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Molecular and Cellular Biology, July 2000, p. 4553-4561, Vol. 20, No. 13
Department of Cell Biology and Genetics, Centre for
Biomedical Genetics, Erasmus University
Rotterdam,1 and Department of Radiation
Oncology, Daniël den Hoed Cancer Center,3
3000 DR Rotterdam, and Laboratory of Health Effects Research,
National Institute of Public Health and the Environment, 3720 BA
Bilthoven,2 The Netherlands
Received 20 December 1999/Returned for modification 16 February
2000/Accepted 5 April 2000
DNA interstrand cross-links (ICLs) represent lethal DNA damage,
because they block transcription, replication, and segregation of DNA.
Because of their genotoxicity, agents inducing ICLs are often used in
antitumor therapy. The repair of ICLs is complex and involves proteins
belonging to nucleotide excision, recombination, and translesion DNA
repair pathways in Escherichia coli, Saccharomyces cerevisiae, and mammals. We cloned and analyzed mammalian
homologs of the S. cerevisiae gene SNM1
(PSO2), which is specifically involved in ICL repair. Human
Snm1, a nuclear protein, was ubiquitously expressed at a very low
level. We generated mouse SNM1 DNA interstrand cross-links (ICLs)
prevent strand separation, thereby physically blocking transcription,
replication, and segregation of DNA. In bacterial and yeast cells, the
presence of one unrepaired ICL can be lethal (36, 40).
Humans are exposed to environmental ICL agents, such as furocoumarins,
from plants and cosmetics (50). Due to their extreme
genotoxicity, agents that induce cross-links in DNA are widely used in
antitumor therapy. Examples include cisplatin, mitomycin C (MMC), and
derivatives of nitrogen mustard, such as melphalan and
cyclophosphamide. Most of these agents cause a number of different
lesions in DNA, including monoadducts, DNA-protein cross-links, and DNA
intrastrand cross-links and ICLs. The latter are the main cause of cell
death, although they constitute only a small percentage of the total
number of adducts (6, 36). Resistance of tumors to
cross-linking agents can be caused by a variety of mechanisms,
including increased repair of ICLs (1, 5, 31). Therefore, an
understanding of how ICLs are repaired is important.
To elucidate the mechanism of ICL repair in yeast, genetic screens have
been performed to find genes specifically involved in ICL repair. A
number of snm and pso mutants have been isolated after screening for strains with increased sensitivity to nitrogen mustard and 8-methoxypsoralen plus UVA light, respectively (25, 48). Some of the mutants isolated are sensitive to several
classes of DNA-damaging agents, while others are almost exclusively
sensitive to ICL agents. The gene mutated in one of these strains is
SNM1, which is allelic with PSO2 and encodes a
nuclear 76-kDa protein (9, 23, 47). snm1 mutants
are sensitive to a number of agents that cause ICLs, but they are only
mildly sensitive to monofunctional alkylating agents and 254-nm UV
light (UV254 nm) and are not hypersensitive to gamma rays
(6, 25, 49). In exponentially growing cells, the expression
of SNM1 can be induced by ICL agents or UV254 nm
(61). Overexpression of SNM1 results in an
increased resistance to nitrogen mustard and cisplatin (22).
Many genes involved in DNA damage repair are conserved between yeast
and higher eukaryotes. One way to elucidate the mechanisms of ICL
repair in mammalian cells is by studying mammalian homologs of
Saccharomyces cerevisiae genes specifically involved in ICL repair. A human homolog of SNM1 has been isolated, and a
comparison of its predicted protein product (hSnm1) to S. cerevisiae Snm1 (ScSnm1) has revealed 39% similarity, excluding
gaps (43). The gene is located on chromosome 10q25, a region
often found to be rearranged in tumors (4, 52, 58). Here, we
report on the characterization of mammalian SNM1 homologs.
hSNM1 cDNA constructs.
The cDNA of human
SNM1 (hSNM1) (kindly provided by N. Nomura) was
used to make several constructs containing tags at the 5' and 3'
termini of hSNM1 (Table 1).
For this purpose, the 5' terminus of hSNM1 was modified to
generate an XhoI restriction enzyme site just in front of
the expected initiation codon, thereby deleting the 900-bp 5'
untranslated region that contains 15 additional ATG sequences. The
following tags were added to hSnm1: an N-terminal histidine tag
(His10; single-letter amino acid code:
MGHHHHHHHHHHGGSR), a C-terminal hemagglutinin tag (HA;
PGGYPYDVPDYAS), and a C-terminal histidine-hemagglutinin tag
(PHHHHHHGGSAYPYDVPDYAS). Parts of the hSNM1 cDNA
were subcloned into pEGFPC vectors (expressing green fluorescent
protein [GFP]; Clontech) (Table 1).
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Disruption of Mouse SNM1 Causes Increased Sensitivity
to the DNA Interstrand Cross-Linking Agent Mitomycin C
ABSTRACT
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
/ embryonic
stem cells and showed that these cells were sensitive to mitomycin C. In contrast to S. cerevisiae snm1 mutants, they were not
significantly sensitive to other ICL agents, probably due to redundancy
in mammalian ICL repair and the existence of other SNM1
homologs. The sensitivity to mitomycin C was complemented by
transfection of the human SNM1 cDNA and by targeting of a
genomic cDNA-murine SNM1 fusion construct to the disrupted
locus. We also generated mice deficient for murine SNM1.
They were viable and fertile and showed no major abnormalities.
However, they were sensitive to mitomycin C. The ICL sensitivity of the
mammalian SNM1 mutant suggests that SNM1
function and, by implication, ICL repair are at least partially
conserved between S. cerevisiae and mammals.
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
TABLE 1.
Characteristics of the mammalian
SNM1 constructs
Subcellular localization of hSNM1. The constructs GFP-hSNM1, GFP-570hSNM1, GFP-1515hSNM1, and GFP-ChSNM1 were transfected into CHO9 cells. To obtain stable transfectants, cells were split after 1 day and subjected to G418 selection (1 mg/ml). Single colonies expressing GFP-hSnm1 proteins were expanded. The GFP-hSNM1 construct was also microinjected into multinucleated fibroblasts according to previously described procedures (27).
Generation of anti-hSnm1 antibodies.
A 354-bp
PstI-HindIII fragment from the
hSNM1 cDNA was subcloned into pTrcHisC (Table 1). The fusion
protein derived from this plasmid contained amino acids 644 to 763 of
hSnm1 fused to a His6 tag and was produced in
Escherichia coli strain DH5
. The protein was present in
the insoluble fraction and was dissolved in 6 M urea. It was purified
on a Ni-nitrilotriacetic acid column, eluted with 200 mM imidazole, and
used to immunize two rabbits. The detection limit of the polyclonal
antibodies was determined by immunoblotting using a range of 1 to 1,000 ng of antigen and was shown to be below 1 ng. The antibodies were
affinity purified using fusion protein immobilized on a nitrocellulose filter.
Expression of recombinant hSnm1. The cDNA encoding His10-hSnm1-HA was subcloned into pFastBac1 (GibcoBRL) (Table 1). The resulting plasmid was used to create recombinant viruses and to produce the protein in Sf21 cells as described by the manufacturer. Protein extracts of these cells were made as described previously (54).
Construction of mSNM1 targeting vectors. A mouse testis cDNA library was hybridized with a 2.7-kb EcoRI hSNM1 cDNA fragment. The resulting murine SNM1 (mSNM1) cDNA clone was sequenced and used to screen a lambda phage genomic library made from mouse strain 129/Sv. Genomic fragments hybridizing to the mSNM1 cDNA were subcloned in pBluescript II KS (Stratagene). The locations and intron and exon borders of the first five exons were determined by restriction analysis and DNA sequencing. Targeting constructs were made by cloning a 4-kb SalI fragment encompassing exons 2 and 3 and a 5-kb HindIII fragment encompassing part of exon 4 and exons 5 to 7 in pBluescript II KS. Between these fragments, a cassette containing either a neomycin (neo) resistance gene or a hygromycin (hyg) resistance gene was inserted. This step resulted in the partial deletion of exon 4. These targeting constructs are referred to as mSNM1neo and mSNM1hyg, respectively (Table 1) (see Fig. 3A).
ES cell culture and electroporation. Embryonic stem (ES) cells were electroporated with the mSNM1hyg targeting construct and cultured on gelatinized dishes as described previously (17). The cells were split 24 h after electroporation, and hygromycin B was added to a final concentration of 200 µg/ml. After 7 to 10 days, colonies were isolated and expanded. Genomic DNA from individual clones was digested with SpeI or HindIII and analyzed by DNA blotting using the flanking exon 1 probe (see Fig. 3A). DNA from targeted clones with the expected hybridization pattern was subsequently digested with SspI and hybridized with the internal intron 5 probe to confirm proper homologous integration. To obtain ES cell lines carrying a disruption in both mSNM1 alleles, an mSNM1hyg-targeted ES cell line was electroporated with the mSNM1neo targeting construct. After selection with G418 (200 µg/ml) for 7 to 11 days, colonies were isolated and expanded. The isolated DNA was digested with HindIII and hybridized with exon 1 DNA to identify cell lines containing two targeted mSNM1 alleles.
Rescue of mSNM1/ cells by
hSNM1 cDNA constructs.
The hSNM1-HA cDNA
was subcloned into pPGK-p(A) to express the gene under the control of
the phosphoglycerate kinase (PGK) promoter (Table 1). This cDNA
expression construct was coelectroporated with a puromycin-expressing
plasmid into mSNM1neo/hyg cells. Clones were
selected with puromycin (1 µg/ml) for 10 days. Integration of the
cDNA construct was confirmed by DNA blotting.
Rescue of mSNM1/ cells by
mSNM1 genomic constructs.
A GFP-mSNM1
targeting fusion construct was made under the control of the
mSNM1 promoter (see Fig. 3B). To obtain the expression of
mSNM1 with 3'-terminal GFP, the 3' terminus of
mSNM1 cDNA was modified by PCR to create an EcoRV
site in front of the stop codon. In this site, the GFP cDNA
was cloned, and the borders were sequenced. Then, the 5.6-kb genomic
mSNM1 HindIII fragment encompassing exons 1 to 4 was cloned
into the cDNA, thereby fusing exon 4 from the genomic fragment with
cDNA exons 4 to 9. Behind the endogenous polyadenylation signal, a
neo cassette and the 5-kb mSNM1 HindIII fragment
were cloned (see Fig. 3B). The construct, named
mSNM1C-GFP, was transfected into
mSNM1hyg-targeted ES cells, and clones were
selected with G418 (200 µg/ml) and expanded. The isolated DNA was
digested with SspI and hybridized with the intron 5 probe to
screen for cell lines containing the homologously integrated target DNA.
Cell survival assays. The sensitivity of ES cells to increasing doses of DNA-damaging agents was determined by measuring their colony-forming ability as described before (17). The cloning efficiency of untreated cells varied between 10 and 30%. Cells were incubated in drug-containing media for 1 h. Sensitivity to 8-methoxypsoralen plus UVA light was determined as follows. Dishes were incubated for 30 min in medium with 8-methoxypsoralen in the dark. Then, the cells were irradiated with 12 kJ of 320- to 380-nm UVA light at 2 mW/cm2 in phosphate-buffered saline containing 8-methoxypsoralen. All measurements were performed in triplicate.
Miscellaneous methods. Reverse transcription (RT)-PCR, immunofluorescence microscopy, generation of mSNM1 mutant mice, and in vivo MMC survival assays were performed according to standard procedures and as previously described (17, 18, 55).
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RESULTS |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Comparison of Snm1 from different species.
A database search
using either ScSnm1 or hSnm1 as a query sequence revealed the existence
of homologs of Snm1 in many different species, including
Schizosaccharomyces pombe, Aspergillus niger, Arabidopsis thaliana, Caenorhabditis elegans,
Drosophila melanogaster, Danio rerio, and
Rattus norvegicus (Fig. 1). We
isolated and sequenced the mouse homolog (mSnm1) as described below. In
addition, we identified part of a second human and mouse Snm1 homolog
that we refer to as hSnm1B or mSnm1B, respectively, and a third human Snm1 homolog, called hSnm1C. Comparison of these different mammalian homologs showed that hSnm1 and mSnm1 displayed the highest degree of
similarity to ScSnm1. hSnm1B and mSnm1B showed a high degree of
similarity to each other but less similarity to ScSnm1. In A. thaliana, three Snm1 homologs were also found, but these were not
clear homologs of hSnm1, hSnm1B, or hSnm1C. The length of the protein
varied between different homologs and species. Some homologs showed
similarity in the N-terminal region of Snm1, including the putative Zn
finger domain present in ScSnm1. Other homologs did not contain this Zn
finger domain and showed no significant similarity in the N-terminal
region. Deletion of the Zn finger in S. cerevisiae did not
render cells more sensitive to nitrogen mustard (23). The
middle part of the protein was least conserved among all species, while
conservation was highest in the C-terminal region. Eight regions stood
out in particular, and we refer to these as motifs (Fig. 1). However,
because these motifs appeared unique for Snm1, no clues regarding their
function could be obtained through database searches.
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Subcellular localization of hSnm1.
The subcellular
localization of hSnm1 was determined by microinjecting human
fibroblasts and by transfecting CHO9 cells with a
GFP-hSNM1 fusion construct (Table 1 and Fig.
2). As a control, the pEGFPC2
vector was transfected. In this case, GFP was diffusely present
throughout the cell. In contrast, the location of GFP-hSnm1 was clearly
restricted to the nucleus after both microinjection and transfection,
as revealed by inspection of the cells under a fluorescence microscope
after one to several days (data not shown). GFP-hSnm1 was excluded from
the nucleolus. In cells expressing large amounts of protein, there was
also weak staining of the cytoplasm. We conclude that hSnm1 is a
nuclear protein. A few days after transfection or microinjection, many
of the cells expressing large amounts of GFP-hSnm1 underwent
morphological changes that were consistent with apoptosis. This finding
indicates that Snm1 could be toxic when overexpressed in cells; this
notion would also explain why extensive attempts to generate stable
protein-expressing clones after G418 selection failed. Transfection of
a series of GFP-hSNM1 fusion constructs revealed that the
hSnm1 nuclear localization signal is located within the N-terminal 190 amino acids, which corresponds to the potential sequence for the
nuclear localization signal at about amino acid 20 (Fig. 2).
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, absent; + through +++, present in increasing amounts).
Generation of mSNM1-disrupted mouse cells.
Screening of a mouse testis cDNA library with the hSNM1 cDNA
yielded a mouse cDNA clone spanning the 3'-terminal 2 kb of
mSNM1. This clone was sequenced and used as a probe to
obtain genomic mouse DNA. Two clones that spanned the first seven exons
of the mSNM1 genomic locus were obtained. The locus was
characterized by restriction site mapping, PCR, and sequencing of the
first two exons (Fig. 3A). A targeting
construct was made by subcloning into pBluescript II KS a 4-kb
SalI fragment encompassing exons 2 and 3 and a 5-kb
HindIII fragment encompassing part of exon 4 and exons 5 to 7 with a selectable marker gene in between. In this way, part of
intron 3 and 25 bp of exon 4 were deleted. This procedure resulted in
elimination of the C-terminal 287 amino acids of mSnm1, which contain
most of the conserved motifs (Fig. 1). Moreover, aberrant RNA splicing
that skips the targeted exon would result in a frameshift mutation.
Disruption constructs were made containing the neo or the
hyg selectable marker gene (mSNM1neo
and mSNM1hyg, respectively).
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and mSNM1/ cells were performed using
primers for exons 2 and 7. RNA from mSNM1+/
cells showed a clear signal from the wild-type mSNM1 allele, and both cell lines showed three weak signals from the knockout allele
(data not shown). These three bands were cloned and sequenced. One
product would result in a frameshift and a premature stop codon. The
other two products, one containing an insert from the neo
cassette and the other splicing around exon 4, used an alternative splice site in exon 5 which restored the reading frame. Both would result in disruption of conserved motifs 2 and 3 of mSnm1
(Fig. 1). No significant difference in growth rate between
mSNM1+/+ ES cells and all
mSNM1+/ and mSNM1/
cell lines was detected. We conclude that, similar to the situation for
S. cerevisiae, disruption of mSNM1 in mouse cells
results in viable cells (47).
mSNM1/ ES cells are sensitive to MMC.
S. cerevisiae snm1 mutants are sensitive to DNA ICL agents
but not to gamma rays and only slightly to UV light (25,
49). We therefore investigated the effects of these
DNA-damaging agents on the survival of
mSNM1/ ES cells. These cells were found to
be approximately twofold more sensitive to MMC than
mSNM1-proficient cells, which were otherwise isogenic
(Fig. 4A). There was no difference
between mSNM1+/+ and
mSNM1+/ cells. In contrast, we did not find
any significant sensitivity to 8-methoxypsoralen plus UVA,
cisplatin, melphalan, UV254 nm, methyl methanesulfonate,
and gamma rays (Table 2). As a control, ERCC1/ ES cells were also treated with these
agents and found to be sensitive to UV254 nm and all ICL
agents tested (Table 2), as expected from the behavior of
ERCC1/ mouse embryonic fibroblasts and
ERCC1 mutant CHO cells (14). ERCC1/ cells were not sensitive to methyl
methanesulfonate and gamma rays (Table 2).
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/ ES
cells was caused exclusively by the disruption of mSNM1,
cDNA rescue experiments were performed.
mSNM1neo/hyg cells were electroporated with a
cDNA construct expressing HA-tagged hSnm1 together with a plasmid
expressing the dominant selectable marker for puromycin.
Puromycin-resistant cell lines that had integrated hSNM1-HA
were fully corrected for the MMC sensitivity of
mSNM1/ ES cells (Fig. 4B).
mSNM1 is expressed at low levels in ES cells.
To
investigate the expression of mSNM1 in ES cells, we
generated a GFP-tagged version of mSNM1 under the control of
the endogenous mSNM1 promoter (Fig. 3B and Table 1).
For this purpose, a targeting construct,
mSNM1C-GFP, was made by cloning mSNM1
cDNA exons 4 to 9 with 3'-terminal GFP behind
genomic exon 4. As a consequence, the mRNA consisted of a
fusion of four genomic exons and the cDNA with
3'-terminal GFP and was transcribed under the
control of the endogenous mSNM1 promoter.
mSNM1C-GFP was transfected into
mSNM1+/ ES cells, and clones containing tagged
and knockout mSNM1 alleles were obtained.
/ ES cells was
complemented by the presence of mSNM1C-GFP (Fig.
4C). Amplification by RT-PCR using an mSNM1 primer and a
GFP primer spanning at least one intron revealed the
expression of mSNM1C-GFP at the RNA level (data
not shown). However, fluorescence microscopy failed to detect the GFP
signal. Given the detection limit, this result implies that there are
less than 10,000 molecules of mSnm1 per cell (45). We were
also unable to detect either endogenous or transfected mSnm1 by
immunoblot analysis with anti-GFP (Clontech) and anti-hSnm1 antibodies,
even though anti-hSnm1 antibodies were highly sensitive, with a
detection limit of about 5,000 Snm1 molecules per cell (data not
shown). We cannot exclude the possibility, however, that our anti-hSnm1
antibodies were unable to recognize mSnm1.
Similarly, we tried to detect the presence of hSnm1-HA expressed
under the control of the PGK promoter in
mSNM1/ ES cells; this construct can rescue
the MMC sensitivity of these cells (Fig. 4B). Although both anti-hSnm1
and anti-HA antibodies recognized the protein when it was overproduced
in Sf21 cells, we could not detect the protein in ES cells by
either immunoblot or immunofluorescence microscopy (data not
shown). Taken together, these results indicate that Snm1 protein levels
in ES cells are very low, even when the protein is expressed under the
control of the relatively strong PGK promoter.
Generation of mSNM1/ mice.
The
experiments described above show that disruption of mSNM1 is
compatible with normal ES cell growth. Subsequently, we investigated whether mSnm1 is required for normal mouse development. Cells from four
targeted ES clones, carrying the mSNM1hyg
allele, were injected into C57BL/6 blastocysts, and 11 chimeric mice
were obtained. The disrupted mSNM1 allele was transmitted to
the mouse germ line. F1 heterozygous offspring were
intercrossed, and F2 offspring were genotyped by DNA
blotting, PCR analysis, or both (data not shown). Among 78 genotyped
animals, 20 mSNM1+/+, 30 mSNM1+/, and 28 mSNM1/ animals were identified. This
outcome is compatible with normal Mendelian segregation of the
disrupted mSNM1 allele. Thus, disruption of mSNM1
does not result in embryonic or neonatal lethality. No statistically
significant difference in weight was observed among mSNM1+/+, mSNM1+/, and
mSNM1/ littermates (data not shown).
Importantly, the mSNM1/ mice exhibited
no macroscopic abnormalities up to at least 12 months of age.
Both mSNM1/ males and females were fertile.
mSNM1/ mice are sensitive to MMC.
We tested whether the sensitivity of mSNM1/
ES cells to the DNA ICL agent MMC was also found in
mSNM1/ mice.
mSNM1+/ and mSNM1/
mice were treated with various doses of MMC, ranging from 7.5 to 15 mg/kg of body weight. Injection of 10 and 15 mg/kg in female mice
resulted in enhanced sensitivity in mSNM1/
mice (Fig. 5). In addition, with 15 mg/kg, a shorter latency period in mSNM1/
mice was observed. In male mice, 7.5 mg/kg was lethal for 30% of
the knockout mice, while all heterozygous mice survived. At 10 mg/kg, only 10% of the mSNM1/ mice survived
treatment, while 70% of the mSNM1+/ mice
survived (Fig. 5). These results show that the hypersensitivity to MMC found in mSNM1/ ES cells is also
present in mSNM1/ mice.
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Characterization of mammalian SNM1.
We have analyzed
mammalian homologs of the S. cerevisiae SNM1 gene, which
specifically provides resistance to ICL agents. Like yeast
snm1 knockout cells, mSNM1/ ES
cells and mice are viable, showing that SNM1 is not
essential for viability (21). The mice do not show any major
abnormalities and are fertile. mSNM1/ ES
cells and mice are sensitive to MMC, but the cells are not sensitive to
UV254 nm, methyl methanesulfonate, or gamma rays, in
agreement with the results obtained with S. cerevisiae snm1 cells (25, 49). These results argue for a specific role of mammalian SNM1 in the cellular response to ICL agents,
similar to the role of yeast SNM1.
/ cells,
although the detection limit of our antibodies was below 1 ng and the
fluorescence microscopy detection limit of GFP is about 10,000 molecules per cell (45). This result suggests that the level
of expression of Snm1 is very low, in agreement with the codon usage
and the lack of a TATA box. S. cerevisiae SNM1 is also
expressed at a very low level (47). Nevertheless, we could
complement the MMC sensitivity of mSNM1/
cells with hSnm1-HA and mSnm1-GFP; thus, the protein levels must have
been sufficient, and the HA and GFP tags at the C terminus of Snm1 must
not have interfered with its function.
Cross-link repair pathways. The formation and repair of ICLs in cells are complex processes, with major differences between ICL agents. The sensitivity of a cell to a certain ICL agent is dependent on all steps in the processing of ICLs, ranging from uptake and metabolization to damage recognition and repair (6). A number of major DNA damage repair pathways are involved in the repair of ICLs. In S. cerevisiae, mutants in the nucleotide excision repair (NER), recombination repair, and translesion repair pathways are sensitive to ICL agents (24). Moreover, a number of ICL-sensitive mutants, such as pso2 to pso4 mutants, do not quite fit into one of these pathways (23). The major repair pathway of ICLs in S. cerevisiae is supposed to start with incision by the NER system, resulting in a DNA double-strand break (DSB), in contrast to the incision in E. coli, which does not result in a DSB (13, 28, 40, 59). The DSB can be repaired by the recombination repair pathway (2, 28). Repair may also occur via the translesion repair pathway, involving RAD6 and RAD18 (51). Yeast translesion repair pathway mutants are not defective in the repair of an ICL on a plasmid, in contrast to recombination repair pathway mutants, suggesting that chromatin structure can have an important influence (39). On the other hand, rad52 mutants, which are impaired in recombination repair, are hardly sensitive to nitrogen mustard, suggesting that the repair pathway used may depend on the ICL agent (51).
In mammals, a large number of mutant cell lines are known to be sensitive to ICL agents (11). Many of the known mutated genes in those cell lines belong to the NER pathway, the recombination repair pathway, and/or the error-prone postreplication repair pathway (11, 17, 32). A number of genes cannot be attributed to one of the known repair pathways (7, 11). The molecular mechanisms of mammalian ICL repair are still unknown, but they are probably similar to those of S. cerevisiae and E. coli ICL repair. Incision of the ICLs is supposed to be performed by some of the NER proteins, as mutations in ERCC1 result in a decrease in the incision of ICLs and reduced repair-associated replication (35). In contrast to the situation in S. cerevisiae, where no significant differences among NER mutants are found, most mammalian NER-deficient cells are only moderately sensitive to ICL agents (11). However, ERCC1/XPF mutants are among the most MMC-sensitive cell lines. Therefore, in addition to their role in NER, ERCC1 and XPF are likely to be involved in the incision of ICLs or subsequent recombination, independent of the other NER genes. The sensitivity of the other NER mutants might be caused solely by a defect in the repair of monoadducts. Hamster cell lines with an MMC sensitivity similar to that of ERCC1 mutants are irs1 and irs1SF (11). The genes mutated in these cell lines are XRCC2 and XRCC3, which are paralogs of the recombination repair gene RAD51 and are important for chromosomal stability and DSB repair (12, 30, 37, 46, 56, 57). The mechanism by which these genes work is still unknown, but they could have an important role in recombination repair of ICLs. Another group of genes involved in the response to ICL agents consists of the Fanconi anemia (FANC) genes. FANC is characterized by developmental abnormalities, pancytopenia of blood cells, and a predisposition to cancer (16). FANC cells are sensitive to ICL agents and to oxidative DNA damage and show cell cycle abnormalities (7). To date, eight complementation groups have been found, and three genes have been cloned, FANCA, FANCC, and FANCG (15, 19, 29, 38, 53). FANCC knockout mice are very sensitive to MMC, although otherwise, their phenotype is very mild (8, 10, 60). The possibility that SNM1 is one of the remaining FANC genes cannot be excluded.Role of Snm1 in ICL repair. The results for S. cerevisiae SNM1 show that Snm1 is not required for all ICL repair. S. cerevisiae snm1 cells show synergism with mutants from both the translesion and the recombination repair pathways with regard to sensitivity to ICL agents, suggesting that Snm1 functions in an alternative pathway (24, 51). Moreover, snm1 cells are capable of repairing an ICL on a plasmid, suggesting that the activity of Snm1 may be related to the modulation of chromosomal structure (39). The sensitivity of snm1 mutants to all ICL agents tested suggests that Snm1 is involved in an ICL agent-independent step and therefore probably after the formation of ICLs (6, 25, 49). This notion is consistent with the fact that snm1 mutants are capable of creating DSBs after treatment with 8-methoxypsoralen plus UVA light, in contrast to NER mutants, which are not able to incise the DNA near the ICL (40, 42). snm1 mutants are, however, epistatic with NER mutants, suggesting that they play a role in this repair pathway (24, 51). Snm1 may be involved in restoring the continuity of the DNA, because snm1 cells are not able to repair the DSBs formed (40). Alternatively, Snm1 could also play a more indirect role, in the regulation of ICL repair.
mSNM1 knockout ES cells are specifically sensitive to MMC but not to cisplatin, melphalan, or 8-methoxypsoralen plus UVA light, in contrast to S. cerevisiae snm1 cells. Therefore, mammalian SNM1 is probably not essential for a general ICL repair pathway. Mammalian cells could have different proteins for the recognition and repair of different ICLs, while yeast cells could depend on a single protein. Alternatively, some translesion repair pathways in mammals could be more efficient than those in yeast, taking over most ICL repair in mSNM1 knockout ES cells but being insufficient to repair MMC-induced ICLs. We cannot exclude the possibility of the presence in our knockout ES cells of some mutated mSnm1 that could fulfill some of the functions of the protein and thereby mitigate the phenotype. However, this notion is not a likely explanation of the mild phenotype, because two highly conserved motifs were deleted. As the sequence of the protein does not give clues to its function, we can only speculate about its role. mSnm1 could be involved in the activation of MMC, decreasing the number of ICLs or other damage caused by this agent. Alternatively, it could be responsible for the recognition of MMC-induced ICLs and other structurally related ICLs. These explanations would be inconsistent with the yeast snm1 phenotype, although a direct comparison is not possible because of the existence of multiple mammalian SNM1 homologs and because MMC has not been tested in S. cerevisiae. It is also possible that Snm1 is involved in a regulatory pathway, influencing the response of the cell to the damage caused by MMC. The most attractive assumption for the function of mSnm1 is a direct role in the repair of ICLs, as suggested by the results for its homolog in S. cerevisiae.Induction of mouse Rad51 foci is normal in
mSNM1/ cells.
In S. cerevisiae, snm1 and rad52 mutants are
synergistic with respect to treatment with ICL agents (24,
51). The RAD52 group of recombination repair genes, of
which the main members are RAD51, RAD52, and
RAD54, is also involved in ICL repair in mammals
(17, 33, 44). On treatment with MMC, mouse Rad51 forms
nuclear foci (20). Immunofluorescence on mSNM1
knockout ES cells after MMC treatment showed normal focus formation of Rad51 (data not shown). In contrast, XRCC3 and mouse
RAD54 (mRAD54) mutants do not show Rad51 foci
after treatment with cisplatin and MMC, respectively (3,
55). These results suggest either that Snm1 is required for the
ICL recombination repair pathway after the involvement of Rad51 or that
it plays a role independent of the recombination repair pathway.
Redundancy of mammalian Snm1 function.
The relatively mild
phenotype of mSNM1/ ES cells and mice is
reminiscent of the phenotype found for
mRAD54/ cells and mice, which are also about
twofold more sensitive to MMC than wild-type cells and mice (17,
18). This mild phenotype can probably be attributed in part to
the existence of parallel pathways for the repair of ICLs. In addition,
known homologs for both mSNM1 and mRAD54 could
take over part of the function (Fig. 1) (26). In fact,
redundancy is a phenomenon that is quite common in mammalian DNA damage
repair pathways. The NER gene RAD23, the translesion repair
gene RAD6, and the recombination repair gene RAD51 have several mammalian homologs (34, 41,
57). These duplications provide cells with the possibility of
functional differentiation in the repair of different types of damage,
in different phases of the cell cycle, or in different cell types. Moreover, the cell can fall back on the alternative repair pathway in
case of dysfunction of one of the proteins involved, a strategy which
may prevent inappropriate repair and cell transformation. It will be
important to analyze the other SNM1 homologs and to look at
the effects of ICL agents on double knockout cells. Furthermore, study
of cells with mutations in SNM1 and other genes functioning in ICL repair, such as mRAD54, will yield information on the
relative importance of the different ICL repair pathways and the level of redundancy between different pathways. Nevertheless, the sensitivity of mSNM1 knockout cells to MMC shows that there is no
complete redundancy in mammalian cells. Both the sequence conservation and the functional conservation with yeast Snm1 underline the significance of SNM1 in ICL repair.
| |
ACKNOWLEDGMENTS |
|---|
We thank W. Vermeulen for performing the microinjection experiment.
This work was supported by grants from The Netherlands Organization for Scientific Research (NWO) and the Dutch Cancer Society. R.K. is a fellow of The Royal Netherlands Academy of Arts and Sciences.
| |
FOOTNOTES |
|---|
* Corresponding author. Mailing address: Department of Cell Biology and Genetics, Erasmus University Rotterdam, P.O. Box 1738, 3000 DR Rotterdam, The Netherlands. Phone: 31-10-4087168. Fax: 31-10-4089468. E-mail: kanaar{at}gen.fgg.eur.nl.
Present address: Department of Biological Chemistry, Howard Hughes
Medical Institute, University of California, Los Angeles, CA
90095-1662.
| |
REFERENCES |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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