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INTRODUCTION |
The dimeric MutS protein is part of the Escherichia
coli MutSLH DNA repair system that corrects mismatches arising in
DNA as biosynthetic errors (1). The mutator phenotype of strains with
an inactivated mutS gene is consistent with this idea (2). In addition MutS also participates in homeologous recombination, transcription-coupled repair, very short patch repair, resolution of
directly repeated DNA sequences, and sensitivity to
cis-platin and alkylating agents (3-6). MutS binds
specifically to mismatched base pairs and insertion/deletion mispairs
of up to four nucleotides as well as adducted base mismatches (7, 8).
The DNA-bound MutS protein binds ATP and in association with dimeric
MutL can form
-looped structures with concomitant hydrolysis of ATP,
suggesting movement of the protein complex on DNA away from the
mismatch (9). An alternative model suggests that ADP stabilizes DNA binding and ATP increases dissociation (10). After addition of MutH,
the MutSLH complex cleaves DNA 5' to hemimethylated -GATC- sequences in
the unmethylated strand. Subsequent excision repair from this nick can
occur in either direction by one of several exonucleases, and
directionality is imparted through interaction of the MutLS complex
with helicase II, the uvrD gene product (11). Resynthesis of
the gapped DNA is accomplished by DNA polymerase III holoenzyme and
subsequent ligation by DNA ligase.
The basic elements of the scheme outlined above have been conserved in
eukaryotes (3-5). This includes MutS and MutL homologues, each of
which shows conservation with the E. coli protein at the amino acid level. Unlike bacteria, however, multiple MutS (MutS homologue) and MutL (MutL homologue, postmeiotic segregation) homologs
are present as heterodimers in eukaryotes. Mutation in these genes
results in microsatellite sequence instability and may dispose
individuals to hereditary or sporadic cancer, especially colon
carcinoma. The mechanism for strand discrimination during the repair
process in eukaryotes is unknown.
At present, there is no structural information for any MutS homologue.
The amino acids that are important for specific and nonspecific contact
with DNA are not known. Similarly the residues important for
dimerization and contacting MutL are unknown. To identify which regions
of MutS are essential for these functions, we have made specific
deletions in the mutS gene and have examined the properties
of the truncated proteins. The results obtained with the E. coli protein should be applicable to other members of the MutS
family given their overall amino acid conservation (4).
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EXPERIMENTAL PROCEDURES |
Bacterial Strains and Culture Conditions--
Strain E. coli K-12 GM5864 is GM4271 lysogenized with phage
DE3. GM4271
is a mutS458::mTn10Kan derivative of
CC106 (12). Bacteria were grown in L broth supplemented with 100 µg/ml ampicillin and/or 10 µg/ml chloramphenicol. The methods for
determining mutation frequency to rifampicin resistance or
Lac+ papillation have been described previously (12).
Construction of Plasmid-borne Mutations and Protein
Expression--
Plasmid pMQ372 is a pET3d (Novagen) derivative that
contains the wild-type mutS gene and lacks the
AvaI site of pET3d. pMQ395 is derived from pMQ372 but
contains the N-terminal His6 tag of pET15b fused to the
mutS coding sequence. Plasmids pMQ372 and pMQ395 fully
complement the mutS458 mutation in strain GM4271, as
determined by monitoring for spontaneous mutation to rifampicin resistance, even in the absence of T7 RNA polymerase induction (see
Table II). Plasmids pMQ382 and pMQ393 are derivatives of pACYC184 and
carry His6-tagged mutS and mutL,
respectively (12).
Deletions in the E. coli mutS gene were made in pMQ372 at
naturally occurring restriction enzyme recognition sites, with
appropriate endonucleases (New England Biolabs), to produce derivatives
with promoter-proximal, promoter-distal, or midgene mutations (see Fig.
1). The fusion joint for each deletion end point is shown in Table
I. The method of constructing the
deletion at the SspI site resulted in the addition of 13 additional amino acids to the mutant protein. Mutations from the pMQ372
series were moved into pMQ395 by fragment swapping to generate proteins
with an N-terminal His6 tag. The His6 tag
wild-type and deleted mutant proteins were prepared as recommended by
the manufacturer (Novagen) in strain GM5864. Although the specific
conditions for culturing cells varied for each construct, in general
GM5864 transformed with a pMQ395 mutant derivative was grown at
37 °C to an A600 of 0.8, shifted to room
temperature, and isopropyl-1-thio-
-D-galactopyranoside (IPTG)1 was added to 50 µM final concentration. Incubation was continued for 2-3
h at room temperature, and the cells were harvested and lysed in a
French pressure cell (Aminco) in lysis buffer (20 mM Tris-HCl, pH 7.9, 5 mM imidazole, 500 mM NaCl)
followed by a brief sonication to reduce viscosity. The lysate was
centrifuged in a Beckman Ti70 rotor at 39,000 rpm for 30 min at
4 °C, followed by filtration through a 0.45-µm syringe filter
(Acrodisc). His6 tag protein was bound to and eluted from
nickel-affinity resin (His-Bind, Novagen) as recommended by the
manufacturer. At least 80% of the wild-type and 10-30% of the mutant
MutS were recovered as soluble protein. All proteins were at least 95%
pure as judged by Coomassie Brilliant Blue staining of polyacrylamide
gels and by Western blotting (e.g. see Fig. 4). Protein
concentration was assayed using the Bradford reagent (Bio-Rad). ATPase
activity was assayed by the method of Weinstock et al. (13),
and percent [
-32P]ATP hydrolysis was measured by
scanning polyethyleneimine chromatography plates using a Molecular
Dynamics PhosphorImager equipped with ImageQuant software.
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Table I
DNA sequences of fusion joints
The plasmid structures are shown in Fig. 1. The underlined sequences
are the fusion joints using the restriction enzymes shown in Fig. 1.
The H after the plasmid name indicates an N-terminal
His6-tag.
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Heteroduplex DNA Construction--
A 154-bp DNA oligonucleotide
with a centrally located G-T mismatch at bp 76 was constructed
as follows. Oligonucleotides MM181 (86-mer, 5'-CGGCGATATTCTAG
ACACAGGCGATGGTTTTGATAGAGCATCTTGGACGATTTGTAACAACTCGGAGTTCATAGATCTCCCATTCG-3') and MM186 (92-mer,
5'-AGAGGATCCGCACTTTAACTTCCGTATGCCTATGGAAGTCAGAGAGAAATTAAAATTCAGAGCGGAGGCGAATGGGAGGTCTATGAACTCCG-3') were synthesized by Dr. Kendall Knight (University of
Massachusetts Medical School). The underlined bases are complementary,
and the mismatched bases are in bold. The DNA sequence of these
oligonucleotides is derived from the phage P22 mnt gene
(14). The oligonucleotides were mixed, annealed in 20 mM
Tris-HCl, pH 7.6, 10 mM EDTA, 5 mM
MgCl2 at 70 °C for 10 min, and then slowly cooled to
room temperature. The single-stranded regions were converted to duplex
DNA in the presence of dNTPs and [
-32P]dATP (800 Ci/mmol) and DNA polymerase I Klenow fragment (New England Biolabs) in
reaction buffer supplied by New England Biolabs. Following the
reaction, labeled double-stranded DNA was recovered after passage
through a Qiaquick (Qiagen) column. Binding of MutS to heteroduplex DNA
was measured by band shifting in polyacrylamide gels as described
previously (8).
MutS Heterodimer Detection and MutS-MutL Protein
Interaction--
To measure MutS heterodimer interaction, strain
GM5864 was transformed with pMQ382, encoding wild-type
His6-tagged MutS, and with a pMQ372 mutS
deletion derivative. MutS-MutL interaction utilized strain GM5864
transformed with pMQ393, which encodes wild-type
His6-tagged MutL, and with a pMQ372 mutS
deletion derivative. For the His6-tagged derivatives listed
in Fig. 1, the same procedure was used except that the wild-type MutS
was not His6-tagged. The strains were grown to
A600 of 0.7 at 37 °C (no IPTG was added) and
harvested, and His6-tagged proteins were isolated as
described above. A portion of each fraction from the affinity column
was subjected to SDS-PAGE. Immunoblotting on Immobilon-P membranes (Millipore) after semi-dry electroblotting (Owl Scientific) employed rabbit polyclonal antiserum to MutS and MutL (BAbCO) using
chemiluminescence according to the manufacturer's (Tropix) instructions.
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RESULTS |
Construction of Deletion Mutations--
The amino acids important
for MutS dimerization, for binding to heteroduplex DNA, and for
interaction with MutL are not known. We have constructed deletion
derivatives in the E. coli mutS gene to determine whether
these functions can be localized to specific regions of the MutS
protein. Because ATP binding and/or hydrolysis is necessary for
heteroduplex DNA-induced MutS conformational change (15) as well as
movement of MutS along the heteroduplex DNA (9) and for MutS-MutL
interaction (16), we constructed deletion mutations in the
mutS gene that retained the coding sequence of the P-loop
motif for nucleoside triphosphate binding (between the HpaI
and SspI sites in Fig. 1). All
mutant proteins derived from the deletion constructs had similar low
ATPase activity (about 0.34 µM ATP
hydrolyzed/µM MutS/min) as the wild type.

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Fig. 1.
Structure and properties of mutS
deletion mutants. Plasmids pMQ372 and pMQ395 have the
wild-type mutS sequence without (open
rectangle) and with (gray box) an
N-terminal His6-tag DNA, respectively. Pertinent
restriction enzyme recognition sequences are shown. The DNA encoding
the P-loop domain for ATP binding is between the HpaI and
SspI sites. The sequence of fusion joints and extent of the
deletions are given in Table I. The gray box at
the 3'-end of p 680-853H and p 680-853 represents DNA encoding 13 additional amino acids. The ATPase activity of the proteins is given as
µM ADP produced/min/µM MutS. MW,
calculated molecular weight; G-T, ability to bind to G-T
mismatched substrate DNA; S-S, extent of dimerization;
S-L, extent of MutS-MutL interaction; ND, not
determined.
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Mutator Phenotype of Cells with Plasmid-borne mutS
Deletions--
Plasmids containing the wild-type and mutant
mutS deletion alleles were introduced into wild-type and
mutS E. coli strains. The presence of the plasmids in the
wild-type strain did not significantly alter the mutation frequency to
rifampicin resistance (Table II) or the
frequency of Lac+ reversion as measured by papillation
(less than 1% of colonies showing one or more papillae). Similarly the
plasmids did not significantly alter the mutation frequency of the
mutS strain to rifampicin resistance (Table II) or
Lac+ papillation (>90% of cells showing papillation). The
deletion mutations, therefore, inactivate the protein for genetic
complementation and do not show dominance in the wild type.
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Table II
Mutation frequency to rifampicin resistance
Bacterial strains containing the plasmids listed above were grown to
saturation in duplicate in L broth with ampicillin but without IPTG.
Portions of the cultures were diluted, if necessary, and spread on
plates containing ampicillin or rifampicin. The plates were scored
after overnight incubation at 37 °C.
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Heteroduplex DNA Binding of Mutant Proteins--
MutS protein
binds specifically to base mismatches and insertion/deletions in DNA
(7, 8). The wild-type and deletion mutant proteins were tested for
their ability to bind to a 154-bp heteroduplex containing a G-T
mismatch at position 76 by monitoring bandshifting in polyacrylamide
gels (Fig. 2). The wild-type protein bound the radioactive substrate oligonucleotide efficiently at low
concentration, and apparent binding was decreased by 85% in the
presence of a 50-fold excess of non-radioactive competitor DNA (Fig.
2). At a wild-type protein concentration that resulted in complete loss
of the free oligonucleotide band, mutant proteins produced by
p
680-853H, p
26-260H, p
261-556H, and p
1-311H showed 9-, 44-, 87-, and 100-fold reduction in specific binding, respectively (Fig. 2). These data indicate that the N-terminal end of the MutS protein is essential for substrate binding.

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Fig. 2.
MutS protein binding to a 154-bp G-T
heteroduplex substrate. A, construction of the 154-bp
DNA fragment substrate. B, the three
lanes for each mutant protein indicate (from left
to right) that fractions eluting at 30, 55, and 80 mM imidazole from the affinity column were tested. The
0 lane represents heteroduplex DNA only. The
amount of wild-type protein was 0.6 pmol; for p 1-311H and
p 261-556H, 1.2 pmol; and for p 680-853H and p 26-260H, 1.6 pmol. The amount of the heteroduplex DNA fragment was 200 fmol. A
50-fold excess of unlabeled competitor DNA was used. nt,
nucleotide.
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MutS Heterodimer Detection--
MutS protein is a dimer in
solution (7). We have used an in vivo assay to test for the
ability of mutant proteins to dimerize with wild-type MutS. Compatible
plasmids were used to express His6-tagged wild-type and
native mutant proteins in the same cell. His6-tagged
protein was purified from cell lysates and subjected to SDS-PAGE. The
proteins were blotted to a membrane and probed with MutS antiserum. If
mutant-wild-type heterodimers are formed, then two reactive bands, at
different molecular weights, should be present (Fig.
3A). Expression of the
wild-type His6-tagged MutS gene in the presence of the
compatible plasmid vector produces predominantly a single protein band
distribution at the predicted wild-type Mr in
eluates from the nickel column (Fig. 3B, vector). A similar result was obtained if both His6-tagged and
non-tagged wild-type proteins were present in cell extracts (Fig.
3B, pMQ372). We assume the lower molecular weight
band in these two figures is because of protein degradation. The
distribution of wild-type and mutant proteins in eluates from the
affinity column was similar if not identical for p
261-556 and
p
1-260 encoded mutant proteins, indicating efficient heterodimer
formation in vivo. Deletion of the N-terminal region of MutS
encompassed by these two mutations (Fig. 1) therefore does not affect
dimerization. For the p
680-853 and p
261-566 encoded proteins,
however, the distributions of wild-type and mutant proteins were not
the same (Fig. 3), indicating less efficient heterodimer formation. The
basis for the altered distribution of the mutant proteins is unknown.
These mutant proteins are as stable and soluble as those made from
p
261-556 and p
1-260, suggesting that lack of heterodimer
formation is not because of these causes. None of the mutant
non-His6-tagged MutS proteins bind to the affinity column
(data not shown). The results suggest that amino acid residues in the
C-terminal end of the protein are responsible for efficient
dimerization.

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Fig. 3.
Analyzing MutS dimerization by affinity
chromatography. A, schematic representation of the
experiment showing the result expected if heterodimerization occurs (2 bands) or not (1 band). B, for each
plasmid construct, fractions eluting at 30, 55, 80, and 105 mM imidazole from the affinity column were tested in
duplicate for MutS immunoblotting after SDS-PAGE. Lanes
m and s are, respectively, marker wild-type MutS
protein and the fraction obtained after stripping the column with
EDTA.
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MutS-MutL Interaction--
MutS protein bound to heteroduplex DNA
is thought to recruit MutL protein into a complex (16). We have been
unable to detect MutS-MutL complexes by
co-immunoprecipitation.2 We
have used, instead, a variation of the technique to detect dimerization
in which His6-tagged MutL was co-expressed in
vivo with non-His6-tagged MutS. Affinity purification
of MutL followed by SDS-PAGE and immunoblotting with MutS antibody
should allow for detection of MutS-MutL complexes (Fig.
4A). The control experiments showed that a band at the expected Mr was
detected in cells expressing MutS (Fig. 4B,
pMQ372) but was not detected in its absence (Fig. 4B, vector). Reduced amounts of mutant MutS were
detected in cells containing the C-terminal and midgene mutS
deletions (Fig. 4B, p
680-853 and
p
261-566). One mutation deleting the
N-terminal end produced almost as much cross-reacting material as
wild-type (Fig. 4B, p
1-260)
whereas another resulted in a vast excess of it (Fig. 4B,
p/
26-260). We assume the latter result indicates a
higher affinity of the mutant protein for MutL than wild type or
binding of more MutS molecules to MutL than normal. The results above
suggest that the MutS-MutL interaction region is at the C-terminal end
of MutS, perhaps overlapping with the P-loop domain of the ATP binding
site (Fig. 1).

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Fig. 4.
Analyzing MutS-MutL interaction by affinity
chromatography. A, schematic representation of the
experiment showing the result expected if MutS-MutL interaction occurs
(1 band) or not (no bands). B, for
each plasmid construct, fractions eluting at 30, 50 (two), and 80 mM (two) from the affinity column were tested for MutS
immunoblotting after SDS-PAGE. As a control, MutL immunoblotting was
also carried out on each fraction and is shown below that for
MutS.
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DISCUSSION |
The mutant proteins we have used all have approximately the same
ATPase specific activity as the wild-type MutS protein, suggesting that
gross structural alterations are not present. This is supported by the
observation that the mutant proteins are able to dimerize and to
interact with MutL unless specifically defective in one or both of
these processes.
The mutS deletion mutations produce little, if any, residual
mismatch repair in vivo, and they do not impart a dominant
phenotype in a wild-type strain (Table II). The latter finding is
surprising for those mutant proteins, such as p
1-311, that fail to
bind DNA but still interact with downstream proteins. It should be noted that IPTG was not added to the cultures to induce the phage T7
RNA polymerase because the wild-type mutS plasmids
complemented the mutator phenotype in its absence (Table II) and
because the plasmid instability increased in its presence. Perhaps the
ratio of mutant protein to wild type is low in vivo in
uninduced cultures because of a lower than expected level of expression
and/or poor solubility of the mutant proteins, thereby precluding
sequestration of sufficient wild-type and mutant monomers into inactive dimers.
The results from Fig. 1 suggest that the N-terminal end of MutS is
important for heteroduplex DNA binding. This is in agreement with the
observation of Malkov et al. (17) that Phe-39 of the Thermus aquaticus (Phe-36 in E. coli) MutS cross-links
to mismatched DNA. We also find, however, that specific DNA binding
occurs at a low level in the mutant deleted for residues 26-260 but is
not detectable if residues 261-556 are deleted (Fig. 2). In
Thermus thermophilus, a proteolytic fragment containing
residues 275-570 of MutS is able to bind double-stranded DNA (18),
confirming the importance of this region for DNA binding.
The ability of MutS to dimerize appears to reside within the region
containing residues 557-853 (Fig. 3). This agrees with the finding by
Alani (19) that in yeast the C-terminal 114 amino acids of Msh2 are
important for interaction with Msh6. Taken together these findings
suggest that the same region of MutS homologs is used for homo- and
heterodimerization. Alani et al. (15) have also described a
mutation (A859E) in yeast Msh2 located in the putative helix-turn-helix
domain, which affected Msh2-Msh6 heterodimer formation. The equivalent
residue in E. coli MutS is Ala-776 that is within the
predicted interaction region (residues 557-853). Because none of the
mutations we tested were completely defective in dimerization, it is
highly probable that additional residues are involved.
The C-terminal region of MutS also seems to be important for MutS-MutL
interaction, and the results in Fig. 4 are the only available
mutational data addressing this question. Given that MutS-MutL
interaction was detected in the presence of MutS, but not in its
absence, and that the extent of the interaction could be varied by
mutation (Fig. 4), we believe the assay detected bonafide interaction.
However, given the high NaCl concentration (500 mM) used in
these experiments, it is probable that the MutS-MutL complexes detected
in these experiments are not bound to heteroduplex DNA. This suggests
that a fraction of the total MutS protein in vivo may be
loosely bound to MutL in the absence of DNA. Even when MutS is bound to
DNA in vitro, its interaction with MutL is not readily
detectable (20). This might explain why the MutS-MutL complexes are
detected in column eluates only at low concentrations of imidazole
(Fig. 4). None of the mutant proteins tested was totally deficient in
MutS-MutL interaction, suggesting that amino acids 556-630, which
include the P-loop motif, are required.
The methods we have used for protein interaction do not allow a
quantitative measurement of the interaction affinities. We have,
therefore, used a qualitative scale to express the results (Fig.
1).
The MutS family of proteins from prokaryotes and eukaryotes is highly
conserved (3-5, 21). The results we have obtained with the E. coli MutS protein should be applicable to other members of the
family. It should now be possible to target individual amino acid
residues in the regions we have identified in MutS or its homologues to
further define those important for DNA binding, dimerization, and MutL interaction.