From the Department of Environmental Oncology, the
¶ Department of Health Policy and Management, and the
§ Second Department of Surgery, University of Occupational
and Environmental Health, 1-1 Iseigaoka, Yahatanishi-ku,
Kitakyushu 807-8555, Japan
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ABSTRACT |
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We have developed a new strategy for the
evaluation of the mutagenicity of a damaged DNA precursor
(deoxyribonucleoside 5'-triphosphate) in Escherichia coli.
8-Hydroxydeoxyguanosine triphosphate (8-OH-dGTP) and
2-hydroxydeoxyadenosine triphosphate (2-OH-dATP) were chosen for this
study because they appear to be formed abundantly by reactive oxygen
species in cells. We introduced the oxidatively damaged nucleotides
into competent E. coli and selected mutants of the
chromosomal lacI gene. Both damaged nucleotides induced lacI gene mutations in a dose-dependent manner,
whereas unmodified dATP and dGTP did not appear to elicit the
mutations. The addition of 50 nmol of 8-OH-dGTP and 2-OH-dATP into an
E. coli suspension induced 12- and 9-fold more substitution
mutations than the spontaneous event, respectively. The 8-OH-dGTP
induced A·T C·G transversions, and the 2-OH-dATP elicited G·C
T·A transversions. These results indicate that the two
oxidatively damaged nucleotides are mutagenic in vivo and
suggest that 8-OH-dGTP and 2-OH-dATP were incorporated opposite A and G
residues, respectively, in the E. coli DNA. This new method
enables the evaluation and comparison of the mutagenic potentials of
damaged DNA precursors in vivo.
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INTRODUCTION |
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Mutations, alterations of genetic information, are believed to
cause various diseases and are generated by many environmental mutagens
such as ROS,1 ultraviolet,
X-, and -rays, and alkylating agents, in addition to mispair
formation during replication. Among them, ROS are believed to be very
important sources of mutations and to be involved in mutagenesis,
carcinogenesis, and aging (1, 2) because ROS are generated endogenously
by normal oxygen metabolism and are also produced by many environmental
mutagens and carcinogens.
Among the forms of oxidative DNA damage reported, 8-OH-Gua (3) is recognized as an important lesion because of its mutagenicity (4-9). This modified base is used widely as a marker of DNA oxidation (3, 10) because of its sensitive detection by an HPLC system connected to an electrochemical detector. An oxidized form of adenine, 2-OH-Ade, is produced by Fenton-type reactions of deoxyadenosine derivatives (11, 12). The yields of 2-OH-Ade are similar to those of 8-OH-Gua in the monomeric form, although its formation in DNA is less efficient. It was reported that the treatment of cultured human cells with H2O2 induces 2-OH-Ade accumulation in DNA (one-fifth of that of 8-OH-Gua) (13). Moreover, 2-OH-Ade possesses mutation inducibility similar to that of 8-OH-Gua in Escherichia coli and mammalian cells (14, 15). Thus, 2-OH-Ade appears to be another important form of DNA damage produced by ROS.
An oxidative DNA lesion is likely to be formed through two pathways. One is the direct oxidation of a residue in DNA, and another is the incorporation of an oxidatively damaged DNA precursor by a DNA polymerase(s). In fact, it was shown that the two pathways contribute almost equally to the formation of 8-OH-Gua in DNA (16). Moreover, the presence of the MutT protein and its mammalian counterpart, which hydrolyze the mutagenic nucleotide 8-OH-dGTP, indicates that the prevention of its incorporation into DNA is important in organisms (17, 18). Thus, it is necessary to determine the mutagenicity of an oxidatively damaged DNA precursor in cells to reveal the overall effects on the mutations induced by ROS.
However, few studies on the mutagenic potential of an oxidatively
damaged DNA precursor have been reported. 8-OH-dGTP is used as a
substrate by DNA polymerases and is inserted opposite C and A in the
template DNA (5, 17). We reported the insertion of 2-OH-dAMP opposite T
and C by the mammalian DNA polymerase (11). The incorporation of
5-hydroxydeoxycytidine 5'-triphosphate and 5-formyldeoxyuridine
5'-triphosphate opposite A and G has also been reported (19, 20).
Minnick et al. (21) used 8-OH-dGTP as a substrate in SV40
origin-dependent DNA replication with the use of a human
cell extract (21). All of these studies were conducted in
vitro, and to our knowledge, the mutagenicity of a damaged
nucleotide in vivo has never been reported.
In this study we introduced two important oxidatively damaged purine
nucleotides, 8-OH-dGTP and 2-OH-dATP (Fig.
1), into E. coli and analyzed
the induction of mutations in the lacI gene on the
chromosomal DNA. 8-OH-dGTP and 2-OH-dATP were similarly mutagenic
in vivo and elicited A·T C·G and G·C
T·A
transversions, respectively. This new method enables the evaluation and
comparison of the mutagenic potentials of damaged DNA precursors
in vivo.
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EXPERIMENTAL PROCEDURES |
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Materials--
All chemicals for the E. coli media
were as described (22-24). The E. coli strains W3110 (wild
type, F) and DH5
(F
,
80d lacZ
M15
(lacZYA-argF)
U169,endA1,recA1,hsdS17
(rk
mk+),deoR,
thi-1, supE44,
,
gyrA96, relA1) were used. Recombinant
Taq DNA polymerase was purchased from Nacalai Tesque Inc.
The dATP used for the preparation of 2-OH-dATP was from Sigma.
Nucleotides for control treatments were from Amersham Pharmacia
Biotech. Digoxygenin-labeled oligonucleotides and other unmodified
oligonucleotides were from Nissinbo (Tokyo, Japan) and from Hokkaido
System Science Co. (Sapporo, Japan), respectively, in purified
forms.
Preparation of Damaged Nucleotides-- 8-OH-dGTP was prepared as described previously (5). 2-OH-dATP was prepared by the treatment of dATP with Fe(II)-EDTA-O2 and was purified by HPLC as described (11).
Introduction of Nucleotides into E. coli W3110 Cells and
Selection of lacI Mutants--
Selection of
lacI
mutants was carried out by the method of
Miller (25). A white colony (the lacI+ genotype)
of W3110 was taken from an 5-bromo-4-chloro-3-indolyl
-D-galactopyranoside minimum plate and was inoculated
into Luria-Bertani (LB) medium. The E. coli culture was
incubated at 37 °C for 2 h, and competent cells were prepared
by treatment with 0.1 M calcium chloride as described (23,
26). To 195 µl of the E. coli suspension, 5 µl of
nucleotide solution was added, and the mixture was placed on ice for 30 min. After heat shock treatment (42 °C for 2 min and then 0 °C
for 2 min), 800 µl of LB medium was added, and the cells were
incubated at 37 °C for 45 min. A portion of the culture was diluted,
transferred onto an LB agar plate, and incubated at 37 °C overnight.
Another portion of the culture was transferred onto a P-gal plate and
was incubated at 37 °C for 3 days. The colonies that grew on the
P-gal plate contain a mutation in either the lacI or
lacO gene and were scored as mutants (25). The mutation frequency (MF) was calculated according to the numbers of colonies on
the P-gal and LB plates. The lacI
mutants were
selected from the mixture of lacI
and
lacOc mutants on the P-gal plates as described (25).
The isolated mutants were inoculated into 0.5 ml of LB medium and were
incubated at 37 °C overnight.
Analysis of Mutations--
The chromosomal DNA was isolated from
the E. coli cells containing the mutated
lacI gene with a SepaGene kit (Sanko Junyaku).
The DNA fragment containing the lacI
gene was
amplified by polymerase chain reaction as described previously (22,
23).
Measurement of 8-OH-Gua Content in Chromosomal DNA in E. coli-- The E. coli W3110 treated with 8-OH-dGTP as described above was incubated at 37 °C for an additional 60 min in LB medium. After centrifugation, the E. coli pellet was washed with ice-cold LB medium to remove unincorporated 8-OH-dGTP. Control E. coli was treated similarly. The chromosomal DNAs were extracted from 8-OH-dGTP-treated and control bacteria using a DNA Extractor WB Kit (Wako Pure Chemicals). The 8-OH-Gua content was measured by the HPLC-electrochemical detector method after complete digestion as described (10).
Transformation of E. coli DH5 Cells in the Presence of
Deoxynucleotide--
Competent DH5
cells were prepared as described
(26). To 100 µl of the E. coli suspension, 5 µl of the
solution containing 1 ng (0.29 fmol) of pMY189 (27) and 25 nmol of a
nucleotide was added. The transformation was carried out by the
standard method (26).
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RESULTS |
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Induction of Mutations in the Chromosomal lacI Gene by Damaged DNA
Precursors--
We treated wild type E. coli W3110 with
various deoxyribonucleotides, and the frequencies of
lacI and lacOc mutations in
the chromosomal DNA were measured. When 50 nmol of either dGTP or dATP
was added to the bacteria, the MF observed was very similar to that of
the control (no addition of nucleotide) (Table
I). On the other hand, the MF was found
to be increased when 50 nmol of either 8-OH-dGTP or 2-OH-dATP was
added. The relative MFs were increased about 2.5-fold over the control
value by the addition of 50 nmol of either 8-OH-dGTP or 2-OH-dATP
(Table I). 2-OH-dATP appeared to induce more mutations than 8-OH-dGTP.
Moreover, the increases in the MF were dependent on the amount of the
nucleotides added (Table I). These results indicate that the two
oxidatively damaged nucleotides were mutagenic in vivo, as
speculated from the results of in vitro experiments (11,
17). No decrease in the survival ratio (number of colonies on LB
plates) was observed by the addition of the damaged nucleotides under
the conditions used (data not shown and see below).
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Analysis of Mutations--
We analyzed the sequences of the
lacI gene in the lacI mutants
obtained by the treatments with 50 nmol of nucleotide (89 and 86 cases
for 8-OH-dGTP and 2-OH-dATP, respectively). 61 mutants obtained with
the control experiments were also analyzed. Table II shows the summary of the sequence
analyses. The addition or deletion of the 5'-TGGC-3' sequence was
detected in 84% of the mutants in the control experiments. This type
of mutation has been reported as the most frequent mutation in the
lacI gene (22, 23, 28, 29). 15% of the mutants contained a
single base substitution.
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Damaged Nucleotides Get into Bacteria-- Competent W3110 cells were treated with 33P-labeled 8-OH-dGTP or 2-OH-dATP, and radioactivities in the treated cells were counted after thorough washing and lysis. We observed that 1.3% and 0.3% of the added 8-OH-dGTP and 2-OH-dATP, respectively, were present in cells. Thus, the damaged nucleotides entered E. coli cells. Moreover, these results indicate that 8-OH-dGTP was incorporated into the cells 4.5-fold more than 2-OH-dATP.
We next measured 8-OH-Gua content in chromosomal DNA of 8-OH-dGTP-treated bacteria. 8-OH-Gua was detected by the HPLC-electrochemical detector, a method with high sensitivity. We found that the 8-OH-Gua level of the treated cells was 1.58/105 Gua. We observed that the 8-OH-Gua level of the E. coli treated without 8-OH-dGTP was 0.57/105 Gua. Thus, 8-OH-Gua level of 1.01/105 Gua over the background was induced by the treatment with 8-OH-dGTP, suggesting that the added damaged nucleotide entered bacterial cells and was incorporated by DNA polymerase III. Note that in these experiments, we incubated the bacteria for an additional 60 min (compared with 45 min in mutagenesis experiments) because of the amount of DNA required. We speculate that the ratio of 8-OH-Gua may be decreased by cell division and repair processes during this incubation period. Thus, the 8-OH-Gua content was estimated to be higher than 1.58/105 Gua when the bacteria were transferred onto LB and P-gal plates in mutagenesis experiments.Cytotoxic Effects of Damaged Nucleotides--
In the experiments
with W3110, the numbers of E. coli colonies on the LB plates
were very similar in all cases (data not shown). Thus, the addition of
an oxidatively damaged nucleotide did not appear to decrease the
survival ratio under the conditions used. This observation may be the
result of a low efficiency of the nucleotide incorporation into the
cells. To know whether the incorporation of an oxidatively damaged
nucleotide was cytotoxic, 2-OH-dATP or 8-OH-dGTP was introduced into
E. coli together with a plasmid containing a selection
marker gene, and the survival ratio was measured. We used competent
E. coli DH5 cells, which are frequently employed for
transfection experiments, and a plasmid containing
-lactamase gene
(pMY189) (27). Competent DH5
cells were treated with pMY189 together
with 108 excess amount (25 nmol) of the damaged nucleotide.
We counted the number of transformants on an agar plate containing
ampicillin as an indicator of the cytotoxicity.
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DISCUSSION |
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8-OH-dGTP and 2-OH-dATP, two major forms of oxidatively damaged
DNA precursors, induced lacI and
lacOc mutations with nearly equal frequencies (Table
I). Taken together with the mutation spectra data (Table II), we
estimated that the frequencies of single base substitution mutations
were increased by 12- and 9-fold with 8-OH-dGTP and 2-OH-dATP,
respectively (see "Results"). Because 8-OH-dGTP got into E. coli 4.5-fold more than 2-OH-dATP, the actual mutagenicity of
substitutions of 2-OH-dATP was calculated to be about 3-fold that of
8-OH-dGTP. These results imply that the mutagenic potential of
2-OH-dATP is as important as that of 8-OH-dGTP, when present in the
nucleotide pool. Since the formation of the 2-OH-Ade base in monomers
by ROS is comparable with that of 8-OH-Gua (11, 12), the production of
2-OH-dATP appears to be similar to that of 8-OH-dGTP. Thus, 2-OH-dATP
and 8-OH-dGTP may contribute nearly equally to mutagenesis pathways in
which the oxidation of DNA precursors is involved.
8-OH-dGTP induced A·T C·G transversions almost exclusively
(Table II). This finding can be interpreted as follows. DNA polymerase III incorporated 8-OH-dGMP opposite A residues in the DNA, and the
polymerase inserted C opposite the 8-OH-Gua bases during the next round
of replication (Fig. 3). Similar results
have been obtained by the combination of in vitro DNA
synthesis and transfection of the synthesized DNA (5, 21). Thus, the
new method appears to be effective for the investigation of the
mutational properties of a damaged DNA precursor in
vivo.
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2-OH-dATP induced G·C T·A transversions in the lacI
gene (Table II). This finding implies that either 2-OH-dAMP was
incorporated opposite G, and then dTMP was inserted opposite the
incorporated 2-OH-Ade residue during the next round of replication
(Fig. 3), or 2-OH-dAMP was incorporated opposite C, and then dAMP was
inserted opposite the incorporated 2-OH-Ade residue during the next
round of replication. Because 2-OH-Ade residues in single-stranded
plasmid vectors are "read" as A with more than 99% probability
(14), the former explanation is most likely. Thus it is probable that the DNA polymerase III of E. coli incorporated 2-OH-dAMP
opposite the G residues in the DNA. This conclusion is in contrast to
our previous finding that the mammalian DNA polymerase
, another replicative DNA polymerase, inserts 2-OH-dAMP opposite the C residues in DNA (11). However, a 2-OH-Ade·G pair is formed when the Klenow fragment of E. coli DNA polymerase I inserts dGMP opposite
2-OH-Ade during in vitro DNA synthesis (30, 31). Moreover,
an A
C transversion is induced by 2-OH-Ade in plasmid vectors in
E. coli (14), whereas this type of mutation is not elicited
in simian cells (15). Thus, prokaryotic DNA polymerases may
characteristically form the 2-OH-Ade·G pair.
The reasons why the mispairing properties of 2-OH-Ade are dependent upon the DNA polymerases are not clear. One possibility is the difference in hydrophobicities of the active site of each polymerase. 2-OH-Ade forms two tautomers, the 2-hydroxy (enol) and 1,2-dihydro-2-oxo (keto) isomers (see Fig. 1). This equilibrium may be affected by the microenvironment around the base (32, 33). Thus, the difference in the hydrophobicity of the active site may affect the enol-keto equilibrium. This putative shift would have an important effect on the formation of a base pair involving 2-OH-Ade (11, 31).
We transfected competent E. coli DH5 cells with a plasmid
containing
-lactamase gene in the presence of 8-OH-dGTP or
2-OH-dATP. The number of transformants on an agar plate containing
ampicillin was less than that of the transformants obtained with the
plasmid alone (Table V). This effect was not due to the inhibition of transformation by the presence of dNTP and/or the increase in ionic
strength because the normal nucleotides did not have any effect (Table
V). These results indicate that either damaged nucleotide, 8-OH-dGTP or
2-OH-dATP, was cytotoxic to cells upon incorporation. These effects may
be the result of mutations in the
-lactamase gene on the plasmid
and/or in other essential gene(s) on the chromosome. Alternatively,
extension reaction from an 8-OH-Gua or 2-OH-Ade residue at the 3'-end
of a primer may block DNA replication. The other possibility is the
replication block during translesional synthesis past these residues in
the templates. This possibility is unlikely for 2-OH-Ade because the oxidized adenine in double-stranded DNA does not induce the replication block (14).
It is very important that 2-OH-dATP elicited the G·C T·A
transversion, which is one of the mutations frequently induced by ROS.
To date, this mutation is thought to be mediated by the formation of
8-OH-Gua in the DNA. However, the formation of 2-OH-dATP in the
nucleotide pool will elicit this kind of mutation in bacteria, as shown
in this study (Table II). In addition, the G·C
T·A transversion
occurs by a lipid peroxidation system without an increase in the
formation of 8-OH-Gua in DNA (34). Thus, the G·C
T·A
transversion and other mutations appear to occur by a variety of
pathways and not just by a single DNA lesion.
The MutT protein hydrolyzes 8-OH-dGTP to produce the cognate monophosphate (17). This function prevents mutations by the damaged nucleotide. Thus, in mutT strains, 8-OH-dGTP will induce more mutations than observed in this study. It is possible that a MutT-like activity may eliminate 2-OH-dATP, which was as mutagenic as 8-OH-dGTP (Table I). Further studies will be necessary to determine the involvement of the putative MutT-like activity in the prevention of mutations induced by various oxidized deoxynucleoside triphosphates.
One of our major objectives was to establish a new method to evaluate the mutagenicity of a damaged DNA precursor (deoxynucleoside 5'-triphosphate) in vivo. To our knowledge, the mispairing properties of an oxidatively damaged DNA precursor have been evaluated by in vitro DNA polymerase reactions and by mutagenesis experiments using vector DNA in which the oxidized precursor is incorporated by in vitro DNA synthesis (5, 11, 17, 19-21). It should be emphasized that the MutT-like activity is a factor in the determination of the mutagenicity of damaged nucleotides in the in vivo method. Thus, our new approach will effectively complement the in vitro method. One may think that this new method resembles experiments in which modified nucleosides are added to the culture medium (35). In this type of experiment, a modified nucleoside must be converted to the cognate triphosphate prior to the incorporation into DNA. The total efficiency of the kination reactions is very different, depending upon the structure of the modified nucleoside. However, our approach eliminates the effects of differences in the enzymatic kination reaction efficiency and directly evaluates the mutagenicity of damaged nucleoside triphosphates.
Another major objective was to investigate the mutational properties of 2-OH-dATP, which is produced by ROS. We demonstrated that 2-OH-dATP was as mutagenic as 8-OH-dGTP (Table I). Moreover, 8-OH-Gua and 2-OH-Ade in double-stranded DNA induce mutations with similar frequencies in E. coli and mammalian cells (5-8, 14, 15). These results indicate that 2-OH-Ade is an important oxidative lesion. Furthermore, 2-OH-Ade in DNA appears to be repaired less efficiently in cells.2 Thus, even if the extent of 2-OH-Ade formation in DNA is low, the 2-OH-Ade might be accumulated in DNA more easily than 8-OH-Gua. 2-OH-Ade might play an important role in the mutagenesis pathways of aging and carcinogenesis.
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FOOTNOTES |
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* This work was supported in part by a grant-in-aid for scientific research on priority areas from the Ministry of Education, Science, Sports, and Culture of Japan.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.
To whom correspondence should be addressed. Tel.:
81-93-691-7468; Fax: 81-601-2199; E-mail: h-kasai{at}med.uoeh-u.ac.jp or
hirokam{at}med.uoeh-u.ac.jp.
1
The abbreviations used are: ROS, reactive oxygen
species; 8-OH-Gua, 8-hydroxyguanine; HPLC, high performance liquid
chromatography; 2-OH-Ade, 2-hydroxyadenine; 8-OH-dGTP,
8-hydroxy-2'-deoxyguanosine 5'-triphosphate; 2-OH-dAMP,
2-hydroxy-2'-deoxyadenosine 5'-monophosphate; 2-OH-dATP,
2-hydroxy-2'-deoxyadenosine 5'-triphosphate; LB, Luria-Bertani; P-gal,
phenyl--D-galactopyranoside; MF, mutation frequency;
8-OH-dGMP, 8-hydroxy-2'-deoxyguanosine 5'-monophosphate.
2 Y. Tsurudome, T. Hirano, H. Kamiya, R. Yamaguchi, S. Asami, H. Itoh, and H. Kasai, unpublished data.
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REFERENCES |
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