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J Biol Chem, Vol. 273, Issue 46, 30398-30405, November 13, 1998
From the Institute of General Microbiology, University of Bern,
Baltzer-Strasse 4, CH-3012 Bern, Switzerland
The mismatch-binding activity Cmb1 of
Schizosaccharomyces pombe was enriched from wild type
cells, and N-terminal sequencing enabled cloning of the respective
gene. The deduced amino acid sequence of cmb1+
contains a high mobility group domain, a motif that is common to a
heterogeneous family of DNA-binding proteins. In crude protein extracts
of a cmb1 gene-disruption strain, specific binding to C/T,
C/A, and C/ DNA can be damaged by a variety of physical and chemical agents.
Most types of lesions are efficiently removed from DNA by repair
mechanisms. The nucleotide excision repair
(NER)1 system is able to
correct a broad spectrum of adducts, including cyclobutane pyrimidine
dimers and 6-4 photoproducts caused by ultraviolet light, intrastrand
cross-links formed by cis-diamine-dichloroplatinum(II) (cisplatin), as well as O6-methylguanine
(O6meG) produced by methylating agents such as
N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) (1-3). The mutHLS-like mismatch-repair pathway
efficiently corrects most base-base mismatches and small DNA loops,
which frequently occur during replication by misincorporation of bases and strand slippage, respectively (4, 5). A defect in the human genes
hMSH2 (a homologue of Escherichia coli MutS),
hMLH1, hPMS1, or hPMS2 (all of which are MutL
homologues) is responsible for the heritable cancer syndrome hereditary
nonpolyposis colon cancer. The clinical symptoms of patients with
homozygously mutated hMSH2, hMLH1, or
hPMS2 genes are accompanied by microsatellite instability
and mismatch repair deficiency, whereas there is no indication that
hPMS1 has a role in mismatch repair (4, 6, 7). The human
mismatch-binding activity hMutS We have identified a mismatch-binding activity in
Schizosaccharomyces pombe crude extracts showing high
affinity to cytosine-containing mismatches (16). Here, we describe the
purification of this activity, Cmb1, and cloning of the respective
gene. DNA sequencing revealed that Cmb1 contains a HMG domain. Cmb1 was
purified from an E. coli overexpression strain and tested
for binding to mismatches, small DNA loops, and substrates containing
either GpG intrastrand cross-links or O6meG. Furthermore,
we addressed the question of whether a cmb1 mutant responds
differently to the chemicals cisplatin, transplatin, and MNNG.
DNA Binding Analyses--
Binding of Cmb1 protein to DNA
substrates was tested with a band shift assay. Substrates and
competitor DNA used to test binding to mismatches, loops, and
O6meG lesions are in the M13mp9 polylinker sequence context
(16). The sequence of the platinated substrates and their unplatinated controls is shown in Fig. 6A. For platination, 1 pmol of
plus strand was incubated for 24 h at 37 °C in 100 µl of 10 mM Tris-HCl (pH 7.5) containing either 0.1 mM
cisplatin or 0.1 mM transplatin (Sigma). Unreacted
cisplatin or transplatin was removed by chromatography on Sephadex G25
(Amersham Pharmacia Biotech). The extent of platinated oligonucleotides
was checked on a 6% polyacrylamide gel and was estimated to be
approximately 60-80%. Modified and unmodified strands were
5'-end-labeled by T4 polynucleotide kinase (Promega) in the presence of
[ Partial Purification of Cmb1 from S. pombe and N-terminal Peptide
Sequencing--
The procedure for obtaining S. pombe cells
for protein extracts was as described (16), except that we used Buffer
A (25 mM Tris-HCl (pH 7.5, adjusted at room temperature),
150 mM NaCl, 1 mM EDTA, 0.5 mM
spermidine, 0.1 mM spermine, 5 mM
For N-terminal sequencing, a 4-ml aliquot of fraction V was
concentrated to 280 µl by 75 min of centrifugation at 3600 × g in two 2-ml Centricon-10 tubes (Amicon Inc.). Proteins
were separated on a 12% SDS-polyacrylamide gel, transferred to an
Immobilon membrane by electroblotting, and stained with Amido Black,
and the 22-kDa protein band (400 pmol) was excised. The N-terminal
sequence EKNGLQKLIPPRLKTIWNQMLVETKGAGN of 29 amino acids was obtained
by Johann Schaller and Urs Kämpfer (Protein Analytical Service,
Institute of Biochemistry, Bern, Switzerland) using a pulsed-liquid
phase Sequenator 477A (Applied Biosystems Inc.).
Polymerase Chain Reactions (PCRs)--
PCRs were performed in a
Perkin-Elmer DNA thermal cycler. Standard reactions of 50 µl
contained 10-100 pmol of each of two primers, 0.1 mM each
dNTP, 1 unit of Taq DNA polymerase (Appligene) in a standard
PCR buffer (Appligene). As templates, either 300 ng of genomic S. pombe DNA (for PCR cloning) or about 10 ng of plasmid DNA (for
hybridization probes, for the construction of the
cmb1::his3+ gene disruption, and for
cloning of cmb1 into the E. coli expression vector pT7-7) were used. Reactions included 5 min at 94 °C,
followed by 30 cycles of 45 s of denaturation at 94 °C, 1 min
of annealing at a temperature depending on the composition of the
primer (annealing temperatures are specified below for individual
experiments), and 1 min of synthesis at 72 °C. Finally, a 10-min
extension step at 72 °C was applied. Differences from this standard
protocol are indicated in the text.
PCR Cloning--
The 29 amino acid sequence determined from the
Cmb1 protein was used to isolate a PCR clone of the
cmb1+ gene from chromosomal S. pombe
DNA. Four degenerate primers derived from the ends (primers CC-1a,
CC-1b, and CC-3) or from a middle part (primer CC-2) of this sequence
were used for PCR: primer CC-1a,
5'-CTCGGATCCAA(A/G)AA(C/T)GG(A/C/G/T)CT(A/C/G/T)CA(A/G)AA-3', primer CC-1b,
5'-CTCGGATCCAA(A/G)AA(C/T)GG(A/C/G/T)TT(A/G)CA(A/G)AA-3' (both
derived from the amino acid sequence KNGLQK), primer CC-2, 5'-CAT(C/T)TG(A/G)TTCCA(A/G/T)AT(A/C/G/T)GT(C/T)TT-3' (derived from
KTIWNQM), and primer CC-3,
5'-CACGGATCCTT(A/C/G/T)CC(A/C/G/T)GC(A/C/G/T)CC(T/C)TT-3' (derived from KGAGN). Primers CC-1a, CC-1b, and CC-3 contain a BamHI restriction site and three additional bases at their
5'-ends. A PCR product of the expected size was obtained with primer
pair CC-1b/CC-3 but not with CC-1a/CC-3. Amplification was performed by
three initial cycles with an annealing temperature of 40 °C, followed by 30 cycles with an annealing temperature of 65 °C. PCR
products were separated on 1.5% agarose gels. A band of the expected size (102 bp) was eluted from the gel (17) and used for
reamplification with primers CC-1b and CC-3 and for PCR with primers
CC-1b and CC-2. This reaction (annealing at 47 °C) produced a 67-bp
fragment, indicating that the 102-bp fragment contained the sequence of
interest. The reamplified 102-bp DNA fragment was digested with
BamHI, cloned into pUC18, and sequenced. One clone (pOL50)
was used for further screening.
Library Screening--
The pOL50-insert was random-prime-labeled
with 32P (Ready to Go DNA labeling kit, Amersham Pharmacia
Biotech) and used to screen a plasmid-based library of S. pombe (18) by colony hybridization. From this library several
clones with a truncated open reading frame (ORF) of
cmb1+ were identified. A region containing 264 bp of the ORF was amplified by PCR with primer CC-6
(5'-CTGTTTGACGCAATGCCTCC-3') and primer CC-7
(5'-GATCTCATCGGCAGTCAAAG-3') (annealing at 54 °C). The PCR product
was radiolabeled with 32P and hybridized to ordered cosmid
and P1 libraries (19, 20). Two cosmids and four P1 clones were obtained
which all map on chromosome I between ras1+ and
cdc3+ (19). From one cosmid (ICRFc60H037), three
fragments from the region of interest were subcloned in pUC18 (pOL54 to pOL59).
Sequence Analysis--
Double-stranded plasmid-DNA (pOL54 to
pOL59) was sequenced by the dideoxy method using a sequencing kit
(United States Biochemical Corp.). The cmb1+ ORF
was determined from both strands with primers derived from pUC18 and
with primers derived from the inserts (not shown). Sequences were
analyzed with the Wisconsin program package, version 9 (Genetics Computer Group, Madison, WI).
Gene Disruption of cmb1+--
The following strategy
resulted in replacement of the complete ORF of
cmb1+ by his3+. A 342-bp
fragment of the 5'-flanking region and a 512-bp fragment of the
3'-flanking region of cmb1+ were amplified by
PCR (annealing at 50 °C) with Pfu DNA polymerase (Stratagene). For the 5'-flanking region, primers DC-1
(5'-ATAGTCTATGGTGAGTAATG-3') and DC-2 (5'- TCAAGTACTGAGTAAATATCG-3')
were used, and for the 3'-flanking region, primers DC-3
(5'-ACACTCTACTTGCCCAGATC-3') and DC-4 (5'-TATTGGGCGTGCAAATTGTG-3') were
used. The primers contain restriction sites at their 5'-ends for
cloning (not shown). PCR products were inserted to the left and right
of the his3+ gene, previously cloned in
pBluescript KS. A 1.5-kb fragment containing this
cmb1::his3+ construction was
transformed into a S. pombe h+
his3-D1 strain (21). His+ transformants were checked
by Southern hybridization for correct gene disruption (data not shown).
Strain OL142 (h+
cmb1::his3+ his3-D1) was stored
for further experiments.
Overexpression of Cmb1 in E. coli and Purification--
The
full-length cmb1 gene was amplified by PCR (annealing at
54 °C) using Pfu polymerase (Stratagene), primer EC1
(5'-CTTGCTAGCCATATGCGTCTGTTTGACGCAATG-3'), and primer EC2
(5'-GTAGGATCCTCATCATCGAAATCCGGCTTC-3'), derived from the 5'- and
3'-ends of the coding region, respectively. A truncated cmb1
gene missing the 5' nucleotide sequence coding for the amino acids
2-41 was amplified with primer EC3
(5'-CTTCATATGGAAAAGAATGGATTACAGAAG-3') and primer EC2 (annealing at
55 °C). Primers EC1 and EC3 contain a NdeI restriction
site, and primer EC2 contains a BamHI restriction site. The
PCR products were digested with NdeI/BamHI and
then ligated with digested pT7-7. The ligation mixtures were
transformed into E. coli strain XL1blue, and plasmids
containing correct inserts were checked for mutations by sequencing.
For expression of the protein, the plasmids were transformed into
E. coli strain BL21/DE3. Stationary phase cultures of the
expression strains were inoculated 1:40 in fresh LB medium containing
100 µg/ml ampicillin and incubated at 37 °C until an
A600 of about 0.7 was reached.
Isopropylthio-
The strain BL21/DE3 containing pT7-7 with the truncated
cmb1 gene was used for purification of the respective
protein, Cmb1 Physiological Tests--
To test sensitivity of S. pombe cells to various chemicals, 1 × 107 cells
derived from stationary phase cultures were incubated with shaking in 2 ml of 0.25% yeast extract; 1.5% glucose, adenine, histidine, and
uracil (each 0.005%); and varying concentrations of cisplatin (0-0.8
mM), transplatin (0-3 mM), or MNNG (0-400 µg/ml). Cells were incubated with either cisplatin or transplatin for
90 min or with MNNG for 30 min. The cells were harvested, washed with 5 ml of 0.85% NaCl, pelleted again, and resuspended in 0.85% NaCl.
Appropriate dilutions were plated on YEA (0.5% yeast extract; 3%
glucose; and 1.5% agar supplemented with adenine, histidine and uracil
(each 0.01%)). Plates were incubated for 3 days at 30 °C before
scoring colonies. The experiments were carried out six times in the
case of cisplatin, two times with transplatin, and six times with MNNG.
Partial Purification of Cmb1 from S. pombe--
An activity
binding to cytosine-containing single-base mismatches was previously
identified in S. pombe crude extracts (16). We enriched this
activity, Cmb1, from extracts of an S. pombe wild type
strain (fraction I) by ammonium sulfate precipitation (fraction II),
followed by chromatography through DEAE-cellulose (fraction III),
double-stranded DNA-cellulose (fraction IV), Heparin-agarose (fraction
V), and MonoQ columns (fraction VI) (see under "Experimental Procedures"). Analysis of the fractions from the last purification step by a band-shift assay revealed a strong enrichment of the mismatch-specific activity in fractions 30 and 31 (Fig.
1A). The intensity of the band
shift correlated well with the intensity of a 22-kDa band visualized in
a SDS-polyacrylamide gel by Coomassie Blue staining (Fig.
1B). The purest fractions also contained a second dominant
protein of about 65 kDa and a few other proteins at low concentrations.
These fractions were tested for their mismatch-binding ability. We
found strong affinity to C/A, T/C, and C/ Cloning of the cmb1+ Gene--
400 pmol of the 22-kDa
Cmb1 protein were used for N-terminal sequencing and resulted in the
determination of 29 amino acids (Fig.
2A). Four degenerate primers
derived from the peptide sequence of Cmb1 were used for PCR (Fig.
2A). A band of the expected size, obtained with primer pair
CC-1b/CC-3, was eluted from the gel and subjected to PCR with primer
pair CC-1b/CC-2. A single 67-bp band was produced, indicating that the
102-bp fragment contained the sequence of interest. Sequencing of the
102-bp fragment from five clones revealed inserts with the coding
capability for the amino acid sequence determined from the Cmb1
peptide. The insert of one clone (pOL50) was used to screen a
plasmid-based S. pombe library (18). Several clones were
identified, but all contained only part of the open reading frame of
cmb1+ at one end of the inserts (Fig.
2B). This truncated ORF (270 bp) contained 126 bp upstream
and 61 bp downstream of the sequence previously determined from the PCR
clones. A PCR product of this ORF was used as hybridization probe for
ordered cosmid and P1 libraries (19, 20). Two cosmids and four P1
clones were obtained that all map on chromosome I between
ras1+ and cdc3+ (19).
From one cosmid (ICRFc60H037), three fragments from the region of
interest were subcloned in pUC18 (Fig. 2C). By sequencing the 3'-flanking region of cmb1+, the
arg3+ gene (22) was found to be located
approximately 600 bp downstream from the ORF of
cmb1+, which is consistent with the mapping of
cmb1+ between ras1+ and
cdc3+. Double-stranded sequencing of
cmb1+ revealed an intronless ORF of 669 bp
encoding a 26-kDa protein. The partially purified Cmb1 has a molecular
mass of 22 kDa. Judged from its N-terminal amino acid sequence used for
cloning, it represents a proteolytic product without the N-terminal 41 amino acids encoded in the ORF of cmb1+.
Cmb1 Is a HMG Domain Protein--
By data base searches with the
deduced amino acid sequence of cmb1+, a HMG
domain was identified in the C-terminal part (Fig.
3). This DNA-binding motif is common to a
large family of HMG domain proteins characterized in various eukaryotes
(13, 23). Originally, HMG-1 and HMG-2 were found to be abundant
non-histone components of chromatin, migrating with high mobility in
gels (28). A number of proteins were then identified that contain one
to six regions of significant homology to regions of HMG-1 and HMG-2
(23, 29). These regions were named HMG domains. Some HMG domain
proteins are transcription factors that recognize specific DNA
sequences, whereas some are known to bind to specific DNA structures,
such as four-way junctions (30, 31), intrastrand cross-links (13, 15)
or intermediates of V(D)J recombination (26). Interestingly, our data
base searches revealed that also hPMS1, a human MutL homolog (24),
contains a HMG domain.
A hydropathy profile of the HMG domain of Cmb1 revealed that it is
extremely hydrophilic, a feature common to other HMG domains (26, 32).
A few amino acids are conserved among sequence-specific HMG domain
proteins, whereas others are present only in non-sequence-specific proteins (23). In this respect, Cmb1 belongs to the subfamily of
non-sequence-specific HMG domain proteins (Fig. 3).
A Second C/C Binding Activity, Cmb2, Is Present in the cmb1 Mutant
Cells--
The cmb1::his3+ disruption
strain OL142 was constructed as described under "Experimental
Procedures." Because the haploid mutant is viable,
cmb1+ has no essential function. We tested
mismatch binding with crude protein extracts from the cmb1
disruption mutant. In contrast to extracts from wild type, in which
strong binding to all cytosine-containing mispairs was detected (Fig.
4, lanes 2-5), no binding to
C/A, C/T, or C/ Expression of Cmb1 in E. coli and Binding to Mismatches and Small
DNA Loops--
The full-length cmb1 ORF and a truncation
were expressed in E. coli. The latter version, encoding a
22-kDa protein, Cmb1
For further binding tests, the recombinant Cmb1 Cmb1 Binds to 1,2 GpG Intrastrand Cross-links When Cytosines Are
Opposite the Modified Bases--
Several HMG domain proteins are known
to bind to 1,2 GpG intrastrand cross-links (13, 15). We were therefore
interested to learn whether Cmb1 also shows specific binding to this
type of lesion (Fig. 6A).
Single-stranded oligonucleotides, either platinated or not, were poor
substrates (Fig. 6B, lanes 1 and 2). A complex
was formed with unplatinated homoduplex (lane 3), which
might indicate a preference of Cmb1 for cytosine-rich sequences. Nevertheless, binding to the cisplatinated substrate cisplatin-GpG/CpC was much stronger (lane 4). Interestingly, only weak binding
was detected to cisplatinated oligonucleotides, where opposite the 1,2 cross-linked guanines, other bases were present instead of the
cytosines (lanes 5-8). Even the change of only one cytosine strongly affected complex formation (lane 8). Binding to the
compound lesions (intrastrand cross-link combined with mismatches) was as low as, or even lower than, to homoduplex (lane 3) and to
a substrate containing a G/T mismatch in the same sequence context (lane 9). No significant affinity to the
trans-diamine-dichloroplatinum(II)-GpTpG/CpApC lesion was
found (data not shown). This 1,3 intrastrand cross-link is produced by
transplatin, an isomer of cisplatin.
S. pombe Cells Defective in cmb1 Are Sensitive to
Cisplatin--
To investigate the cellular role of Cmb1 in repair of
lesions produced by either cisplatin or transplatin, the cytotoxicity of these drugs was tested in wild type and the cmb1 mutant.
The cmb1 strain was significantly more sensitive to
cisplatin than wild type. In the presence of 0.8 mM
cisplatin, cell survival was about 17-fold reduced (Fig.
6C). On the other hand, when cells were treated with
transplatin at concentrations between 1 and 3 mM, no
difference in survival was found between wild type and the
cmb1 mutant (data not shown).
Cmb1 Binds to O6meG:C but Not to
O6meG/T--
The cytotoxic action of MNNG is predominantly
due to methylation of guanines. O6meG can equally pair with
cytosine and thymine. Thus a persisting O6meG:C site in
DNA is frequently changed during replication to O6meG/T,
resulting in fixation of a mutation (33). We tested binding of Cmb1 to
both types of lesions. When a cytosine was present opposite the
modified guanine, a complex was formed that was of comparable strength
to that seen with the substrate containing a C/C mismatch (Fig.
7A, lanes 2 and 3).
In contrast, binding to O6meG/T was as weak as to G:C
homoduplex DNA and to G/T containing substrate (lanes 1, 4, and 5). When S. pombe cells, defective in
cmb1, were treated with different amounts of MNNG, no
difference from wild type cells was found in survival rates (Fig.
7B).
Cmb1 Is a HMG Domain Protein--
We have previously described the
identification of an activity in S. pombe crude extracts
that efficiently binds to the cytosine-containing mismatches C/C, C/A,
T/C, and C/
The Cmb1 protein contains an HMG domain at the C-terminal end (Fig. 3).
Various HMG domain proteins are known to recognize specific DNA
structures, such as V(D)J recombination signals (26), cruciform DNA
(30, 31), and intrastrand cross-links (13, 15). These features suggest
a role in DNA recombination and DNA repair. Interestingly, hPMS1 also
contains an HMG domain (Fig. 3). hPMS1 is one of the three human MutL
homologues which, if defective, causes a predisposition to the colon
cancer syndrome hereditary nonpolyposis colon cancer (7, 24).
Cmb1 Binds to Cytosine-containing Mismatches--
The purification
of Cmb1 from S. pombe cells resulted in highly active
fractions, which predominantly contained the Cmb1 protein but also a
few other proteins (Fig. 1). Although the strength of mismatch binding
correlated well with the concentration of the Cmb1 protein in the
various fractions from the last purification step, it could not be
ruled out that mismatch binding was provided by another protein in the
fractions. Efforts to separate Cmb1 from the remaining S. pombe proteins by additional chromatographic steps failed (data
not shown). We therefore decided to test substrate binding with
recombinant Cmb1 protein purified from E. coli.
In a first step, the full-length cmb1+ ORF, as
well as a truncated version, Cmb1
We then purified Cmb1 A Second C/C Binding Activity, Cmb2, Exists in S. pombe--
A
cmb1 gene disruption strain was tested for the presence of
the mismatch-binding activity. Specific binding to C/A, C/T, and C/ Cmb1 Specifically Binds to 1,2 GpG Intrastrand Cross-links and to
O6meG When Cytosines Are Opposite the Damaged
Bases--
1,2 GpG intrastrand cross-links, produced by cisplatin, are
substrates of NER proteins, of MutS-type proteins, and of HMG domain
proteins (1, 10, 13, 15). We found that this lesion is also bound by
the HMG domain protein Cmb1. Our data further suggest that binding by
Cmb1 requires the cytosines opposite the cisplatinated guanines.
Consistently, O6meG:C but not O6meG/T is
recognized by Cmb1. Thus, like other HMG domain proteins, MutS-type
proteins, and the NER complex, Cmb1 recognizes a series of DNA
distortions. It is not well understood how the various recognition
proteins discriminate between normally paired bases and the different
types of altered DNA structures. In the case of Cmb1, the presence of
cytosines in mismatches as well as in DNA lesions might be crucial for
recognition. hMutS
Although Cmb1 specifically binds to an O6meG:C lesion (Fig.
7A), no significant difference between wild type and the
cmb1 mutant was found when cells were treated with MNNG
(Fig. 7B). This may be due to efficient repair of
O6meG by an O6-methylguanine-DNA
methyltransferase. However, more experiments should be done to clarify
whether Cmb1 has a role in removal of O6meG from DNA.
Genetic experiments with cmb1 disruption strains revealed
changes of the pattern of meiotic mismatch repair but no alteration in
mitotic mismatch repair.2 The
observed effects on meiotic mismatch repair were rather weak. This may
be due to redundancy of Cmb1 with other proteins of similar function.
One candidate is Cmb2, because it is able to bind to C/C mismatches
(Fig. 4). Cmb1 may act as an accessory protein binding to chemically
modified DNA and mismatches and thereby marking such sites for repair.
A candidate repair pathway is the NER system, which can restore bases
damaged by cisplatin and MNNG (1, 3). Our recent data show that NER
genes of S. pombe are involved in mismatch repair in a
MutS/L-independent pathway.2 The key characteristic is the
repair of C/C mismatches that cannot be repaired by a MutS/L-type
system (40, 41).
We thank Wolf-Dietrich Heyer and Primo
Schär for helpful discussions and Edgar Käslin, Ursula
Reinhard, and Vladimir Bashkirov for advice on protein purification. We
are also grateful to Elmar Maier and Nadja Pohl for kindly providing
cosmid/P1 libraries and clones.
*
This work was supported by the Swiss National Science
Foundation.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AJ002513.
The abbreviations used are:
NER, nucleotide
excision repair; cisplatin or cis-DDP, cis-diamine-dichloroplatinum(II); O6meG, O6-methylguanine; MNNG, N-methyl-N'-nitro-N-nitrosoguanidine; O4meT, O4-methylthymine; HMG, high mobility
group; transplatin or trans-DDP, trans-diamine-dichloroplatinum (II); PCR, polymerase chain
reaction; bp, base pair(s); kb, kilobase pair(s); ORF, open reading
frame; Cmb, cytosine mismatch binding.
2
O. Fleck and J. Kohli, unpublished data.
The High Mobility Group Domain Protein Cmb1 of
Schizosaccharomyces pombe Binds to Cytosines in Base
Mismatches and Opposite Chemically Altered Guanines*
,
ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References
was abolished. Weak binding to C/C revealed the presence
of a second mismatch-binding activity, Cmb2. Cmb1, enriched from
S. pombe and purified from Escherichia coli,
bound specifically to C/C, C/T, C/A, T/T, and C/ but showed little or no affinity to other mismatches and small loops. Cmb1 recognizes 1,2 GpG intrastrand cross-links, produced by the chemotherapeutic drug
cisplatin, when two cytosines are opposite the cross-linked guanines
but not when other bases are present. Consistently,
O6-methylguanine:C but not O6-methylguanine/T
lesions were bound. Thus, cytosines in mismatches and opposite
chemically modified guanines are the preferred target of Cmb1
recognition. cmb1 mutant cells are more sensitive to
cisplatin than wild type cells, indicating a role of Cmb1 in repair of
cisplatin-induced DNA damage.
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
, a heterodimer of the two MutS
homologous proteins hMSH2 and GTBP (8, 9), also recognizes DNA lesions
containing O6meG, O4-methylthymine, and
cisplatin-induced 1,2 GpG intrastrand cross-links (10). Thus,
mismatch-repair proteins and NER proteins might compete in repair of
some types of DNA lesions. Whereas repair mediated by the NER system
normally restores wild type information, the action of mismatch-repair
proteins on lesions rather contribute to cytotoxicity (11, 12). Several
high mobility group (HMG) proteins are known to bind to GpG intrastrand
cross-links (13). Binding by HMG-1 shields the lesion from excision by
the human excinuclease protein complex (14). Consistently, a
Saccharomyces cerevisiae mutant, defective in the HMG-box
gene IXR1, is more resistant to cisplatin than wild type
(15).
EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
-32P]ATP and annealed with their complementary
strands as described (16). To test binding to the various substrates,
20-µl reactions were performed for 15 min at 4 °C in 25 mM Tris-HCl (pH 7.5), 0.5 mM dithiothreitol, 4 mM spermidine, 0.5 mM EDTA, 10% glycerol, 0.01 mM ZnCl2, 50-200 mM NaCl in the
presence of 40 fmol radiolabeled substrate. The amount of unlabeled
homoduplex DNA as competitor and the source of protein samples are
indicated in the text. Reaction samples were separated by
electrophoresis (110 V, 4 °C) in 6% nondenaturing polyacrylamide
gels in 40 mM Tris-HCl (pH 7.5), 20 mM sodium
acetate, 1 mM EDTA. Subsequently, gels were subjected to autoradiography.
-mercaptoethanol, 0.1 mM phenylmethylsulfonyl fluoride,
1 mM dithiothreitol) for washing and suspending cells. To
estimate the degree of purity of Cmb1, protein samples from different
purification steps were separated on 12-13% SDS-polyacrylamide gels
by electrophoresis and analyzed after staining with silver nitrate or
Coomassie Blue. For protein purification, all steps were done at
4 °C. 485 g of cells (720 ml), previously frozen in liquid
nitrogen and stored at 
70 °C, were thawed. A 400-ml chamber for a
Bead beater (Biospec Products) loaded with 350 g of acid-washed
glass beads (0.5 mm in diameter) was used for four consecutive steps of
cell disruption. Each step included ten 30-s intervals, with at least 2 min of cooling on ice between each interval. The suspensions were
pooled and centrifuged for 1 h at 32,000 rpm in a Ti45 rotor
(Beckman). The 510-ml supernatant was separated from cell debris and
lipids (fraction I). (NH4)2SO4 was
added to a final concentration of 50%, and the precipitate was
collected by 20 min of centrifugation at 13,000 × g,
suspended in Buffer B150 (25 mM Tris-HCl (pH 7.5, adjusted
at room temperature), 150 mM NaCl, 0.1 mM EDTA,
10% glycerol (v/v), 5 mM -mercaptoethanol, 0.1 mM phenylmethylsulfonyl fluoride) and dialyzed twice
against 5.5 liters of Buffer B150 (fraction II). The 310-ml fraction II
was loaded at a flow rate of 140 ml/h on two parallel DEAE-cellulose
columns (DE52, Whatman, 20 cm2 × 22 cm), equilibrated with
Buffer B150. The flow-through (420 ml of fraction III) was loaded at a
flow rate of 43 ml/h on a double-stranded DNA-cellulose column (Sigma,
4.5 cm2 × 20 cm), equilibrated with B150. Bound proteins
were eluted by a 480-ml linear gradient from 100% B300 to 100% B700
(same buffers as B150, except that they contain 0.3 and 0.7 M NaCl, respectively). Active fractions (between 0.4 and
0.6 M) were pooled and dialyzed twice against 4 liters of
Buffer B150 to give 290 ml of fraction IV. Fraction IV was loaded at a
flow rate of 27 ml/h on a heparin-agarose column (type I, Sigma, 1.8 cm2 × 7 cm), equilibrated with Buffer B150. Bound proteins
were eluted step-wise with 2 column volumes of B400, B550, and B700,
respectively (buffers differ from Buffer B150 in NaCl concentrations of
0.4, 0.55, and 0.7 M, respectively). The mismatch-binding
activity Cmb1 eluted at 0.55 M NaCl. A 4-ml aliquot was
stored for N-terminal peptide sequencing (see below). The remainder of
the fractions were pooled, dialyzed twice against 2 liters of B150 (18 ml of fraction V) and then loaded on a 1 ml MonoQ column (HR5/5,
Amersham Pharmacia Biotech) at a flow rate of 0.5 ml/min. Bound
proteins were eluted by 2 ml of B300 followed by a 14-ml linear
gradient from 0.3 to 1 M NaCl. Cmb1 peaked at 0.4 M NaCl of the gradient but was also detected in the
flow-through and in the fractions eluted by 0.3 M NaCl,
indicating that the MonoQ column was overloaded. Nevertheless, the use
of this column enabled the identification of the mismatch binding
activity as a 22-kDa protein (Fig. 1). Active fractions were separately
dialyzed against Buffer S (25 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.1 mM EDTA, 50% glycerol (v/v), 5 mM -mercaptoethanol, 0.2 mM
phenylmethylsulfonyl fluoride) and stored at 20 °C (fraction VI).
-D-galactoside was added to a final
concentration of 1 mM. After 5 h of induction, cells
were harvested by 10 min of centrifugation at 8300 × g
and suspended in Buffer A. After brief freezing in liquid nitrogen, cells were stored at 70 °C until further use. To prepare crude protein extracts containing Cmb1 or Cmb141, Buffer B150 was added to
the thawed cells (
of the total volume). Cells were disrupted
by sonication on ice (10 times for 30 s, with at least 1 min
cooling on ice between each round). After 20 min of centrifugation at
10,000 × g, the supernatant was removed (crude protein
extracts, fraction I).
41. Fraction I (137 mg of protein in 28 ml) was
obtained from a 750-ml E. coli culture and loaded at a flow
rate of 19 ml/h on a Heparin-agarose column (type I, Sigma, 1.8 cm2 × 11 cm) previously equilibrated with Buffer B150.
Bound proteins were eluted by 20 ml of Buffer B300 followed by a 100-ml
gradient from 300 to 800 mM NaCl. Cmb141 peaked at 580 mM. The purest fractions were collected and dialyzed in
B150 to give fraction II (2.8 mg of protein in 7 ml). Fraction II was
loaded at a flow rate of 8 ml/h on a P11 phosphocellulose column
(Whatman, 0.64 cm2 × 4.7 cm). Bound proteins were eluted
with 10 ml of B350 (Buffer B containing 350 mM NaCl),
followed by a 60-ml gradient from 350 to 800 mM NaCl.
Cmb141 eluted between 400 and 550 mM and peaked at 480 mM. At this step, a few faint bands of other proteins were detected in early fractions (around 400 mM) but not in the
other fractions containing Cmb141. However, an additional
purification step was included with later fractions. These fractions
were pooled and the NaCl concentration was adjusted to 300 mM by addition of Buffer B0 (same as Buffer B150 but
without NaCl). The resulting fraction III (1.9 mg of protein in 7.3 ml)
was loaded at a flow rate of 7 ml/h on a double-stranded DNA-cellulose
column (United States Biochemical Corp., 0.64 cm2 × 4.7 cm), equilibrated with B300. After washing, a 40-ml gradient from 300 to 600 mM NaCl was applied. Cmb141 peaked at 400 mM. No other proteins could be detected in a silver-stained
SDS-polyacrylamide gel. Cmb141 was then dialyzed against Buffer S
and stored in aliquots at 20 °C (fraction IV, 1.8 mg of protein in
7 ml).
RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References
and some binding to C/C
and T/T. No or weak binding was observed to homoduplex and to
substrates with a T/G, A/A, G/G, or G/A mismatch (Fig. 1C).
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Fig. 1.
Purification and characterization of
Cmb1. A, MonoQ fractions were tested for
mismatch-binding activity by band-shift analysis. The reactions
contained radiolabeled C/ substrate and 55 mM NaCl. No
competitor DNA was added. Mismatch-binding activity peaked in fractions
30 and 31, which eluted at about 0.4 M NaCl from the
column. B, Coomassie-stained 12% polyacrylamide gel,
containing the same fractions as in A. The intensity of the
22-kDa protein band (marked by an arrow) correlates well
with the intensity of the mismatch-specific complex shown in
A. M indicates size marker proteins (Bio-Rad).
C, mismatch binding specificity of partially purified Cmb1.
MonoQ fraction 29, containing partially purified Cmb1, was tested using
a 120-fold excess of unlabeled homoduplex as competitor. The NaCl
concentration in the reaction was 80 mM. An activity was
detected that showed high affinity to all cytosine-containing
mismatches and T/T, but no or only weak binding to other mismatches and
homoduplex.
View larger version (13K):
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Fig. 2.
Cloning of the cmb1+
gene. A, 29 amino acids were determined from purified
Cmb1 by N-terminal peptide sequencing. Four primers derived from the
amino acid sequence were used for PCR (see under "Experimental
Procedures"). B, a PCR fragment obtained with primers
CC-1b/CC-3 was used to screen a plasmid-based library (18). Several
clones containing a truncated ORF of cmb1+ were
identified. The putative start codon (ATG) is indicated. The insert
end, a Sau3A restriction site, is marked with GATC. The
amplification product obtained with primers CC-6/CC-7 was used to
identify cmb1+ on ordered cosmid/P1 filters (19,
20). C, DNA fragments were isolated from cosmid ICRFc60H037
and cloned into pUC18 to give plasmids pOL54 to pOL59. Identical
fragments cloned in different orientations are shown below the
chromosomal map (e.g. pOL54/pOL55). The
restriction map was constructed by Southern hybridization with
chromosomal DNA, with cosmid DNA, and by restriction analysis of the
plasmid clones pOL54-pOL59. The left end, containing a
NsiI and SalI site, is not drawn to scale.
Abbreviations for restriction sites: Bg, BglII;
EV, EcoRV; N, NsiI;
Sa, SalI; and Sc, ScaI.
During sequencing of cmb1+, the
arg3+ gene was identified about 600 bp
downstream. Open reading frames are indicated by black
arrows.
View larger version (47K):
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Fig. 3.
Comparison of the Cmb1 HMG domain with other
HMG domains. Only a selection of HMG domains from proteins
belonging to the subgroup of non-sequence-specific HMG domain proteins
is shown. The proteins in this subfamily, like the HMG domain of Cmb1,
have a proline at position 4, a serine at position 9, a tyrosine at
position 70, and no proline at position 75 (23). Pro4,
Ser9, and Tyr70 are shown above the aligned
sequences. The alignment was created by the Pile-Up program (GCG
software package, version 9, University of Wisconsin) with a gap weight
of 9 and a gap length weight of 4. The aligned sequences were arranged
with the Boxshade program (Bioinformatic Group, ISREC, Lausanne,
Switzerland). Identical amino acids are shown in black and
similar amino acids in gray. The HMG domains shown are from
human hPMS1 (24), mouse tsHMG (25), mouse T160 protein (26), soybean
SB11 (27), S. cerevisiae Ixr1 (15), and S. pombe
Cmb1 (amino acids 144-223). Two HMG domains of the same protein are
distinguished by A and B.
mismatches was observed with the mutant extracts
(lanes 8-10). Surprisingly, weak binding to C/C persisted
(lane 7). Thus a second activity (termed Cmb2) for binding
to C/C exists in S. pombe. Cmb2 does not bind to other
mismatches, whereas Cmb1 binds specifically to C/C, C/A, T/C, C/,
and T/T (Fig. 1C).
View larger version (62K):
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Fig. 4.
Identification of Cmb2. Band-shift
analysis was performed with crude extracts from wild type (lanes
1-5) and from the cmb1 mutant (lanes
6-10). A 40-fold excess of unlabeled homoduplex was included in
the reactions. Specific binding to C/A, C/ , and C/T was not detected
with cmb1 extracts (lanes 8-10). Complex
formation with C/C in cmb1 extracts (lane 7)
revealed the existence of an additional C/C binding protein, Cmb2
(marked by an arrow).
41, corresponds to the proteolytic peptide
enriched from S. pombe cells. Crude extracts of both
overexpression strains were tested for mismatch binding. Both Cmb1- and
Cmb141-containing extracts exhibited an activity that bound to the
mismatches C/C, C/A, C/T, C/, and T/T but only weakly to other
mismatches and to homoduplex (data not shown). The complex was not
observed when uninduced strains or a strain containing the empty
expression vector were used. No differences in gel migration and
mismatch specificity between Cmb1, Cmb141, and the proteolytic Cmb1
peptide enriched from S. pombe were found (data not shown).
Noticeably, overexpression of Cmb141 was about 20 times higher than
overexpression of the full-length protein.
41 protein was
extensively purified (see under "Experimental Procedures"). Cmb141 was first tested for its binding ability to substrates containing base-base mismatches, single unpaired nucleotides and small
loops with two or four nucleotide insertions (Fig.
5). A strong protein-DNA complex was
formed when C/C or C/ mismatches were present (Fig. 5, lanes
2 and 3). Weak or no interaction was found with T/-,
G/-, A/-, C2/-, C4/-, and
T2/-containing substrates (lanes 4-9). Thus,
one unpaired cytosine is well recognized, but not an unpaired thymine,
guanine, or adenine. On the other hand, recognition is not dependent on
the presence of mispaired or unpaired cytosines per se, as
two or four cytosines in a loop were only weakly bound by Cmb141
(Fig. 5, lanes 7 and 8).
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Fig. 5.
Substrate specificity of Cmb1 protein
purified from E. coli. Band-shift assays were
performed to test the specificity of Cmb1 to various mismatches and
small loops. The reactions contained 2.5 pmol of Cmb1 41 protein, 40 fmol of radiolabeled substrates as indicated, a 120-fold excess of
unlabeled homoduplex as competitor, and 240 mM NaCl in a
standard reaction buffer. Strong binding was only observed with the
substrates C/C and C/ (lanes 2 and 3).
View larger version (17K):
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Fig. 6.
Cmb1 binds to cis-DDP-GpG/CpC
intrastrand cross-links and confers resistance to cisplatin.
A, structure of oligonucleotides used for the binding assay.
The homoduplex containing GpG/CpC (underlined) at the
central position is shown in full. For other oligonucleotides, only the
differences at the central positions are given. cis-DDP-GpG
substrates contain a 1,2 intrastrand cross-link between the two
adjacent guanines of the plus strand and were obtained after cisplatin
treatment. cis-DDP-GpG/GpG is shown as an example for the
substrates in which the two cytosines opposite the
cis-DDP-GpG lesion were changed.
trans-DDP-GpTpG/CpApC contains a 1,3 intrastrand cross-link
between the two guanines and was produced by treatment with
transplatin. B, band shift analysis to test binding of Cmb1
to GpG intrastrand cross-links. The reactions contained 3.7 pmol of
Cmb1 41, 40 fmol of substrate, a 200-fold excess of unlabeled
homoduplex DNA, and 50 mM NaCl in a standard reaction
buffer. Cmb1 strongly bound to double-stranded substrates containing a
cis-DDP-GpG lesion when two cytosines were opposite the
intrastrand cross-link (lane 4) but only weakly when other
bases were present (lanes 5-8). Also, binding to a G/T
mismatch in the same sequence context (lane 9) was as low
as binding to homoduplex (lane 3). Note that the
cis-DDP-GpG containing substrates run more slowly in the gel
than the unmodified substrates. No specific binding to the
transplatinated substrate was detected (data not shown). C,
cytotoxic effect of cisplatin on cell survival of S. pombe
wild type (
) and cmb1 (
) strains. The experiment was
carried out six times (see under "Experimental Procedures"). It
should be noted that we found some variations in cell survival between
different experiments. However, in all tests, cmb1 was more
sensitive to cisplatin than was wild type. Points are the
average of three independent experiments; bars represent
S.D.
View larger version (18K):
[in a new window]
Fig. 7.
Cmb1 binds strongly to O6meG:C
but only weakly to O6meG/T. A, band shift
analysis to test binding of Cmb1 to O6meG lesions. The
reactions contained 3.3 pmol of Cmb1 41 protein, 40 fmol of
radiolabeled substrates as indicated, a 120-fold excess of unlabeled
homoduplex as competitor, and 120 mM NaCl in a standard
reaction buffer. B, cytotoxic effect of MNNG on cell
survival of S. pombe wild type () and cmb1
() strains. The experiment was carried out six times (see under
"Experimental Procedures"). Points are the average of
three independent experiments; bars represent S.D.
DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
and weakly binds to T/T (16). We now report partial
purification of the 22-kDa activity, Cmb1, from S. pombe
wild type cells and cloning of the gene. The ORF of
cmb1+ encodes a 26-kDa protein with 223 amino
acids, indicating that a proteolytic peptide was enriched from S. pombe. However, we found no differences in mismatch specificity
between the full-length 26-kDa Cmb1 and the 22-kDa peptide when
overproduced in E. coli.
41, which is missing the first 41 amino acids, like the proteolytic peptide purified from yeast, were
overexpressed in E. coli and tested for mismatch binding. No
difference in specificity was found between the two recombinant Cmb1
peptides and Cmb1 enriched from S. pombe. In the E. coli expression strain containing the empty pT7-7 vector, the
specific mismatch-binding complex was not detected. It is concluded
that Cmb1 is able to bind to mismatches without the need of other
S. pombe proteins.
41 from E. coli cells and tested
binding to mismatches and small DNA loops. Cmb1 recognizes C/C, C/A, C/T, T/T, and a C/ insertion but shows no or weak affinity to other
types of mismatches and insertions (Fig. 5 and data not shown).
Although one unpaired cytosine is strongly bound by Cmb1, two or four
unpaired cytosines are poor substrates (Fig. 5). Thus, cytosine-containing base mismatches (and T/T) represent a DNA structure
irregularity that is recognized by a new type of HMG domain protein.
This binding pattern is different from E. coli MutS and
eukaryotic MutS, which are able to bind to almost all types of base
mismatches (34-38). The difference is most pronounced for C/C
mismatches, which are a poor substrate for binding and repair by
E. coli and yeast MutS proteins. A human mismatch-binding activity was identified that binds to A/C, C/T, and T/T mismatches without requirement of the MutS homologue hMSH2 (39). This specificity is similar to that of Cmb1 (Fig. 1) except that C/C mismatches were
poorly bound by the human activity.
was abolished, but some binding to C/C remained (Fig. 4). We conclude
that a second activity, Cmb2, exists in S. pombe that
recognizes C/C but not other mismatches. Thus, C/C mismatches are
recognized redundantly by Cmb1 and Cmb2.
and some HMG domain proteins are thought to
confer cellular sensitivity in response to the drug cisplatin. Whereas
binding by the HMG domain proteins HMG-1 and Ixr1 is proposed to shield
cisplatin adducts from repair (14, 15), it is likely that binding of Cmb1 facilitates repair, as indicated by enhanced sensitivity of the
cmb1 mutant to cisplatin. Transplatin is an isomer of
cisplatin that is inactive as a chemotherapeutic drug, and cross-links
produced by transplatin are not bound by HMG domain proteins (13).
Consistently, an ixr1 mutant treated with transplatin showed
no difference from wild type in cell survival (15). Similarly, Cmb1
does not bind to a 1,3 GpTpG intrastrand cross-link produced by
transplatin, and in comparison to wild type, the cmb1 mutant
is not more sensitive to transplatin (data not shown).
ACKNOWLEDGEMENTS
FOOTNOTES
Supported by a Forschungsstipendium of the Deutsche
Forschungsgemeinschaft. To whom correspondence should be addressed.
Tel.: 41-31-631-4656; Fax: 41-31-631-4684; E-mail:
fleck{at}imb.unibe.ch.
REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.
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