Overexpression of Ogg1 in mammalian cells: effects on induced and spontaneous oxidative DNA damage and mutagenesis
Stephan Hollenbach,
Andreia Dhénaut1,
Inge Eckert,
J. Pablo Radicella1 and
Bernd Epe2
Institute of Pharmacy, University of Mainz, D-55099 Mainz, Germany and
1 CEA, UMR217 CNRS/CEA, Département de Radiobiologie et Radiopathologie, F-92265 Fontenay-aux-Roses, France
 |
Abstract
|
---|
Chinese hamster ovary cell lines (AA8 and AS52) were stably transfected to overexpress hOgg1 protein, the human DNA repair glycosylase for 7,8-dihydro-8-oxoguanine (8-oxoG). In the transfectants, the repair rate of 8-oxoG residues induced by either potassium bromate or the photosensitizer [R]-1-[(10-chloro-4-oxo-3-phenyl-4H-benzo[a]quinolizin-1-yl)-carbonyl]-2-pyrrolidinemethanolplus light was up to 3-fold more rapid than in the parental cells. However, the improved repair had little effect on the mutagenicity of potassium bromate in the guanine phosphoribosyl transferase (gpt) locus of the OGG1-transfected AS52 cells. The steady-state (background) levels of DNA base modifications sensitive to Fpg protein, which include 8-oxoG, in cells not exposed to a damaging agent were not reduced by the overexpression of Ogg1 protein. Moreover, the spontaneous mutation rates in the gpt locus were similar in OGG1-transformed and vector-only-transformed cells. The results demonstrate the potential of Ogg1 protein to remove its substrate modifications from most of the chromosomal DNA. They indicate, on the other hand, that the Ogg1 protein alone may not be rate limiting for the repair of the residual substrate modifications observed in cells under normal growth conditions.
Abbreviations: gpt, guanine phosphoribosyl transferase; 8-oxoG, 7,8-dihydro-8-guanine; Ro19-8022, [R]-1-[(10-chloro-4-oxo-3-phenyl-4H-benzo[a]quinolizin-1-yl)-carbonyl]-2-pyrrolidinemethanol.
 |
Introduction
|
---|
7,8-Dihydro-8-oxoguanine (8-oxoG) is an oxidative DNA modification that has been shown to be pre-mutagenic, giving rise predominantly to GC
TA transversions (13). 8-OxoG is generated in high yields in the reaction of apparently all oxidants with DNA. Relatively high concentrations of 8-oxoG have also been observed in the nuclear and mitochondrial DNA of all types of untreated cells, although the absolute values of these background levels are still controversial (4). The 8-oxoG levels in the DNA of untreated cells are assumed to reflect the balance (steady-state) between a continuous generation of 8-oxoGprobably by reactive oxygen species formed in the cellular oxygen metabolismand its removal, which is predominantly or exclusively accomplished by specific base excision repair (58).
A gene encoding a human repair glycosylase for 8-oxoG, hOGG1, has recently been cloned (915). Similar to most enzymes that initiate base excision repair, the hOgg1 protein has an associated endonuclease activity, generating DNA single-strand breaks at substrate modifications. The hOgg1 protein has relatively high sequence homology to the corresponding yeast enzyme, but is not structurally related to the functionally analogous enzyme in bacteria, the Fpg protein. Defects of the yOGG1 gene in yeast and of the fpg gene in Escherichia coli have been shown previously to increase the spontaneous mutation frequencies in these species, generating pronounced mutator phenotypes (16,17). If hOGG1 would be similarly important for the genetic stability in human cells, the hOGG1 expression is expected to influence the risk of malignant transformation in a way analogous to that observed for other genes involved in the repair of DNA, e.g. hMSH2 and hMLH1 (18,19). Indeed, mutations in the hOGG1 gene have been found in human lung and kidney tumours, in support of the assumption that an Ogg1 deficiency may predispose to malignant changes (20).
Here we describe the overexpression of hOGG1 in mammalian cells. We report on the effects of the overexpression on the repair and mutagenicity of DNA damage in cells exposed to oxidants, on the one hand, and on the steady-state (background) levels of oxidative DNA modifications and the spontaneous mutation rates in untreated cells, on the other.
 |
Materials and methods
|
---|
Cells, repair endonucleases and chemicals
AS52 chinese hamster ovary cells, which carry the bacterial guanine phosphoribosyl transferase (gpt) gene for analysis of mutations (21), were obtained from W.J.Caspary (Research Triangle Park, USA) and cultured in Ham's F12 medium with 5% fetal calf serum, supplemented with penicillin (100 U/ml) and streptomycin (100 µg/ml). In the case of transfected cells, 200 µg/ml geneticin sulfate (G418) was added to the medium. Formamidopyrimidine-DNA glycosylase (Fpg protein) from E.coli was prepared as described previously (22).
[R]-1-[(10-chloro-4-oxo-3-phenyl-4H-benzo[a]quinolizin-1-yl)-carbonyl]-2-pyrrolidinemethanol (Ro19-8022) was a gift from HoffmannLaRoche AG (Basel, Switzerland). Most other chemicals and enzymes were purchased from Sigma (Deisenhofen, Germany).
Transfection of hOGG1 into AS52 and AA8 cells
The open reading frame for the hOGG1 gene was amplified by PCR from the cDNA using the primers 5'-CGGAATTCATGCCTGCCCGCGCGCTTC and 5'-GCCCAAGCTTCCATCTAGCCTTCCGGCCC.
The PCR products were digested with EcoRI/HindIII and purified by agarose gel electrophoresis. The 1 kb resulting DNA fragment was then ligated into the EcoRI/HindIII restriction site of vector pcDNA3.1() (Invitrogen, Carlsbad, CA). This construct, pPR65, allows the constitutive expression of hOGG1 driven by the cytomegalovirus promoter in mammalian cells.
A total of 3x105 cells in 5 ml culture medium were plated in culture dishes (60 mm). After incubation for 16 h, 1 µg pPR65 or pcDNA3.1() (vector-only control) dissolved in 97 µl culture medium plus 3 µl FuGene6 transfection reagent (Boehringer Mannheim, Mannheim, Germany) was added to the cells and the incubation continued for another 24 h. Subsequently, aliquots of 1% of the cells were plated in culture dishes (100 mm). Geneticin sulfate (G-41; final concentration 800 µg/ml) was added after 48 h. Single clones were isolated after 10 days, propagated in 24 well plates and analysed for OGG1 expression as described below.
Analysis of transfectants for hOGG1 expression and enzyme activity
A total of 5x106 cells were suspended in 0.35 ml lysis buffer (20 mM TrisHCl pH 8.0, 1 mM EDTA, 250 mM NaCl, 0.8 µg /ml antipain, 0.8 mg/ml leupeptin, 0.8 µg/ml aprotinin). The cell suspension was sonicated during 8 s at 4°C with pulses of 1 s each leaving 10 s intervals. After centrifugation at 4°C for 45 min at 85 000 g the supernatant was recovered, its protein content was determined (Bradford) and aliquots were used for the detection of hOgg1 by western blot or for enzymatic activity measurements. For western blot analysis, 50 µg protein was separated by SDSPAGE and transferred to a nitrocellulose membrane. To detect hOgg1, a polyclonal rabbit antibody against the whole protein was used (C.Dhérin, J.P.Radicella and S.Boiteux, unpublished data). Fapy glycosylase activity was determined using [3H]Fapy-poly(dGdC) as described (22). The 8-OxoG repair activity was determined on 34mer oligonucleotides containing a single 8-oxoG at position 16, which were synthesized as described previously (23). The sequence used in this study was: 5'-GGCTTCATCGTTATT(8-oxoG)ATGACCTGGTGGATACCG-3'.
The 34mer oligonucleotide containing the modified base was labelled at its 5' end using [
32P]ATP and T4 polynucleotide kinase. The 32P-labelled strand was hybridized with the complementary oligonucleotide carrying a cytosine opposite 8-oxoG by incubation at 90°C for 10 min followed by slow cooling to room temperature. The assay mixtures (14 ml final volume) contained 50 fmol of 32P-labelled DNA duplex and cell extracts (3 µg of protein) in NTE buffer (70 mM NaCl, 2 mM Na2EDTA, 25 mM TrisHCl pH 7.6). The reactions were performed at 37°C for 30 min and the products were separated by 20% denaturing PAGE in the presence of 7 M urea.
Treatment of AS52 and AA8 cells with oxidants
Cells were exposed to various concentrations of KBrO3 in Ca2+- and Mg2+-free PBS (140 mM NaCl, 3 mM KCl, 8 mM Na2HPO4, 1 mM KH2PO4 pH 7.4) at 37°C (106 cells/ml) for 5 min (to measure repair kinetics) or 15 min (for the mutation assay). The exposure of the cells to the photosensitizer Ro19-8022 in the presence of visible light from a 1000 W halogen lamp (Philips PF811) at a distance of 38 cm was carried out in Ca2+- and Mg2+-free PBS on ice. Illumination for 10 min corresponded to 166 kJ/m2 between 400 and 800 nm, equivalent to 14 kJ/m2 between 400 and 500 nm. To remove the damaging agents, the treated cells were washed three times.
Quantification of endonuclease-sensitive modifications by alkaline elution
The alkaline elution protocol followed the method of Kohn et al. (24) with modifications (25,26). The sum of modifications sensitive to Fpg protein and single-strand breaks was obtained from experiments, in which the cellular DNA was incubated for 60 min at 37°C with Fpg protein (1 µg/ml) immediately after cell lysis. Under these conditions, the incision by the enzyme at endonuclease-sensitive modifications has been shown to be saturated (26). The numbers of modifications incised by the repair endonuclease were obtained by subtraction of the number of single-strand breaks observed in experiments without endonuclease treatment. Elution curves obtained with
-irradiated cells were used for calibration, assuming that 6 Gy generate one single-strand break per 106 bp. When induced modifications (rather than background levels) were to be quantified, the slopes observed with untreated control cells were subtracted.
Quantification of induced mutation frequencies
The AS52 cell-line and its transfectants were cultured in cleansing medium (containing 11 µg/ml thymidine, 219 µg/ml xanthine, 22 µg/ml adenine, 1.2 µg/ml aminopterin and 8.8 µg/ml mycophenolic acid) for 1 week to eliminate spontaneous gpt mutants. A sample of 0.5x106 cells were incubated in recovery medium (containing 1.2 µg/ml thymidine, 11.5 µg/ml xanthine, 3 µg/ml adenine) for 48 h. Cells were exposed to KBrO3 or Ro19-8022 plus light as described above and subsequently cultured in full medium for 1 week (expression time). A total of 2x105 cells were diluted in 10 ml culture medium and plated in tissue culture dishes (100 mm). After 2 h, 6-thioguanine was added to each plate (final concentration 2.5 µg/ml) for selection. To determine the cloning efficiencies, 200 cells in 5 ml culture medium were plated in culture dishes (60 mm). After incubation for 7 to 9 days, the medium was replaced by NaCl solution (0.9%, w/v), the cell colonies were fixed with methanol (20°C) for 15 min and stained with Giemsa (10% in H2O) for 15 min. The subsequent quantification of 6-thioguanine-resistant cells and the determination of cytotoxicity (ratio of the plating efficiencies of treated and untreated cells directly after exposure to KBrO3 or Ro19-8022 plus light) was carried out according to the protocol of Tindall et al. (27).
Quantification of spontaneous mutation rates
The determination of spontanous mutation rates was carried out as described by Glaab and Tindall (28). AS52 cells were cleared of gpt mutants by culture in cleansing medium (see above) and subsequently kept in regular medium under exponential growth conditions. When cells were replated at various times, the number of cell divisions after the removal of the cleansing medium was determined by cell counting and correction for the plating efficiency (0.89 ± 0.07), and aliquots of the cells (1x106) were analysed for the fraction of 6-thioguanine-resistant cells as described above.
 |
Results
|
---|
Overexpression of OGG1 in mammalian cells
An expression vector carrying the open reading frame of hOGG1 was transfected by means of a lipid-based reagent into AA8 and AS52 Chinese hamster ovary cells. The latter cell line carries the bacterial gpt gene for mutation analysis. Stably transfected clones were selected by their geneticin resistance. Protein extracts of several clones were analysed for the expression of hOGG1 by western blots and for the endonuclease and glycosylase activity of hOgg1 protein (cleavage of an 8-oxoG-containing oligomer, release of modified bases from poly(dGdC) treated with methyl methanesulfonate plus alkali). Compared with the wild-type cells, the enzyme activity was ~10-fold higher in clones AS52-ogg12 (Figure 1
) and AA8-ogg119. Several other clones had intermediate glycosylase activities.

View larger version (26K):
[in this window]
[in a new window]
|
Fig. 1. Expression of hOGG1 in transfected and parental AS52 cells. Total protein extracts were analysed for (i) hOgg1 protein by western blotting (upper panel); (ii) cleavage of a 32P-labelled oligonucleotide containing a single 8-oxoG-C base pair by gel electrophoresis and subsequent autoradiography (central panel); and (iii) the release of radioactive bases (Fapy lesions) from poly(dGdC) modified by [3H]methyl methanesulfonate and alkali (lower panel).
|
|
Repair of base modifications induced by KBrO3 and Ro19-8022 plus light
To induce oxidative DNA damage, the OGG1-transfected and parental cells were exposed to KBrO3 or the photosensitizer Ro19-8022 plus light. Both agents were shown previously to generate cellular DNA damage profiles in which base modifications recognized by Fpg protein, the functional analogue of Ogg1 in bacteria, are the prevailing lesions (26,29,30). For both oxidants, a high percentage of the Fpg-sensitive modifications (70 ± 11% in the case of bromate and 60 ± 5% in the case of Ro19-8022) was identified as 8-oxoG by means of HPLC with electrochemical detection (31). Other types of DNA modification such as strand breaks, sites of base loss and oxidative pyrimidine modifications are generated by KBrO3 and photoexcited Ro19-8022 in relatively low yields.
For the determination of the repair kinetics, the numbers of Fpg-sensitive modifications in the cellular DNA were determined by means of the alkaline elution technique (25,26) at various time points after exposure to the oxidants. The damaging conditions were chosen to generate ~0.3 Fpg-sensitive modifications per 106 bp. The induction of this low level of DNA damage was not associated with cytotoxicity, i.e. it had no effects on the cloning efficiency of the cells. The results (Figures 2 and 3
) indicate that the OGG1-transfected AS52 and AA8 cells repaired the Fpg-sensitive base modifications induced by KBrO3 or photosensitization much more rapidly than the parental cells. The repair rates in the various clones correlated well with the degree of overexpression estimated from the western blots and enzyme activity measurements in the cellular extracts (data not shown). It is noteworthy that repair rates in the parental AS52 cells were slightly, but significantly, lower than in the AA8 cells.

View larger version (19K):
[in this window]
[in a new window]
|
Fig. 2. Upper panel: repair of Fpg-sensitive DNA modifications induced by 0.02 µM Ro19-8022 plus light (14 kJ/m2 between 400 and 500 nm) at 0°C in OGG1-transfected (clone AS52-ogg12) and parental AS52 cells. The numbers of lesions detected at various times after the treatment were corrected for the number of lesions in untreated control cells. The number of induced Fpg-sensitive modifications (100%) was ~0.3/106 bp for both cell lines. Data represent the means ± SD of two to three independent experiments. Lower panel: repair after 2 h in parental AS52 and AA8 cells and various OGG1-transfected clones. The degree of hOGG1 expression observed in the cellular extracts is indicated above the columns.
|
|

View larger version (17K):
[in this window]
[in a new window]
|
Fig. 3. Upper panel: repair of Fpg-sensitive DNA modifications induced by 20 mM KBrO3 (5 min) in parental and OGG1-transfected AS52 cells (clone AS52-ogg12). The numbers of lesions detected at various times after the treatment were corrected for the number of lesions in untreated control cells. The number of induced Fpg-sensitive modifications (100%) was ~0.35/106 bp for both cell lines. Data represent the means ± SD of two to three independent experiments. Lower panel: repair after 3 h in parental AS52 and AA8 cells and various OGG1-transfected clones. The degree of hOGG1 expression observed in the cellular extracts is indicated above the columns.
|
|
Mutation frequencies induced by photoexcited Ro19-8022 and KBrO3
In order to test whether the more rapid repair of 8-oxoG in the OGG1-transfected cells results in a reduced mutagenicity of oxidants, we determined the mutation frequency in the gpt locus of wild-type and OGG1-transfected AS52 cells exposed to photoexcited Ro19-8022 and KBrO3.
In the case of Ro19-8022 plus light, cytotoxicity prevented the detection of pronounced mutagenicity (Figure 4
). Thus, exposure conditions that induced approximately eight Fpg-sensitive modifications per 106 bp reduced the cloning efficiency to 1%. The mutation frequency in the wild-type AS52 cells was only 2-fold higher than the spontaneous mutation frequency under these conditions, but indeed appeared to be even lower in the OGG1-overexpressing cells (Figure 4
, upper panel).

View larger version (14K):
[in this window]
[in a new window]
|
Fig. 4. Induction of gpt mutants (upper panel) and cytotoxicity (lower panel) in parental and OGG1-transfected AS52 cells by various concentrations of Ro19-8022 plus light (14 kJ/m2 between 400 and 500 nm). Cytotoxicity data were taken from ref. 32.
|
|
KBrO3 proved to be much more mutagenic than Ro19-8022 plus light at moderately toxic concentrations (Figure 5
). However, the mutation frequencies in the OGG1-overexpressing AS52 cells were only slightly lower than in the wild-type cells. Similarly, the cytotoxicity of KBrO3 was the same in both strains (Figure 5
, lower panel).

View larger version (16K):
[in this window]
[in a new window]
|
Fig. 5. Induction of gpt mutants (upper panel) and cytotoxicity (lower panel) in parental and OGG1-transfected AS52 cells by various concentrations of KBrO3 (15 min at 37°C). The data points represent the means ± SD of six independent experiments.
|
|
Steady-state levels of Fpg-sensitive modifications
The steady-state (background) levels of Fpg-sensitive modifications determined in untreated OGG1-overexpressing and parental AS52 and AA8 cells are shown in Figure 6
. No significant differences were observed between the various strains.

View larger version (26K):
[in this window]
[in a new window]
|
Fig. 6. Steady-state (background) levels of DNA modifications recognized by Fpg protein (1 µg/ml) in various parental and OGG1-tranfected AS52 and AA8 cells.
|
|
Spontaneous mutation rates
The numbers of spontaneous gpt mutants observed in AS52 cells and several tranformant clones at various times (cell generations) after an incubation in a cleansing medium, which selects for an intact gpt activity and thus eliminates pre-existing mutants, are shown in Figure 7
. Linear increases of the mutation frequencies (constant mutation rates) were observed for all cell clones over ~70 generations (~8 weeks). The spontaneous mutation rates calculated from these data are given in Table I
. The mutation rates in the two OGG1-overexpressing clones AS52-ogg12 and AS52-ogg13 were significantly lower than in the parental AS52 cells. However, reduced mutation rates were also observed in a vector-only transfected AS52. A similarly low mutation rate was also observed in another clone that did not significantly express hOGG1 (data not shown). It has to be concluded that the OGG1 overexpression has no signficant influence on the spontanous generation of mutation in the gpt locus of AS52 cells cultured under normal conditions.
The presence of geneticin, which selects for transfected cells, had no influence on the mutation rates (data not shown). The doubling time of the transfectants (18.3 ± 2.2 h for clone AS52-ogg12) was not significantly different from that of the parental AS52 cells (18.9 ± 2.5 h).
 |
Discussion
|
---|
The results described above demonstrate that the human 8-oxoG DNA glycosylase Ogg1 can be overexpressed in mammalian cells. The overexpression results in a several-fold higher enzyme activity in cellular extracts (Figure 1
) and correspondingly to an up to 3-fold more rapid cellular repair of oxidative base modifications sensitive to Fpg protein, the bacterial functional analogue of Ogg1 protein (Figures 2 and 3
). Similar effects were observed for Fpg-sensitive modifications induced by KBrO3, a rodent carcinogen which requires activation by glutathione and possibly generates DNA damage via bromine radicals or related species (29), and Ro19-8022, a relatively polar photosensitizer which induces DNA damage at least in part via singlet oxgen (32). Repair rates observed in the parental AS52 and AA8 cells are similar to values reported previously for the removal of 8-oxoG or Fpg-sensitive modifications in several types of rodent and human cells (26,3336). The observation that a 10-fold higher enzyme activity (Figure 1
) is associated with an only 3-fold higher repair rate (Figures 2 and 3
) might be an indication that the cellular repair involves additional components that become rate-limiting at high OGG1 protein concentrations.
Since the repair kinetics observed in the overexpressing cells are not biphasic (Figures 2 and 3
), all or most of the induced Fpg-sensitive modifications are also substrates of the hOgg1 protein. This is consistent with previous observations that the Fpg-sensitive modifications generated by both KBrO3 and photoexcited Ro19-8022 are predominantly 8-oxoG residues (29,31,32).
The effect of the OGG1 overexpression on the mutagenicity of the oxidants could not be demonstrated unambiguously. The cytotoxicity of photoexcited Ro19-8022, most probably caused by damage to other cellular targets than DNA, did not allow determination of mutation frequencies for concentrations that induced more than eight Fpg-sensitive base modifications per 106 bp. Due to the low number of mutations induced under these conditions (Figure 4
), the difference between wild-type and OGG1-overexpressing AS52 cells was not significant. The low mutation frequency in the gpt gene at this level of DNA damage is, however, consistent with the relatively low mutation frequencies (<2%) observed when DNA containing single 8-oxoG residues was replicated in mammalian cells (37,38). KBrO3 generated a much higher mutation frequency than Ro19-8022 plus light, but the difference between the parental and OGG1-overexpressing cells was only marginal (Figure 5
). It is likely that the mutagenicity of KBrO3 at high concentrations (>20 mM) is not caused by Fpg-sensitive modifications. The assumption is supported by a non-linear dose response (Figure 5
), which is an indication that the damaging mechanism and therefore the types and ratios of DNA modifications at the highly mutagenic concentrations may be different from those observed previously at low KBrO3 concentrations (29). The findings suggest that 8-oxoG is also not the DNA modification responsible for the carcinogenicity of KBrO3 in rodents.
Despite the much higher repair capacity for Fpg-sensitive modifications, the steady-state (background) levels of these modifications were unchanged in all transfected clones (Figure 6
). The result is an indication that the activity of Ogg1 protein in the cells is not rate-limiting for the removal of those substrate modifications that are present in untreated cells, possibly because these are not accessible to the enzyme. A more trivial explanation for the finding would be that the background levels detected by Fpg protein result from artifactual oxidation during cell lysis or unspecific incisions by the enzyme. This possibility cannot be completely excluded, although there are several indications that the observed steady-state levels are indeed correct, as summarized previously (26,31). In particular, an artifactual oxidation by hydroxyl radicals (in the absence of repair) would generate a damage profile different from that observed in the untreated cells (30), and the differences observed between the steady-state levels in various cell types or between proliferating and arrested cells are difficult to reconcile with a significant number of unspecific incisions.
The overexpression of Ogg1 protein did not reduce the spontaneous mutation rate observed in the gpt locus of AS52 cells, either because the steady-state levels of the Ogg1-sensitive modifications were unchanged as suggested by the results described above or because the substrate modifications of Ogg1 protein do not contribute significantly to the overall spontaneous mutation rate in this locus. Surprisingly, the mutation rates in all tranfectants, including the vector-only transfected cells, were lower than in the parental AS52 cells. A putative explanation is that the selection for stable transfection also selects against genetically unstable (hypermutable) cells.
In conclusion, the data presented here indicate that an overexpression of hOgg1 protein alone has no effect on the levels of spontaneous oxidative DNA damage and mutation rates, although it accelerates the repair of substrate modifications induced by exogenous oxidants. Experiments with knock-out mice will show whether defects of OGG1 enhance the steady-state levels of oxidative DNA damage and the spontaneous mutation rates and predispose to cancer development.
 |
Acknowledgments
|
---|
The authors would like to thank Dr Serge Boiteux for his support and fruitful discussions. This work was supported by the Deutsche Forschungsgemeinschaft (SFB 519), the Commissariat à l'Energie Atomique (CEA) and by a grant from the Association pour la Recherche sur le Cancer (ARC no. 9200).
 |
Notes
|
---|
2 To whom correspondence should be addressed Email: epe{at}mail.uni-mainz.de 
 |
References
|
---|
-
Wood,M.L., Dizdaroglu,M., Gajewski,E. and Essigmann,J.M. (1990) Mechanistic studies of ionizing radiation and oxidative mutagenesis: genetic effects of a single 8-hydroxyguanine (7-hydro-8-oxoguanine) residue inserted at a unique site in a viral genome. Biochemistry, 29, 70247032.[ISI][Medline]
-
Cheng,K.C., Cahill,D.S., Kasai,H., Nishimura,S. and Loeb,L.A. (1992) 8-Hydroxyguanine, an abundant form of oxidative DNA damage, causes G
T and A
C substitutions. J. Biol. Chem., 267, 166172.[Abstract/Free Full Text]
-
Moriya,M. (1993) Single-stranded shuttle phagemid for mutagenesis studies in mammalian cells: 8-oxoguanine in DNA induces targeted GC
TA transversions in simian kidney cells. Proc. Natl Acad. Sci. USA, 90, 11221126.[Abstract]
-
Collins,A.R., Cadet,J., Epe,B. and Gedik,C. (1997) Problems in the measurement of 8-oxoguanine in human DNA. Report of a workshop, DNA oxidation, held in Aberdeen, UK, 1921 January, 1997. Carcinogenesis, 18, 18331836.[Abstract]
-
Demple,B. and Harrison,L. (1994) Repair of oxidative damage to DNA: enzymology and biology. Annu. Rev. Biochem., 63, 915948.[ISI][Medline]
-
Wood,R.D. (1996) DNA repair in eukaryotes. Annu. Rev. Biochem., 65, 135167.[ISI][Medline]
-
Cunningham,R.P. (1997) DNA glycosylases. Mutat. Res., 383, 189196.[ISI][Medline]
-
Boiteux,S. and Radicella,J.P. (1998) Excision repair of 8-oxoguanine in eukaryotes: the OGG1 proteins. In Dizdaroglu,M. and Karakaya,A. (eds) Advances in DNA Damage and Repair. Plenum Press, New York, NY, pp. 3545.
-
Arai,K., Morishita,K., Shinmura,K., Kohno,T., Kim,S.R., Nohmi,T., Taniwaki,M., Ohwada,S. and Yokota,J. (1997) Cloning of a human homolog of the yeast OGG1 gene that is involved in the repair of oxidative DNA damage. Oncogene, 14, 28572861.[ISI][Medline]
-
Aburatani,H., Hippo,Y., Ishida,T., Takashima,R., Matsuba,C., Kodama,T., Takao,M., Yasui,A., Yamamoto,K., Asano,M., Fukasawa,K. et al. (1997) Cloning and characterization of mammalian 8-hydroxyguanine-specific DNA glycosylase/apurinic, apyrimidinic lyase, a functional mutM homologue. Cancer Res., 57, 21512156.[Abstract]
-
Bjoras,M., Luna,L., Johnsen,B., Hoff,E., Haug,T., Rognes,T. and Seeberg,E. (1997) Opposite base-dependent reactions of a human base excision repair enzyme on DNA containing 7,8-dihydro-8-oxoguanine and abasic sites. EMBO J., 16, 63146322.[Abstract/Free Full Text]
-
Lu,R., Nash,H.M. and Verdine,G.L. (1997) A mammalian DNA repair enzyme that excises oxidatively damaged guanine maps to a locus frequently lost in lung cancer. Curr. Biol., 7, 397407.[ISI][Medline]
-
Radicella,J.P., Dherin,C., Desmaze,C., Fox,M.S. and Boiteux,S. (1997) Cloning and characterization of hOGG1, a human homolog of the OGG1 gene of Saccharomyces cerevisiae. Proc. Natl Acad. Sci. USA, 94, 80108015.[Abstract/Free Full Text]
-
Roldan-Arjona,T., Wei,Y.F., Carter,K.C., Klungland,A., Anselmino,C., Wang,R.P., Augustus,M. and Lindahl,T. (1997) Molecular cloning and functional expression of a human cDNA encoding the antimutator enzyme 8-hydroxyguanine-DNA glycosylase. Proc. Natl Acad. Sci. USA, 94, 80168020.[Abstract/Free Full Text]
-
Rosenquist,T.A., Zharkov,D.O. and Grollman,A.P. (1997) Cloning and characterization of a mammalian 8-oxoguanine DNA glycosylase. Proc. Natl Acad. Sci. USA, 94, 74297434.[Abstract/Free Full Text]
-
Michaels,M.L., Cruz,C., Grollman,A.P. and Miller,J.H. (1992) Evidence that MutY and MutM combine to prevent mutations by an oxidative damaged form of guanine. Proc. Natl Acad. Sci. USA, 89, 70227025.[Abstract]
-
Thomas,D., Scot,A.D., Barbey,R., Padula,M. and Boiteux,S. (1997) Inactivation of OGG1 increases the incidence of G:C
T:A transversions in Saccharomyces cerevisiae: evidence for endogenous oxidative damage to DNA in eukaryotic cells. Mol. Gen. Genet., 254, 171178.[ISI][Medline]
-
Fishel,R., Lescoe,M.K., Rao,M.R.S., Copeland,N.G., Jenkins,N.A., Garber,J., Kane,M. and Kolodner,R. (1993) The human mutator homolog MSH2 and its association with hereditary nonpolyposis colon cancer. Cell, 75, 10271038.[ISI][Medline]
-
Modrich,P. (1994) Mismatch repair, genetic stability and cancer. Science, 266, 19591960.[ISI][Medline]
-
Chevillard,S., Radicella,J.P., Levalois,C., Lebeau,J., Poupon,M.-F., Oudard,S., Dutrillaux,B. and Boiteux,S. (1998) Mutations in OGG1, a gene involved in the repair of oxidative DNA damage, are found in human lung and kidney tumours. Oncogene, 16, 30833086.[ISI][Medline]
-
Tindall,K.R. and Stankowski,L.F.Jr (1989) Molecular analysis of spontaneous mutations at the gpt locus in Chinese hamster ovary (AS52) cells. Mutat. Res., 220, 241253.[ISI][Medline]
-
Boiteux,S., O'Connor,T.R., Lederer,F., Gouyette,A. and Laval,J. (1990) Homogeneous Escherichia coli Fpg protein. J. Biol. Chem., 265, 39163922.[Abstract/Free Full Text]
-
Girard,P.M., D'Ham,C., Cadet,J. and Boiteux,S. (1998) Opposite base-dependent excision of 7,8-dihydro-8-oxoadenine by the Ogg1 protein of Saccharomyces cerevisiae. Carcinogenesis, 19, 12991305.[Abstract]
-
Kohn,K.W., Erickson,L.C., Ewig,R.A.G. and Friedman,C.A. (1976) Fractionation of DNA from mammalian cells by alkaline elution. Biochemistry, 15, 46294637.[ISI][Medline]
-
Epe,B. and Hegler,J. (1994) Oxidative DNA damage: endonuclease fingerprinting. Methods Enzymol., 234, 122131.[ISI][Medline]
-
Pflaum,M., Will,O. and Epe,B. (1997) Determination of steady-state levels of oxidative DNA base modifications in mammalian cells by means of repair endonucleases. Carcinogenesis, 18, 22252231.[Abstract]
-
Tindall,K.R., Stankowski,L.F.Jr, Machanoff,R. and Hsie,A.W. (1986) Analysis of mutation in pSV2 gpt-transformed CHO cells. Mutat. Res., 160, 121131.[ISI][Medline]
-
Glaab,W.E. and Tindall,K.R. (1997) Mutation rate at the hprt locus in human cancer cell lines with specific mismatch repair-gene defects. Carcinogenesis, 18, 18.[Abstract]
-
Ballmaier,D. and Epe,B. (1995) Oxidative DNA damage induced by potassium bromate under cell-free conditions and in mammalian cells. Carcinogenesis, 16, 335342.[Abstract]
-
Epe,B. (1995) DNA damage profiles induced by oxidizing agents. Rev. Physiol. Biochem. Pharmacol., 127, 223249.[ISI]
-
Pflaum,M., Will,O., Mahler,H.C. and Epe,B. (1998) DNA oxidation products determined with repair endonucleases in mammalian cells: types, basal levels and influence of cell proliferation. Free Rad. Res., 29, 585594.[ISI][Medline]
-
Will,O., Gocke,E., Eckert,I., Schulz,I., Pflaum,M., Mahler,H.C. and Epe,B. (1999) Oxidative DNA damage and mutations induced by a polar photosensitizer, Ro19-8022. Mutat. Res., in press.
-
Kasai,H., Crain,P.F., Kuchino,Y., Nishimura,S., Ootsuyama,A. and Tanooka,H. (1986) Formation of 8-hydroxyguanine moiety in cellular DNA by agents producing oxygen radicals and evidence for its repair. Carcinogenesis, 7, 18491851.[Abstract]
-
Jaruga,P. and Dizdaroglu,M. (1996) Repair of products of oxidative DNA damage in human cells. Nucleic Acids Res., 24, 13891394.[Abstract/Free Full Text]
-
Dally,H. and Hartwig,A. (1997) Induction and repair of oxidative DNA damage by nickel(II) and cadmium(II) in mammalian cells. Carcinogenesis, 18, 10211026.[Abstract]
-
Will,O., Schindler,D., Boiteux,S. and Epe,B. (1998) Fanconi's anaemia cells have normal steady-state levels and repair of oxidative DNA base modifications sensitive to Fpg protein. Mutat. Res., 409, 6572.[ISI][Medline]
-
Klein,J.C., Bleeker,M.J., Saris,C.P., Roelen,H.C.P.F., Brugghe,H.F., van den Elst,H., van der Marel,G.A., van Boom,J.H., Westra,J.G., Kriek,E. and Berns,A.J.M. (1992) Repair and replication of plasmids with site-specific 8-oxodG and 8-AAFdG residues in normal and repair-deficient human cells. Nucleic Acids Res., 20, 44374443.[Abstract]
-
Le Page,F., Guy,A., Cadet,J., Sarasin,A. and Gentil,A. (1998) Repair and mutagenic potency of 8-oxoG:A and 8-oxoG:C base pairs in mammalian cells. Nucleic Acids Res., 26, 12761281.[Abstract/Free Full Text]
Received April 6, 1999;
revised June 7, 1999;
accepted June 8, 1999.