From the 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
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ABSTRACT |
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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.
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
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
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.
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
[
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 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]110 Structure Determination of Taq MutS Complexed with Mismatch DNA
and ADP·BeFx--
Taq MutS (1-768 amino
acids) was co-crystallized with the 23
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 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
(99 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), ATP 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 ATP
To test whether phosphate analogs could be used to identify a
MutS·ATP-like complex, we performed ATPase assays on Taq
MutS incubated with [
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 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·110 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
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 ATP 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 ATP 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 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.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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
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.
-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.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]ATP. When indicated, a 23-bp mismatch substrate
containing an unpaired T (T insertion; 23
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 [
-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).
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
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]110
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 110
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.
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.
T), two ADP·BeF
Data collection and Refinement statistics
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).
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
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
-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).
View larger version (23K):
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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 [ -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 [
-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 [
-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
[
-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.
-phosphate hydrolysis analyses to show that in the absence of DNA, ADP
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.
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·110
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·110
T complex (Fig. 2B).
View larger version (39K):
[in a new window]
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 110 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]110
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]110
T DNA in
the absence of MutS. *, mobility of unbound substrate. **, mobility of
E. coli MutS·DNA complex.
-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.
View larger version (45K):
[in a new window]
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 1.0
. 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.
S; we
hypothesize that this is due to the inhibition of ATP hydrolysis
because ATP and ATP
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.
View larger version (54K):
[in a new window]
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 ATP 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
99
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 99
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
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
-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).
-phosphate group in ATP, the less
negatively charged BeF
![]() |
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.
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.
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