Download Oxidative nucleotide damage: consequences and prevention

Oncogene (2002) 21, 8895 – 8904
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Oxidative nucleotide damage: consequences and prevention
Mutsuo Sekiguchi*,1 and Teruhisa Tsuzuki2
1
Biomolecular Engineering Research Institute, Suita, Osaka 565-0874, Japan; 2Department of Medical Biophysics and Radiation
Biology, Faculty of Medical Sciences, Kyushu University, Fukuoka 812-8582, Japan
8-Oxoguanine (8-oxo-7,8-dihydroguanine) is produced in
DNA, as well as in nucleotide pools of cells, by reactive
oxygen species normally formed during cellular metabolic processes. 8-Oxoguanine nucleotide can pair with
cytosine and adenine nucleotides with an almost equal
efficiency, then transversion mutation ensues. MutT
protein of Escherichia coli and related mammalian
protein MTH1 specifically degrade 8-oxo-dGTP to 8oxo-dGMP, thereby preventing misincorporation of 8oxoguanine into DNA. The bacterial and mammalian
enzymes are close in their size and share a highly
conserved region consisting of 23 residues with 14
identical amino acids. Following saturation mutagenesis
of this region, most of these residues proved to be
essential to exert 8-oxo-dGTPase activity. Gene targeting was done to establish MTH1-deficient cell lines and
mice for study. When examined 18 months after birth, a
greater number of tumors were formed in the lungs,
livers, and stomachs of MTH17/7 mice, as compared
with findings in wild-type mice. These proteins protect
genetic information from untoward effects of threats of
endogenous oxygen.
Oncogene (2002) 21, 8895 – 8904. doi:10.1038/sj.onc.
1206023
Keywords: DNA replication fidelity; oxygen radicals;
oxidized guanine base; mutagenesis; carcinogenesis;
genomic stability
Introduction
Reactive oxygen species, such as superoxide, hydrogen
peroxide, hydroxyl radical and singlet oxygen, are
produced through normal cellular metabolism, and
formation of such radicals is further enhanced by
ionizing radiations and by various chemicals (Ames
and Gold, 1991; Henle and Linn, 1997). Nucleic acids
exposed to oxygen radicals generate various modified
bases, and more than 20 different types of oxidatively
altered purines and pyrimidines have been detected
(Gajewski et al., 1990; Demple and Harrison, 1994).
Among them, 8-oxo-7, 8-dihydroguanine (8-oxoguanine) is the most abundant, and seems to play critical
roles in mutagenesis and in carcinogenesis (Kasai and
*Correspondence: M Sekiguchi; E-mail: [email protected]
Nishimura, 1984; Fraga et al., 1990). Unlike other
oxidative DNA damage, such as thymine glycol and 5’,
8-purine cyclodeoxynucleoside (Evans et al., 1993;
Brooks et al., 2000; Kuraoka et al., 2000), 8oxoguanine does not block DNA synthesis, rather it
induces base mispairing. 8-Oxoguanine can pair with
both cytosine and adenine during DNA synthesis, and
this mispairing could contribute significantly to
spontaneous mutations in genomic DNA (Shibutani
et al., 1991; Smith, 1992).
Studies on Escherichia coli mutator mutants revealed
that cells possess elaborate mechanisms that can
prevent mutations caused by oxidation of guanine
residues of DNA. 8-Oxoguanine residues in DNA can
be removed by an enzyme that is coded by the mutM
gene (Cabrera et al., 1988; Chung et al., 1991;
Michaels et al., 1991; Bessho et al., 1992), while
MutY protein removes adenine from adenine : 8oxoguanine pairs, which are produced as a result of
misincorporation of adenine opposite to 8-oxoguanine
(Au et al., 1988, 1989; Nghiem et al., 1988; Michaels et
al., 1992). Thus, two proteins, MutM and MutY, act
consecutively at the site of the oxidized guanine
residue in DNA to prevent the occurrence of specific
transversion mutation (Tchou and Grollman, 1993). In
higher organisms, similar enzyme activities have been
detected, and may account for the rapid elimination of
8-oxoguanine from chromosomal DNA. These include
two types of MutM-related proteins, OGG1 and
AtMMH, which carry 8-oxoguanine-DNA glycosylase
activity (van der Kemp et al., 1996; Nash et al., 1996;
Ohtsubo et al., 1998; Boiteux and Radicella, 1999),
and MYH protein that excises adenine paired with 8oxoguanine in DNA (Yeh et al., 1991; Slupska et al.,
1996).
Oxidation of guanine proceeds also in the cellular
nucleotide pool, and 8-oxo-dGTP, the oxidized form of
dGTP, is the mutagenic substrate for DNA synthesis.
It can be incorporated opposite adenine or cytosine
residues of template DNA, the result being A : T to
C : G and G : C to T : A transversions (Maki and
Sekiguchi, 1992; Cheng et al., 1992). However, in
normally growing cells, the frequency of these types of
mutations remains low, owing to the action of enzymes
degrading such mutagenic substrates (Schaaper et al.,
1986; Mo et al., 1991). The MutT protein of E. coli
hydrolyzes 8-oxo-dGTP to 8-oxo-dGMP, thereby
preventing misincorporation of 8-oxoguanine into
DNA (Maki and Sekiguchi, 1992). A similar enzyme
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activity has been detected in mammalian cells, and the
protein responsible was named MTH1 (Mo et al., 1992;
Sakumi et al., 1993; Furuichi et al., 1994).
It has been proposed that one early step in the
progression of human cancers is elevation of the rate
of spontaneous mutation, that is, acquisition of a
mutator phenotype (Ionov et al., 1993; Jackson and
Loeb, 1998; Stoler et al., 1999). If changes in
spontaneous mutation rates are indeed involved in
carcinogenesis, it is expected that defectiveness in the
MTH1 or related genes would increase the likelihood
of tumor occurrence. To address this question, targeted
disruption of the MTH1 as well as the OGG1 gene has
been done (Klungland et al., 1999; Minowa et al.,
2000; Tsuzuki et al., 2001). These studies are
significantly important in terms of multiple defence
mechanisms against induction and progression of
tumors in animal bodies.
MutT-related error avoidance mechanism for DNA
synthesis
There are at least three steps required to prevent errors
during DNA replication; (1) selection of a nucleotide
complementary to the template by DNA polymerase,
(2) removal of a misincorporated non-complementary
nucleotide by an editing nuclease, associated with
DNA polymerase, and (3) correction of a misincorporated nucleotide by the post-replicational mismatch
repair system (Kornberg and Baker, 1992). Recently,
another process for preventing replication errors was
revealed. This error-avoiding mechanism functions at
the pre-replicational step by degrading a naturally
occurring mutagenic substrate for DNA synthesis
(Maki and Sekiguchi, 1992). Here, MutT protein plays
a major role while MutM and MutY participate in the
process for providing a unique mutational specificity to
the mutT7 mutant (Tajiri et al., 1995).
Among the many mutators found in E. coli, mutT
has drawn particular attention. MutT is one of the first
mutators found in organisms (Treffers et al., 1954) and
specifically induces A : T to C : G transversion mutations (Yanofsky et al., 1966). As a consequence of this
unidirectional mutator activity, mutT7 cells have
increased GC content in the chromosomal DNA
(Cox and Yanofsky, 1967). Akiyama et al. (1987)
cloned the mutT gene and, based on sequence analysis,
identified a protein with 129 amino acid residues. The
MutT protein was purified to physical homogeneity
and was shown to have nucleoside triphosphatase
activity (Bhatnagar and Bessman, 1988). Using an in
vitro DNA synthesis system, Akiyama et al. (1989)
demonstrated that the MutT protein specifically
prevented misincorporation of dGMP onto the
poly(dA)/oligo(dT)20 template-primer. Subsequently
Maki and Sekiguchi (1992) found that the nucleotide
misincorporated opposite the adenine residue of the
template is not dGMP but rather its oxidized form, 8oxo-dGMP. Although the guanine base assumes almost
exclusively anti conformation in DNA, in the case of 8oxoguanine, there exists almost the same quantity of
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anti and syn forms, the former pairing with cytosine
and the latter pairing with adenine. When 8-oxo-dGTP
was added to an in vitro DNA replication system, 8oxo-dGMP was indeed incorporated opposite cytosine
and adenine residues of the template, with almost equal
frequencies. The MutT protein possesses enzyme
activity which hydrolyzes 8-oxo-dGTP to 8-oxodGMP, thereby preventing the misincorporation of 8oxoguanine into DNA.
Here, one must emphasize the importance of eliminating the oxidized form of guanine base from DNA. 8Oxoguanine was first described to be a minor modified
base found in DNA treated with heated glucose (Kasai
and Nishimura, 1984). This modified base was later
revealed to be present in untreated DNA, albeit at levels
no higher than approximately three molecules of 8oxoguanine/106 guanine residues in the chromosomal
DNA (Tajiri et al., 1995). It thus seems that the level of
reactive oxygen species produced by cellular metabolic
intermediates may be sufficient to oxidize the guanine
base of the nucleotide pool as well as that of DNA, even
in normally growing cells.
Spontaneous mutagenesis caused by the oxidation of
guanine nucleotides can be separated into two pathways: one starting from oxidation of guanine in DNA
and the other from oxidation of guanine nucleotide in
the precursor pool (Figure 1). The oxidation of DNA
forms the 8-oxoG : C pair which would induce a
G : C?T : A transversion if the 8-oxoG is not corrected
by MutM (Fpg) protein. When 8-oxoG remains
unrepaired until DNA polymerase arrives at the lesion,
dAMP would be inserted opposite the mutagenic
lesion. However, in most cases, the resulting 8-oxoG : A
pair is reversed back to 8-oxoG : C by the action MutY
protein which removes the adenine base from the 8oxoG : A mispair. These dual defence mechanisms
against mutagenesis were first proposed by Michaels
et al. (1992), based on findings that the combination of
mutM and mutY mutations resulted in synergistic
mutator effects specific for G : C?T : A transversion
and that a multi-copy plasmid carrying the mutM gene
suppressed the mutator effect of the mutY7 mutant.
The high rate of G : C to T : A mutation in the mutM
mutY double mutator strain likely reflects the high level
of 8-oxoG content in the DNA (Tajiri et al., 1995).
While the mutation caused by the oxidation of DNA
is unidirectional, incorporation of 8-oxo-dTP into
DNA would result in base substitutions in two
directions. In E. coli cells, the MutT protein seems to
prevent both A : T to C : G and G : C to T : A
transversions by eliminating 8-oxo-dGTP from the
nucleotide pool. For the spontaneous mutagenesis
caused by 8-oxo-dGTP, MutM and MutY proteins
also have a role, but their functions in the A : T to
C : G pathway differ from those in the G : C to T : A
pathway. The repair enzymes, especially the MutY
protein, promote the fixation of A : T to C : G
transversion whereas the MutM and MutY proteins
cooperate to suppress the G : C to T : A transversion, in
the same manner as they do for oxidative DNA, as
illustrated in Figure 1c.
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M Sekiguchi and T Tsuzuki
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Figure 1 8-Oxoguanine-related mutagenesis. (a) Base substitution mutations, classified into transition and transversion according
to orientations of the base changes. Among two types of transition and four types of transversion mutations, mutT deficiency causes
A : T?C : G transversion while deficiencies in mutM and mutY induce G : C?T : A transversion. (b) Distribution of types of mutations, as measured in the rpsL gene of E. coli (Tajiri et al., 1995). Blue bars represent G : C?T : A transversion and yellow bars
represent A : T?C : G transversion. All six types of base substitutions appearing though the mutation frequency of wild-type cells
is 100 times less than those for mutant strains (Mo et al., 1991). (c) 8-Oxoguanine-related mutagenesis pathways leading to induction of specific types of mutations. The guanine residue in DNA as well as dGTP in the nucleotide pool can be oxidized to 8-oxoguanine. As shown in the left side surrounded by a blue line, 8-oxoguanine produced in DNA induces G : C?T : A transversion,
which is prevented by the actions of MutM and MutY proteins. Incorporation of 8-oxoguanine-containing nucleotide into DNA
could induce A : T?C : G and G : C?T : A transversions, both of which can be prevented by MutT protein, by hydrolyzing 8oxo-dGTP. In mutT7 mutant cells, occurrence of G : C?T : A transversion is specifically prevented by functions of MutM and
MutY proteins, resulting in a preferential occurrence of A : T?C : G transversion, as surrounded by a yellow line
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Mammalian MTH1 protein with 8-oxo-dGTPase
activity
In search of an activity similar to E. coli MutT in
mammalian cells, Jurkat cells (a human T-cell leukemia
cell line) were found to contain a high level of such
activity. Taking advantage of this high level of activity,
the enzyme was purified to apparent physical homogeneity (Mo et al., 1992; Sakumi et al., 1993). Substrate
specificity of the enzyme was examined using a-32Plabeled dNTPs. Although dGTP and dATP were also
hydrolyzed to the corresponding nucleoside monophosphates, product yields were about 5% of those with 8oxo-dGTP. Neither TTP nor dCTP was hydrolyzed by
the enzyme. The apparent Km for hydrolysis of 8-oxodGTP was 70 times lower than that for the degradation
of dGTP, whereas the maximal reaction rates observed
with both substrates were similar.
Based on the partial amino acid sequence determined
with the purified human 8-oxo-dGTPase protein, a
cDNA for human enzyme was cloned and the
nucleotide sequence determined (Sakumi et al., 1993).
The molecular mass of the protein, as calculated from
the predicted amino acid sequence, was 17.9 kDa, a
value close to that estimated from analysis of SDS –
PAGE. By placing the cDNA under control of the lac
promoter, a high level of 8-oxo-dGTPase activity was
induced in the presence of isopropyl-1-thio-b-Dgalactopyranoside. On expression of cDNA in E. coli
mutT7 cells the increased level of spontaneous
mutation frequency was considerably reduced. Similar
but more striking suppressive effects were observed
when mouse or rat cDNA was expressed in the mutT7
cells (Kakuma et al., 1995; Cai et al., 1995). Thus,
mammalian 8-oxo-dGTPase functions in E. coli cells to
prevent mutations caused by the accumulation of 8oxo-dGTP in the nucleotide pool.
The mammalian gene for 8-oxo-dGTPase has been
named MTH1 for mutT homolog 1. Transfection of
human MTH1 cDNA brought about a significant
reduction in 8-oxoguanine content of DNA in mouse
embryonic fibroblasts as well as in tumor cells, with or
without H2O2 treatment (Colussi et al., 2002). As MTH1
protein decreases both steady-state and oxidant-induced
8-oxoguanine levels in DNA, endogenous oxidation of
the deoxynucleotide pool is a definite source of DNA
damage and the deoxynucleotide pool is a significant
target for exogenous oxidative damage.
To elucidate structure of the gene, human genomic
libraries were screened using cDNA as a probe
(Furuichi et al., 1994). The gene which spans
approximately 8 kb consists of five exons, and the
coding sequence resides on exons 2, 3, 4 and 5 (Oda et
al., 1997, 1999). As will be described below, some of
the exons are composed of differentially processed
segments. Using fragments of genomic DNA as probes,
chromosal assignment of the gene was made. These
fragments derived from different regions, consistently
hybridized to a single locus on the short arm of
chromosome 7 at p22. Thus, the MTH1 gene is located
on human chromosome 7p22 (Furuichi et al., 1994).
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Northern blot analysis revealed that MTH1 mRNA
is present in almost all tissues of adult mouse, among
which expression levels are exceedingly high in thymus
and testis (Oda et al., 1997). Fetal tissues (brain and
liver) contained large amounts of MTH1 mRNA. In
peripheral blood lymphocytes, the level of MTH1
mRNA was significantly increased after concomitant
treatment with phytohemagglutinin and interleukin-2.
Analyses of the 5’ regions of the MTH1 transcripts
revealed that seven types of MTH1 mRNA, which may
be produced by transcription initiation at different sites
and/or alternative splicing (Figure 2). In most or all of
human cells and tissues, type 1 mRNA which lacks the
exon 2 sequence is present as a major form. More than
one ATG initiation codons in-frame were found in the
5’ regions of some of the MTH1 mRNAs and,
moreover, there is a polymorphic alteration at the 5’
splicing site located in exon 2. Thus, regulation of
expression of the human MTH1 gene is complex and
Figure 2 Genomic structure and alternative splicing of the human MTH1 gene (Oda et al., 1997). In the upper part, the overall
structure of the gene is shown, in which exons 1 to 5 are indicated
by filled boxes. In the lower part, seven types of MTH1 mRNA
are shown, which can be produced by transcription initiation at
different sites and alternative splicing. Hatched boxes represent
exons
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may be subjected to cell type-specific modification (Oda
et al., 1999).
In eukaryotic cells, a pool of dNTP for nuclear
DNA replication is present mainly in the cytosol
(Bestwick et al., 1982). Mitochondria, which preserve
a pool of dNTP for mitochondrial DNA synthesis,
consist of more than 10% of the total intracellular
dNTP. The mitochondrial respiratory chain located on
inner membranes is a major site for the initiation of
lipid peroxidation, which can lead to oxidation of the
guanine to 8-oxoguanine. In addition, the mitochondrial respiratory chain produces superoxide, which can
be converted to hydroxyl radicals via hydrogen
peroxide. The hydroxyl radical is the main species of
reactive oxygen that attacks the guanine base. Thus,
DNA and dNTP in the mitochondrial pool may be
exposed to a greater oxidative stress than is the case in
the nucleus. In this context, the intracellular location of
8-oxo-dGTPase was determined (Kang et al., 1995). In
human Jurkat cells, most of the enzyme activity was
found in cytosolic and the mitochondrial fractions. The
specific activity of 8-oxo-dGTPase in the mitochondrial
fraction was about 17% of that in the cytosolic
fraction; almost no enzyme activity was found in
nuclear and microsomal fractions. Electron microscopic immunocytochemistry, using a specific
antibody against MTH1 protein, showed that MTH1
protein was localized in the mitochondrial matrix. An
identical molecular form of MTH1 protein appears to
be present in the cytosol and in the mitochondria.
Structural features of MTH1-related proteins
Human MTH1 and E. coli MutT proteins are similar
in size and there is a certain degree of sequence
homology in these proteins. Genes for analogous
functions were isolated from Proteus vulgaris and
Streptococcus pneumoniae, bacteria distantly related to
E. coli (Kamath and Yanofsky, 1993; Bullions et al.,
1994). The products of the latter two genes carry
enzyme activity specifically degrading dGTP to dGMP
and are structurally and functionally related to the E.
coli MutT protein. Most of the identical residues are in
a region corresponding to the 23 residues from Gly37
to Gly59 of E. coli MutT, known as the MutT
signature (Bessman et al., 1996). Homologs of human
MTH1 protein were identified in mice and rats, based
on isolated cDNAs (Kakuma et al., 1995; Cai et al.,
1995). Both proteins comprise 156 amino acid residues,
as was the case for the human MTH1 protein, and
amino acid sequences are highly conserved.
Alignment of the sequences of these six proteins shows
that all carry a highly conserved sequence in nearly the
same region, corresponding to amino acids 36 to 58 for
human MTH1 (Figure 3a). Ten of 23 amino acid residues
in this region are identical, hence this probably
constitutes an active center for the enzyme.
The secondary structure of MutT protein has been
determined by heteronuclear multi-dimensional NMR
(Abeygunawardana et al., 1993, 1995; Weber et al.,
1993, Frick et al., 1995) and the conserved region was
found to consist of a loop – helix – loop motif which
may contribute to nucleotide binding and nucleotide
pyrophosphohydrolase activity. NMR studies were
further extended to determine the roles of conserved
amino acid residues in the possible active site of the
MutT protein, in conjunction with site-directed
mutagenesis analyses (Lin et al., 1996, 1997). Recently,
NMR analysis of human MTH1 protein has been
made (Mishima et al., unpublished result). Structure of
MutT protein, both free and Mn-associated forms, was
resolved by X-ray crystallographic analysis (Yamagata
et al., unpublished result).
The 23-residue sequence is a sole conserved sequence
among all MutT and MTH1 homologs with 8-oxodGTPase, and of the many other proteins with the
MutT signature so far identified, some hydrolysing
various nucleotide derivatives, such as dATP, diadenosine oligophosphates, NADH, ADP-ribose, and
GDP-mannose (O’Handley et al., 1996, 1998; Sheikh
et al., 1998). Furthermore, a diphosphoinositol polyphosphate phosphohydrolase that hydrolyzes a nonrelated polyphosphate also contains the 23-residue
(Safrany et al., 1998). A chimeric protein MTH1-Ec,
in which the 23-residue sequence of MTH1 was
replaced with that of MutT, retains its capacity to
hydrolyze 8-oxo-dGTP (Fujii et al., 1999), thereby
indicating that the 23-residue sequences of MTH1 and
MutT are functionally and structurally equivalent and
constitute functional modules (Figure 3b).
Among these 23 residues in the putative active site
region, 10 amino acids are conserved through bacteria
to human. To establish the functional significance of
these residues, saturation mutagenesis of the 10
conserved residues of MutT and MTH1 proteins were
done, in combination with negative and positive
mutant screening, using mutagenesis primers that
contain a completely degenerated codon for each
residue (Figure 3c). Shimokawa et al. (2000) found
that Gly37, Gly38, Glu44, Arg52, Glu53, and Glu57 in
MutT protein could not be replaced by any other
residue without losing activity.
Replacement of the corresponding residues of MTH1
protein also caused loss of function (Fujii et al., 1999).
In the latter case two additional residues were found to
be essential, though there is the possibility that
formation of the human protein in heterologous cell
(E. coli) might cause a more severe effect on expression
of the function. It seems that these essential amino acid
residues interact with the substrate nucleotide and
Mg++ to exert catalytic functions.
Tumorigenesis and mutagenesis in mice lacking MTH1
To obtain insight into the role of MTH1 protein in
terms of spontaneous tumorigenesis as well as
mutagenesis caused by the oxygen-induced DNA
damage, it is necessary to establish cell and mouse
lines defective in the MTH1 gene. This was achieved
recently using gene targeting techniques.
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M Sekiguchi and T Tsuzuki
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Figure 3 Comparison of structures of MutT family proteins. (a) The 23-residue modules of MutT-related proteins from various
organisms, as shown by hatched boxes. Conserved amino acid residues are indicated by bold letters. (b) Secondary structures of
E. coli MutT, human MTH1 and a chimeric protein MTH1-Ec. In the chimeric MTH1 protein, the 23-residue module is replaced
with that of E. coli MutT protein. (c) Targeted mutagenesis analyses of E. coli MutT and human MTH1 protein. Amino acid residues conserved through the six MutT-related proteins are indicated by bold letters and those that could not be replaced by any
other residue without losing function are placed in the yellow boxes. The data were taken from Fujii et al. (1999) and Shimokawa et
al. (2000)
The mouse MTH1 gene is composed of five exons
and spans about 10 kb (Igarashi et al., 1997). Using
the isogenic genomic DNA fragment, the entire area of
the third exon containing the initiation codon and the
adjacent intron regions were replaced with a neo
cassette (Tsuzuki et al., 2001). The resulting construct
was electroporated into ES cells, and cells showing
resistance to both G418 and ganciclovir were selected
to obtain MTH1+/7 cells. MTH17/7 cells were
generated from the MTH1+/7 cells by growing in the
presence of 1.5 mg/ml of G418, a concentration which
is six times higher than that used for the isolation of
MTH1+/7 cells.
MTH17/7 cells thus obtained were used to examine
the effect of MTH1 deficiency on spontaneous
mutagenesis. Mutations in the Hprt gene, located on
the X chromosome in the mouse genome, render cells
resistance to 6-thioguanine and, in this forward mutation assay, two independently isolated MTH17/7 cell
lines exhibited an approximately twofold higher
mutation rate, compared with the value of MTH1+/+
cells. Thus, MTH1 may have the potential to prevent
the occurrence of mutations under normal growth
conditions.
It should be noted, however, that degree of increase
in spontaneous mutation frequency, due to the loss of
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MutT-related functions, differs considerably in mouse
and E. coli cells. Increases in Hprt mutations detected
in mouse MTH17/7 cells is twofold compared with
that in MTH1+/+ cells, whereas the value of mutation
frequency for E. coli mutT7 cells is 100 times over that
in wild-type cells (Tajiri et al., 1995). Several
hypotheses to explain this difference may be considered, among which the most plausible is that
mammalian cells may possess an enzyme(s) capable
of degrading 8-oxo-dGTP in addition to MTH1. This
notion was strengthened by the recent finding that even
E. coli, despite its predominant MutT activity,
possesses an additional enzyme activity that degrades
8-oxo-dGTP. GTP cyclohydrase II, encoded by the
ribA gene of E. coli, can hydrolyze 8-oxo-dGTP to the
corresponding nucleoside monophosphate. In the
mutT7 background, ribA7 cells showed two times
higher spontaneous mutation frequencies as compared
with ribA+ cells (Kobayashi et al., 1998). It is unlikely
that a GTP cyclohydrase II-type enzyme is present in
mammalian cells because the biosynthesis of riboflavin,
in which this type of enzyme functions, does not take
place in animals. Recently, a mouse cDNA clone that
considerably suppresses the high mutability of E. coli
mutT7 cells was isolated (Cai et al., unpublished
result). The protein encoded by this cDNA carried
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M Sekiguchi and T Tsuzuki
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activity to degrade 8-oxo-dGTP, and could act as an
MTH1 redundancy factor.
MTH1 homozygous mutant mice were generated by
microinjecting MTH1+/7 ES cell clones into blastocysts and subsequently crossing the resulting chimera
and heterozygous mice. MTH17/7 mice have a normal
physical appearance, but have a high susceptibility for
spontaneous tumorigenesis (Tsuzuki et al., 2001).
Around 18 months after birth, many tumors were
found in lungs and livers of MTH17/7 mice, but there
were few in MTH1+/+ mice. The elevated incidence of
tumor formation in the liver of MTH17/7 mice
correlated well with the highest content of MTH1
protein in this organ of the wild-type mouse (Kakuma
et al., 1995). As generally observed in spontaneous and
carcinogen-induced hepatocarcinogenesis in rodents
(Poole and Drinkwater, 1996), there is a high
susceptibility of male mice to liver tumorigenic events,
compared with their female counterparts. It has been
suggested that the hormonal environment of the host
affects the development of many types of tumors,
especially those in the liver. More tumors tend to form
in the stomach of MTH17/7 mice, as compared with
MTH1+/+ mice, although the statistical significance is
weak. Indeed, as there are different profiles of
antioxidant enzyme, such as superoxide dismutases
and catalases, different susceptibility to tumorigenesis
among organs may reflect the metabolic balance of
oxidative stress, inducing lesions in DNA as well as in
the nucleotide pool. Altogether, it can be concluded
that the intracellular level of MTH1 protein is an
important factor in determining susceptibility of mice
to tumor induction by endogenous oxidative damage.
To examine in vivo mutation events due to the
MTH1-deficiency, a reporter gene, rpsL of E. coli, was
introduced into MTH17/7 mice (Egashira et al., 2002).
Interestingly, the net frequency of rpsL7 forward
mutants showed no apparent increase in MTH17/7
mice as compared to findings in MTH1+/+ mice.
However, there are differences between these two
genotypes in class- and site-distributions of the rpsL7
mutations recovered from the mice. Unlike MutTdeficient E. coli, an increase in frequency of
A : T?C : G transversion was not evident in MTH1
nullizygous mice. Nevertheless, the frequency of singlebase frameshifts at mononucleotide runs was 5.7-fold
higher in spleens of MTH17/7 mice than in those of
wild-type mice. Since the elevated incidence of singlebase frameshifts at mononucleotide runs is a hallmark
of the defect in MSH2-dependent mismatch repair
system, this weak site-specific mutator effect of
MTH17/7 mice could be attributed to a partial
sequestration of the mismatch repair function that
may act to correct mispairs with the oxidized
nucleotides.
Studies of OGG1-defective mice would provide much
more insight into the matter. OGG17/7 mice accumulate abnormally high levels of 8-oxoguanine in their
genomes and exhibit a moderately elevated spontaneous mutation rates in their tissues (Klungland et al.,
1999; Minowa et al., 2000). Nevertheless, there was no
sign of development of malignancy during about 12
months after birth. Alternative mechanisms might
function to minimize the effects of an increased load
of 8-oxoguanine in mammalian genomes.
Metabolism of oxidized nucleotides
Figure 4 summarizes enzymatic reactions for interconversion of 8-oxoguanine-containing nucleotides in
mammalian cells. Enzymatic conversion of ribonucleotides to deoxyribonucleotides occurs at the level of
nucleoside diphosphate, and ribonucleotide reductase,
the enzyme responsible, has a relatively broad substrate
specificity. Four types of naturally occurring ribonucleotides, ADP, GDP, CDP and UDP, are converted
to the corresponding deoxyribonucleotides by a single
species of reductase enzyme (Thelander and Reichard,
1979). However, this enzyme is inactive on the 8oxoguanine-containing nucleotide, as revealed with
mouse ribonucleotide reductase (Hayakawa et al.,
1999). This implies that 8-oxoguanine-containing
deoxyribonucleotides must be generated in the cellular
deoxyribonucleotide pool.
Human cells contain nucleoside diphosphate kinase,
an enzyme activity which phosphorylates various
nucleoside diphosphates to the corresponding nucleoside triphosphates (Kornberg and Baker, 1992). This
enzyme can convert 8-oxo-dGDP to 8-oxo-dGTP,
although the rate of phosphorylation of 8-oxo-dGDP
was one-third that of dGDP (Hayakawa et al., 1995).
Thus, in addition to direct oxidation of dGTP, 8-oxodGTP may be generated by the phosphorylation of 8oxo-dGDP.
Once 8-oxo-dGTP is produced, this can be incorporated into DNA. Various DNA polymerases from
eukaryotes and prokaryotes have the potential to
utilize 8-oxo-dGTP as substrate (Cheng et al., 1992;
Maki and Sekiguchi, 1992; Pavlov et al., 1994; Minnick
et al., 1994). Thus, the action of 8-oxo-dGTPase is a
pre-requisite for high fidelity of DNA replication.
8-Oxo-dGMP, produced by the action of 8-oxodGTPase, cannot be re-phosphorylated by cellular
enzymes. Human guanylate kinase, which phosphorylates both GMP and dGMP to the corresponding
nucleoside diphosphates, is totally inactive for 8-oxodGMP (Hayakawa et al., 1995). This would provide
another basis for excluding this mutagenic substrate
from the DNA precursor.
8-Oxo-dGMP is dephosphorylated to yield the
corresponding
nucleoside,
8-oxodeoxyguanosine.
Nucleosides are readily transported through the cell
membrane, and extracellular nucleosides can be
excreted into the urine. Dephosphorylation of 8-oxodGMP, therefore, may be an essential step for
excretion of 8-oxoguanine-containing compounds. The
enzyme that catalyzes this reaction, 8-oxo-dGMPase,
was partially purified from an extract of human Jurkat
cells, and the mode of action was elucidated
(Hayakawa et al., 1995). 8-Oxo-dGMP is the preferred
substrate of the enzyme, and other nucleoside monoOncogene
Oxidative nucleotide damage
M Sekiguchi and T Tsuzuki
8902
Figure 4 Interconversion of 8-oxoguanine-containing nucleotides in mammalian cells. This scheme is based on the results of Hayakawa et al. (1995, 1999). (1) Ribonucleotide reductase; (2) nucleoside diphosphate kinase; (3) 8-oxo-dGTPase (MTH1); (4) guanylate
kinase; (5) DNA polymerase; (6) 8-oxo-dGMPase. O. denotes oxidative reaction
phosphates are cleaved albeit at significantly lower
rates.
As discussed above, MTH1 and MutT are key
enzymes for sustaining high fidelity of DNA replication, in oxygenated stated of living systems. These
enzymes of diverged origins share a common character
to degrade 8-oxo-dGTP to 8-oxo-dGMP, thereby
eliminating the mutagenic substrate from the DNA
precursor pool. In spite of this common feature, they
exhibit certain significant differences. First, MTH1 and
MutT differ in the capacity to degrade 8-oxoguaninecontaining ribonucleotides. E. coli MutT protein
cleaves 8-oxoGTP as efficiently as does 8-oxo-dGTP
(Taddei et al., 1997). On the other hand, human
MTH1 is hardly capable of hydrolyzing 8-oxoGTP; its
potential to degrade 8-oxoGTP is only 2% that for 8oxo-dGTP (Hayakawa et al., 1999). These apparent
differences in efficiency of degrading an error-evoking
substrate may be counterbalanced by the capacity of
RNA polymerases to discriminate the unfavorable
substrate. E. coli RNA polymerase can utilize 8oxoGTP as substrate at a rate 10% that of GTP
whereas mammalian RNA polymerase II incorporates
little of 8-oxoguanine into RNA (Hayakawa et al.,
1999).
The second point is that the mammalian enzyme has
a broader substrate specificity as compared with the
bacterial enzyme. MTH1 hydrolyzes 8-oxo-dATP, 2hydroxy-dATP and 2-hydroxyATP as well as 8-oxodGTP (Sakai et al., 2002). This is in contrast with
MutT, which acts on 8-oxoguanine-containing nucleotides alone. Recognition of 8-oxo-dGTP and 2hydroxy-dATP appears to depend on different residues
of MTH1 protein. Transgenic mice expressing engineered MTH1 proteins lacking each one of the
activities would be useful to reveal the biological
significance of these two activities.
Acknowledgements
We thank Drs H Hayakawa, H Maki, Y Nakabeppu, Y
Yamagata, Y Nakatsu and H Shimokawa for discussion
and helpful advice, and M Ohara for useful comments on
the manuscript.
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