear antigen.

Three major models have been developed to explain how MMR factors initiate and execute DNA excision steps that can occur up to several kilobases away from a recognized mismatch. Each of these models incorporates a role for ATP binding and hydrolysis by the MSH and MLH proteins. In the first model, binding of MSH proteins to a mismatch substrate induces an ATP-dependent conformational change that allows recruitment of the MLH proteins. The resulting MSH·MLH complex uses the energy of ATP hydrolysis to translocate along DNA to encounter downstream repair factors such as endonucleases, exonucleases, and helicases (20, 21). In a second model, ATP acts as a molecular switch analogous to G-proteins (22, 23). In the absence of DNA the MSH proteins are bound to ADP. Mismatch binding provokes ADP Right-arrow  ATP exchange, which allows MSH proteins to form an ATP hydrolysis-independent sliding clamp that slides along DNA to encounter downstream MMR components. The MLH proteins are thought to regulate the MSH proteins by modulating hydrolysis and/or ADP Right-arrow  ATP exchange activities (22). Finally, the MutS ATPase activity has been hypothesized to play a proofreading role in MMR by both verifying mismatch recognition and authorizing repair (24). This model hypothesizes that MSH proteins have to simultaneously bind ATP and the mismatched DNA to recruit MutL and initiate repair, thus enhancing the specificity of mismatch recognition and avoiding mismatch-independent initiation of repair. In this model, the MSH proteins activate downstream repair functions while remaining bound to the mismatch site.

Crystal structure analysis of both the Taq and E. coli MutS·DNA complexes suggested that mismatch recognition occurs through an induced-fit mechanism between functionally asymmetric DNA binding domains in the MutS homodimer and kinked mismatched DNA (25, 26). Regions were also identified in MutS that could serve as transmitters between the ATP binding and mismatch recognition domains. Although MutS· DNA·ATP complexes have been refractory to x-ray crystallography analysis, both Taq MutS·DNA·ADP and E. coli MutS· DNA·ADP complexes have been solved. In the Taq MutS·DNA structure, ADP was localized to both ATP-binding sites; in E. coli MutS only the A subunit, which directly interacts with the mispaired base, bound ADP. Conformational differences between the Taq MutS·DNA and MutS·DNA·ADP structures have provided a model to explain how ATP might influence mismatch binding by MutS, even though the two binding sites are separated by ~70 Å (24). The small changes in the ATP-binding site that resulted from ADP binding appeared to be amplified by a long lever arm that links the ATP and DNA binding domains, resulting in a rotation of the two protein subunits toward each other and a pronounced increase in the mobility of domain IV in subunit B that stabilizes the mispaired base in the major groove.

The Taq and E. coli MutS·DNA structures both contain a disordered six amino acid region in each subunit (629-634, SDDLAG, in Taq MutS). The first of the two aspartate residues in this region is highly conserved among MutS homologs. These residues, which map immediately adjacent to a conserved nucleotide-binding N2 motif, remained disordered even in the MutS·DNA structures containing bound ADP (24-26). Junop et al. (24) hypothesized that these residues participate in conformational changes upon ATP binding that are amplified throughout the MutS molecule in a manner analogous to that suggested by analysis of the MutS·DNA·ADP complex. Because the surrounding residues of this disordered loop are in the vicinity of the ATP-binding site, the ordering of these residues may depend on the presence of an intact ATP including the gamma -phosphate. Previous mutational studies (24) indicated that the MutS-bound ATP is at a high energy state favoring hydrolysis unless a nearby conserved carboxylate (Glu-694 in E. coli MutS) is replaced by alanine. It was also found that the E694A mutant MutS, which fails to hydrolyze ATP, is still able to mediate DNA-dependent activation of MutH.

In this study, we searched for ATP analogs that can bind to Taq MutS with the goal of visualizing the disordered loop surrounding the ATP-binding site and elucidating the role of ATP in recruiting downstream mismatch repair components. We report here the inhibition of the ATPase activity of the Taq, E. coli, and S. cerevisiae MutS proteins by ADP·beryllium fluoride (BeFx) (ABF), the crystallographic studies of Taq MutS complexed with mismatched DNA and ABF, and biochemical analyses of the effect of ABF on mismatch recognition by Taq, E. coli, and S. cerevisiae MutS homolog proteins.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Protein Purification-- Taq MutS proteins, the large fragment of 1-768 amino acids and full-length, and E. coli MutS and MutL were overexpressed in E. coli and purified as described (2, 24, 25). MSH2·MSH6 and MLH1·PMS1 were overexpressed and purified as described in Alani (27) and Hall et al. (28), respectively.

ATPase Assays-- Taq MutS ATPase activity was measured in 10-15 µl reactions containing 20 mM Tris, pH 8.0, 1 mM DTT, 5 mM MgCl2, 60-90 mM KCl, and 20-1000 µM [alpha -32P]ATP. When indicated, a 23-bp mismatch substrate containing an unpaired T (T insertion; 23Delta T; Ref. 25) was included at 1 µM, and nucleotide analogs were included as described. Reactions were initiated by MutS addition (0.5-2.0 µM final concentration) and were incubated at 37 °C for 15 min. E. coli MutS ATPase activity was measured under similar conditions with the exception that a 35-bp mismatch substrate containing a T:G mismatch (35GT) (5'-CCCTGTGCGACGCTAGCGTGCGGCCTCGTCTGTCC-3', and the complementary strand except for a G opposite the underlined T) was used, and reactions were incubated at 22 °C for 60 min. MSH2·MSH6 ATPase activity was measured in 10 µl reactions containing 20 mM Tris, pH 7.5, 2.0 mM MgCl2, 1.0 mM DTT, 100-160 mM NaCl, and 20-100 µM [alpha -32P]ATP. When specified, a 37-bp mismatch substrate containing a single A insertion (37-+1, Ref. 27) was included at 1 µM, and ADP and ADP·BeFx was included at 80 and 250 µM. The reactions were initiated by addition of MSH2·MSH6 (0.1-0.4 µM final concentration) and were incubated at 30 °C for 15 min. ATP hydrolysis reactions were terminated by the addition of EDTA, pH 8.0, to 25 mM, and the extent of ATP hydrolysis was determined using polyethyleneimine cellulose F (Merck) thin layer chromatography (2) followed by phosphorimaging quantification (Molecular Dynamics). Sodium fluoride, aluminum chloride, and beryllium chloride were purchased from Fluka. Sodium tungstate was obtained from ICN. Orthovanadate (Sigma) solution was prepared according to the Salmon laboratory web site (www.bio.unc.edu/faculty/salmon/lab/salmonprotocol.html).

Throughout this paper X µM ADP·BeFx (where "X" refers to any concentration) consists of X µM ADP, X µM BeCl2, and 5× µM NaF. NaF (0.8 M, stored at -70 °C) and BeCl2 (0.2 M, stored at 4 °C) stock solutions were prepared by dissolving each compound in double-distilled water in plastic tubes. ADP, BeCl2, and NaF were mixed immediately before inclusion in the ATPase assay or crystal soaking experiments (see below). ABF is thought to act as an ATPase inhibitor as follows. In the presence of NaF, the chloride atoms in BeCl2 are thought to be displaced by fluorides to form beryllofluoride (BeFx) with BeF<UP><SUB>3</SUB><SUP>−</SUP></UP>·H20 the predominating species in aqueous solution (29). BeFx is thought to inhibit the ATPase activity of some ATPases by tightly binding in conjunction with ADP to the active site. The active site acts as a template to facilitate coordinate covalent bonding between ADP and BeFx (29).

Measurement of the Half-life of Taq and E. coli MutS·DNA Complexes-- Binding of Taq MutS to mismatched DNA was performed for 5 min at room temperature in a reaction containing 0.2 µM MutS, 1 nM [32P]110Delta T substrate (containing a single T insertion in the middle of the substrate, Ref. 30), 20 mM HEPES, pH 7.8, 50 mM NaCl, 1 mM DTT, 0.1 mg/ml BSA, 100 µM ADP. At t = 0, a 37-bp T bulge substrate (30) was added at 2 µM in combination with either no nucleotide or 80 µM ADP, ATP, or ABF. At t = 15, 30 45, 60, and 75 s, aliquots were removed and electrophoresed in a 5% native polyacrylamide gel as described by Schofield et al. (30). After electrophoresis, gels were dried and quantified on a Fuji BAS-1500 phosphorimager. The dissociation of E. coli MutS·DNA complexes was assayed as follows. 50 nM MutS dimer was preincubated with 2 nM [32P]110Delta T in 20 mM HEPES, pH 7.8, 50 mM NaCl, 5 mM MgCl2, 1 mM DTT, 0.1 mg/ml BSA, 5% sucrose for 5 min at room temperature. At t = 0, 100 nM unlabelled 110Delta T and 100 µM ATP, ADP, or ABF were added. At t = 15 and 300 s, aliquots were removed and electrophoresed in a 4% Tris-glycine gel containing 5 mM MgCl2.

Structure Determination of Taq MutS Complexed with Mismatch DNA and ADP·BeFx-- Taq MutS (1-768 amino acids) was co-crystallized with the 23Delta T substrate in buffer C (25% polyethylene glycol 4000, 100 mM cacodylate, pH 5.9, 100 mM ammonium sulfate, 10 mM MgSO4, 1 mM DTT, 3% 2-methyl-2,4-pentanediol) as described previously (25). Protein-DNA binary complex crystals were then soaked for 1 h in buffer C containing 1 mM ADP and then transferred into buffer C containing 1 mM ADP, 2 mM BeCl2, and 10 mM NaF (buffer D) for 24 h to form the ternary complex of MutS·DNA·ABF. Co-crystals were then successively soaked in buffer D containing 5% and then 10% ethylene glycol prior to cryopreservation. Attempts to co-crystallize all three components using ~500 screening conditions yielded only very small crystals unsuitable for x-ray diffraction.

The ABF-soaked MutS·DNA co-crystals diffracted X-rays to 3.1-2.7 Å resolutions (see Table I). Several data sets were collected in-house using a phosphorimaging plate mounted on a Rigaku RU200 x-ray generator. Data were processed using HKL (31), and molecular replacement was successfully carried out using the MutS·DNA·ADP structure as a search model by CNS (32). The crystal diffracting to 3.1 Å resolution showed the most complete electron density map for ADP·BeF<UP><SUB>3</SUB><SUP>−</SUP></UP> and was chosen for further refinement. The final refined model contains two Taq MutS subunits, both of which contain 1-765 amino acids, one DNA duplex (23Delta T), two ADP·BeF<UP><SUB>3</SUB><SUP>−</SUP></UP> with full occupancy, two structural SO4 ions, and 99 water molecules. The R and Rfree were refined to 20.9 and 25.9% using CNS (32). Coordinates reported in this paper have been deposited in the Protein Data Bank with ID code of 1NNE.


                              
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Table I
Data collection and Refinement statistics

MSH2·MSH6 DNase I Footprinting-- 20 µl binding reactions were performed at 30 °C for 4 min in 20 mM HEPES, 40 µg/ml acetylated BSA, 1 mM CaCl2, 1 mM MgCl2, 25 mM NaCl, 250 nM MSH2·MSH6, and 10 nM 32P-labeled 99 bp substrate containing a T insertion (99Delta T). This DNA substrate was prepared by annealing the following PAGE purified oligonucleotides (IDT): 5'-GCATTGCTATCTGATTTGGCGCACCGGATCCTGACTGGAATCGTGGC GATACCGAGCTCCTGATGGCCATAGACGCATTGCTATCTGATTTGGCGCACCG and 5'-CGGTGCGCCAAATCAGATAGCAATGCGTCTATGGCCATCAGGAGCTCG GTATCGCCCGATTCCAGTCAGGATCCGGTGCGCCAAATCAGATAGCAATGC. ATP (400 µM), ADP (400 µM), and ABF (400 µM) were included as indicated. Following a 4 min incubation at 30 °C 0.5 µM cold 37-+1 substrate (27) was added for 2 min at room temperature followed by addition of DNase I (Stratagene) at 0.02 units/µl. The DNase I reaction was quenched after a 2 min incubation at room temperature by the addition of 100 µl of stop solution (85% ethanol, 1.5 M ammonium acetate, 20 µg/ml yeast tRNA). Samples were allowed to precipitate on dry ice for at least 30 min, then spun for 25 min at 15,000 rpm washing once with 75% ethanol. Dried samples were resuspended in 4 µl 90% formamide, 50 mM EDTA and denatured at 92 °C for 2 min and snap cooled on ice. Samples were run at 75 watts on a denaturing 12% PAGE for one hour. Gels were dried and visualized using a phosphorimaging system and the Imagequant program (Molecular Dynamics).

Ternary Complex Formation Assay-- The formation of a ternary complex containing MSH2·MSH6, MLH1·PMS1, mismatched DNA, and ATP, ADP, or ABF was examined in gel shift assays. 15 µl reactions were set up on ice in 20 mM HEPES, pH 7.6, 40 µg/ml AcBSA, 8% sucrose, 1 mM MgCl2, and 25 mM NaCl. 32P-labeled mismatched duplex DNA (+1 48-mer, Ref. 33) was present at 133 nM and ATP (Amersham Biosciences), ADP (Amersham Biosciences), ATPgamma S (Sigma), and ABF were diluted in 20 mM HEPES, pH 7.6, and included at a final concentration of 400 µM. Following MSH2·MSH6 (133 nM) and MLH1·PMS1 (82 nM) addition, binding reactions were incubated at room temperature for 8 min. Samples were then electrophoresed at 130 V for 50 min at room temperature in 4% (w/v) native polyacrylamide gels containing 0.5× Tris borate EDTA buffer. Gels were dried and then visualized using a phosphorimaging system and analyzed using Imagequant (Molecular Dynamics).

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

ABF Is a Strong Inhibitor of the Taq MutS, E. coli MutS, and S. cerevisiae MSH2·MSH6 ATPase Activities-- Attempts to obtain the crystal structures of Taq MutS in an ATP-bound state using non-hydrolyzable ATP analogs, such as ATPgamma S or AMPNP, have been unsuccessful. Only MutS·ADP complexes were identified, suggesting that the ATP-bound state of MutS was unstable (24). We therefore searched for ATP analogs that can strongly inhibit the Taq MutS ATPase activity as an indication that it competes well for the ATP-binding site and with the goal of capturing Taq MutS bound with both mismatch DNA and an ATP analog. Biochemical and structural studies have suggested that in the presence of ADP, phosphate analogs such as orthovanadate, tungstate, and aluminum tetrafluoride mimic the metastable ADP·Pi transition state that occurs after hydrolysis, whereas ADP·beryllium fluoride mimics ATP (29, 34, 35). Consistent with this idea was the observation that the E. coli MutL·ADP·BeF<UP><SUB>3</SUB><SUP>−</SUP></UP> complex displayed a conformation as measured in x-ray crystallographic analysis that was indistinguishable from the MutL·AMPPNP complex.2

To test whether phosphate analogs could be used to identify a MutS·ATP-like complex, we performed ATPase assays on Taq MutS incubated with [alpha -32P]ATP in the presence of ATP analogs. As shown in Fig. 1A, ABF most strongly inhibited the Taq MutS ATPase, with tungstate displaying no inhibition and vanadate and aluminum fluoride showing only weak inhibition. The inhibition by ABF was concentration-dependent, and under conditions where ATP and ABF concentrations were identical, the Taq MutS ATPase activity was reduced to ~5% of the uninhibited activity (Fig. 1B). Similar inhibition profiles were observed for both the E. coli MutS (Fig. 1C) and S. cerevisiae MSH2·MSH6 ATPases (Fig. 1D), suggesting a conserved mechanism of inhibition. It is important to note that the inhibition of the E. coli and Pseudomonas aeruginosa MutS ATPase activities by ortho- and decavanadate was recently reported by Pezza et al. (36).


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Fig. 1.   The effect of ATP analogs on the Taq MutS, E. coli MutS, and S. cerevisiae MSH2·MSH6 ATPase activities. A, inhibition of Taq MutS ATPase by ATP analogs. Taq MutS and [alpha -32P]ATP were included at 2.0 and 250 µM, respectively, and ADP (250 µM), tungstate (0.5 mM), vanadate (0.5 mM), BeCl2 (0.5 mM), AlCl3 (0.5 mM), and NaF (2.5 mM) were included as indicated. B, ATPase assays performed with 0.5 µM Taq MutS and the indicated concentrations of [alpha -32P]ATP, ADP, and ABF. C, inhibition of the E. coli MutS ATPase activity by ADP and ABF in the presence and absence of mismatched DNA. ATPase assays performed with 1.0 µM E. coli MutS and ADP, ABF (80 µM), and 35GT mismatch substrate (2.0 µM) were included as indicated. D, inhibition of the MSH2·MSH6 ATPase by ABF. 0.4 µM MSH2·MSH6 was incubated with the indicated concentrations of [alpha -32P]ATP. ADP (80 µM) and ABF (80 µM) were included in the reactions as indicated. E, inhibition of the MSH2·MSH6 ATPase activity by ABF in the presence and absence of mismatched DNA. 0.1 µM MSH2·MSH6 incubated with 50 µM [alpha -32P]ATP. ADP (80 or 250 µM), ABF (80 or 250 µM), and 1.0 µM 37-+1 substrate were included in the ATPase reactions as indicated.

Previous studies with human MSH2·MSH6 (22) and E. coli MutS proteins (37) suggested that the rate-limiting step in MSH protein ATP hydrolysis is altered by mismatch DNA. Gradia et al. (22) utilized ADP exchange and single turnover gamma -phosphate hydrolysis analyses to show that in the absence of DNA, ADP Right-arrow  ATP exchange by the human MSH2·MSH6 ATPase was rate-limiting. However, in the presence of mismatch DNA, nucleotide exchange was dramatically increased. Consistent with this observation, Bjornson et al. (37), using pre-steady state chemical quench analysis, found that in the absence of DNA the rate-limiting step of the E. coli reaction was after hydrolysis, indicating that under saturating conditions MutS is predominantly in an ADP-bound form. In the presence of DNA, however, the rate-limiting step shifted to either prior or during hydrolysis. Together, these studies suggest that the presence of mismatch DNA shifts the rate-limiting step from ADP (product) release to ATP binding or hydrolysis (product formation). If ABF inhibits the MSH ATPases by inhibiting nucleotide exchange, then inhibition by this analog might be reduced in the presence of mismatch DNA because nucleotide exchange would no longer be rate-limiting. The effect of ABF on the ATPase activity of E. coli MutS (Fig. 1C) and S. cerevisiae MSH2·MSH6 (Fig. 1E) was significantly reduced, but not eliminated, in the presence of mismatch DNA. For MSH2·MSH6, the presence of homoduplex DNA, which stimulated ATPase activity to a lesser extent than mismatch DNA, also reduced the inhibition by ABF. The reduction in inhibition, however, was less than was observed with mismatch DNA (data not shown). Together, these studies provide additional support that ABF inhibits the ATPase activity of MutS family members through a common mechanism.

ABF Does Not Induce the Dissociation of Taq or E. coli MutS from Mismatch DNA-- Previous studies (20-22, 38, 39) have shown that ATP promotes the dissociation of MSH proteins from mismatched DNA. We tested whether ABF conferred a similar effect on the Taq and E. coli MutS proteins. The half-life of the Taq MutS·110Delta T complex was investigated in the presence and absence of nucleotide using gel shift assays (Fig. 2A, Ref. 30). When challenged with as low as 10 µM ATP, the half-life of a Taq MutS·110Delta T complex was reduced to less than 8 s; an ADP challenge, however, yielded a half-life of 140 s. As shown in Fig. 2A, ABF and ADP challenges were indistinguishable. A similar result was observed for the E. coli·MutS·110Delta T complex (Fig. 2B).


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Fig. 2.   The effect of ABF on the stability of the Taq and E. coli MutS·DNA complexes. A, half-life of Taq MutS·DNA complexes. Taq MutS was preincubated with 32P-labeled 110Delta T DNA followed by the addition at time 0 of excess cold competitor mismatched DNA and 80 µM ADP, ATP, or ABF as indicated. Data were quantified and plotted as fraction bound versus time from which half-lives were determined. B, half-life of E. coli MutS-DNA complexes. E. coli MutS was preincubated with [32P]110Delta T DNA followed by the addition at time 0 of excess cold competitor DNA and 100 µM ADP, ATP, or ABF as indicated. Lane C contains [32P]110Delta T DNA in the absence of MutS. *, mobility of unbound substrate. **, mobility of E. coli MutS·DNA complex.

Crystal Structure of the Taq MutS·DNA·ABF Complex-- The overall structures of the MutS·DNA·ABF and MutS·DNA·ADP complexes were quite similar (Fig. 3). The major differences were: 1) both ADP·Mg2+ moieties in the MutS·DNA·ADP structure were replaced in the MutS·DNA·ABF complex by ADP·BeF<UP><SUB>3</SUB><SUP>−</SUP></UP>, and the two SO4 in the MutS·DNA·ADP structure, which resembled the hydrolyzed gamma -phosphates were no longer observed; (2) a disordered loop, residues 629-634 in the MutS B subunit, was traceable in the MutS·DNA·ABF structure, whereas the equivalent loop in the A subunit of the structure remained disordered; (3) the DNA binding domains of MutS together with the DNA was shifted upon ABF binding.


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Fig. 3.   Crystal structure of a MutS·DNA·ABF complex. A and B, the ABF bound to the A and B subunits shown with the 2(Fo-Fc) electron density map contoured at 1.0sigma . The newly formed loop in the B subunit (629-634) shown in green (including 628-638 amino acids) is close to the ABF bound to the A subunit as indicated. C, ribbon diagram of the MutS·DNA·ABF complex. The A subunit is shown in blue, the B subunit in green, DNA duplex in orange, and the loop 629-634 formed in the B subunit is in red. The two ABF and SO4 ions are shown as ball-and-stick in yellow and pink, respectively. The 629-634 loop of the B subunit is in close contact with residue Arg-267 (shown as light blue ball-and-stick) in the A subunit. Arg-267 is located on a variable loop between residues 263 and 271, which may play a role in communicating between subunits. In addition, DNA associated with MutS·ABF is shifted toward the exit of the DNA-binding channel relative to the DNA associated with MutS·ADP.

In the crystal structure, ABF binding was accompanied by the ordering of a loop (629-634) located in the B subunit (Fig. 3, A and B). The densities corresponding to the loop in the B subunit allowed tracing of the polypeptide backbone but did not allow the assignment of individual side chains. The tracing of this loop (Fig. 3C) rules out the possibility that domains V of the MutS dimer were swapped (40). The ordered loop in the B subunit pointed toward the ATP-binding site of the A subunit, and the closest approach (~4 Å) of this loop to the ATP is between residues 634-635 and the sugar ring of the ATP. It is not clear how this loop is affected by the presence of ABF or why residues 629-634 of the A subunit remain disordered despite full occupancy of the ATP site in the B subunit by ABF. It is not surprising, however, that the two subunits are asymmetric because only the A subunit directly interacts with the mismatched base and only the A subunit of E. coli MutS is bound with ADP (26). It remains a possibility that the loop in the B subunit captured in the crystal is only a remnant of the active ATP-bound form of the MutS·DNA ternary complex.

The root mean square deviation between the MutS proteins bound with ADP and with ABF was only 0.2 Å, which is about the accuracy of these structural models. However, after superimposing the entire ~1500 protein residues between the two structures, it is apparent that the DNA associated with MutS·ABF was shifted by ~0.4 Å toward the exit of the DNA-binding channel relative to the DNA associated with MutS·ADP (see Alani et al. _movie2.gif in Supplementary Material). ATP is known to dissociate DNA from MutS, and ABF can dissociate DNA shorter than a 30 mer from MutS in solution,3 such as the one we used in the crystal (Fig. 2). In the MutS·DNA·ABF crystal structure, the newly formed loop in the B subunit was in close contact with residue Arg-267 in domain III of the A subunit. We hypothesize that the presence of ABF is communicated between the two MutS subunits through the contact between the loop (629-634) and domain III in addition to the helical lever arm linking the ATPase and DNA binding domains within one subunit.

A Ternary Complex Containing MSH2·MSH6, MLH1·PMS1, and Mismatch DNA Forms in the Presence of ABF-- In the presence of ATP, MSH2·MSH6 and MLH1·PMS1 form a ternary complex on mismatched DNA that is thought to reflect an early step in mismatch repair (11). We tested the ability of ABF (100-400 µM) to mediate ternary complex formation in native acrylamide gels containing +1 mismatch substrate, MSH2· MSH6, and MLH1·PMS1 (see "Experimental Procedures"). At all concentrations, ABF promoted ternary complex formation to roughly the same extent that was observed with ATP (Fig. 4A and data not shown). At these ABF concentrations, MSH2· MSH6 remained bound to the mismatch as measured in a DNase I protection assay, whereas the same range of ATP concentrations resulted in a significant loss of mismatch-specific binding (Fig. 4B and data not shown). The finding that ABF can induce ternary complex formation without releasing MSH2·MSH6 from the mismatch site is consistent with ternary complex formation occurring at the mismatch site. Higher levels of ternary complex were consistently observed in the presence of ATPgamma S; we hypothesize that this is due to the inhibition of ATP hydrolysis because ATP and ATPgamma S yielded similarly high levels of ternary complex in reactions performed in the absence of MgCl2 (data not shown). An analogous interaction between E. coli MutS, MutL, and mismatched DNA (30) could not be tested due to technical problems associated with including ABF in the gel and buffer during electrophoresis steps.


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Fig. 4.   A ternary complex containing MSH2·MSH6, MLH1·PMS1, and mismatched DNA forms in the presence of ABF. A, binding reactions and gel shift assays were performed as described under "Experimental Procedures." Reactions contained ADP, ABF, ATP, and ATPgamma S as indicated. *, unbound DNA substrate; **, Msh2·Msh6·DNA complex, ***, ternary complex. B, the MSH2·MSH6 footprint is maintained in the presence of ABF. The 99Delta T substrate was incubated with MSH2·MSH6 and 400 µM ATP, ADP, BF (400 µM BeCl2, 2 mM NaF) or ABF, as indicated, and then subjected to DNase I protection analysis (see "Experimental Procedures"). In the absence of nucleotide, MSH2·MSH6 protected 24 nucleotides on the bottom strand of 99Delta T, from nucleotides 41-64 (counting 5' to 3'). Note that the T insertion residue is located between nucleotides 56 and 57 as indicated by the asterisk.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In this study we showed that ABF inhibited the ATPase activity of both the bacterial and yeast MSH proteins. This inhibition, however, was partially relieved by mismatch DNA, suggesting that ABF inhibits the MSH ATPases at a post-hydrolytic step in the ATPase reaction cycle. Pre-steady state, ADP exchange, and single turnover studies performed with the E. coli and human MSH proteins suggested that in the absence of DNA, ADP release was the rate-limiting step and that the MSH proteins were predominantly in an ADP-bound form under ATP saturating conditions (22, 37). The experiments presented, and the finding that BeFx in the absence of exogenous ADP acted as a strong inhibitor of the Taq MutS ATPase (Fig. 1 and data not shown), suggest that the inhibition of the MSH ATPase activity can be achieve either by addition of preformed ABF or BeFx alone. In the latter case, inhibition is thought to occur through the association of BeFx with the ATP hydrolysis product ADP to form ABF, which further slows down product release. The finding that ABF was a much weaker inhibitor in the presence of mismatch DNA can be explained by the shifting of the rate-limiting step in the ATPase reaction cycle to steps that occur prior to or during hydrolysis (22, 37). A pre-steady state analysis of the effect of ABF on the MutS ATPase activity in the presence or absence of mismatch DNA should allow a direct test of this hypothesis.

The MSH proteins belong to a family of ABC-ATPases including RAD50, the histidine permease HisP, and the ribose transporter RbsA, that display cooperative ATP hydrolysis activities that are often regulated by ligand binding (41-43). Hopfner et al. (43) solved the structure of the RAD50 catalytic domain in the presence and absence of ATPgamma S. From this analysis they suggested that a conserved signature motif in the ATP binding domain of one RAD50 subunit acted as a sensor for the presence of an ATP gamma -phosphate located in the domain of the other subunit and vice versa. In RAD50 this sensing mechanism is hypothesized to link ATP binding to large structural changes in the ATP binding domain including displacement of the signature motifs, RAD50 dimer association, and the formation of a positively charged groove between the RAD50 dimers that acts as a DNA-binding surface. This elegant cooperativity model provides an explanation for how RAD50 protein binds DNA in an ATP-dependent fashion (44).

The MutS proteins share many of the characteristics of the RAD50 family of proteins. For example, the MutS ATPase active site is a composite that requires dimerization for function (45). In addition, binding of its DNA substrate, i.e. mismatches, by MutS is also modulated by ATP binding and hydrolysis (24, 26, 45). MutS, although sharing some characteristics, displays features that appear different from those described for RAD50. First, in MutS the interactions between the DNA binding domains and mismatched DNA are asymmetric (25, 26). Second, crystallographic analysis is consistent with an asymmetry in the ATPase domains. In E. coli MutS only the A subunit was bound to ADP; in Taq MutS, ABF only ordered the B subunit loop that is in close proximity to the ATP binding domain in the A subunit. Analysis of the eukaryotic MSH proteins also suggests an asymmetry with respect to the ATPase activity of each subunit. For example, the MSH6 ATPase of the S. cerevisiae MSH2·MSH6 complex appears to respond to mismatch binding, whereas the MSH2 subunit appears insensitive (33, 38, 39).

Our analyses of the MutS·DNA·ADP and MutS·DNA·ABF complexes argue against the idea that large conformational changes occur in the Taq MutS ATP binding domain upon ATP binding. In the Taq MutS·DNA·ADP structure, ADP binding resulted in only a small (0.4 Å) change in the ATP-binding site. However, this change appeared to be amplified by the long lever arm, resulting in a pronounced change in the conformation of the domains that contact the mismatched DNA (25). Analysis of the Taq MutS·DNA·ABF structure suggested that the presence of ABF is communicated between the two MutS subunits through contacts between the ordered loop in subunit B and the ATPase and III domains in subunit A. These interactions are then transmitted to the DNA mismatch through domain II and the lever arm. As shown in Fig. 3, the ABF-mediated changes in the MutS·DNA·ADP structure were subtle but suggested conformational changes that were directed toward DNA release. This inter-subunit communication may be similar to that observed for RAD50. The subtle changes observed in the Taq MutS·DNA·ABF complex compared with the Taq MutS·DNA·ADP complex may not be an unreasonable depiction of the active state of the complex. Biochemical studies have shown that ADP can also promote dissociation of MutS from mismatch DNA, though to a much lesser extent than with ATP (21, 30), and we observed the dissociation of MutS in the presence of ABF from smaller (< 30 bp) but not larger mismatch substrates.

Why does ABF fail to mediate the dissociation of the MSH proteins from mismatch DNA? One possibility is that the MutS·ATP·mismatch complex is very unstable. Relative to the gamma -phosphate group in ATP, the less negatively charged BeF<UP><SUB>3</SUB><SUP>−</SUP></UP> could allow ABF to bind to the MutS·DNA binary complex in the crystal structure and induce some but not all of the conformational changes required for MMR. Such an idea would explain why ABF did not mediate dissociation from mismatch substrates yet allowed MSH·MLH interactions. Alternatively, the inter-subunit contacts that we observed in a relatively low resolution crystal structure could have been more pronounced if the MutS·DNA·ABF complex was not trapped in the crystal lattice; thus the possibility remains that the fully "active" form was not revealed. Regardless, the observed conformational changes suggest that the loop formation plays an important role in regulating mismatch binding and in mediating MSH·MLH interactions. Mutagenesis of the loop region, in particular the highly conserved aspartate residue, as well as residues such as Arg-267 that come in close proximity to the ordered loop in the MutS·DNA·ABF structure, could provide a direct test of this hypothesis.

In conclusion, this study suggests that ABF is a strong inhibitor of the ATPase activity of E. coli, Taq, and S. cerevisiae MSH proteins and that this inhibition appears significantly stronger than that observed for other ATP analogs such as ortho-vanadate and aluminum tetrafluoride (Fig. 1 and data not shown). For E. coli MutS and S. cerevisiae MSH2·MSH6, the ATPase inhibition by ABF was greatly attenuated when assays were performed in the presence of mismatch DNA. In addition, ABF prevented the dissociation of all three MSH family mismatch complexes. These data suggest that the mode of communications between the DNA- and ATP-binding sites propagated through the structure of MutS is conserved among the MSH family proteins. Because of technical obstacles encountered in the gel shift studies utilizing E. coli MutS, MutL, and ABF, our data leave open the possibility that conformational changes required to recruit downstream repair factors (e.g. ternary complex formation) may not be as extensively conserved. We would not be surprised if ABF is only able to mediate such interactions in the eukaryotic system because the bacterial and eukaryotic MSH and MLH factors differ with respect to subunit organization and DNA substrate specificity.

    ACKNOWLEDGEMENTS

We thank Fred Dyda, Phillip Cole, Kyoshi Mizuuchi, Alba Guarne, Jennifer Surtees, Hong Ling, James Hu, and Kelly Rausch for technical advice and many helpful discussions.

    FOOTNOTES

* 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.

This work is dedicated to the memory of Stanley A. Racoosin.

The atomic coordinates and the structure factors (code 1NNE) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

The on-line version of this article (available at http://www.jbc.org) contains a movie that compares the structures of the Taq MutS·DNA·ADP and MutS·DNA·ABF complexes.

Supported by National Institutes of Health Grant GM53085.

|| To whom correspondence may be addressed: 459 Biotechnology Bldg, Ithaca, NY 14853-2703. Tel.: 607-254-4811; Fax: 607-255-6249; E-mail: eea3@cornell.edu.

Dagger Dagger To whom correspondence may be addressed: 9000 Rockville Pike, Bldg. 5, Rm B1-03, Bethesda, MD 20892. Tel.: 301-402-4645; Fax: 301-496-0201; E-mail: wei.yang@nih.gov.

Published, JBC Papers in Press, February 11, 2003, DOI 10.1074/jbc.M213193200

2 J. Hu and W. Yang, unpublished data.

3 J. Y. Lee and W. Yang, unpublished data.

    ABBREVIATIONS

The abbreviations used are: MMR, mismatch repair; ABF, ADP·beryllium fluoride; DTT, dithiothreitol; BeFx, beryllium fluoride; BSA, bovine serum albumin.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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