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INTRODUCTION |
The bacterial MutS protein and the eukaryotic heterodimeric
complexes of MutS homologue proteins, the MSH2·MSH6 (MutS
) and MSH2·MSH3 (MutS
) complexes, are mispair recognition factors that function in MMR1 (for review,
see Refs. 1-4). These proteins have a higher affinity for mispairs as
compared with base pairs and form a specific footprint at the site of a
mispair (5-14). The MutS protein recognizes both base:base and
insertion/deletion mispairs (5). In contrast, the MSH2·MSH6 complex
appears to recognize both base:base and insertion/deletion mispairs,
whereas the MSH2·MSH3 complex appears relatively specific for
insertion/deletion mispairs (7-11, 14-17). The difference in affinity
of these proteins in vitro for different mispairs
versus base pairs is small and has generally been reported to be not more than 30-fold (5, 7, 9, 12, 14). When either the MutS
homodimer, the MSH2·MSH6 heterodimer, or the MSH2·MSH3 heterodimer
binds a mispair, the protein complex forms a ring around the DNA (7, 9,
12, 18-22). On binding ATP, these protein rings undergo a
conformational change, are released from the mispair, and slide along
the DNA (7-9, 12, 20-23). A number of potential functions of these
sliding clamps have been postulated, although their exact role in MMR
is not understood.
The recognition of mispaired bases by the MutS family of
proteins in vivo may be more complex than mispair
recognition in vitro. Using MMR-defective mutants in
Saccharomyces cerevisiae, one can calculate from spontaneous
mutation rates in coding sequences like CAN1 and
URA3, or even in rare hypermutable sequences, that MMR
likely has the ability to recognize a single mispaired base per genome
of ~107 base pairs (4, 9, 15, 16). It seems unlikely that
a ~30-fold affinity difference for mispairs versus base
pairs can alone account for this in vivo MMR specificity.
There are several other mechanisms that may contribute to the
specificity of MMR. First, the MutS family of proteins forms complexes
with other MMR proteins, and these complexes could have increased
mispair specificity. Examples include the interaction of MSH2 with EXO1 (24), the interaction of the MSH2·MSH6 and MSH2·MSH3 complexes with
PCNA (25-27), and the interaction of MutS with the
clamp subunit of DNA polymerase III (28). Second, mispair binding by the MutS
family proteins could drive mispair-specific conformational changes
required for MMR. Examples include the assembly of MutL onto MutS at a
mispair (6, 29), the assembly of the eukaryotic MLH1-containing
heterodimeric complexes with either the MSH2·MSH6 or MSH2·MSH3
complexes (30-33) at a mispair, and even the
mispair-dependent conformational changes induced by ATP
binding (7-9, 12, 13, 20-23). Finally, the interaction between the
MutS family proteins and either PCNA or the
clamp subunit of DNA
polymerase III has suggested that MMR may be coupled to replication
(25-28). The observation that hMSH6 and hMSH3 co-localize with PCNA in
replication foci (27) and that MutS is targeted to replication regions
in bacteria is consistent with this idea (34). Such coupling could
occur either by targeting MMR proteins to regions of replicating DNA or
by incorporating MMR proteins as part of the replication machinery.
PCNA has been suggested to function in MMR at both an early step and
during the DNA resynthesis step (35, 36). One role of PCNA in MMR may
be mediated by interactions between PCNA and the MSH2·MSH6 and
MSH2·MSH3 complexes through PCNA interaction motifs present in MSH6
and MSH3 (25-27). Mutations that alter amino acids in these PCNA
interaction motifs cause a partial MMR defect in vivo and
eliminate the in vitro interaction between PCNA and the
MSH2·MSH6 and MSH2·MSH3 complexes (25, 26). Similarly, mutations in
the gene encoding PCNA that cause MMR defects often eliminate the
in vitro interaction between PCNA and the MSH2·MSH6 complex (26, 37). PCNA increases the in vitro
mispair binding specificity of the MSH2·MSH6 complex, and
this is eliminated by amino acid substitutions in PCNA that cause MMR
defects (26). These results suggest PCNA may play a role in MMR at the
mispair recognition step. To further understand the role of PCNA in
mispair recognition, we investigated the mispair binding properties of the PCNA·MSH2·MSH6 complex. The results presented here demonstrate that the PCNA·MSH2·MSH6 complex can form a ternary complex with fully base-paired DNA, but when a mispair is present PCNA is excluded from the complex. These results suggest an in vivo model of
mispair recognition whereby MSH2·MSH6 binds to PCNA on the DNA and is then transferred to the mispair.
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MATERIALS AND METHODS |
DNAs--
The DNA duplexes used in this study
have been described previously (9, 12). In brief, the
oligonucleotide 5'-ATTTCCTTCAGCAGATAGGAACCATACTGATTCACAT-3' (bottom
strand) was annealed with either the
5'-ATGTGAATCAGTATGGTTCCTATCTGCTGAAGGAAAT-3' (G/C top strand)
oligonucleotide or the 5'-ATGTGAATCAGTATGGTTTCTATCTGCTGAAGGAAAT-3' (G/T top strand) oligonucleotide to yield the homoduplex (G/C) or
the heteroduplex (G/T) DNAs, respectively. The annealed products were
purified by high pressure liquid chromatography using a GEN-PAK FAX column (Waters Corp.) and then 5'-end-labeled with
[
-32P]ATP and T4 polynucleotide kinase if necessary.
Protein Purification--
MSH2·MSH6 was purified using a
S. cerevisiae overexpression system as described previously
(8, 9, 12), and PCNA was purified using an Escherichia coli
overexpression system as described previously (38). All protein
preparations were greater than 98% pure as judged by SDS-PAGE.
Sedimentation Analysis--
Sedimentation analysis
was performed as described previously (26, 37). In brief, protein
samples (80 µl) were loaded onto the top of 4-ml, 15-30% glycerol
gradients and centrifuged at 45,000 rpm at 4 °C for 20 h in a
Beckman SW60 rotor. Thirty-one fractions were collected from the bottom
of each gradient. The amount of radioactivity present in 15 µl of
each fraction was determined by liquid scintillation counting. Aliquots
(12.5 µl) of selected fractions were used for subsequent Western blot
analysis. Alternatively, 127 µl of each fraction was concentrated
using a Microcon YM 30 Centrifugal Filter (Millipore Corp.) and
analyzed by SDS-PAGE using 4-15% gradient gels, which were
subsequently stained with Coomassie Blue. The following amounts of
proteins or DNAs (individually or mixtures thereof) were incubated on
ice for 30 min and sedimented through the gradients: 7.5 µg of
MSH2·MSH6, 2.5 µg of PCNA, 680 ng of G/C DNA plus 3.7 ng of
-32P-labeled G/C DNA, 680 ng of G/T DNA plus 3.7 ng of
-32P-labeled G/T DNA.
Co-precipitation--
PCNA was biotinylated
according to the ECL protein biotinylation kit (Amersham Biosciences).
MSH2·MSH6 (1.5 pmol of heterodimer) and biotinylated PCNA (1.5 pmol
of homotrimer) was mixed with either no DNA, G/T DNA, or G/C DNA in
buffer A (50 mM NaCl, 50 mM Tris-HCl, pH 7.5, 10% glycerol, 1 mM EDTA, 1 mM dithiothreitol, and 0.01% (octylphenoxy)polyethoxyethanol (Sigma)) and incubated on
ice for 30 min. The total reaction volume was 40 µl, and the amounts
of DNA added are indicated in individual experiments. For each
reaction, 80 µl (40 µl of settled resin) of streptavidin-agarose (Novagen) was washed once in buffer A and centrifuged at 500 × g for 1 min at 4 °C. The supernatant was removed, and the
reaction mixture was added to the streptavidin-agarose. The mixture was incubated on ice for 30 min with gentle mixing every 5 min. The agarose
beads were then washed three times by centrifugation at 500 × g
for 1 min at 4 °C followed by resuspension in 40 µl of buffer A. After the last resuspension, SDS loading buffer was added to the
mixture, and the mixture was incubated for 10 min at 95 °C. 15 µl
of the supernatant was analyzed by SDS-PAGE and Western blotting. For
the precipitation experiments with and without nucleotide, MSH2·MSH6
(1.5 pmol of heterodimer) and biotinylated PCNA (1.5 pmol of
homotrimer) was mixed with either no DNA, G/T DNA (7.5 pmol), or G/C
DNA (7.5 pmol) in buffer A or buffer A supplemented with either ATP
(250 µM) or ATP
S (250 µM).
Western Blotting--
Protein samples were transferred to
immunoblot polyvinylidene difluoride membranes (Bio-Rad) in 25 mM Tris base, 192 mM glycine, and 20% (v/v)
methanol and analyzed by Western blotting essentially as described
previously using polyclonal rabbit antibodies raised against either
full-length MSH2, MSH6, or PCNA (26) and the ECL Plus protocol
(Amersham Biosciences). The resulting films were quantified by densitometry.
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RESULTS |
Sedimentation Analysis of MSH2·MSH6·PCNA·DNA
Complexes--
Using sedimentation analysis, earlier studies showed
that the MSH2·MSH6 protein complex interacted with wild-type PCNA but did not interact with several MMR-defective mutant PCNAs to the extent
seen with wild-type PCNA (26, 37). Here we studied the interactions
between MSH2·MSH6, PCNA, and either homoduplex or heteroduplex DNA.
As observed previously (26, 37), the addition of PCNA to
MSH2·MSH6 caused MSH2·MSH6 to sediment further into the
gradient (lower fraction numbers) than MSH2·MSH6 alone (Fig.
1A). PCNA alone was found in
fractions 17 and above, whereas addition of MSH2·MSH6 caused PCNA to
sediment further into the gradient such that PCNA was now observed in
the same fractions as the shifted MSH2·MSH6 (Fig. 1B).
This shift of MSH2·MSH6 and PCNA to larger sedimentation
coefficients is consistent with the formation of a
MSH2·MSH6·PCNA complex (26, 37). Addition of either homoduplex
(G/C) or heteroduplex (G/T) DNA to MSH2·MSH6 caused both MSH2·MSH6
and the DNA to sediment at a larger sedimentation coefficient than either MSH2·MSH6 (Fig. 1A) or the DNA
alone (Fig. 1, C and D) (the peak fraction for
each DNA sedimented alone was fraction 25). This is consistent with
previous observations that MSH2·MSH6 can bind to oligonucleotide
duplexes that do or do not contain mispaired bases (7-12, 14). The
addition of PCNA to either homoduplex (G/C) or heteroduplex (G/T) DNA
did not alter the sedimentation of either PCNA (Fig. 1B) or
the DNA (Fig. 1, C and D). This is consistent
with the observation that PCNA does not stably bind to linear DNA
(26).

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Fig. 1.
Sedimentation analysis of in vitro
interactions between PCNA, MSH2·MSH6, and either homoduplex
(G/C) or heteroduplex (G/T) DNA. Reactions containing PCNA,
MSH2·MSH6, homoduplex (G/C) DNA, and heteroduplex (G/T) DNA, either
individually or in mixtures (as indicated on the right side
of the figure), were mixed, sedimented through glycerol gradients,
separated into fractions, and analyzed as described under "Materials
and Methods." Fraction 1 is the bottom of each gradient.
A, Coomassie Blue staining of MSH2 (bottom band)
and MSH6 (top band) present in each fraction. B,
Coomassie Blue staining of PCNA present in each fraction. C,
percentage of radiolabeled homoduplex (G/C) DNA present in each
fraction. D, percentage of radiolabeled heteroduplex (G/T)
DNA present in each fraction.
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When MSH2·MSH6, PCNA, and the homoduplex (G/C) DNA were combined and
sedimented, MSH2·MSH6 (Fig. 1A), PCNA (Fig.
1B), and the homoduplex (G/C) DNA (Fig. 1C) all
sedimented further into the gradient than observed with any combination
of two. These results suggest that MSH2·MSH6, PCNA, and the
homoduplex (G/C) DNA can form a stable complex. Interestingly, the same
results were not seen when MSH2·MSH6, PCNA, and the heteroduplex
(G/T) DNA were combined and sedimented. Under these conditions,
MSH2·MSH6 (Fig. 1A) and the heteroduplex (G/T) DNA
(Fig. 1D) co-sedimented to the same position as the
preformed MSH2·MSH6·heteroduplex (G/T) DNA complex, while all
of the PCNA appeared to sediment to the same position as PCNA alone.
Moreover, no PCNA was observed in the fractions containing the
MSH2·MSH6·heteroduplex (G/T) DNA complex (as observed by staining
with Coomassie Blue). The inability to observe co-sedimentation
of PCNA with the MSH2·MSH6·heteroduplex (G/T) DNA complex was
independent of the order of addition of the proteins and DNA, including
preincubation of MSH2·MSH6 with PCNA prior to the addition of the
heteroduplex (G/T) DNA (data not shown). These results suggest that
when an MSH2·MSH6·PCNA complex binds to the heteroduplex (G/T) DNA,
PCNA is excluded from the complex.
To substantiate these findings, Western blotting was used to provide
more sensitive detection of PCNA (Fig.
2). After sedimentation and fractionation
of reactions that contain PCNA, MSH2·MSH6, and a DNA (either G/C or
G/T), peak fractions (fractions 11 and 12) that contain MSH2·MSH6 and
DNA were analyzed to detect PCNA. The same fractions from reactions
containing PCNA mixed with MSH2·MSH6 or PCNA mixed with either the
homoduplex (G/C) or the heteroduplex (G/T) DNA were analyzed as
controls. PCNA was present in the two fractions when MSH2·MSH6 or
MSH2·MSH6 and the homoduplex (G/C) DNA were incubated with PCNA prior
to sedimentation. However, PCNA was not detected when either the
homoduplex (G/C) DNA alone, the heteroduplex (G/T) DNA alone, or
MSH2·MSH6 with the heteroduplex (G/T) DNA were incubated with PCNA
prior to sedimentation. This strengthens the above finding that PCNA
forms a complex with MSH2·MSH6 and homoduplex (G/C) DNA but not with
MSH2·MSH6 and heteroduplex (G/T) DNA.

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Fig. 2.
Sedimentation analysis and detection of PCNA
by Western blot analysis. Sedimentation analysis was performed as
described in Fig. 1 legend and under "Materials and Methods."
A, percentage of radiolabeled DNA, either homoduplex (G/C)
or heteroduplex (G/T) (as indicated), in each fraction. B,
PCNA present in fractions 11 and 12 from different reactions as
indicated, visualized by Western blotting.
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Co-precipitation Analysis of MSH2·MSH6·PCNA·DNA
Complexes--
Co-precipitation experiments were performed to analyze
the formation of MSH2·MSH6·PCNA·DNA complexes. Biotinylated PCNA
was incubated with MSH2·MSH6 and either no DNA or increasing amounts of the homoduplex (G/C) DNA or the heteroduplex (G/T) DNA. The resulting complexes were then precipitated with streptavidin-agarose, and the bound proteins were detected by SDS-PAGE and Western blotting. The addition of the heteroduplex (G/T) DNA caused a large decrease in
the amount of MSH2 and MSH6 that co-precipitated with the biotinylated PCNA (Fig. 3A); the amount of
MSH2 and MSH6 bound was reduced by ~70% compared with the levels
observed when no DNA was added. In contrast, the addition of the
homoduplex (G/C) DNA caused a small ~10% decrease in the amount of
MSH2 and MSH6 that co-precipitated with the biotinylated PCNA compared
with no added DNA (Fig. 3A). These results suggest that the
addition of the heteroduplex (G/T) DNA causes MSH2·MSH6 to partition
into MSH2·MSH6·DNA complexes thus inhibiting the formation of
MSH2·MSH6·PCNA complexes, whereas the addition of homoduplex DNA
does not appear to inhibit the formation of MSH2·MSH6·PCNA
complexes to the same extent.

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Fig. 3.
Co-precipitation of MSH2 and MSH6 with
PCNA. A, biotinylated PCNA (1.5 pmol of trimer) was
incubated with MSH2·MSH6 (1.5 pmol of heterodimer) and either no DNA
or increasing amounts of heteroduplex (G/T) or homoduplex (G/C) DNA (as
indicated), and the PCNA-binding proteins were precipitated and
analyzed by Western blotting as described under "Materials and
Methods." B, co-precipitation experiments were performed
as described above except biotinylated PCNA (1.5 pmol of trimer) was
incubated with MSH2·MSH6 (1.5 pmol of heterodimer) and either no DNA,
heteroduplex (G/T) (7.5 pmol), or homoduplex (G/C) DNA (7.5 pmol) in
buffer containing either no nucleotide, ATP (250 µM), or
ATP S (250 µM).
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Co-precipitation experiments were also used to examine the effect of
ATP and ATP
S on the interactions between PCNA and MSH2·MSH6 in the
presence or absence of a DNA. When there was no DNA present, the
addition of ATP or ATP
S caused only a small ~10% decrease in the
amount of MSH2 and MSH6 that co-precipitated with the biotinylated PCNA
when compared with samples with no nucleotide added (Fig. 3B). In the presence of the homoduplex (G/C) DNA, the
addition of ATP and ATP
S caused a small ~15 or 19% increase,
respectively, in the amount of MSH2 or MSH6 bound to PCNA compared with
reactions with no nucleotide added (Fig. 3B). In the
presence of the heteroduplex (G/T) DNA, the addition of ATP or ATP
S
caused a larger increase, ~70 and ~90%, respectively, in the
amount of MSH2 and MSH6 that co-precipitated with the biotinylated PCNA
compared with samples with no nucleotide added. It has been shown that
addition of ATP or ATP
S to MSH2·MSH6 in the presence or absence of
PCNA causes MSH2·MSH6 to dissociate from heteroduplex DNA (7-9, 12,
20, 22, 26). Our results are consistent with the idea that when ATP or
ATP
S is added to mixtures containing MSH2·MSH6, PCNA, and the
heteroduplex (G/T) DNA, MSH2·MSH6 has a lower affinity for the
heteroduplex (G/T) DNA thereby increasing the MSH2·MSH6 available
to complex with PCNA.
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DISCUSSION |
In the present study, we observed that MSH2·MSH6 and
MSH2·MSH6·PCNA complexes each formed a complex with homoduplex
(G/C) DNA and that MSH2·MSH6 formed a complex with heteroduplex (G/T) DNA. In contrast to the results observed with homoduplex (G/C) DNA,
when MSH2·MSH6, PCNA, and heteroduplex (G/T) DNA were incubated together, only an MSH2·MSH6·heteroduplex (G/T) DNA complex was obtained. Consistent with this, heteroduplex (G/T) DNA inhibited the
formation of a co-precipitable MSH2·MSH6·PCNA complex, whereas homoduplex (G/C) DNA had essentially no effect on the formation of a
co-precipitable MSH2·MSH6·PCNA complex. These results indicate that
the MSH2·MSH6·PCNA complex can bind nonspecifically to DNA, but
when MSH2· MSH6 binds to a mispair, binding of PCNA to MSH6 is
disrupted. This suggests that either binding to a mispair
alters the conformation of MSH2·MSH6 such that MSH6 cannot strongly
interact with PCNA or that the position of the mispaired DNA within the MSH2·MSH6 ring may exclude PCNA from the complex. To gain further insight into this observation, we are developing approaches to analyze
the structure of MSH2·MSH6· PCNA·DNA complexes and are analyzing the effect of other MMR proteins on complex formation. In
previous studies using gel mobility shift assays, we observed that
addition of PCNA to MSH2·MSH6 increased mispair binding specificity of MSH2· MSH6 but did not alter the mobility of the
mispair·MSH2·MSH6 complex, whereas the nonspecific
protein·DNA complexes formed often had a somewhat reduced
mobility and a more heterogeneous nature (see Fig. 3E of Ref. 26).
These results suggest that the conformational changes that occur during
the transition between the nonspecific MSH2·MSH6·PCNA·DNA complex
and the MSH2·MSH6·mispair complex in which PCNA is excluded may
modestly improve mispair binding specificity.
A number of studies have shown that the MSH2·MSH6 and
MSH2·MSH3 heterodimers bind to PCNA through a conserved motif found in MSH6 and MSH3 and that these interactions are important for MMR (25,
26). These observations provide molecular insights into how PCNA might
function at an early step in MMR and suggest that PCNA may play a role
in mispair recognition. Three different types of models have been
proposed for how PCNA might function early in MMR (25-27, 35-37).
First, it has been suggested that because a PCNA trimer has three
distinct protein docking sites, PCNA may assemble a higher order
complex containing both MMR and replication proteins at replicating
regions of DNA. However, analysis of the interaction of multiple
proteins with PCNA has indicated that one PCNA-interacting protein
displaces a second from PCNA rather than assembling into a complex with
more than one protein bound to PCNA (39-43). Second, it has been
suggested that MSH2·MSH6 and MSH2·MSH3 form sliding clamps at
mispairs that migrate to the replication DNA polymerase and induce
dissociation of the replication complex (27). Constraints on this model
are that it needs a mechanism by which the MSH2·MSH6 and MSH2·MSH3
clamps can catch up to the rapidly progressing DNA polymerase and then
overcome the structural barrier of encountering PCNA on the opposite
face on which it is thought to bind (28, 44). Finally, because PCNA is
naturally loaded onto 3'-primer termini in replication intermediates
and is then left on the DNA when replication proteins dissociate (45),
PCNA would be expected to decorate replicating regions of DNA. This
could then target MSH2·MSH6 and MSH2·MSH3 to replicating regions of
DNA (26) as has been suggested for other proteins (46). Such a
mechanism would target the MSH2·MSH6 and MSH2·MSH3 mispair
recognition proteins to newly replicated regions of DNA where mispaired
bases would normally be found. Our findings support this latter
hypothesis and are consistent with a model where MSH2·MSH6 associates
with PCNA that is tethered to DNA to form a nonspecific
PCNA·MSH2·MSH6·PCNA complex, and once this complex encounters a
mispair, the MSH2·MSH6 is transferred from PCNA to the mispair, thus
activating MMR. Such a mechanism would increase the intracellular
mispair recognition affinity of MSH2·MSH6 at newly replicated DNA.