Mismatch DNA recognition protein from an extremely thermophilic bacterium,
Thermus thermophilus
HB8
Mismatch DNA recognition protein from an extremely thermophilic bacterium, Thermus thermophilus HB8
Satoko
Takamatsu
,
Ryuichi
Kato
and
Seiki
Kuramitsu*
Department of Biology, Faculty of Science, Osaka University, 1-1 Machikaneyama-cho, Toyonaka,
Osaka
560,
Japan
Received October 31, 1995
;
Revised and Accepted January 8, 1996
DDBJ accession no. D63810
ABSTRACT
The
mutS
gene, implicated in DNA mismatch repair, was cloned from an extremely
thermophilic bacterium,
Thermus thermophilus
HB8. Its nucleotide sequence encoded a 819-amino acid protein with a molecular mass of 91.4 kDa. Its predicted amino
acid sequence showed 56 and 39% homology with
Escherichia coli
MutS and human hMsh2 proteins, respectively. The
T.thermophilus
mutS
gene complemented the hypermutability of the
E.coli
mutS
mutant, suggesting that
T.thermophilus
MutS protein was active in
E.coli
and could interact with
E.coli
MutL and/or MutH proteins. The
T.thermophilus
mutS
gene product was overproduced in
E.coli
and then purified to homogeneity. Its molecular mass was estimated to be 91 kDa
by SDS-PAGE but
~
330 kDa by size-exclusion chromatography, suggesting that
T.thermophilus
MutS protein was a tetramer in its native state. Circular dichroic measurements
indicated that this protein had an
[alpha]
-helical content of
~
50%, and that it was stable between pH 1.5 and 12 at 25
o
C and was stable up to 80
o
C at neutral pH.
Thermus thermophilus
MutS protein hydrolyzed ATP to ADP and Pi, and its activity was maximal at 80
o
C. The kinetic parameters of the ATPase activity at 65
o
C were
K
m = 130
[mu]
M and
k
cat = 0.11 s
-1
.
Thermus thermophilus
MutS protein bound specifically with G-T mismatched DNA even at 60
oC.
INTRODUCTION
All living organisms have DNA repair systems to counteract DNA damage caused by
sunlight, chemical agents and DNA replication errors (
1
). DNA repair systems involve photoreactivation, base excision repair,
nucleotide excision repair, mismatch repair and recombinational repair (
1
). Mismatched base pairs are produced by either genetic recombination, DNA
damage or DNA replication errors. In
Escherichia coli
, they are repaired by the
mutH
,
L
and
S
gene products (
2
). MutS homologues have been found not only in other bacteria, but also in
yeast,
Xenopus
, mouse and human (
3
-
9
). Moreover, MutL homologues have also been found in organisms ranging from
bacteria to the higher eukaryotes (
10
-
12
). These findings suggest that the mismatch repair system is ubiquitous for all
living organisms (
1
,
3
).
In humans, kindred analysis of hereditary nonpolyposis colorectal cancer (HNPCC)
has implicated four genes in the development of the disease (
8
-
11
,
13
). These are
hMSH2
, which is a
MutS
homologue, and
hMLH1
,
hPMS1
and
hPMS2
, which are
MutL
homologues in man. A defect in one of these genes apparently predisposes to
tumor formation. Molecular analysis of the mismatch repair system is required
in order to understand the mechanism of tumor development.
E.coli
has been extensively used as a model to study the mismatch repair system at the
molecular level (
2
). MutS protein specifically binds to mismatched base pairs in the DNA. MutL
protein then interacts with MutS protein to increase the stability of the MutS-mismatch DNA complex. MutH protein, a single-strand endonuclease, binds to the complex and then recognizes and nicks
the unmethylated (i.e. newly replicated) strand of DNA at the hemi-methylated GATC site. The newly replicated strand is degraded,
resynthesized and then the mismatched base pair is repaired. In the above
model, MutS protein recognizes mismatched base pairs in the DNA, although the
details of the reaction mechanism by which this recognition process occurs
remain to be elucidated.
Of the three proteins involved in the mismatch repair process, MutS protein is
the most important because of its initial role in mismatched base pair
recognition. Therefore, we decided to analyze MutS protein in detail. In order
to understand the structure-function relationships for this protein at the molecular level, enzymatic
and physicochemical analysis is required. Proteins from thermophilic bacteria
are particularly useful for study because they are stable and easily
crystallized. The extremely thermophilic bacterium,
Thermus thermophilus
HB8, is an aerobic, rod-shaped, non-sporulating Gram-negative eubacterium, which can grow at temperatures in excess
of 75oC (
14
). In this report, we describe the cloning, sequencing and overexpression of the
mutS
gene from
T.thermophilus
. We also describe the purification and characterization of MutS protein and
demonstrate that it was stable at temperatures <= 80oC, it had ATPase activity, and that it could recognize G-T mismatches. These results help to define the molecular role of
MutS protein during substrate recognition.
Ap
r
; pBR322 derivative, vector for gene expression
(44)
pLysE
Cm
r
; pACYC184 derivative, represses the expression of T7 RPase
(44)
pTS3
Ap
r
; pUC118 derivative, carrying the entire
T.thermophilus mutS
gene
This work
pSS1
Ap
r
; pET3a derivative, a plasmid for expression of the
T.thermophilus mutS
gene
This work
a
Another name for this strain is CC106.
MATERIALS AND METHODS
Bacterial strains, media, plasmids and chemicals
All of the
E.coli
strains and plasmids used in this study are listed in Table
1
. They were grown in LB medium or Terrific broth at 37oC (
15
).
Thermus thermophilus
HB8 was grown at 75oC as described elsewhere (
16
). DNA manipulations were carried out using standard procedures (
15
). The reagents were purchased as follows: DEAE-cellulose DE52 from Whatman
Biochemicals; Phenyl-Toyopearl 650M from Tosoh; Sephacryl S-300 HR from Pharmacia. The DNA oligomers used were synthesized by a
Cyclone Plus DNA synthesizer or purchased from Kurabo or Nihon Seifun. All
other reagents used in this study were of the highest grade commercially
available.
Cloning, sequencing and overexpression of the
T.thermophilus
mutS
gene
Two highly conserved regions taken from five MutS homologues (
E.coli
MutS,
Salmonella typhimurium
MutS,
Streptococcus pneumoniae
HexA,
Saccharomyces cerevisiae
Msh2 and human DUC-1) (
4
) were used as the bases for the designed synthesis of mixed oligonucleotide
primers for the polymerase chain reaction (PCR). The left and right mixed
oligonucleotide primers were 5'-AC(C/G)GG(C/G)CC(C/G)AACATGGG(C/G)GG(C/GAT)- AA-3' and 5'-AA(G/A)CG(G/C/A)TG(G/C)GTGATGAA(G/A)- CT(C/T)-3', respectively.
Using
T.thermophilus
HB8 genomic DNA as a template, PCR was carried out under the condition
described previously (
16
). Genomic DNA from
T.thermophilus
was digested with
Pst
I and then subjected to Southern hybridization using the
32
P-labelled PCR fragment as a probe. The DNA fragments which hybridized with
the probe were purified and ligated into pUC119 to produce a mini gene bank of
T.thermophilus
genomic DNA.
Escherichia coli
DH5[alpha] cells transformed with the mini gene bank were screened by colony
hybridization. Among the positive signals, the DNA fragment (pTS3) containing
the entire
T.thermophilus
mutS
gene was obtained. The nucleotide sequence of the
Xba
I-
Bam
HI region of pTS3 was determined on both strands by the dideoxy method (Applied
Biosystems, Taq cycle sequencing system) using model 373A ABI automated DNA
sequencer.
An
Nde
I restriction site was created at the first ATG codon of the
T.thermophilus
mutS
gene by PCR-available site-directed mutagenesis. Using the
Nde
I site,
T.thermophilus
mutS
gene was cloned into the plasmid pET3a. The resulting plasmid, named pSS1, was
used to transform the
E.coli
strain JM109(DE3) harboring the plasmid pLysE. The transformant was cultivated
at 37oC in LB medium containing 50 [mu]g/ml ampicillin and 25 [mu]g/ml chloramphenicol until the density of the culture reached 4 * 10
8
cells/ml. The cells were then incubated for a further 2 h in the presence of 50
[mu]g/ml IPTG and then harvested by centrifugation and stored at -80oC.
Purification of
T.thermophilus
MutS protein
Frozen cells (100 g) were thawed, suspended in buffer I [50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 5 mM [beta]-mercaptoethanol and 25% (w/v) sucrose] and disrupted by
sonication on ice. Brij-58 was added to a final concentration of 0.5% (w/v) and then the cell
extract was stirred for 30 min at 4oC. Following this, the cell lysate was incubated at 70oC for 10 min and centrifuged (15 000
g
) for 40 min at 4oC. A DEAE-cellulose DE52 column (bed volume 500 ml) was equilibrated with
buffer II [50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 5 mM [beta]-mercaptoethanol and 10% (v/v) glycerol] before the
supernatant was loaded. The column was then washed once with 150 ml of buffer
II and eluted with a 3000 ml gradient of 0-500 mM NaCl in buffer II. Ammonium sulfate was added to the MutS fraction
to give a final concentration of 15% (w/v) and then it was loaded onto a Phenyl-Toyopearl 650M column (bed volume 60 ml) previously equilibrated with 15%
(w/v) ammonium sulfate in buffer II. The column was washed with 50 ml of the
same buffer and then eluted with a 600 ml gradient of 15-0% (w/v) ammonium sulfate in buffer II. Fractions containing the protein
were loaded onto a Sephacryl S-300 HR column (bed volume 400 ml) previously equilibrated with buffer III
[50 mM Tris-HCl (pH 7.5), 100 mM KCl, 0.1 mM EDTA, 5 mM [beta]-mercaptoethanol and 10% (v/v) glycerol]. The column was
eluted with buffer III, the fractions containing MutS protein were concentrated
and then stored at 4oC. At each step, the column fractionation pattern was analyzed by SDS-PAGE.
The final amount of purified MutS protein was 40 mg and the yield was 0.4 mg
protein/g cells.
The N-terminal amino acid sequence of the purified protein was analyzed using an
automated protein sequencer (ABI, model 473A). The molar extinction coefficient
of
T.thermophilus
MutS protein was calculated to be 51 000 M
-1
cm
-1
at an absorption maximum of ~278 nm, using the procedure described previously (
17
).
ATPase assay
Hydrolysis of [[alpha]-
32
P]ATP by
T.thermophilus
MutS protein was assayed by thin-layer chromatography (TLC) between 4 and 95oC (
18
). The radioactive counts from ATP and its hydrolysis product, ADP, were
quantified using a BAS2000 image analyzer (Fuji photo film).
Mismatch DNA binding assay
Mismatch DNA binding was measured by gel retardation assay. Complementary
strands of a 16mer or a 37mer oligonucleotide were synthesized (Fig.
6
). Each top strand was labeled at the 5' end with [[gamma]-
32
P]ATP using with polynucleotide kinase. The oligonucleotides were annealed at 70oC (16mer) or 95oC (37mer) for 10 min, allowed to cool to 30oC (at 1oC/min), and then placed on ice until required. When the
assay was carried out using 37 bp duplex DNA as a substrate, each binding assay
reaction mixture (total volume 10 [mu]l), comprising 0.2 pmol oligonucleotide duplex, the assay buffer [50 mM Tris-HCl (pH 7.5), 100 mM KCl, 0.1 mM EDTA, 5 mM [beta]-mercaptoethanol, 10% (v/v) glycerol and 20 mM MgCl
2
] and
T.thermophilus
MutS protein, was incubated at 35 or 60oC for 30 min. After adding 2 [mu]l 50% (w/v) sucrose, the binding assay reaction mixtures were loaded
onto a 6% (w/v) acrylamide gel containing 20 mM magnesium acetate and run at 35oC for 90 min or 60oC for 70 min at 76 V in 1* TAE buffer (
15
) containing 20 mM magnesium acetate. The gel was then dried and placed in
contact with an imaging plate. The bands were analyzed using a BAS2000 image
analyzer. When the assay was carried out using 16 bp duplex DNA, concentration
of magnesium ion was adjusted to 5 mM in the assay buffer and 1 mM in the
running buffer, respectively.
RESULTS
Cloning, sequencing and primary structures of the
T.thermophilus
mutS
gene
PCR was used to amplify a part of the
T.thermophilus
mutS
gene using primers designed from highly conserved regions of five MutS
proteins. The entire length of the
mutS
gene was cloned, as described in Materials and Methods, and its nucleotide
sequence was determined. The G+C content of the DNA fragment was 68% (data not
shown), which agrees well with that of genomic DNA from
T.thermophilus
HB8 (69%) (
14
). One open reading frame was found consisting of 2457 nucleotides, which
encoded a protein comprising 819 amino acid residues with a molecular mass
estimated at 91 379 Da. The amino acid sequence of
T.thermophilus
MutS protein is similar to those of the other MutS homologues (Fig.
1
). It showed 56 and 39% homology with
E.coli
MutS (
19
) and human hMsh2 (
8
) proteins respectively and common to all MutS homologues, the sequence around
Gly596 containing the Walker's A-type nucleotide binding motif (
20
), was also conserved in
T.thermophilus
MutS protein.
Complementation of the high mutagenicity of an
E.coli mutS
mutant by the
T.thermophilus
mutS
gene
A complementation test was carried out to determine whether
T.thermophilus
MutS protein had a similar activity to that of
E.coli
. The spontaneous mutation rates of wild-type and
mutS
E.coli
strains, containing the appropriate plasmids, were assessed by measuring the
frequency of rifampicin resistance mutations in each strain. As summarized in
Table
2
, the mutability of
mutS
mutant harboring the plasmid carrying the
T.thermophilus
mutS
gene was only a quarter of that for the
E.coli
mutS
mutant
strain, GM4271. Thus,
T.thermophilus
MutS protein would appear to play the same function that of
E.coli
.
Overproduction and purification of
T.thermophilus
MutS protein
In order to analyze the biochemical properties of
T.thermophilus
MutS protein, it was overproduced in
E.coli
and purified. The original clone of
T.thermophilus
mutS
gene from pTS3 was subcloned into the expression vector pET3a. As shown in
Figure
2
, an IPTG-induced band, observed at ~91 kDa, was identical to the molecular mass calculated from the amino
acid sequence of
T.thermophilus
MutS protein.
T.thermophilus
MutS protein was purified from the
E.coli
cells, which overproduced the protein. After heat treatment, which removed most
of the endogenous
E.coli
proteins, the protein was purified to homogeneity by sequential column
chromatography on DEAE-cellulose DE52, Phenyl-Toyopearl 650M and Sephacryl S-300 HR (Fig.
2
). The N-terminal amino acid sequence was found to be G-G-Y-G-G-V-K-M-E-G-M-L-K-G-E-G-P-G-P-L-. The sequence was identical to that expected after translation from the nucleotide sequence of the
T.thermophilus mutS
gene expect that the N-terminal Met residue was truncated.
Physicochemical characteristics of
T.thermophilus
MutS protein
Molecular size.
We attempted to characterize the purified
T.thermophilus
MutS protein using various physicochemical approaches. First, we used size-exclusion chromatography to estimate the molecular size of
T.thermophilus
MutS protein in its native state. Its molecular mass was calculated to be ~330 kDa (Fig.
3
), which was 3.6 times larger than its monomer (91.4 kDa). This indicated that
T.thermophilus
MutS protein exists as tetramer in solution.
Biochemical characteristics of
T.thermophilus
MutS protein
ATPase activity
. The Walker's A-type nucleotide binding motif was conserved in MutS proteins (Fig.
1
) and ATPase activity has been shown in
S.typhimurium
MutS (
24
),
E.coli
MutS (
25
) and
S.cerevisiae
Msh1 (
26
). In order to find out whether the purified
T.thermophilus
MutS protein also has ATPase activity, we incubated it with [[alpha]-
32
P]ATP and separated out the reaction products using TLC.
T.thermophilus
MutS protein hydrolyzed ATP to ADP and Pi (data not shown) and its activity was
maximal at 80oC (Fig.
5
). To determine the
K
m
and
k
cat
values of
T.thermophilus
MutS protein, ATPase activity was measured in the presence of various
concentrations of ATP. At 65oC, the values for
K
m
and
k
cat
were calculated to be 130 [mu]M and 0.11 s
-1
, respectively.
DISCUSSION
MutS protein from
T.thermophilus
The
mutS
gene from an extremely thermophilic bacterium,
T.thermophilus
HB8, was cloned and its nucleotide sequence determined. It encoded a 91.4 kDa
protein which had 56 and 39% homology with
E.coli
MutS and human hMsh2 proteins, respectively. This is the first time that the
mutS
gene has been isolated from a thermophilic bacterium. We found that the
T.thermophilus
mutS
gene could complement the high mutability of the
E.coli
mutS
mutant. This suggests that there is a mismatch repair system in
T.thermophilus
which is similar to that found in
E.coli
and that the functions of MutS protein from both types of bacteria are the
same.
The purified
T.thermophilus
MutS protein, overproduced in
E.coli
, maintained its secondary structure (Fig.
4
A) and activities (Figs
5
and
7
). We predicted the secondary structures of the MutS homologues (Fig.
1
), based on the observation that the three-dimensional structure of homologous proteins are almost identical (
22
). The [alpha]-helical content of the MutS proteins was estimated to be 49%, and
this value agreed well with that predicted from the CD measurement (47%). It
should be noted that the predicted secondary structure of the N-terminal region for MutS proteins are similar even though the homology of
this region is relatively low.
Measurements of its secondary structure and ATPase activity have shown that
T.thermophilus
MutS protein was heat stable at temperatures <= 80oC. This thermostability could be related to its amino acid sequence.
The following amino acid residues, known to be chemically unstable at high
temperatures, were decreased in number in
T.thermophilus
MutS protein when compared to those of
E.coli
MutS protein: Cys (6 to 2), Asn (27 to 7), Gln (42 to 17) and Met (25 to 14).
Whereas the number of Pro residues was increased from 40 in
E.coli
MutS protein to 47 in
T.thermophilus
. Pro residues have the highest [beta]-turn potential of all the amino acids and play an important role in
peptide folding and globular structure formation. They are considered to
decrease the entropy of the protein in its denatured state or increase the
conformational enthalpy in its native state, thereby increasing protein
stability (
27
). Similar changes have also been observed in other thermostable proteins (
16
,
28
,
29
).
Protein-protein interaction of MutS protein
Escherichia coli
MutS protein interacts with MutL homodimers forming a ternary complex (
30
). Therefore, the result of the complementation test strongly suggests that
T.thermophilus
MutS protein could be capable of interacting with
E.coli
MutL protein to produce functional repair complexes. Incomplete complementation
could be due either to a low level of
T.thermophilus
mutS
gene expression in
E.coli
, to weak interactions between inter-species Mut proteins and/or to submaximal activity of
T.thermophilus
MutS protein in
E.coli
cultured at 37oC.
The results of the size-exclusion chromatography strongly suggested that
T.thermophilus
MutS protein exists as a tetramer in its native state.
E.coli
MutS protein is known to exist as both monomer and oligomer (
31
), however, human hMsh2 protein forms heterodimers with GTBP (G-T binding
protein), which is a new MutS homologue in eukaryotes (
32
,
33
). On the other hand, hMsh2 protein forms a protein-mismatch DNA complex where
it can exist as either the monomer, dimer or multimer (
34
). For MutL homologues,
E.coli
MutL forms a homodimer (
30
), and human MutL homologues form heterodimers (
35
,
36
). These reports suggest that the oligomerization of the mismatch repair
proteins is conserved between prokaryotes and eukaryotes. Therefore, eukaryotic
MutS homologues may also be capable of forming tetramers.
Although at neutral pH,
T.thermophilus
MutS protein was a tetramer in the absence of SDS, it was highly aggregated
between pH 4 and 6, most probably due to isoelectric precipitation. The
isoelectric point of
T.thermophilus
MutS protein, calculated from its amino acid sequence, is 5.6 and therefore,
its net charge will approach zero at pH 4-6. The region of
T.thermophilus
MutS protein responsible for protein-protein interaction may be hydrophobic, as reported for many other
proteins (
37
).
The amino acid sequences of
T.thermophilus
MutS protein and its counterparts in prokaryotes and eukaryotes are similar,
and the ATP binding motifs are conserved in all of these proteins. Since it has
been shown that
S.typhimurium
MutS (
24
),
E.coli
MutS (
25
) and
S.cerevisiae
Msh1 (
26
) proteins have ATPase activity, it seemed reasonable to expect that
T.thermophilus
MutS protein would also have this activity. Our results show that
T.thermophilus
MutS protein was able to hydrolyze ATP to ADP and Pi. The kinetic parameters (
k
cat
and
K
m
) and catalytic efficiencies (
k
cat
/
K
m
) of its ATPase activity are summarized in Table
3
. The catalytic efficiencies for many well evolved enzymes are similar over a
range of 1 * 10
6
to 3 * 10
8
s
-1
M
-1
(
38
), however, the values for MutS proteins were all ~10
3
s
-1
M
-1
which are three to five orders of magnitude lower than those of the well
evolved enzymes. Since the catalytic efficiencies of MutS proteins were similar
at the different physiological temperatures, a low rate constant must be a
characteristic of these MutS proteins. This low rate constant suggests the
presence of the slow conversion step in the MutS protein-substrate complex. The
K
m
value of
T.thermophilus
MutS protein at 65oC was more than one order of magnitude higher than that of other
prokaryotic MutS protein at 37oC (Table
3
). The value of
K
m
may become relatively high as the temperature rises because the binding step is
exothermic ([Delta]
H
< 0) reaction.
Thermus thermophilus
MutS protein bound efficiently and with high specificity to the G-T mismatched heteroduplex DNA than it did to homoduplex DNA. This
mismatch binding activity is similar to those of MutS homologues from other
species (
31
,
34
,
39
-
42
) and suggests that
T.thermophilus
MutS may also play a role in mismatch repair initiation by providing a target
for the excision process. The finding of which the non-specific binding of this protein to homoduplex DNA was weaker at 60oC than at 35oC, suggests either that mismatched base pair recognition ability
of
T.thermophilus
MutS protein is more efficient at higher temperature or that non-specific DNA-protein complex dissociate more easily. More efficient specific
binding at 35oC than 60oC suggests that the MutS-mismatched DNA complex is more unstable at
higher temperature. It is interesting to speculate on the nature of the protein-DNA interaction in thermophiles. At higher temperatures, the secondary
structure of their DNA alters, therefore, the interaction of proteins with DNA
at these temperatures must be stabilized in such a way as to allow their
association. The stable complex formed between
T.thermophilus
MutS protein and DNA is a useful model in which to study the molecular
mechanism of mismatch recognition.
ACKNOWLEDGEMENT
We are grateful to Dr M. G. Marinus (University of Massachusetts Medical School)
for providing the
E.coli
strains GM4244 and GM4271.
REFERENCES
1 Friedberg,E.C., Walker,G.C. and Siede,W. (1995) DNA Repair and Mutagenesis. ASM Press, Washington, DC.
