(Received for publication, July 5, 1995; and in revised form, October 18, 1995)
From the
Two distinct mismatch binding activities are detected using
bandshift assays with human cell extracts and DNA with mispairs at
defined positions. One requires hMSH2 protein and is absent from
extracts of LoVo cells, which contain a partial deletion of the hMSH2
gene. The second activity is independent of hMSH2 and is present at
normal levels in LoVo and three other cell lines, which are defective
in in vitro hMSH2-dependent binding. The two mismatch
recognition activities are distinguished by their sensitivity to
polycations and can be resolved by chromatography on MonoQ.
hMSH2-independent activity has been purified extensively from wild-type
cells and from a cell line deficient in hMSH2-dependent binding. The
purified material preferentially recognizes AC, some
pyrimidine
pyrimidine mismatches, and certain slipped mispaired
structures. Binding exhibits some sequence preferences. The similar
properties of the two mismatch binding activities suggest that they
both contribute to mismatch repair.
The recognition and correction of non-Watson-Crick base pairs increases the fidelity of DNA replication (1) and prevents recombination between partially diverged (homeologous) DNAs(2) . In Escherichia coli, a single mismatch recognition protein, MutS, initiates both the correction of DNA replication errors and the abortion of homeologous recombination intermediates(3) . Mismatch repair is more complex in eukaryotes, and Saccharomyces cerevisiae expresses at least four functional MutS homologs(4) . There is some specialization among these, and yeast MSH1 (for MutS Homolog) is mitochondrial, MSH2 participates in repair of nuclear genomic DNA, while MSH3 and MSH4 are involved in recombination(4) . Yeast cells express multiple homologs of a second E. coli mismatch correction protein, MutL, which participates with MutS in the initial steps of repair. In this case, genetic and biochemical evidence indicates that the yeast MutL function is carried out by a heterodimer of MutL homologs, the products of the MLH1 and PMS1 genes(5) .
Human mismatch correction proteins are particularly important because of their association with hereditary non-polyposis colorectal carcinoma (HNPCC), a syndrome in which there is a familial clustering of colorectal and other carcinomas(6) . Germ-line mutations in three homologs of E. coli MutL, hMLH1, hPMS1, and hPMS2, have been implicated in HNPCC(7, 8, 9) . A heterodimer between two of these gene products, hMLH1 and hPMS2, constitutes the human MutL function(10) . Other human MutL-related genes have been identified, but their functions remain undefined, and there is no known association with disease(8) . To date, four human MutS homologs have been described. Duc-1 is the product of a divergent transcript from the dihydrofolate reductase promoter(11) . hMSH4 was identified on the basis of its sequence similarity to E. coli MutS. The functions of these proteins are unknown, and neither has been implicated in disease. Two other MutS-like proteins, hMSH2 and GTBP, a 160-kDa protein, participate together in mismatch binding(12, 13) . The former is of particular importance since germ-line mutations in hMSH2 may underlie about 40% of hereditary non-polyposis colorectal carcinoma cases(14) .
It
is likely that the extended repertoire of mismatch repair proteins in
human cells reflects functional specialization. Biochemical assays
provide an alternative to comparisons of sequence homology in the
search for putative mismatch repair proteins. Using a bandshift assay
with mismatched DNA molecules, we identified a human mismatch binding
function that could be distinguished from a known GT mismatch
recognition activity (15) by its apparent preference for
A
C and pyrimidine
pyrimidine mismatches(16) . The
G
T binding reaction is now known to be mediated by hMutS
, a
GTBP
hMSH2 heterodimer(12, 13) . Here, we
describe the extensive purification of the other mismatch binding
function. The activity is independent of hMSH2. It is present in
several cell lines defective in hMSH2-dependent binding, including one
that is homozygous for a partially deleted hMSH2 gene. Extracts of
cells deficient in hMSH2-dependent binding but containing apparently
normal hMSH2-independent (A
C) binding activity, are unable to
repair A
C mismatches in vitro, suggesting that the
A
C binding function does not simply replace hMutS
in the
repair of A
C mismatches. A
C binding is carried out by a
protein complex of similar size to hMutS
. The two activities are
resolved following extensive purification, and the partially purified
binding functions are affected differently by polycations. Preferential
binding by both mismatch recognition activities is influenced to some
extent by the sequence context of the mismatch. The substrate
preferences of the purified A
C binding activity provide clues to
its possible role in mismatch repair, and the similarities between
hMSH2-dependent and -independent binding suggest that the two might
play complementary roles.
The generic 34-mer substrates for bandshift assays are shown below. The precise sequences of mismatched duplexes (oligonucleotides 1-7) are given in Table 1.
Oligonucleotides were end-labeled and annealed as described previously to generate either paired or mismatched duplexes. For most substrates, oligonucleotides synthesized in two or more independent batches were used. No differences in the ability to serve as substrates for binding were observed between batches.
The assay mixtures (20 µl) are based on
that of Holmes et al.(21) and contained 20 mM TrisHCl, pH 7.6, 5 mM MgCl
, 1 mM glutathione, 50 µg/ml bovine serum albumin, 0.1 mM each dNTP, 1.5 mM ATP, 70 mM KCl, and 10 ng of
heteroduplex DNA. The reaction was started by addition of extract (70
µg) and continued for 60 min at 37 °C. Reactions were
terminated by the addition of 30 µl of 25 mM EDTA, 0.5%
SDS, and proteinase K (50 µg/ml). After a further 15 min at 37
°C, the mixture was extracted with phenol:chloroform followed by
chloroform, and DNA was recovered by ethanol precipitation.
Precipitated DNA was dissolved in buffer for digestion with AlwNI followed by the appropriate diagnostic restriction
enzyme (at high concentrations, some diagnostic enzymes were able to
cut mismatched DNA, and each batch of mismatched substrate was
therefore titrated with the appropriate enzyme prior to assay to
determine the minimum effective concentration and thereby avoid
over-digestion). For analysis with XmnI, for which there are
two sites in HK7 outside the heteroduplex cassette region, digestion
with AlwNI was omitted. Digestion products were separated on
agarose gels, denatured, and transferred to Hybond N
filters (Amersham International), which were subsequently probed
with radiolabeled random-primed (Boehringer) M13 DNA. Radioactive DNA
was localized by autoradiography.
SDS-polyacrylamide gel analysis was performed using 6 or 8% gels.
Staining was Coomassie or Silver Stain Plus (Bio-Rad). Protein
concentrations were determined by the Bradford method or estimated from A.
Figure 1:
AC
mismatch binding by colorectal carcinoma cell extracts. a,
extracts (12 µg) of the human colorectal carcinoma cell lines
indicated were combined with radiolabeled A
C-mismatched duplex
34-mer oligonucleotide as described under ``Experimental
Procedures.'' Bound and free oligonucleotides were separated by
electrophoresis on a 6% polyacrylamide gel and located by
autoradiography. Complex A is the A
C-specific complex. b, extracts of Raji or LoVo cells were preincubated with a
30-fold excess of the non-radioactive duplex oligonucleotide indicated.
Radioactively labeled A
C substrate was then added, and complex A
formation was analyzed by gel
electrophoresis.
Figure 2:
In vitro correction of AC
mismatches. a, the substrates for the reaction were form II
duplex molecules constructed by annealing viral (v) and
mismatched complementary (c) strands as described under
``Experimental Procedures.'' Each substrate contained the
mismatch shown within a heteroduplex cassette sequence. The position of
the mismatch is designated 0. The numbers in parentheses indicate the distance (kilobases) measured in a clockwise
direction from the mismatched site. Correction of the A
C mismatch
in the nicked strand (left) generates a unique MluI
site. Correction of the C
A mismatch in the nicked strand (right) introduces an additional XmnI site. b, correction assay. Substrates (10 ng) were incubated with
cell extract (70 µg) for 1 h at 37 °C. The DNA was recovered
and digested with AlwNI plus MluI (left
panel) or with XmnI (right panel). Digestion
products were separated on agarose gels, transferred by Southern
blotting, and probed with radiolabeled M13 DNA. The fragment sizes in
kilobases are indicated next to the arrows.
Figure 3:
Effect of spermine on GT and
A
C mismatch binding. a, wild-type cells. The standard
bandshift assay was carried out with the active mismatch binding
fraction purified from the parental Raji cells by ammonium sulfate
precipitation followed by heparin-Sepharose and DNA-cellulose
chromatography. A
C and G
T substrates were assayed in the
presence of the concentrations of spermine as indicated. b, as
in a with partially purified material from RajiF12 cells. Lanes designated M indicate the positions of
complexes A and B formed by partially purified wild-type extract on
A
C and G
T substrates,
respectively.
AC and G
T mismatch binding
activities were resolved by chromatography on MonoQ. An extract of Raji
cells enriched for both activities by sequential ammonium sulfate and
DNA-cellulose fractionation was applied to a MonoQ column in buffer
containing 0.1 M NaCl. The bound protein was eluted by a
gradient of NaCl to 0.5 M. Fractions were assayed for both
A
C and G
T mismatch binding. A
C binding activity
eluted early in the gradient at approximately 0.2 M NaCl. It
was detected by the formation of complex A (Fig. 4a)
with an A
C substrate. No G
T binding (complex B) was
observed in these early fractions. The peak of A
C binding was
partially coincident with the formation of a complex that migrated
close to the well of the gel. This complex was not mismatch-specific
and was formed to the same extent with both A
C and G
T
substrates. In fractions containing very high levels of A
C
binding activity, a small amount of complex A was also formed with the
G
T substrate, suggesting that while formation of this complex
with A
C mismatches is highly preferred, this preference is not
absolute. Similar behavior has been observed with unfractionated
extracts(16) . G
T binding (complex B) eluted after the
A
C and this nonspecific binding activity. There was a slight
overlap with the tail of the A
C binding peak (fraction
13, Fig. 4a), but the later G
T binding
fractions did not bind detectably to A
C. Two other rapidly
migrating complexes (C and D) were also seen. These complexes, which
have been noted previously in crude extracts, are not mismatch specific
and were formed to similar extents with both substrates. Thus, in
addition to their differential susceptibility to spermine, the two
binding activities also exhibit different behavior on ammonium sulfate
fractionation and can be physically resolved on MonoQ.
Figure 4:
Separation of AC and G
T
binding activities on MonoQ. a, wild-type cells. Mismatch
binding activity purified by ammonium sulfate precipitation and
DNA-cellulose chromatography was applied to MonoQ. Elution conditions
were as described under ``Experimental Procedures.'' Each
fraction was assayed for A
C (left lane for each
fraction) and G
T (right lane) binding. Complexes A and B
are the A
C and G
T complexes, respectively. Complexes C and
D are nonspecific complexes. b, As in a with material
from RajiF12 cells.
AC
binding activity was purified from extracts of the G
T
binding-defective RajiF12 cells by the same procedure. Fig. 4b shows the mismatch binding activity in MonoQ
fractions. Complex A was detected with an A
C substrate in
fractions 9-14 and again partially overlapped fractions forming
the slowly migrating nonspecific complex. No complex B was detected in
any fraction. The nonspecific binding (complex D) that partially
overlapped the G
T binding activity in wild-type preparations was
also observed. This serves as an internal control for these fractions.
Similar data were obtained from MonoQ chromatography of extracts of
LoVo cells (data not shown). Thus, the A
C binding activity, which
is present at wild-type levels in G
T binding-defective cells, has
the same purification properties as the wild-type A
C binding
activity. This suggests that the two mismatch binding activities are
independent of one another.
Figure 5:
Size determination of AC binding
activity. MonoQ fractions containing A
C binding activity were
applied to a Superose 12 column. Each fraction was assayed for A
C
binding (complex A). The fractions corresponding to the elution
position of dextran blue (V
), ferritin
(440 kDa, F), and catalase (232 kDa, C) are
indicated.
SDS-polyacrylamide gel analysis of several preparations indicated
that active AC binding fractions (complex A) were associated with
prominent silver-staining protein bands of estimated M
= 110,000 and 140,000. Examples of two preparations,
purified by slightly different procedures, are shown in Fig. 6.
Although several stained bands are apparent in the purified fraction,
two relatively intense bands at 110 and 140 kDa were observed in these
and several other independent preparations. A prominent protein larger
than 250 kDa was observed in the fractions catalyzing the formation of
the slowly migrating nonspecific complex. The intensity of this band
from different column fractions was proportional to the extent of
formation of the nonspecific complex but not of the A
C
mismatch-specific complex A. We conclude that this protein is not
responsible for A
C mismatch recognition. Since gel filtration
indicates that the native M
of the A
C
binding activity is around 250,000, a likely possibility is that the
A
C binding complex is a heterodimer of approximately 250 kDa
comprising the prominent 140- and 110-kDa proteins that were
consistently observed in the purified fractions.
Figure 6:
Proteins involved in AC binding. Two
independent preparations of A
C binding activity from Raji cells
were analyzed by SDS-polyacrylamide gel electrophoresis followed by
silver staining. The positions of the molecular weight standards are
indicated on the right side of the figure. The arrows on the left side indicate the positions of the two most
prominent silver-stained bands that were reproducibly observed in
several preparations. Lane 1, Raji activity purified by
ammonium sulfate (5-55%) fractionation followed by successive
double-stranded DNA-cellulose, MonoQ, MonoS, and Superose 12
chromatography as described under ``Experimental
Procedures.'' Lane 2, Raji activity purified by an
identical procedure except that a 5-35% ammonium sulfate
fractionation was used, and the MonoS chromatography step was
omitted.
Figure 7:
Substrate preferences of AC binding
activity. a, The standard bandshift assay was carried out
using 34-mer duplexes containing the single base mispair indicated (Table 1) and the purified A
C binding activity. The
A
C complex (A) is shown with an arrow. The
substrates containing the AT and CA loops are shown under
``Experimental Procedures'' (oligonucleotides 9 and 10). b, the standard bandshift assay was carried out using the
radiolabeled 34-mer duplex containing an A
C mispair (Table 1, oligonucleotide 2). Non-radioactive competitor 34-mer
duplexes were included in 30-fold excess as
indicated.
Unpurified cell extracts also form complex A on
certain looped mismatched structures(23) . The same 40-mer
substrate containing the sequence (AT) in which one
dinucleotide repeat is displaced from the duplex (oligonucleotide 9)
was also recognized by the purified fraction, and complex A was formed (Fig. 7a). An analogous substrate (oligonucleotide 10)
in which a CA loop displaced from (CA)
replaced the AT loop
was not detectably recognized by the purified A
C binding activity (Fig. 7a). This substrate was bound, however, by a
late-eluting fraction from MonoQ that contained the G
T binding
activity (data not shown). These data provide further evidence for the
different preferences for mismatch recognition by the two binding
functions and suggest that some slipped mispaired intermediates are
also substrates for the A
C binding activity.
A third set of
mismatched substrates was based on the heteroduplex cassette sequence
in the in vitro mismatch correction assay (oligonucleotides 11
and 12). Surprisingly, the purified AC binding activity did not
efficiently recognize a 48-mer duplex containing a single A
C
mismatch at the same position as the repair substrate (Fig. 2a). Complex A was formed but only to a limited
extent, and binding was similar to that seen with a G
T (Table 1, oligonucleotide 3) substrate (Fig. 8a).
When a MonoQ fraction enriched for G
T binding, but containing a
small amount of A
C binding activity (far right lane, Fig. 8a), was used with the A
C mismatched 48-mer,
complex B was formed. This preferential binding by the G
T
activity to the A
C mismatch in this substrate was confirmed with
unfractionated extracts (data not shown). Neither binding activity
detectably recognized a substrate containing an AT loop in an
(AT)
sequence placed in the heteroduplex cassette region.
Thus, recognition of both single base mismatches and slipped/mispaired
structures by one or either of the two mismatch binding activities
exhibits some dependence on the sequence context of the mispairs.
Figure 8:
Effect of sequence context on AC and
G
T binding activities. a, the bandshift assay was
carried out using the standard A
C mismatched 34-mer
(oligonucleotide 2) (A
C, first and last lanes),
the standard G
T mismatched 34-mer (oligonucleotide 3) (G
T),
an A
C mismatched substrate based on the sequence in the
heteroduplex cassette of the in vitro mismatch repair assay
(A
C, lanes 3 and 6) (oligonucleotide 10), or an
AT loop in the heteroduplex cassette sequence (oligonucleotide 12). The
A
C and G
T complexes (A and B) are shown
with arrows. Binding in the four left lanes is by the
purified A
C binding activity. Binding in the four right lanes is by a MonoQ fraction enriched for G
T binding activity but
which contains a low level of A
C binding activity. b,
the MonoQ fraction containing both A
C and G
T binding
activities assayed with the standard A
C mismatched 34-mer
(A
C) (oligonucleotide 2), the standard G
T mismatched 34-mer
(G
T) (oligonucleotide 3), or the standard 34-mer in which the
A
C mismatch was inverted (oligonucleotide 7 in Table 1).
The A
C and G
T complexes (A and B) are
shown with arrows.
Another striking example of sequence effects on mismatch recognition
was provided by the standard 34-mer substrate. When the AC
mismatch in this substrate was inverted (Table 1, oligonucleotide
7), thus altering its surrounding sequence context, the A
C
binding activity no longer recognized the A
C mismatch. This
mismatch was also a substrate for the partially purified G
T
binding activity, and complex B was formed (Fig. 8b).
The complexity of mismatch repair in eukaryotic cells is
reflected in the abundance of different human mismatch repair proteins.
Since there are at least four MutS homologs in yeast, some
diversification of mismatch recognition is also likely in human cells.
It is interesting to note that the constituents of hMutS, hMSH2
and GTBP, are both MutS homologs (12, 13) and so each
may contribute to mismatch recognition. The A
C binding activity
may be an example of the diversity in mismatch recognition.
Not all
human mismatch recognition proteins are necessarily involved in
mismatch repair. Mg and ATP-independent mismatch
binding detected by bandshift assays is associated with mammalian DNA
topoisomerase I(25) . Topoisomerase I can be excluded as a
candidate for the A
C binding activity on the basis of its size
and its substrate preferences. It is a single polypeptide of M
= 95,000, which is sometimes purified as
a proteolysis product of approximate M
=
80,000, whereas the A
C binding activity is purified as an
apparently stable complex of estimated M
250,000. Topoisomerase I acts on all single base mismatches, whereas
the purified A
C binding complex is more fastidious in its
recognition. Two other human proteins that bind to mismatches and can
be detected by the bandshift assay have been described. One of these,
hMutY, the human homolog of the E. coli MutY protein, is a DNA
glycosylase that removes A residues from A
G,
A
7,8-dihydro-8-oxoguanine, and, with 30-fold less efficiency,
A
C mismatches(26) . hMutY and the recently described
deoxyinosine 3`-endonuclease (27) can be excluded as candidates
for the A
C binding activity both on the basis of their sizes (65
and 25 kDa, respectively) and of their substrate preferences. In
initial fractionation studies of human cell extracts, A
C binding
activity was associated with a protein, or complex, of approximately
100 kDa(16) . It is possible that this reflects an autonomous
binding activity of the smaller component of an A
C complex
dissociated at relatively low protein concentrations.
Our data
suggest that the AC binding complex is a dimer of proteins of
approximately 110 and 140 kDa. This composition is similar to that of
the G
T binding complex of hMSH2 and GTBP (12, 13) (104 and 160 kDa). Despite this similarity in
size, the complexes formed with A
C and G
T substrates are
resolved in the bandshift assay. The faster migration of the A
C
complex suggests that the A
C binding proteins may be more acidic.
A difference in surface charge is consistent with separation of the
A
C and G
T binding activities on MonoQ and may also underlie
the different effect of spermine on A
C and G
T binding.
Polyamines can affect protein-DNA interactions in several ways,
including inducing DNA bending (28) and altering the site
occupancy by binding proteins(29) . A contribution of one or
more of these other properties to the different effect of spermine on
A
C and G
T binding cannot be excluded.
The presence of
AC binding activity in RajiF12, LoVo, and DLD1 extracts, all of
which have no detectable G
T binding, indicates that A
C
binding is a separate function. This is supported by our partial
purification of the A
C binding activity from RajiF12 and LoVo
cells and the demonstration that its properties are indistinguishable
from the wild-type activity. Since LoVo and DLD1 contain mutations in
hMSH2 (14, 30) and GTBP(31) , respectively,
both of which inactivate G
T binding, it appears that neither
hMSH2 nor GTBP is required for the A
C binding reaction.
Although we have no direct evidence that the AC binding
activity participates in mismatch correction, their similarities in
size and general properties suggest that the G
T and A
C
binding complexes may play complementary roles. We previously reported
that the A
C binding activity in unfractionated cell extracts
efficiently recognizes T
C and T
T mismatches but binds to
C
C only poorly(16) . These substrate preferences are
apparent in the purified A
C binding complex. Since of all single
base mismatches, C
C mispairs are corrected least efficiently in vitro and in vivo(21, 32) ,
these observations are consistent with an involvement of the A
C
binding activity in mismatch repair. The purified binding complex also
recognizes AT dinucleotides displaced from a (AT)
sequence.
This ability to bind to displaced dinucleotides is analogous to the
recognition of displaced CA dinucleotides by the G
T binding
function (23) and suggests that the A
C binding activity
might also be involved in stabilizing microsatellite sequences.
Recognition by the AC mismatch binding activity does not
exhibit an absolute dependence on DNA sequence context because the
A
C complex is formed on A
C mispairs and AT loops in
completely unrelated sequences. The context of the mismatch can,
however, influence its recognition. We observed that neither A
C
mismatches nor AT loops in a third, unrelated, sequence were recognized
by the purified A
C binding complex. Instead, these mismatches
were substrates for the G
T binding activity. Several other
observations are also consistent with a degree of overlap in mismatch
recognition by the A
C and G
T binding activities. At high
concentrations, the purified A
C complex binds to G
T
mismatches. This can be seen in the MonoQ fractions that are highly
enriched for A
C binding activity. In addition, purified
hMutS
binds (albeit rather poorly) to A
C
mismatches(22) . The rules that govern recognition cannot be
inferred from these few data, but local sequence determinants may
contribute to the probability that mismatches are recognized by one or
other of the binding heterodimers.
Data from the in vitro mismatch correction assays indicate, however, that mismatches
cannot simply be divided into two groups, one comprising substrates for
hMutS and the other the mismatches recognized by the A
C
complex. LoVo cell extracts cannot repair several mismatched structures in vitro, implicating hMSH2 in their correction. Our data
indicate that the repair deficiency of LoVo extends to A
C (and
C
A) mismatches, at least in the particular context of the
heteroduplex cassette. The inability of LoVo, DLD1, and RajiF12 cell
extracts to correct A
C mismatches in the in vitro assay
is paralleled by the absence of recognition by the A
C binding
activity of A
C mismatches in the heteroduplex cassette sequence.
A
C mismatch recognition in this particular context is mediated by
the G
T complex, which is absent from these cells. hMutS
and
the A
C mismatch binding activity may turn out to have
complementary but partially overlapping roles in mismatch recognition.
The in vitro assay provides a good indication as to whether
particular mismatches are repaired. These assays are building up a
picture of the requirements for hMutS in the correction reaction.
A caveat should be added, however. The assays provide a strand
discrimination signal for correction in the form of a nick in the
substrate DNA. The possibility that the mismatch binding activities
participate in strand selection during correction of their preferred
mispairs has not been ruled out. Provision of a pre-incised substrate
may obviate the need for this selective recognition and may miss an
important property of the mismatch recognition complexes.
Many DNA
repair functions are partially duplicated by back-up activities with
similar specificities. The AC binding activity might be one of
these and may serve a complementary function to the hMutS
complex
in mismatch recognition and initiation of repair.