(Received for publication, August 7, 1995; and in revised form, December 6, 1995)
From the
Recognition of mispaired or unpaired bases during DNA mismatch
repair is carried out by the MutS protein family. Here, we describe the
isolation and characterization of a thermostable MutS homolog from Thermus aquaticus YT-1. Sequencing of the mutS gene
predicts an 89.3-kDa polypeptide sharing extensive amino acid sequence
homology with MutS homologs from both prokaryotes and eukaryotes.
Expression of the T. aquaticus mutS gene in Escherichia
coli results in a dominant mutator phenotype. Initial biochemical
characterization of the thermostable MutS protein, which was purified
to apparent homogeneity, reveals two thermostable activities, an ATP
hydrolysis activity in which ATP is hydrolyzed to ADP and P and a specific DNA mismatch binding activity with affinities for
heteroduplex DNAs containing either an insertion/deletion of one base
or a GT mismatch. The ATPase activity exhibits a temperature optimum of
approximately 80 °C. Heteroduplex DNA binding by the T.
aquaticus MutS protein requires Mg
and occurs
over a broad temperature range from 0 °C to at least 70 °C. The
thermostable MutS protein may be useful for further biochemical and
structural studies of mismatch binding and for applications involving
mutation detection.
Mismatch repair plays a critical role in maintaining genetic stability in both prokaryotes and eukaryotes (reviewed in (1) ). The mismatch repair pathway removes mismatches arising from errors of replication that escape proofreading functions, from chemical modification of bases, notably the deamination of 5-methylcytosine, and from homologous recombination between divergent sequences giving rise to heteroduplex DNA containing mispaired or unpaired bases. Mismatch repair also serves as a barrier to interspecies recombination by inhibiting recombination between divergent sequences(2, 3, 4) . The most extensively studied long-patch mismatch repair systems are the methyl-directed mismatch repair pathway of Escherichia coli and Salmonella typhimurium(5) and the Hex-dependent mismatch repair pathway of Streptococcus pneumoniae(6) . The E. coli methyl-directed mismatch repair pathway has been reconstituted in vitro using purified components and has been shown to require 10 proteins, MutS, MutL, MutH, DNA helicase II, SSB, exonuclease I, exonuclease VII, RecJ, DNA polymerase III holoenzyme, and DNA ligase (7, 8) .
Among the proteins required for methyl-directed mismatch repair, MutS alone recognizes and binds to heteroduplex DNA containing mispaired or unpaired bases. The in vitro affinity of MutS protein for mismatches and small insertion/deletions of one to four bases roughly parallels the in vivo efficiency of repair(9, 10) . In the presence of ATP, a complex consisting of MutS bound to a mismatch site together with MutL activates the MutH endonuclease which incises the unmethylated strand at hemimethylated d(GATC) sites. In this way, repair in E. coli is directed to the newly synthesized strand (reviewed in (11) ). In S. pneumoniae, mismatch repair is initiated by MutS and MutL homologs, HexA and HexB, respectively; however, no MutH equivalent has been identified, and repair is thought to be directed by single-strand breaks(6) .
Genetic and biochemical studies of mismatch repair establish that many of the essential features of mismatch repair in prokaryotes have been conserved in eukaryotes. A number of eukaryotic MutS and MutL homologs have been identified including at least five MutS homologs (12, 13, 14, 15) and three MutL homologs (16, 17, 18) in yeast. Defects in genes encoding some of these homologs in humans have been implicated in hereditary nonpolyposis colon cancer in which tumor cells are characterized by instability of microsatellite repeats(19, 20, 21, 22, 23) .
Elucidating the molecular mechanism of mismatch repair is important given the essential nature of the process in virtually all organisms. Studies of prokaryotic mismatch repair enzymes may be advantageous in certain respects due to their relative simplicity. For example, whereas repair in prokaryotes involves a single MutS species and a single MutL species, repair in eukaryotes involves complex interactions among different MutS and MutL homologs. Studies of two human MutS homologs, hMSH2 and GTBP/p160, indicate that a heterodimer of both proteins is required for optimal binding and correction of mismatches and insertions/deletions of 1 or 2 bases in vitro(24, 25) , a finding that has been substantiated in vivo(26) .
In this paper, we characterize a MutS homolog from the thermophilic eubacterium Thermus aquaticus YT-1. The highly conserved sequence of the thermostable MutS protein and its heteroduplex binding properties, namely the involvement of a single polypeptide species and mismatch binding over a wide temperature range up to 70 °C, indicate that it will be a useful reagent in biochemical and structural studies directed toward a mechanistic understanding of mismatch repair. In addition, the thermostable MutS protein may be useful in applications involving mutation detection.
Figure 1: Nucleotide sequence of the T. aquaticus YT1 mutS gene. The sequence of the mutS gene is shown along with flanking 5` and 3` sequences. Underlined peptide sequences correspond to regions to which the PCR primers were directed. RBS, putative ribosome binding site.
Purification of
the MutS protein was monitored by the presence of a 90-kDa
IPTG-inducible polypeptide on SDS-polyacrylamide gels. The relative
size of the MutS polypeptide was deduced from its migration on
SDS-polyacrylamide gels relative to molecular weight standards
(Bio-Rad, broad range standards). Frozen cells were thawed and
disrupted by sonication. A crude cell extract was obtained by
centrifugation for 60 min at 100,000 g. The
supernatant was heated to 70 °C for 45 min and centrifuged at
16,000
g for 20 min. The supernatant was brought to
30% saturated (NH
)
SO
and
centrifuged for 20 min at 16,000
g. The pellet
containing MutS protein was resuspended in Buffer A, 20 mM potassium phosphate, pH 8.0, 100 mM NaCl, 10% (v/v)
glycerol, 0.1 mM EDTA, 0.1 mM dithiothreitol, 1
mM phenylmethylsulfonyl fluoride, and 10 µg/ml each
pepstatin and leupeptin, and dialyzed extensively against Buffer A. The
protein sample was then applied to a Mono Q column (Pharmacia)
equilibrated in Buffer A and eluted with a linear gradient of 100
mM to 640 mM NaCl in Buffer A. MutS protein eluted at
approximately 260 mM NaCl. Fractions containing MutS protein
were pooled and dialyzed against Buffer A and stored at -20
°C. From 1 liter of culture, we obtained approximately 2 mg of MutS
protein. Scanning densitometry of Coomassie-stained SDS-polyacrylamide
gels containing the Mono Q-purified MutS protein fraction revealed that
the preparation was in excess of 90% pure. Throughout the paper, MutS
protein refers to the Mono Q-purified protein fraction. Protein
concentrations were determined by a modified Bradford assay (Bio-Rad)
using bovine serum albumin as a standard.
The 348-bp fragment was used to probe restriction endonuclease digests of T. aquaticus genomic DNA in Southern blots. The probe hybridized to a single 2.67-kb BamHI fragment which was subsequently cloned from a genomic library of T. aquaticus (see ``Materials and Methods''). A probe derived from the 5` end of the BamHI fragment was used to isolate a 1.0-kb SacI fragment containing the 5` end of the putative mutS gene.
DNA sequencing revealed that the full-length T. aquaticus mutS gene has a single, large open reading frame of 2,436 bp predicting a polypeptide of 811 amino acids corresponding to 89.3 kDa (Fig. 1). The DNA sequence has a 69% GC content reflecting the high overall GC content, 67.4%, of T. aquaticus(32) . A presumptive ribosome binding site, 5`GAGG3`, is located just upstream of the first AUG start codon.
Sequence comparison of the translated open reading frame of T. aquaticus mutS with E. coli, Saccharomyces
cerevisiae, and human MutS homologs predicts extensive sequence
homology, 63% identity with E. coli MutS, 56% identity with S. cerevisiae MSH2, and 55% identity with human MSH2 (Fig. 2). Within the putative nucleotide binding region
corresponding to the region amplified by PCR, the sequence conservation
is even more extensive with T. aquaticus MutS sharing 80%
identity with E. coli MutS. The consensus sequences for the
Mg-ATP binding site, GKS/T and DE invariant sequences (33, 34) , are present in the T. aquaticus translated sequence at positions Gly
and
Asp
, respectively, corresponding to positions C
and T
, respectively, in the nucleotide sequence
shown in Fig. 1. In addition, a putative helix-turn-helix domain (35) is present near the carboxyl terminus of the T.
aquaticus protein at positions Gly
through
Ala
.
Figure 2: Comparison of predicted amino acid sequence of T. aquaticus MutS with MutS homologs. Sequences were aligned using the PILEUP program (Genetics Computer Group, Madison). Boxed residues indicate conserved positions. Bold indicates positions conserved among all four MutS proteins. Abbreviations are: TaqMutS, T. aquaticus MutS; EcoMutS, E. coli MutS; Msh2, S. cerevisiae MSH2; hMsh2, human hMSH2. Sequences for E. coli and eukaryotic MutS proteins are from GenBank.
The presence of the T. aquaticus mutS gene in repair-proficient E. coli conferred a dominant mutator phenotype as judged by elevated frequencies of spontaneous mutation to rifampicin resistance (Table 1). Whereas the pET3a vector alone yielded a mutation frequency similar to that of the host E. coli BL21, the presence of T. aquaticus mutS resulted in a 14.3-fold increase in the frequency of spontaneous mutation. This result is analogous to that previously observed for other heterologous mutS genes from humans and S. pneumoniae(19, 36) although the magnitude of the increase in the spontaneous mutation frequency is lower in the case of T. aquaticus mutS.
Not surprisingly, we were unable to demonstrate
direct complementation of an E. coli mutS strain, GW3732 (mutS201::Tn5) (37) by
expression of the heterologous T. aquaticus mutS gene cloned
in an IPTG-inducible expression vector, pTrc 99 A (data not
shown). Such complementation is unlikely given the temperature optimum
of the thermophilic protein, see below, as well as the requirement in E. coli for MutS-mediated activation of MutH cleavage at
hemimethylated d(GATC) sites which are absent in T. aquaticus.
Figure 3:
High level expression of the cloned T.
aquaticus mutS gene in E. coli. A 2.9-kb genomic fragment
containing the coding region of T. aquaticus mutS was cloned
into the T7 expression vector pET3a and transformed into E. coli BL21(DE3) as described under ``Materials and Methods.''
Upon induction of T7 polymerase with IPTG, a 90-kDa polypeptide was
synthesized at high levels as judged by electrophoresis on a 12%
SDS-polyacrylamide gel (lanes 1 and 2). The 90-kDa
polypeptide was partially purified from crude cell extracts by
centrifugation at 100,000 g followed by a heat
denaturation step, precipitation in ammonium sulfate, and
chromatography on a Mono Q anion exchange column (lanes
3-6). A molecular mass standard, lysozyme, 14 kDa,
co-migrated with the tracking dye to within 1.6 cm of the bottom of the
gel.
Identification of the 90-kDa protein as that encoded by the cloned T. aquaticus mutS gene was determined by N-terminal amino acid sequencing of the polypeptide excised from an SDS gel. Sequencing of the first 15 amino acid residues yielded an exact match with the predicted sequence shown in Fig. 2.
Figure 4:
Thermostable ATP hydrolysis by T.
aquaticus MutS. A, ATP hydrolysis assays were carried out
for 15 min at 70 °C in the presence of increasing amounts of MutS
protein and [-
P]ATP and analyzed by thin
layer chromatography as described under ``Materials and
Methods.'' Lanes 1-9 contained 0, 0.1, 0.2, 0.4,
0.6, 0.8, 1.0, 1.5, and 3.0 µg of protein, respectively. Arrows mark the migration positions of unlabeled ATP and ADP standards. B, quantitation of ATPase assays shown in A. C, ATP hydrolysis as a function of time. Reactions containing
770 ng of protein were incubated for varying lengths of time at 70
°C. Results are the average of at least two independent trials;
standard deviations are indicated by error
bars.
The thermostability of the ATPase activity of T. aquaticus MutS was assessed over a wide temperature range (Fig. 5). Consistent with the 70-75 °C temperature optimum for T. aquaticus growth in vivo(32) , the temperature optimum for the ATPase activity as judged by the extent of ATP hydrolysis after 15 min was 80 °C although ATP hydrolysis mediated by MutS occurred at lower temperatures. Thus, 72% of the ATP was hydrolyzed at 80 °C, whereas 22% of the ATP was hydrolyzed at 37 °C. At 90 °C, the extent of ATP hydrolysis was reduced to only 15%. In a control experiment, nonenzymatic hydrolysis of ATP at 80 °C in the absence of MutS protein was less than 4% (data not shown). These assays were conducted in the presence of 20 mM Tris-HCl, pH 7.5 at 25 °C. At 70 °C, the pH would be expected to drop about 1.3 pH units to pH 6.2. Subsequent ATPase experiments have been repeated at 70 °C in the presence of 20 mM Hepes buffer, pH 7.5 at 25 °C, instead of Tris-HCl with no discernible effect on the extent of ATP hydrolysis (data not shown). Reactions carried out in the presence of Hepes buffer would be expected to show a considerably smaller effect of temperature on pH, pH 6.8 at 70 °C, as opposed to pH 7.5 at 25 °C(38) .
Figure 5:
Temperature optimum of ATP hydrolysis.
ATPase assays containing 770 ng of T. aquaticus MutS protein
were carried out at various temperatures as described under
``Materials and Methods.'' Control experiments indicate that
spontaneous hydrolysis of [-
P]ATP in the
absence of protein was less than 4% after 15 min at 80 °C. Results
are the average of at least two independent trials. Standard deviations
are indicated by error bars.
Figure 6: DNA substrates used in ATPase and heteroduplex binding assays. Duplex substrates were made by annealing gel-purified oligodeoxynucleotides and were subsequently purified from polyacrylamide gels as described under ``Materials and Methods.'' Sequences for the 36-bp substrates were described previously(30) .
Figure 7:
T. aquaticus MutS binds to GT
heteroduplexes. A, the ability of MutS protein to bind
specifically to a mismatch was assessed in filter binding assays.
Reactions were initiated by the addition of 6.4 nMP-labeled homoduplex (AT) or heteroduplex (GT) DNA
and 3 µg of MutS protein and were carried out for 15 min at 60
°C in 20 mM Tris-HCl, pH 7.5 (25 °C), 0.1 mM dithiothreitol, 0.1 mM EDTA, 50 µg/ml bovine serum
albumin, 5% (v/v) glycerol, 20 µg/ml poly(dI)-poly(dC), and the
indicated amounts of MgCl
in a 20-µl reaction volume as
described under ``Materials and Methods.'' Reactions were
filtered through BA85 nitrocellulose and NA45 DEAE-cellulose membranes. B, quantitation of the filter binding assays shown in A as described under ``Materials and
Methods.''
Figure 8:
Preferential binding of MutS to a
heteroduplex containing a deletion of one base. A and B, MutS binding to a 36-bp heteroduplex containing a deletion
of one base, 1, was compared to binding of a GT heteroduplex in
filter binding assays as described in Fig. 7. C, DNA
filter binding assays were carried out as described above with
P-labeled, 36-bp DNA substrates, AT homoduplex control, GT
mismatch, or
1 deletion, in the presence or absence of a 100-fold
molar excess of unlabeled competitor DNA as indicated. D,
specific binding to the
1 substrate by MutS protein was
demonstrated in band mobility shift experiments. Binding assays
containing 2.8 µg of MutS protein and 6.4 nM 36-base
single-strand DNA control, 36-bp homoduplex AT control, or 36-bp
1
heteroduplex were carried out at 60 °C as described above except
that a 133-fold molar excess of cold competitor DNA was added as
indicated at the start of the reaction. Binding reactions were
electrophoresed on 6% nondenaturing polyacrylamide gels in 5 mM MgCl
.
The specificity of binding of MutS to heteroduplex
DNAs was assessed in competition experiments (Fig. 8C).
Binding to a P-labeled GT mismatch substrate, 62%
substrate bound, was poorly competed by a hundredfold molar excess of
AT homoduplex competitor, 52% substrate bound. In contrast, binding to
the
P-labeled GT heteroduplex was competed efficiently by
a hundredfold molar excess of unlabeled GT heteroduplex, 7%
heteroduplex bound. The binding by MutS to a single-base deletion
heteroduplex likewise exhibited great specificity since a hundredfold
molar excess of AT duplex failed to reduce binding to the
P-labeled
1 substrate, 89% and 85% heteroduplex bound
in the absence and presence of homoduplex competitor, respectively. In
contrast, a hundredfold molar excess of unlabeled
1 heteroduplex
greatly reduced binding, 16% heteroduplex bound.
The specificity of
binding of MutS to the 36-bp 1 heteroduplex containing the
single-base deletion was also monitored in a band mobility shift assay (Fig. 8D). DNA binding assays were carried out
essentially as described for filter binding assays except that, after
incubation of MutS protein and DNA substrates for 15 min at 60 °C,
the reactions were electrophoresed on a nondenaturing polyacrylamide
gel in the presence of MgCl
at room temperature. As
expected, no significant binding to either single-strand DNA or
homoduplex DNA was observed. In contrast, MutS protein efficiently
bound to the
P-labeled
1 substrate resulting in the
formation of a single, slower migrating species. The formation of a
MutS-
1 DNA complex was not affected by the presence of a 133-fold
molar excess of unlabeled AT homoduplex competitor, but was essentially
abolished by the presence of a 133-fold molar excess of unlabeled
1 heteroduplex. Under these same conditions, we are unable to
demonstrate a gel shift with the GT mismatch substrate although
MutS-mismatch complexes are formed efficiently as judged by filter
binding (data not shown). This result suggests that MutS-
1
complexes are more stable than MutS-GT complexes under these
electrophoretic conditions.
Figure 9:
Heteroduplex binding as a function of
temperature. Filter binding assays were carried out as described in Fig. 7for 15 min at various temperatures in the presence of 1.4
µg of MutS protein and 6.8 nMP-labeled,
61-bp homoduplex or the corresponding 61-bp I1 heteroduplex containing
an insertion of one base (see Fig. 6). C, incubation
without MutS protein at 60 °C. Results are the average of two
trials.
We describe the cloning and overexpression of a MutS homolog from T. aquaticus YT1 and the initial biochemical characterization of the thermostable MutS protein. The gene encodes a 90-kDa polypeptide with DNA mismatch binding and ATPase activities that are active at temperatures as high as 70-80 °C. The deduced amino acid sequence of the T. aquaticus MutS protein exhibits extensive sequence homology with MutS proteins from both prokaryotes and eukaryotes and shares 63% overall identity with E. coli MutS protein. Highly conserved regions include the Walker consensus sequence for nucleotide binding (33, 34) as well as a helix-turn-helix motif near the carboxyl terminus of the protein(12, 39) .
Expression of T. aquaticus mutS in mismatch-proficient E. coli resulted in a dominant mutator phenotype as judged by the frequency of spontaneous mutation to rifampicin resistance. By analogy to other MutS homologs, notably S. pneumoniae hexA(36) and human hMSH2(19) , the dominant mutator phenotype conferred by T. aquaticus mutS suggests a role for T. aquaticus mutS in mismatch repair in vivo although it has not been shown directly. The basis for the dominant mutator phenotype conferred by the T. aquaticus gene is unknown. The thermostable MutS protein may bind to heteroduplex DNA in the E. coli genome and thereby prevent the endogenous repair machinery from gaining access to mismatches. Alternatively, the thermostable MutS protein may interact nonproductively with other components of the E. coli mismatch repair machinery, for example, the MutL protein.
As others have
observed for the S. pneumoniae hexA gene(36, 39) , we were unable to demonstrate
complementation of the mutator phenotype of an E. coli
mutS mutant by the introduction of the T.
aquaticus mutS gene. This is not unexpected given that
methyl-directed mismatch repair in E. coli responds to the
state of methylation at dam methylation sites. Such a
methylation system is not known to exist in thermostable bacteria, and,
as in the case of S. pneumoniae and eukaryotic systems, it is
unlikely that the T. aquaticus MutS protein can activate E. coli MutH, the endonuclease that incises at hemimethylated
d(GATC) sites. In addition, a heterologous MutS protein may not be able
to facilitate the large number of protein-protein interactions required
for mismatch repair in E. coli best documented by interactions
between MutS, MutL, and MutH proteins (reviewed in (5) ).
The presence in the T. aquaticus mutS gene of highly
conserved sequence motifs found in a large number of proteins that bind
and hydrolyze nucleotides including the MutS family (33, 34) predicted that the thermostable MutS protein
would have an ATP hydrolytic activity. Such an ATPase activity has been
demonstrated by others in MutS proteins from both prokaryotes (30, 40) and eukaryotes(41, 42) .
Here, we demonstrate that T. aquaticus MutS protein exhibits a
thermostable ATPase activity in the absence of exogenous DNA resulting
in the cleavage of ATP to ADP and P (Fig. 4). The
ATPase activity exhibits a temperature optimum of 80 °C although
substantial activity is also detected at 60 °C (Fig. 5).
T. aquaticus MutS preferentially binds to a heteroduplex
DNA containing a single GT mismatch. Binding exhibited a requirement
for magnesium ions such that no binding to a GT heteroduplex occurred
in the absence of MgCl; however, mismatch binding was
substantial upon the addition of 2.5 mM MgCl
.
Raising the magnesium concentration from 2.5 mM to 10 mM had no significant effect on either the extent of binding or the
selectivity of mismatch binding (Fig. 7). The thermostable MutS
differs from the S. cerevisiae MSH2 (43) and human
hMSH2 (44) proteins with respect to a requirement for
Mg
. Neither the MSH2 protein nor hMSH2 requires
divalent metal ions for heteroduplex binding, and increasing the
concentration of magnesium abolishes the preferential binding of yeast
MSH2 to mismatched DNAs as opposed to homoduplex DNA. In contrast,
magnesium ions, while not essential, enhance heteroduplex binding by
the human GTBP protein(41) .
Specific heteroduplex binding mediated by the T. aquaticus MutS protein over a wide temperature range, the extensive sequence homology shared between T. aquaticus MutS and other MutS proteins, and the relative ease with which large amounts of the thermostable MutS protein can be obtained make this protein particularly suitable for mechanistic and structural studies of mismatch repair. In addition, studies of mismatch binding at high temperature may yield novel insights into structural determinants that stabilize such interactions. The properties of the T. aquaticus MutS protein may also be useful in practical applications. For example, methods have been developed for mutation detection that exploit the ability of E. coli MutS protein to bind specifically to heteroduplex DNA(45, 46) . Another application is the use of MutS-mediated mismatch recognition to facilitate genetic linkage mapping(47) . Use of the thermostable T. aquaticus protein may be beneficial where enhanced stability of MutS-mismatch complexes is required or where mutation detection at elevated temperatures is desirable.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U33117[GenBank].