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INTRODUCTION |
Mismatch repair (MMR)1
corrects mutagenic mispaired bases that arise in DNA due to
misincorporation of nucleotides during DNA replication or as a result
of chemical damage to DNA and DNA precursors. MMR also repairs
mispaired bases present in recombination intermediates and acts to
prevent recombination between divergent DNAs (for selected reviews, see
Refs. 1-6). The importance of MMR to the cell is illustrated by the
observation that inactivation of some human MMR genes underlies both
inherited and sporadic cancers (for selected reviews, see Refs. 7-9).
The most progress in identifying MMR proteins has been made in
Escherichia coli (for selected reviews, see Refs. 2 and
4-6), where MMR has been reconstituted with the MutH, MutL, MutS, and
UvrD (helicase II) proteins along with DNA polymerase III holoenzyme,
DNA ligase, single-strand DNA binding protein, and one of the
exonucleases: exonuclease I, exonuclease VII RecJ protein, or
the recently implicated exonuclease X (10). In eukaryotes, MMR is known
to require homologs of the MutS and MutL proteins as well as DNA
polymerase 
, proliferating cell nuclear antigen, replication factor
C, replication factor A, and exonuclease I, although other MMR
proteins remain to be identified or definitively shown to act in MMR
(for selected reviews, see Refs. 2-6).
Central to MMR is the bacterial MutS protein and its eukaryotic
homologs Msh2, Msh3, and Msh6. In bacteria, MutS appears to function as
a dimer that recognizes mispaired bases in DNA and coordinates the
mismatch repair reaction (11-14). In eukaryotes, the MutS equivalents
are the Msh2-Msh6 (MutS
) and Msh2-Msh3 (MutS
) heterodimers, which
have different but overlapping mispair recognition specificity
(15-19). The observation that the unique subunits of these
heterodimers (Msh3 and Msh6) determine mispair recognition specificity
rather than the common subunit (Msh2) suggested that the heterodimers
and mispair recognition are asymmetric in nature (3, 4, 15). Studies on
the interaction between ATP and the Msh complexes or MutS bound to a
mispair have indicated that a conformational change induced by ATP
binding results in the formation of a ring-like complex that is clamped
on the DNA and can slide along it (20-24). These views were borne out
by the crystal structure of MutS bound to a mispaired base,
demonstrating an induced fit mode of binding resulting in the formation
of a protein ring in which only one monomer is primarily responsible
for interaction with the mispaired base (12, 13, 25). Mispair
recognition by MutS involves stacking of a Phe residue onto a ring of
the mispaired base, kinking of the DNA facilitated by a Glu residue that contacts the DNA, and other specific amino acid-DNA contacts. Interactions between mispair and ATP binding are evident in the crystal
structure. Genetic studies have shown that many of the residues
critical for mispair binding of MutS are conserved in Msh6 and are
required for mismatch recognition, supporting the asymmetric view of
mispair recognition by both MutS and Msh2-Msh6 (26-32). However,
mispair recognition by the Msh complexes cannot entirely mimic that by
MutS because the msh6-E339A mutation has little effect on
MMR, whereas the equivalent E. coli mutation (muts-E38A) eliminates MMR (29, 32). In addition, Msh3
clearly lacks the equivalent of E. coli Phe36
that is critical for mispair binding by MutS (12, 13, 27).
Several models have been proposed for how MutS and the Msh complexes
coordinate MMR. In one model, MutS binding results in the assembly of a
higher order complex of proteins that translocate along the DNA to
coordinate with the MutH endonuclease activity, which is then activated
to initiate MMR (22, 33). The MutS ring structure likely facilitates
this translocation. In a second model, the MutS complex and the MutH
incision protein recognize their binding sites independently, and
looping facilitates the interaction between the two complexes and other
proteins, leading to the transactivation of MutH and hence MMR (34).
Finally, it has been suggested that the Msh complexes and MutS undergo a conformational change upon ATP binding, converting them to a clamp
structure. The clamp then slides along the DNA and signals other
proteins involved in MMR (20, 21, 24, 35). Integral to these models is
the ability of the MutS or Msh complex to interact with and, in some
cases, activate other proteins that function in MMR. Many such
interactions have been characterized, including the interaction of MutL
and the Mlh1-Pms1 (Pms2 in humans) complex with mispair-bound MutS and
the Msh complexes (36-41), respectively; the interaction of
proliferating cell nuclear antigen with Msh3 and Msh6 (42-44); the
interaction of exonuclease I with Msh2, Msh3, and Mlh1 (45-47); the
interaction of helicase II with MutL (48, 49); and the activation of
MutH by MutS and MutL (34, 50, 51). The identification of these
interactions has identified potential steps in MMR, but has not,
however, shed much light on the overall mechanism of MMR and, in
particular, the mechanism of eukaryotic MMR.
We have been performing a number of genetic studies in
Saccharomyces cerevisiae aimed at better understanding the
mechanism of MMR. In a previous study, we described novel mutations in
MSH6 that appeared not only to inactivate the
Msh2-Msh6 complex, but also to inactivate the Msh2-Msh3 complex (26).
Here, we describe the biochemical properties of four of these mutant
Msh2-Msh6 complexes. Three of them form a much more stable complex with
a mispaired base in the presence of ATP compared with the wild-type
complex. In the case of the fourth mutant complex, a mispaired base is unable to activate the ATPase activity of the mutant complex to the
extent seen for the wild-type complex. These results support the view
that dynamic interactions of Msh2-Msh6 at a mispair are critical for
MMR.
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MATERIALS AND METHODS |
DNA Substrates--
All oligonucleotides use in the studies
described here were purified by reverse-phase HPLC after synthesis and
were obtained from Annovis (Aston, PA). The sequences of the 37-, 38-, 39-, and 41-mer oligonucleotides used for construction of substrates were as follows: 5'-ATG TGA ATC AGT ATG GTT CCT ATC TGC TGA
AGG AAA T (top strand for the GC homoduplex), 5'-ATG TGA ATC AGT ATG GTT TCT ATC TGC TGA AGG AAA T (top strand for the GT
heteroduplex), 5'-ATG TGA ATC AGT ATG AGT TCC TAT CTG CTG
AAG GAA AT (top strand for the +A insertion), 5'-ATG TGA ATC AGT ATG
ATG TTC CTA TCT GCT GAA GGA AAT (top strand for the +AT
insertion), 5'-ATG TGA ATC AGT ATG ATG CGT TCC
TAT CTG CTG AAG GAA AT (top strand for +ATGC insertion), and 5'-ATT TCC
TTC AGC AGA TAG GAA CCA TAC TGA TTC ACA T-3' (bottom strand,
synthesized with or without a biotin at the 3'-end).
Double-stranded DNA (dsDNA) substrates used for ATPase assays were
prepared as follows. Oligonucleotides were annealed in equimolar
concentrations in 1× annealing buffer (50 mM Tris-HCl (pH
7.5), 100 mM NaCl, and 10 mM
MgCl2), and the dsDNA substrates were HPLC-purified on a
Waters Gen-Pak FAX column as previously described (52, 53).
Oligonucleotides of the same sequences were used for the preparation of
dsDNA substrates for use in the IAsys binding studies. A 30% excess of
upper strand oligonucleotide was annealed to the 3'-biotinylated bottom
strand in 1× annealing buffer. After binding to the
streptavidin-coated surface of the IAsys chamber,
non-biotinylated residual single-stranded DNA was removed by
extensive wash cycles with PBST buffer (phosphate-buffered saline + 0.05% Tween 20).
Overproduction and Purification of Msh2-Msh6 Complexes--
Msh2
was coexpressed with wild-type or mutant MSH6 genes
under the control of a GAL10 promoter. The Msh2 expression
vector pRDK354 contains the GAL10-Msh2 fusion on a
2µ URA3 plasmid (54). The
pRDK945-GAL10-Msh6-WT (where WT is wild-type),
pRDK942-GAL10-Msh6-S1036P, pRDK851-GAL10-Msh6-G1067D,
pRDK852-GAL10-Msh6-H1096A, and
pRDK944-GAL10-Msh6-G1142D plasmids all consist of two DNA
fragments joined to each other: the HindIII-BlpI
vector backbone of pRDK568 (15), which consists of the
HindIII-BamHI vector backbone of pRS425
(2µ LEU2) and a BamHI-BlpI fragment containing the
GAL10 promoter fused to the N-terminal coding sequence of
Msh6 (the BlpI site is located at base pair 855 of
MSH6) and the BlpI-HindIII fragment of
MSH6 containing the C terminus of the gene containing the
allele of interest. The C-terminal fragments were obtained from the
wild-type Msh6 plasmid pRDK441, and the mutant fragments were obtained
from pRDK828-Msh6-S1036P, pRDK737-Msh6-G1067D, pRDK721-Msh6-H1096A, and
pRDK495-Msh6-G1142D (26). All of the plasmids were sequenced to ensure
that they contained no mutations other than the desired mutation. The
overexpression strains were constructed by cotransforming the S. cerevisiae strain RKY2418 (MAT ura3-52 leu2
1
his3200 pep4::HIS3 prb11.6R can1 msh2::hisG msh6::hisG),
and wild-type and mutant Msh2-Msh6 proteins were purified from cell
extracts of galactose-induced cells. Briefly, cells were grown at
30 °C in a fermentor (New Brunswick Scientific) in 10 liters of
synthetic complete dropout medium lacking uracil and leucine and
containing 3% glycerol and 0.02% lactate to an A600 of
0.8. The production of Msh2 and Msh6 proteins was induced by addition
of galactose to a final concentration of 2% for 10 h.
Galactose-induced cells (50 g) suspended in an equal volume of buffer
containing 50 mM Tris-HCl (pH 7.5), 1 mM EDTA,
10% glycerol, and 0.01% IGEPAL 20 were lysed with glass beads (Sigma)
in a bead beater (Biospec Products, Inc., Bartlesville OK); the
Msh2-Msh6 complexes were purified by sequential chromatography on
Polybuffer Exchanger 94 resin, single-stranded DNA-cellulose,
and Q-Sepharose essentially as previously described (53); and
the final protein preparation was frozen in aliquots with liquid
nitrogen. All protein preparations contained an equimolar ratio of Msh2
and Msh6 subunits and were >98% pure. Protein concentrations were
determined using the Bradford assay (Bio-Rad) and gel filtration
protein size standards (Bio-Rad) as protein concentration standards.
The yield from 50 g of cells ranged from 0.7 to 1.8 mg.
ATPase Assays--
ATPase activity was measured following a
previously published method (55, 56) in reactions (20 µl) consisting
of 25 mM HEPES (pH 7.8), 10 mM
MgCl2, 0.1 mM EDTA, 100 mM NaCl, 1 mM dithiothreitol, 10% glycerol, 0.001% IGEPAL 40, 75 µg/ml acetylated bovine serum albumin (Promega), 250 µM
[
-32P]ATP, and the indicated dsDNA substrates at 200 nM. The reactions were started by addition of the Msh2-Msh6
complex (wild-type or mutant Msh6) to 120 nM, followed by
incubation at 30 °C for the times indicated in individual
experiments. To stop the reactions, 400 µl of 10% (w/v) activated
charcoal (Norit A) in 40 mM NaH2PO4 was added, and the samples were incubated on ice for 10 min. The tubes
were then centrifuged at 14,000 rpm in a tabletop centrifuge to
sediment the charcoal, and the radioactivity present in duplicate 100-µl portions of the supernatant was determined by liquid
scintillation counting (Beckman LS6000SC). All values were determined
by subtracting the value obtained for a no-protein control that
had been incubated for the same time as the experimental reaction.
DNA Binding Analysis--
Total internal reflectance
measurements using the IAsys Auto Plus system (Affinity Sensors) were
performed to monitor the binding of wild-type and mutant Msh2-Msh6
protein complexes to dsDNA substrates (57). The IAsys Auto Plus
microcuvettes (Affinity Sensors) contained two reaction cells coated
with biotin, which were subsequently bound with excess streptavidin
(Prozyme). Unbound streptavidin was removed from the cuvette by
extensive washing with PBST buffer. One µg of the indicated annealed
dsDNA substrate containing a single 3'-biotin was added to the
streptavidin-coated reaction cells, and excess unbound single-stranded
DNA was removed by washing with PBST buffer. Reaction cells were then
equilibrated with binding buffer (25 mM HEPES (pH 7.8), 5 mM MgCl2, 110 mM NaCl, 1 mM dithiothreitol, 2% glycerol, and 0.05% IGEPAL 40). Purified Msh2-Msh6 proteins were diluted in binding buffer to final
concentrations of between 3 and 150 nM (the standard
condition was 50 nM) and then added to the cells. Protein
binding was monitored for 3-5 min or until equilibrium was reached.
Dissociation studies were performed by replacing the protein solution
with binding buffer. Addition of nucleotides to the association buffer
(protein dilution) or the dissociation buffer (binding buffer) was as
indicated in individual experiments. Following each measurement, the
protein was removed from the chip with 3 M NaCl to
regenerate the DNA-bound surface. The values for
KD, Ka, and
Kd were determined with GraFit5 software (Affinity
Sensors) as described (57). All experiments were performed at
25 °C.
Molecular Modeling--
The molecular models for the Msh2-Msh6
complex were based on the structural coordinates of E. coli
and Thermus aquaticus MutS protein (12, 13, 34). The ribbon
diagram rendition was created with MOLSCRIPT and RASTER3D, whereas the
stick diagram was created with XFIT and RASTER3D (58-60).
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RESULTS |
Structural Effects of MSH6 Amino Acid Substitutions--
In a
previous study, we described a genetic screen for mutations in
MSH6 that cause an increase in the rate of accumulating frameshift mutations when the mutation is present at the chromosomal MSH6 locus or when present on a 2µ vector in an otherwise
wild-type strain (26). The phenotype caused by the chromosomal alleles is a stronger mutator phenotype than caused by null mutations in
MSH6 (15, 17) and was suggested to result from the formation of a mutant Msh2-Msh6 complex that was capable of interfering with the
function of the Msh2-Msh3 complex (26). The dominant phenotype observed
on high copy also suggested that the mutant Msh2-Msh6 complexes formed
might be capable of titrating out downstream components of the MMR
pathway. Molecular modeling based on the MutS structure (12, 13)
indicates that three of the dominant msh6 mutations that
cause the strongest phenotypes (S1036P, G1067D, and G1142D) alter amino
acid residues in the region of the ATP-binding sites of the Msh2-Msh6
heterodimer, as does an amino acid substitution that causes a weaker
and qualitatively different phenotype (H1096A) (Fig.
1). Two of the substitutions (S1036P and
G1067D) are located near the end of two -helices and lie at the
Msh6-Msh2 dimer interface close to the P-loop of the Msh2 ATP-binding
site. Gly1067 is positioned to affect the mobility of
Msh2-Msh6 protein-protein interactions, whereas homology to other ABC
ATPases suggests that Ser1036 functions to directly
hydrogen bond the -phosphate of ATP in the Msh2 ATP-binding site and
may also affect Msh2-Msh6 protein-protein interactions (61-63). The
third substitution (G1142D) is located in the Msh2-Msh6 dimer interface
near the Msh6 ATP-binding site at the end of an -helix at the
junction between the -helix and a connector to a -sheet; this is
the first residue of what has been called the "helix-U-turn-helix"
motif in T. aquaticus MutS, which is involved in
dimerization of the two MutS subunits (14). With the possible exception
of S1036P, these substitutions do not change residues previously
thought to be critical for ATP binding or hydrolysis, but rather affect
the Msh2-Msh6 interface and might affect the conformational transitions
predicted to occur at this interface in response to ADP-ATP exchange
(61-63). The fourth substitution (H1096A) is predicted to alter a
conserved amino acid that coordinates a water molecule and likely
activates it to attack the -phosphate of ATP (62), and this
substitution would be expected to affect ATP hydrolysis by the Msh6
ATP-binding site (61, 63). To better understand the effect of these
amino acid substitutions on the activity of Msh2-Msh6, the ATPase
activity and DNA-binding properties of the wild-type Msh2-Msh6 protein and the mutant complexes Msh2-Msh6-S1036P, Msh2-Msh6-G1067D,
Msh2-Msh6-H1096A, and Msh2-Msh6-G1142D have been investigated.
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Fig. 1.
Molecular modeling of the amino acid
substitutions caused by dominant msh6 mutations.
A, ribbon diagram of the Msh2-Msh6
nucleotide-binding sites and associated dimer interface viewed from the
bottom (outside) of the complex. Msh2 and its bound ADP are in
gold, and Msh6 and its bound ADP are in blue. The
amino acid residues altered by the four mutations described in this
study are indicated in brown. B, stick diagram of
the P-loop region of the Msh6 nucleotide-binding site indicating the
magnesium ion, a coordinated water, and bound ATP.
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Analysis of the ATPase Activity of the Wild-type and Mutant
Msh2-Msh6 Complexes--
To begin to understand the interaction of the
Msh2-Msh6 complex with ATP and DNA, a time course of ATP hydrolysis and
a titration of ATP hydrolysis versus NaCl concentration for
the wild-type complex were performed. Experiments carried out in the
absence of DNA or in the presence of an oligonucleotide duplex with or without a +A insertion/deletion mispair are shown in Fig.
2. The results of these experiments show
that the wild-type complex is an ATPase in the absence of DNA and that
this ATPase activity is stimulated by DNA without a mispair and further
stimulated by DNA with a mispair. Stimulation of the ATPase by the +A
mispair showed a salt optimum of ~100 mM NaCl. Similar
results were obtained when the oligonucleotide duplex contained a GT
mispair (data not shown). The effect of DNA without a mispair or with a
+A mispair on the ATPase activity of Msh2-Msh6 in the presence of 100 mM NaCl was examined in seven independent experiments using
two different Msh2-Msh6 preparations and incubation times of 20, 30, and 40 min. The observed -fold stimulation of ATPase activity by DNA either without a mispair or with a +A mispair versus no
added DNA was 2.94 ± 0.27 and 4.79 ± 0.44, respectively,
and the -fold stimulation by DNA with a +A mispair versus
DNA without a mispair was 1.64 ± 0.20. Similarly, the -fold
stimulation of ATPase activity due to adding 100 mM NaCl to
the reactions versus no NaCl (5 mM due to
reaction components) was 1.15 ± 0.06, 0.96 ± 0.06, and 1.53 ± 0.02 in the absence of DNA or in the presence of DNA
without a mispair or with a +A mispair, respectively. These results are in contrast with previously published results for the S. cerevisiae Msh2-Msh6 complex, where mispair stimulation was either
not observed, or a +A mispair stimulated the ATPase activity by 25%,
but only at NaCl concentrations of 300-500 mM (28,
64-66). However, our results are similar to published results for the
human Msh2-Msh6 complex (56).
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Fig. 2.
Time course and NaCl dependence of ATP
hydrolysis by the wild-type Msh2-Msh6 complex in the absence of DNA or
in the presence of dsDNA with or without a +A insertion.
A, reactions were performed for the indicated times in the
presence of the indicated DNA substrates under standard conditions as
described under "Materials and Methods." The NaCl concentration in
the reactions was 100 mM. B, reactions were
performed in the presence of the indicated NaCl concentrations and DNA
substrates under standard conditions as described under "Materials
and Methods." The time of incubation was 30 min. Note that the lowest
NaCl concentration possible was 5 mM due to NaCl present in
the protein stock preparation.
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To analyze the ATPase activity of the mutant proteins, ATPase assays
were performed under conditions in which the ATPase activity was linear
with respect to time of incubation and protein concentration. Two
independent experiments were performed in which the ATPase activity of
the wild-type and mutant proteins was measured in the absence of DNA
and in the presence of an oligonucleotide duplex that was fully
base-paired or that contained a GT mispair or a +A, +AT, or +ATGC
insertion/deletion mispair. The absolute ATPase activity for each
experimental condition for one of the experiments is shown in Fig.
3; the second experiment yielded
essentially the same results (data not shown). The ratio of ATPase
activity in the presence of the different mispaired base-containing DNA substrates to ATPase activity with fully base-paired DNA and the ratio
of ATPase activity in the presence of fully base-paired DNA to ATPase
activity in the absence of DNA were then calculated for both
experiments. The average value for each ratio and the associated error
are presented in Table I. In the case of
the wild-type complex, the base-paired duplex stimulated ATP hydrolysis ~3-fold. The GT, +A, and +AT duplexes further stimulated the
reaction, consistent with the genetic view that these mispairs are
recognized by the Msh2-Msh6 complex (3, 4, 15, 17, 53, 67). The +ATGC
duplex did not stimulate the reaction above that seen with the
base-paired duplex, consistent with the genetic view that 4-base
insertion/deletion mispairs are not well recognized by the Msh2-Msh6
complex (3, 4, 15, 17, 53, 67).
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Fig. 3.
Mispair stimulation of ATP hydrolysis by the
wild-type and mutant Msh2-Msh6 complexes. Reactions were performed
with the indicated Msh2-Msh6 preparations and DNA substrates under
standard conditions as described under "Materials and Methods." The
NaCl concentration in the reactions was 100 mM, and the
time of incubation was 30 min. The data from one experiment are
presented. However, two independent experiments were performed, and the
values obtained in the two experiments did not deviate from the average
by >10%. See Table I for more comments on reproducibility.
WT, wild-type.
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Table I
Stimulation of mutant and wild-type ATPase activities by different DNA
substrates
ATPase assays were performed with the indicated substrates as described
in the legends to Figs. 2 and 3. The results reported are the ratios of
the amounts of ATP hydrolyzed in the presence of the indicated DNA
substrates. The average values from two independent duplicate
experiments are presented. NA, not applicable (the value of this ratio
is 1.0).
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All four mutant proteins had significant ATPase activity in the absence
of DNA that ranged from 55 to 188% of the activity of the wild-type
protein. These results indicate that the amino acid substitutions did
not greatly affect the intrinsic ATPase activity of the Msh2-Msh6
complex. This is consistent with the fact that three of the amino acid
substitutions (S1036P, G1067D, and G1142D) affect residues that are
generally not thought to be critical for ATP binding and/or hydrolysis,
whereas one of the amino acid substitutions (H1096A) likely affects
hydrolysis by the Msh6 ATP-binding site, but not by the Msh2
ATP-binding site. However, all of the mutant protein complexes showed
significantly altered ATPase activity in the presence of DNA with or
without a mispaired base. The mutant proteins fell into three basic
groups in regard to the effect of DNA on ATPase activity. The
Msh2-Msh6-H1096A complex showed a reduced stimulation by DNA compared
with the wild-type complex, and the mutant complex showed a modestly
reduced, although still significant, relative stimulation by
mispaired bases compared with the wild-type complex. These
data indicate that the ATPase activity of the Msh2-Msh6-H1096A complex
is generally attenuated in response to DNA, even though it binds DNA
and recognizes mispaired bases (see below). The Msh2-Msh6-S1036P and
Msh2-Msh6-G1067D ATPase activity was increased by some (but not all) of
the DNA substrates and not to the extent of the wild-type complex.
Addition of the GT mispair substrate almost completely inhibited the
ATPase activity of these two complexes, whereas the +A and +AT mispairs stimulated the ATPase activity of these complexes relative to DNA
without a mispair. The extent of stimulation by the +A and +AT mispairs
was the same for the Msh2-Msh6-S1036P and wild-type complexes, whereas
the stimulation of the Msh2-Msh6-G1067D complex was not as high as seen
for the wild-type complex. Finally, the Msh2-Msh6-G1142D ATPase
activity showed a different response to added DNA. DNA without a
mispair did not stimulate the ATPase activity, and the GT, +A, and +AT
mispairs inhibited the ATPase activity relative to DNA without a
mispair; in this case, the relative inhibition by the +A and +AT
mispairs was close to that seen with the GT mispair.
Interaction of the Wild-type and Mutant Msh2-Msh6 Complexes with
Mispaired Bases--
The mispair recognition properties of the mutant
Msh2-Msh6 complexes were then investigated using an IAsys biosensor
system. The method was calibrated by analyzing the binding properties of the wild-type complex (Fig. 4). In the
absence of a nucleotide cofactor, the wild-type complex was found to
bind duplex DNA without a mispair to a low extent and to bind GT and +A
mispairs to a significantly greater extent; somewhat greater binding
was observed with the GT mispair relative to the +A mispair. Addition
of ADP to the binding buffer reduced the binding to the GT and +A
mispairs by ~10 and ~40%, respectively. In contrast, addition of
ATP to the binding buffer reduced the binding to the GT and +A mispairs by ~90%. Next, the wild-type complex was allowed to associate with
the three different substrates in the absence of nucleotide cofactor
and then allowed to dissociate in the absence of nucleotide cofactor or
in the presence of ADP or ATP (Fig. 5).
ADP modestly increased the dissociation from the duplex and GT
substrates and more significantly increased the dissociation from the
+A substrate. In all cases, ATP markedly increased the dissociation of
the Msh2-Msh6 complex. Indeed, relative to no nucleotide in the
dissociation buffer, ATP reduced the time for half-dissociation from
the GT and +A substrates from 375 to 4 s and from 107 to 4 s,
respectively. We did not observe that binding of Msh2-Msh6 to a mispair
in the presence of ATP yielded a form resistant to ATP-induced
dissociation (data not shown), as has been observed for E. coli MutS (23). The binding of Msh2-Msh6 as a function of protein
concentration in the absence of nucleotide and the presence of ATP was
determined to calculate the various binding and dissociation constants
that describe binding; Fig. 6 shows an
example of the analysis of mispair binding versus protein
concentration (Msh2-Msh6-S1036P binding to the GT substrate is shown),
and Table II lists the relevant affinity
constants. This analysis confirmed that the relative binding affinity
of the different DNA substrates in the absence of nucleotide was
duplex < +A < GT, consistent with a 10-20-fold greater
affinity for a mispair relative to a base pair. This analysis also
showed that ATP reduced the binding affinity for a mispair to the level
seen for DNA without a mispair.
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Fig. 4.
Biosensor analysis of the association of
wild-type and mutant Msh2-Msh6 complexes with dsDNA and dsDNA
containing a GT mispair or a +A insertion. Shown is the
association phase over 5 min for DNA binding reactions performed with
the indicated Msh2-Msh6 preparations and DNA substrates under standard
conditions as described under "Materials and Methods." The
concentration of wild-type (WT) and mutant Msh2-Msh6
complexes used was 50 nM. The black lines
indicate the absence of nucleotide in the binding buffer; the red
lines indicate the presence of 250 µM ATP in the
binding buffer; and the blue lines indicate the presence of
250 µM ADP in the binding buffer.
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Fig. 5.
Biosensor analysis of the dissociation of
wild-type and mutant Msh2-Msh6 complexes from dsDNA and dsDNA
containing a GT mispair or a +A insertion. Association of the
indicated Msh2-Msh6 preparations with the indicated DNA substrates was
performed in the absence of nucleotide in the binding buffer for 5 min
exactly as described in the legend to Fig. 4. The protein solution was
replaced with binding buffer, and dissociation was monitored over 4 min. The black lines indicate the absence of nucleotide in
the dissociation buffer; the red lines indicate the presence
of 250 µM ATP in the dissociation buffer; and the
blue lines indicate the presence of 250 µM ADP
in the dissociation buffer. WT, wild-type.
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Fig. 6.
Protein concentration dependence of the
association of the Msh2-Msh6-S1036P complex with dsDNA containing a GT
mispair. A, association of the different concentrations
of Msh2-Msh6-S1036P with the GT substrate in the absence of nucleotide
was performed exactly as described in the legend to Fig. 4.
B, shown is a plot of Kon
versus Msh2-Msh6-S1036P concentration for the data in
A calculated using GraFit5 software.
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Table II
Binding constants for the Msh2-Msh6 and Msh2-Msh6-S1036P complexes
DNA binding analysis using the Iasys biosensor system and data analysis
were performed as described under "Materials and Methods." The GC,
GT, and +A substrates used; the protein used; and the absence or
presence of 250 µM ATP in the binding buffer are as
indicated.
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To characterize the mispair-binding properties of the mutant Msh2-Msh6
complexes, a comparison of the association phase of binding to the
duplex, GT, and +A substrates was performed in the absence of added
nucleotide and in the presence of ADP or ATP (Fig. 4). Binding of the
Msh2-Msh6-H1096A complex to the three different DNA substrates in the
absence of nucleotide or in the presence of ADP or ATP was
essentially indistinguishable from that of the wild-type protein.
Binding of the Msh2-Msh6-S1036P complex to the three substrates in the
absence of nucleotide or in the presence of ADP was similar to that of
the wild-type complex. However, addition of ATP had a significantly
different effect on binding of the Msh2-Msh6-S1036P complex compared
with the wild-type complex. Binding of the Msh2-Msh6-S1036P complex to
the GT mispair was not inhibited by ATP in comparison with almost
complete inhibition of the wild-type complex. Surprisingly, binding of
the Msh2-Msh6-S1036P complex to the +A mispair was almost as inhibited
by ATP as binding of the wild-type complex (36% of steady-state
binding with ATP versus without ATP for
Msh2-Msh6-S1036P compared with 32% for the wild-type complex).
The binding properties of the Msh2-Msh6-S1036P complex were verified by
determining the various association and dissociation constants for
mispair binding (Table II). These results show that, in the absence of
nucleotide, the Msh2-Msh6-S1036P complex has a similar binding affinity
for DNA without a mispair or with a GT or +A mispair compared with the
wild-type complex in the absence of nucleotide. In contrast, a high
binding affinity of the Msh2-Msh6-S1036P complex for a GT mispair was
observed in the absence of nucleotide or in the presence of ATP,
whereas ATP reduced the binding affinity of the Msh2-Msh6-S1036P
complex for DNA without a mispair or with +A mispair, similar to the
results obtained with the wild-type complex. Binding of the
Msh2-Msh6-G1067D complex to the GT mispair was also not inhibited by
ADP or ATP, and binding of the Msh2-Msh6-G1067D complex to the +A
mispair was not inhibited by ADP and was only partially inhibited by
ATP (66% of steady-state binding with ATP versus without
ATP for Msh2-Msh6-G1067D compared with 32% for the wild-type complex).
The effect of nucleotides on the binding of the Msh2-Msh6-G1142D
complex was somewhat different. ADP did not inhibit binding to either
the GT or +A mispair, whereas ATP caused a similar partial inhibition
of binding to both the GT and +A substrates, but the level of binding
seen in the presence of ATP was significantly higher than that seen for
the wild-type complex (44% +ATP/
ATP for Msh2-Msh6-G1142D
versus 21% for the wild-type complex for the GT substrate
and 65% +ATP/ATP for Msh2-Msh6-G1142D versus 32% for
wild-type complex for the +A substrate).
The interaction of the mutant Msh2-Msh6 complexes with mispaired bases
was further characterized by measuring the dissociation of the
complexes from the different DNA substrates (Fig. 5). As discussed
above, ADP and, to a much greater extent, ATP induced the dissociation
of the wild-type Msh2-Msh6 complex from the GT and +A substrates. As
already pointed out, ATP decreased the half-time of dissociation from
the GT and +A substrates from 375 to 4 s and from 107 to 4 s,
respectively, compared with no nucleotide in the dissociation buffer.
The dissociation behavior of the Msh2-Msh6-H1096A complex was
essentially the same as that of the wild-type complex for all three
substrates. In the case of the Msh2-Msh6-S1036P complex bound to the GT
mispair, dissociation in the presence of no nucleotide or ADP was
essentially indistinguishable from that of the wild-type complex,
whereas ATP-induced dissociation was much slower, with a half-time of
74 s compared with a half-time of 4 s for the wild-type
complex. In the case of the Msh2-Msh6-S1036P complex bound to the +A
mispair, the dissociation behavior in the absence of nucleotide or in
the presence of ADP or ATP was virtually indistinguishable from that of
the wild-type complex. The dissociation of the Msh2-Msh6-G1067D complex
from both the GT and +A mispairs was indistinguishable from that of the
wild-type complex in the absence of nucleotide, but was significantly
slowed in the presence of ADP or ATP; the half-times of dissociation from the GT and +A substrates in the presence of ATP were 140 and
23 s compared with 4 and 4 s for the wild-type complex,
respectively. In addition, dissociation of Msh2-Msh6-G1067D from the +A
mispair did not go to completion and reached a stable plateau at
~40% of the protein bound compared with >90% dissociation for the
wild-type complex. The dissociation behavior of the Msh2-Msh6-G1142D
complex resembled that of the Msh2-Msh6-G1067D complex, with some
differences. These differences were that the Msh2-Msh6-G1142D complex
dissociated more slowly in the presence of ADP than in the absence of
nucleotide; and in the presence of ATP, it dissociated more rapidly
that the Msh2-Msh6-G1067D complex, but more slowly than the wild-type
complex; the half-times of dissociation from the GT and +A substrates
in the presence of ATP were 9 and 9 s compared with 4 and 4 s
for the wild-type complex, respectively. In addition, a fraction of the
Msh2-Msh6-G1142D complex remained stably associated with both the GT
(20% bound) and +A (20% bound) mispairs in the presence of ATP.
Overall, these data support the conclusion that the Msh2-Msh6-S1036P, Msh2-Msh6-G1067D, and Msh2-Msh6-G1142D complexes form more stable complexes with mispaired bases compared with the wild-type Msh2-Msh6 complex under physiological conditions in which ATP is present.
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DISCUSSION |
In a previous study, we described mutations in MSH6
that behave as if they interfere with both Msh6- and
Msh3-dependent MMR (26). It was suggested that these
mutations result in mutant Msh2-Msh6 complexes that either bind to
mispairs in a way that occludes access by Msh2-Msh3 or sequester other
MMR proteins or protein complexes, rendering mismatch-bound Msh2-Msh3
nonfunctional for MMR. In the present study, we have analyzed the
ATPase activity, mispair-binding specificity, and effect of ADP and ATP
on binding to and dissociation from mispaired bases for three of the
complexes encoded by mutants with the strongest phenotypes observed and for one encoded by a mutant with a weaker phenotype. All of the mutant
proteins appeared to bind mispaired bases with normal affinity and to
discriminate them from base pairs. In addition, all of the mutant
complexes retained intrinsic ATPase activity. However, all of the
mutant complexes appeared to have some defect in the modulation of
mispair binding and dissociation by ATP and the modulation of ATP
hydrolysis by mispaired bases. In contrast to the wild-type complex,
the ATPase activity of three of the mutant complexes (Msh2-Msh6-S1036P,
Msh2-Msh6-G1067D, and Msh2-Msh6-G1142D) was inhibited by the presence
of one or more types of mispaired bases; each of these mutant complexes
showed increased binding to one or more types of mispaired bases in the
presence of ATP; and the mispair-bound complex was refractory to
dissociation upon addition of ATP to an extent that depended on the
type of mispaired base. The mispair-binding properties of the
Msh2-Msh6-S1036P, Msh2-Msh6-G1067D, and Msh2-Msh6-G1142D complexes are
in some ways similar to those of the S. cerevisiae and
human Msh2-Msh6 complexes containing G987D and K1140R substitutions,
respectively, which alter a conserved residue of the P-loop of the Msh6
ATP-binding site required for ATP hydrolysis (66, 68). These two mutant complexes show higher levels of steady-state binding to a mispaired base in the presence of ATP than seen for the wild-type complex, although not as high as seen for the mutant or wild-type complexes in
the absence of ATP. It was not determined whether the amino acid
substitutions alter the association or dissociation kinetics or both,
although in the case of the human mutant complex, the effect was
attributed to decreased binding of ATP. However, although these latter
two mutations are loss-of-function mutations, in neither case has it
been demonstrated that they interfere with Msh2-Msh3 function, in
contrast to the four novel msh6 mutations characterized here.
The altered mispair-binding dynamics of the Msh2-Msh6-G1067D and
Msh2-Msh6-G1142D complexes for both GT and +A mispairs are consistent
with the hypothesis that these two mutant complexes bind to these
mispairs with increased stability and occlude the Msh2-Msh3 complex
from interacting with insertion/deletion mispairs, resulting in a
dominant phenotype (26). The Msh2-Msh6-S1036P complex showed altered
binding dynamics with the GT mispair, but not with the +A mispair.
Because the Msh2-Msh3 complex can function in the repair of +1-base
insertion/deletion mispairs, but not base base mispairs, we
hypothesize that the dominant behavior of the Msh2-Msh6-S1036P complex
results from formation of an unusually stable complex with base
base mispairs that sequesters other MMR components, limiting
their ability to interact with Msh2-Msh3 (26). The mispair-binding
dynamics of the Msh2-Msh6-H1096A complex were indistinguishable from
those of the wild-type complex, although mispair stimulation of its
ATPase activity was modestly attenuated. This suggests that some
ATP-mediated downstream interaction may be partially defective in the
mutant Msh6-H1096A complex.
A number of studies have indicated that binding of a MutS dimer or
Msh2-Msh6 to a mispaired base causes a conformational change in the
protein complex such that subsequent ATP binding results in a further
conformational change, upon which the protein is released from the
mispair (11, 20-24, 53, 67-69). Given this view, how then do the
Msh2-Msh6-S1036P, Msh2-Msh6-G1067D, and Msh2-Msh6-G1142D complexes form
stable complexes with mispaired bases in the presence of ATP? The data
presented here indicate that these complexes should bind ATP and
hydrolyze it to ADP because they each retain the intrinsic
DNA-independent ATPase activity. Then the ADP-bound form binds the
mispair (20, 21, 56), forming a complex in which either the ADP-ATP
exchange is defective or the ADP-ATP exchange occurs, but the
ATP-induced conformational change is insufficient to provoke rapid
release from the mispair. Interestingly, at least for the
Msh2-Msh6-G1067D and Msh2-Msh6-G1142D complexes, ADP binding results in
increased stability of the protein-mispair complex compared with the
wild-type protein complex. This mode of mispair binding and altered
mispair-binding dynamics is consistent with the observation that the
S1036P, G1067D, and G1142D substitutions change amino acids located in
the Msh2-Msh6 dimer interface adjacent to the ATP-binding sites (Fig.
1). Based on studies of related proteins such as Rad50 (61-63), these
amino acid substitutions would be predicted to affect conformational
changes across the interface induced by ADP-ATP exchange and to alter
interactions with mispaired base-induced conformational changes
communicated by the transmitter region between the mispair binding and
ADP-ATP binding regions (12, 13, 25, 34). The Msh2-Msh6-H1096A complex
appears to differ from the other three mutant complexes, as it shows
normal mispair binding and dissociation, but fails to show complete
mispair activation of the ATPase. Reduced mispair activation of the
ATPase is consistent with modeling based on the MutS structure
indicating that the Msh6-H1096A substitution alters an amino acid that
is likely important for ATP hydrolysis by the Msh6 ATP-binding site.
These results suggests that the weak dominant phenotype caused by the
Msh6-H1096A substitution likely stems from the reduced activation of
the ATPase, which might in turn result in inappropriate interactions
between Msh2-Msh6 and other MMR proteins.
The crystal structure of MutS bound to a mispaired base indicates that
mispair recognition by MutS involves stacking of a Phe residue onto a
ring of the mispaired base, kinking of the DNA facilitated by a Glu
residue that contacts the DNA, and other specific amino acid-DNA
contacts (12, 13, 25, 32, 34). The same mode of recognition was
demonstrated for both base base mispairs and insertion/deletion
mispairs. Genetic and biochemical studies have shown that Msh6 is the
mispair recognition subunit of Msh2-Msh6 and is the functional
equivalent of the MutS A-subunit of the asymmetric MutS dimer that
forms when MutS is bound to a mispair (12, 13, 26-32). Based on the
conservation of critical amino acids and mispair cross-linking data,
several studies have suggested that MutS and Msh2-Msh6 may share the
same mispair recognition mode (29, 32). In light of the MutS structure,
the observation that the Msh2-Msh6-S1036P complex shows normal
mispair-binding dynamics and mispair-stimulated ATPase activity with a
+A mispair (and the +AT mispair, where tested) and highly altered
mispair-binding dynamics and mispair inhibition of ATPase activity with
a GT mispair is surprising. These results raise two possibilities.
First, Msh2-Msh6 recognizes base base and insertion/deletion
mispairs in the same way, but the two different mispairs induce
somewhat different conformational changes through the transmitter
region that interacts with the ATP-binding site. And second, different
mispair recognition mechanisms exist for base base and
insertion/deletion mispairs, and this induces somewhat different
conformational changes through the transmitter region that interacts
with the ATP-binding site. The observation that both the Msh3- and
Msh6-containing complexes can interact with insertion/deletion mispairs
even though Msh3 lacks the Phe residue thought to be critical for
mispair recognition by MutS and Msh6 suggests that different mispair
recognition mechanisms could exist.
In this study, we performed an initial biochemical characterization of
four mutant Msh2-Msh6 complexes, the genetic properties of which
suggest that they might interfere with the action of the Msh2-Msh3
complex. Overall, the results presented here support the view that
dynamic interactions of Msh2-Msh6 at a mispair modulated by nucleotide
binding are critical for MMR and imply that there might be multiple
modes of mispair recognition. To further exploit these mutant proteins
as tools for studying the biochemical mechanisms of MMR, we are
presently performing more detailed studies of the ADP-ATP exchange
reaction and analyzing the interaction between the mutant complexes and
other MMR proteins. In addition, we are attempting to isolate
additional mutations that differentially affect the base base
versus insertion/deletion mispair recognition properties of
the Msh2-Msh6 complex.
, and
TOP
ABSTRACT
INTRODUCTION
Present address: engeneOS, 40 Bear Hill Rd., Waltham, MA 02451.
