Mutagenic Bypass of 8-Oxo-7,8-dihydroguanine (8-Hydroxyguanine

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Mutagenic Bypass of 8‑Oxo-7,8-dihydroguanine (8-Hydroxyguanine) by DNA Polymerase κ in Human Cells Hiroyuki Kamiya* and Masahiro Kurokawa Graduate School of Science and Engineering, Ehime University, 2-5 Bunkyo-cho, Matsuyama 790-8577, Japan ABSTRACT: The formation of 8-oxo-7,8-dihydroguanine (GO, 8-hydroxyguanine) in DNA and in the nucleotide pool results in G:C→T:A and A:T→C:G substitution mutations, respectively, since GO can pair with both C and A. In this study, the role of DNA polymerase κ in the mutagenicity of GO was investigated, using a supF shuttle plasmid propagated in human U2OS cells. This translesion synthesis DNA polymerase was knocked down by siRNA, and plasmid DNAs containing GO:C and GO:A pairs were transfected into the knock-down cells. The supF plasmid DNAs replicated in the cells were then introduced into Escherichia coli. Mutation analyses indicated that the knock-down of DNA polymerase κ by siRNA decreased the frequency of G:C→T:A mutation caused by GO:C, although no effects of the DNA polymerase κ reduction were observed for the A:T→C:G substitution induced by GO:A. These results suggested that DNA polymerase κ is involved in the mutagenic bypass of GO in living human cells, when the damaged base is generated by direct DNA oxidation.

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DNA pols η and ζ enhanced the G:C→T:A transversion mutation induced by the GO:C pair in living human cells. Thus, these TLS DNA pols were likely to promote the error-free incorporation of dCTP opposite GO. These results raised the question of whether other TLS DNA pols contribute to the induction of mutagenesis by GO. Human DNA pol κ is errorprone against GO and efficiently inserts dATP opposite the base in vitro,33−37 suggesting that this pol might play a crucial role in the mutagenic bypass of GO in living human cells. However, the mammalian replicative DNA pols α and δ bypass the GO base and are error-prone against this oxidized base in vitro.9−14 DNA pol κ does not act on GO unless this TLS DNA pol is recruited to the replication fork including GO. Thus, it is important to determine whether this DNA pol actually contributes to the mutagenic bypass of the oxidized G base in living cells. Moreover, the knock-down of DNA pol η was shown to cause a decrease in the frequency of A:T→C:G transversion induced by the GO:A base pair.32 It would also be interesting to know whether DNA pol κ is involved in the mutagenic pathway of GO:A that would be formed by the incorporation of dGOTP in living cells. In this study, double-stranded plasmid DNA containing a GO:C pair was transfected into human cells in which DNA pol κ was knocked down. Moreover, plasmid DNA containing a GO:A pair, as the intermediate in the mutagenesis process of dGOTP, was also introduced into the knock-down cells. We found that the knock-down of DNA pol κ reduced the frequency of G:C→T:A transversion mutation induced by the GO:C pair and did not affect the A:T→C:G transversion

INTRODUCTION Replication fidelity is highly important for genomic integrity in organisms. Spontaneous mutation rates have been estimated to be 10−10 to 10−12 errors per base pair per generation in eukaryotes (e.g., ref 1). Many factors are known to disturb correct 2′-deoxyribonucleotide incorporation during DNA replication. DNA damage and damaged 2′-deoxyribonucleotides generated by endogenous and exogenous (environmental) factors are important sources of mutations.2,3 Reactive oxygen species (ROS) are pivotal mutagens, and the oxidized bases in DNA and in the nucleotide pool cause mutagenesis, carcinogenesis, neurodegeneration, and aging.4,5 8-Oxo-7,8-dihydroguanine (GO, also known as 8-hydroxyguanine) is one of the major oxidized bases produced by ROS, and more than 100 GO residues are generated per cell per day.6−8 The GO base is miscoding, since DNA polymerases (pols) incorporate dATP in addition to dCTP opposite the oxidized base.9−14 The base is highly mutagenic in Escherichia coli and mammalian cells, and induces G:C→T:A transversion mutations with an error frequency of 10−3−10−2.15−23 When dGTP is oxidized by ROS, 8-oxo-7,8-dihydro-dGTP (dGOTP) is formed.24,25 This oxidized dGTP is also mutagenic and specifically induces A:T→C:G transversions in living cells.26−28 The A:T→C:G mutations are apparently induced by the incorporation of dGOTP opposite A in the template DNA, resulting in the formation of GO:A base pairs, and the incorporation of dCTP opposite GO during the next round of replication.29 To bypass DNA lesions, mammalian cells contain specialized DNA pols.30,31 We previously examined the involvement of some translesion synthesis (TLS) DNA pols in the GO-induced mutagenesis.32 We demonstrated that the knock-downs of © 2012 American Chemical Society

Received: June 8, 2012 Published: July 17, 2012 1771

dx.doi.org/10.1021/tx300259x | Chem. Res. Toxicol. 2012, 25, 1771−1776

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mutation induced by the GO:A pair. These results suggested that this TLS DNA pol conducts the mutagenesis processes of GO generated by the direct oxidation of G bases in DNA.

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(Life Technologies) and was treated with RNase-free DNase I (Takara) to degrade the genomic DNA in these samples. First-strand cDNA synthesis was performed on 1 μg of total RNA with an Applied Biosystems High Capacity RNA-to-cDNA Kit (Life Technologies), according to the manufacturer’s instructions. Each of the mRNA transcripts was measured by the quantitative PCR method with a BioRad CFX96 Real Time PCR System and EvaGreen chemistry (BioRad), using the following primers: pol κ upper, 5′d G G GC A T T G C T TT C T C T C CT T - 3′ ; p o l κ l o w e r , 5′ dTTCCTCTCTCCATCCCTCGT-3′. Data were expressed as the ratio to the GAPDH mRNA, which was determined using the following primers: GAPDH upper, 5′-dAACTTTGGTATCGTGGAAGG-3′; GAPDH lower, 5′-dGTCTTCTGGGTGGCAGTGAT3′. Statistical Analysis. Statistical significance was examined by the Student’s t-test. Levels of P < 0.05 were considered to be significant.

EXPERIMENTAL PROCEDURES

Materials. Phosphorylated oligodeoxyribonucleotides containing GO and their control oligodeoxyribonucleotides38 were purchased from Nihon BioService (Asaka, Japan) and were purified by HPLC, as described previously.39 Other oligodeoxyribonucleotides were obtained from Hokkaido System Science (Sapporo, Japan) and Sigma Genosys Japan (Ishikari, Japan) in purified forms. The Stealth Select 3 RNAi was obtained from Life Technologies (Carlsbad, CA). The following RNAs were used: κ sense, 5′-UAGCAAUGGCAAAUAUUUCUUCUGC-3′; κ antisense, 5′-GCAGAAGAAAUAUUUGCCAUUGCUA-3′. Stealth RNAi Negative Control Low duplex (Life Technologies) was used as the negative control, according to the recommended GC contents. The KS40/pOF105 E. coli strain was provided by Professor Tatsuo Nunoshiba, of International Christian University, and was used as an indicator strain for the supF mutants.40 Plasmid DNAs containing GO. The following oligodeoxyribonucleotides were synthesized and chemically phosphorylated at their 5′ends on the support during synthesis: T-96, 5′-dGCAGACTCTAAATCTGCCGTCAT-3′; GO-96, 5′-dGCAGACTCGOAAATCTGCCGTCAT-3′; G-122, 5′-dCGACTTCGAAGGTTCGAATCC-3′; GO-122, 5′-dCGACTTCGAAGGOTTCGAATC-3′. The single-stranded form of pZ189-107K/402E was obtained by superinfection of E. coli JM109 containing pZ189-107K/402E with the helper phage VCS-M13 (Agilent Technologies, Santa Clara, CA), and polyethylene glycol precipitation and subsequent deproteinization, as described.41−43 Double-stranded plasmid DNAs containing GO were constructed, essentially as described.44 Namely, the single-stranded forms of pZ189-107K/402E (5 μg, 2.9 pmol) and the GOoligodeoxyribonucleotides (16 pmol) were annealed and treated with T4 DNA pol (2 units, F. Hoffmann-La Roche, Basel, Switzerland) and T4 DNA ligase (350 units, Takara, Otsu, Japan), followed by treatment with Dam methylase (16 units, New England Biolabs, Ipswich, MA) that methylates the N6-position of adenine in the 5′GATC-3′ sequences (to restore the bacterial methylation pattern). Mutagenesis Experiments. U2OS cells (3.0 × 104 cells) were plated onto 24-well dishes and were cultured in Dulbecco’s modified Eagle's medium, supplemented with 10% fetal bovine serum, at 37 °C under a 5% CO2 atmosphere for 24 h. The siRNA (10 pmol) was mixed with Lipofectamine 2000 (0.5 μL, Life Technologies) in serumfree medium (50 μL) and introduced into U2OS cells according to the supplier’s recommendations. After 24 h, the plasmid DNAs containing GO (100 ng, 29 fmol) were transfected with Lipofectamine (1 μL, Life Technologies). After 48 h of culture, the plasmid DNA amplified in the cells was recovered by the method of Stary and Sarasin.45 The recovered plasmid DNA was treated with DpnI, which recognizes the methylated 5′-GATC-3′ sequences to digest unreplicated plasmids. Determination of supF Mutant Frequency. The DpnI-treated DNAs were electroporated into E. coli KS40/pOF105, using a Gene Pulser II transfection apparatus with a Pulse Controller II (Bio-Rad, Hercules, CA). The supF mutant frequency was calculated according to the numbers of white and faint blue colonies on Luria−Bertani agar plates containing nalidixic acid (50 μg/mL), streptomycin (100 μg/ mL), ampicillin (150 μg/mL), chloramphenicol (30 μg/mL), 5bromo-4-chloro-3-indolyl-β-D-galactopyranoside (80 μg/mL), and isopropyl-β-D-thiogalactopyranoside (23.8 μg/mL), and the numbers of colonies on agar plates containing ampicillin and chloramphenicol, as described previously.28,40 For the GO:A plasmid experiments, the supF mutant frequency was determined as the ratio of white and faint blue colonies to total colonies on agar plates containing ampicillin, chloramphenicol, 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside, and isopropyl-β-D-thiogalactopyranoside. Quantitative Reverse Transcription-PCR Analysis of mRNA. Total RNA was extracted from U2OS cells with the TRIzol reagent

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RESULTS Plasmid Construction and Knock-Down of DNA Pol κ. We constructed plasmid DNAs containing the GO base. The parental plasmid, pZ189-107K/402E, contains the supF gene, the mutant (E107K and D402E) SV40 large T antigen gene, and the SV40 origin of replication.42 The mutant SV40 large T antigen is unable to bind p53 and Rb but retains its replication activity, and pZ189-107K/402E replicates normally in cultured cells.42,46 GO was introduced into positions 122 and 96 of the sense strand of the supF gene for GO:C and GO:A, respectively. Position 96 was chosen because it was one of the A:T→C:G hot spots in the supF gene when dGOTP was introduced.27,28 Plasmid DNAs containing G:C and T:A at positions 122 and 96, respectively, were used as control plasmids. The siRNA against DNA pol κ was introduced into U2OS cells (expressing wild-type p53 and Rb), and the knock-down of DNA pol κ was measured by quantitative reverse transcriptionPCR (Table 1). The treatment of U2OS cells with siRNA Table 1. Amounts of DNA Pol κ mRNA in Knocked-Down U2OS Cellsa 24 h

48 h

72 h

0.21 ± 0.01

0.34 ± 0.01

0.31 ± 0.06

a

The amount of mRNA was measured by quantitative reverse transcription-PCR at 24, 48, and 72 h after siRNA introduction. The amount of mRNA was normalized relative to the amount of human GAPDH mRNA present in each sample. Values relative to those in U2OS cells treated with the control siRNA are shown. Experiments were performed three times. Data are expressed as the means ± SEM.

reduced the amount of the DNA pol κ mRNA. The knockdown efficiency was 79% at 24 h after siRNA introduction, which was the time point when the plasmid DNAs were transfected. Knock-Down of DNA Pol κ Reduced the Frequency of G:C→T:A Mutation Induced by the GO:C Pair. We first examined the effects of the knock-down of DNA pol κ by siRNA on the mutation induced by GO:C. The pZ189-107K/ 402E plasmid DNAs, containing single G:C and GO:C pairs at position 122, were transfected into cultured U2OS cells with knocked-down DNA pol κ. The replicated DNA was recovered from the treated cells and was then introduced into the indicator E. coli strain, KS40/pOF105,40 for the calculation of the mutant frequencies of the supF gene. KS40/pOF105 cells containing supF mutant plasmid are resistant to nalidixic acid and streptomycin, and form white or faint blue colonies in the presence of 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside 1772

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and isopropyl-β-D-thiogalactopyranoside because of the lacZ− phenotype. Typically, ∼250 supF mutant colonies on selection plates were obtained (10-fold dilution), and ∼2000 total colonies were formed on titer plates (1000-fold dilution), when GO:C-derived plasmid DNA recovered from the knock-down cells was electroporated into KS40/pOF105. The mutant frequency was determined according to the numbers of the mutant and total colonies, as described in the Experimental Procedures section. This determination was performed for each transfection experiment, and the transfection into U2OS cells was independently conducted four times. Similar supF mutant frequencies were observed for U2OS cells treated with the control and DNA pol κ siRNAs in the experiments using the G:C plasmid (∼5 × 10−4, Figure 1, white

(54% and 52%, respectively; Table 2). To calculate the frequencies of the targeted G:C→T:A transversion, the total supF mutant frequencies were multiplied by the percentages of the G:C→T:A mutation. The targeted G:C→T:A frequencies were 1.0 and 0.7 × 10−3 in the control and pol κ experiments, respectively. Thus, we confirmed that the incidence of the G:C→T:A mutation was decreased by the knock-down of DNA pol κ. This result suggested that DNA pol κ enhances GO:Cmediated mutagenesis by incorporating dATP opposite the oxidatively damaged base. In contrast, no G:C→T:A mutation at position 122 was observed upon the transfection of the control G:C plasmid DNA (Table 2). This result was expected since no modified base was introduced at this position. In other studies, among more than 100 supF mutant colonies obtained by transfection of the G:C plasmid DNA into U2OS cells, only two contained G:C→T:A mutation at position 122 (unpublished results). Similar results were obtained using a different human cell line.32,38 The G:C→A:T and G:C→C:G mutations were found in four of the 52 colonies obtained by transfection of the GO:C plasmid into the cells in which DNA pol κ was knocked down (Table 2). The induction of these mutations by GO:C was reported previously in mammalian cells.19,21 Effects of DNA Pol κ Knock-Down on GO:A-Induced Mutation. An oxidized form of dGTP, dGOTP, is mutagenic and specifically induces A:T→C:G transversions in living cells.26−28 The A:T→C:G mutation is considered to be triggered by the incorporation of dGOTP opposite A in the template DNA, resulting in the formation of GO:A base pairs, and the incorporation of dCTP opposite GO.29 Thus, this mutagenic pathway is considered to contain two steps in which TLS DNA pols may be involved: the incorporation of dGOTP opposite A to form GO:A pairs and that of dCTP opposite GO. Next, we examined the effects of the knock-down of DNA pol κ on the mutation induced by GO:A, the mutation that would reflect the latter incorporation step. The plasmid DNAs containing single T:A and GO:A pairs at position 96 of the supF gene were prepared and transfected into the U2OS cells with knocked-down DNA pol κ. The treatment with the siRNAs against DNA pol κ did not affect the supF mutant frequencies in the experiments using the T:A plasmid (7−8 × 10−4, Figure 2A). When the plasmid DNA with the GO:A pair at position 96 was introduced into U2OS cells treated with the control siRNA, the supF mutant frequency was 92 × 10−2 (92%), which is quite high (Figure 2B). This high mutant frequency was due to the MUTYH protein, which removes A opposite GO (see Discussion).38 The supF mutant

Figure 1. Effects of the knock-down of DNA pol κ in U2OS cells on the mutant frequencies induced by GO:C. White bar, control plasmid containing G:C at position 122; black bar, plasmid containing GO:C at position 122. Experiments were performed four times. Data are expressed as means + SEM *P < 0.05 vs control siRNA.

columns), indicating that the reduced amount of this DNA pol had little influence on the background mutant frequency in this experimental system. When the plasmid with the GO:C pair was introduced into U2OS cells treated with the control siRNA, the supF mutant frequency was increased to ∼1.8 × 10−3 (Figure 1), indicating the mutagenicity of GO in DNA. The knockdown of DNA pol κ reduced the supF mutant frequency to ∼1.3 × 10−3, which was statistically significant (Figure 1). This result suggested that the TLS DNA pol, pol κ, is involved in the mutagenic bypass of the GO lesion. We analyzed the sequences of the supF genes in the colonies on the selection agar plates (Table 2). As expected, the targeted G:C→T:A transversion was the mutation found most frequently in the progeny plasmid DNAs of the GO:C plasmid replicated in the control siRNA-treated and knock-down cells

Table 2. Mutations at Position 122, Found in Progeny Plasmids Replicated in U2OS Cells with Knocked-Down DNA Pol κa G:C mutation single base substitutions transition G:C→A:T transversion G:C→T:A G:C→C:G others no mutation total colonies analyzed a

G°:C pol κ

control

control

pol κ

0 (0)

0 (0)

1 (2)

2 (4)

0 (0) 0 (0) 0 (0) 15 (100) 15 (100)

0 (0) 0 (0) 0 (0) 18 (100) 18 (100)

22 (54) 0 (0) 0 (0) 18 (44) 41 (100)

27 (52) 2 (4) 0 (0) 21 (40) 52 (100)

All data are represented as cases found (%). 1773

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reduction in the targeted G:C→T:A frequency (1.0 and 0.7 × 10−3 in the control and pol κ experiments) suggested involvement of other DNA pols (such as replicative DNA pols α and δ) in addition to this TLS pol, in erroneous dATP incorporation opposite the GO base. However, further studies are necessary to elucidate detailed role(s) of DNA pol κ in mutagenic bypass of GO. Previously, we analyzed the effects of the knock-downs of other TLS DNA pols in human cells.32 The knock-downs of DNA pols η and ζ increased the mutagenesis caused by GO paired with C, while those of DNA pol ι and REV1 did not affect the mutation frequency. Thus, at least three TLS DNA pols bypass the GO lesions during DNA replication: DNA pol κ in error-prone bypass and DNA pols η and ζ in (relatively) error-free bypass. The GO base is not a highly blocking lesion for replicative DNA pols in vitro.9,12−14 However, the participation of TLS DNA pols in the bypass of GO in human cells has been shown (ref 32 and this study). Proliferating cell nuclear antigen (PCNA) provides a scaffold to which TLS DNA pols bind for access to the replication complex stalled at a lesion site, and PCNA ubiquitination is important for the switch from replicative to TLS DNA pols at stalled forks.30,31 Lin suggested interaction of DNA pol κ, SHPRH, RAD18, and polyubiquitinated PCNA, and function of DNA pol κ at stalled forks.47 Moreover, the various TLS DNA pols interact with each other.48 The mechanisms of the DNA pol switch at the GO site, which is not a blocking lesion, remain to be elucidated. We also examined plasmid DNA containing GO:A, as an intermediate in the mutagenesis process of dGOTP. If DNA pol κ also plays a pivotal role in dATP incorporation opposite GO of the GO:A pair formed by dGOTP incorporation, then the frequency of the targeted A:T→C:G transversion that is induced by dCTP incorporation opposite GO would increase upon the knock-down of DNA pol κ. However, we did not observe any effects of the knock-down (Figure 2 and Table 3). The occurrence of the A:T→C:G mutation is highly dependent on MUTYH activity, which removes A from GO:A pairs.38 The removal of A results in disappearance of the original genetic information and the dCTP/dATP incorporation ratio opposite GO in the gap by DNA pols determines the mutation frequency. DNA pols λ and β reportedly perform gap-filling.49,50 In addition, DNA pol η plays a role in the A:T→C:G transversion since its knock-down slightly decreased the mutation frequency.32 The results obtained in the previous and present studies indicated that two TLS DNA pols, λ and η, but not DNA pol κ, are involved in the pathway generating the A:T→

Figure 2. Effects of the knock-down of DNA pol κ in U2OS cells on the mutant frequencies induced by GO:A. (A) Mutant frequencies upon transfection of the control plasmid containing T:A at position 96. (B) Mutant frequencies upon transfection of the plasmid containing GO:A at position 96. Experiments were performed three (T:A) and four (GO:A) times. Data are expressed as means + SEM.

frequency was similar (93 × 10−2) in the cells in which DNA pol κ was knocked down, suggesting that this DNA pol plays a minimal, if any, role in the GO:A-induced mutations. We analyzed the sequences of the supF genes in colonies on the selection agar plates (Table 3). As expected, the A:T→C:G transversion was found in all progeny plasmid DNAs of the GO:A plasmid. No A:T→C:G mutation at position 96 was observed for the transfection of the control T:A plasmid (Table 3).

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DISCUSSION Human DNA pol κ is error-prone against GO and inserts dATP and dCTP opposite the oxidized base in an 8 to 2 ratio in vitro.37 Thus, this TLS DNA pol might be crucial in the mutagenic bypass of GO in living human cells. However, the mammalian replicative DNA pols α and δ are also error-prone against GO.9−14 Therefore, we knocked down DNA pol κ, to examine its involvement in the mutagenesis by GO in living human cells. As shown in Figure 1, the reduction of DNA pol κ significantly decreased the frequency of mutation induced by GO paired with C in DNA. This result suggested that DNA pol κ enhances the mutagenesis by GO:C in cells. A subsequent mutation analysis indicated that the frequency of G:C→T:A transversion mutation at the modified position was reduced by the pol κ knock-down. Thus, DNA pol κ is at least partly responsible for the G:C→T:A transversions caused by GO, generated by the direct oxidation of DNA. The ∼30%

Table 3. Mutations at Position 96, Found in Progeny Plasmids Replicated in U2OS Cells with Knocked-Down DNA Pol κa T:A mutation single base substitutions transition A:T→G:C transversion A:T→T:A A:T→C:G others no mutation total colonies analyzed a

G°:A pol κ

control

control

pol κ

0 (0)

0 (0)

0 (0)

0 (0)

0 (0) 0 (0) 0 (0) 16 (100) 16 (100)

0 (0) 0 (0) 0 (0) 17 (100) 17 (100)

0 (0) 21 (100) 0 (0) 0 (0) 21 (100)

0 (0) 35 (100) 0 (0) 0 (0) 35 (100)

All data are represented as cases found (%). 1774

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C:G transversion mutations induced by GO:A pairs. Note that the dCTP incorporation opposite GO in the gap-filling reaction would occur independently of DNA replication and that G:C→ T:A mutations would be caused by GO:C during replication. Thus, reduction of the G:C→T:A mutation caused by GO:C and no effects on the A:T→C:G mutation caused by GO:A observed in the pol κ-knock-down cells do not contradict each other. The total supF mutant frequency was 92 × 10−2, in the case of U2OS cells treated with the control siRNA (Figure 2B). This value was comparable to that observed in human 293T cells (88 × 10−2).32 Again, the A:T→C:G mutation is strongly dependent on MUTYH activity, and this activity would be high in these cell lines. Although DNA pol κ did not seem to act in the dCTP/dATP incorporation step in the mutagenesis process of dGOTP, this DNA pol might be involved in the dGOTP incorporation step. Thus, additional studies are required to elucidate this possibility. As described above, at least three TLS DNA pols bypass the GO lesions during DNA replication. The experiments using living cells, as described in this article, could reveal the significant proteins involved in the mutagenic pathways of GO. Since the GO base is recognized as one of the important oxidatively damaged DNA bases that cause mutations, from bacterial to mammalian cells, mechanisms that prevent the incorporation of dATP opposite the lesion during DNA replication are important. In fact, DNA pols η and ζ seem to function as mutation suppressors for GO.32 In contrast, DNA pol κ enhanced the GO-caused mutation, as shown in this study (Figure 1). Thus, the recruitment of DNA pol κ to the replication fork including GO might be regulated by unknown mechanism(s). In conclusion, the knock-down of DNA pol κ decreased the mutagenicity of GO paired with C in human U2OS cells. This result suggested that this DNA pol performs the error-prone bypass of the oxidized G base formed in DNA. In contrast, the knock-down did not affect the mutagenicity of GO paired with A. In addition to DNA pols, other cellular enzymes play crucial roles in the mutagenesis processes of damaged DNA and DNA precursors.51 Experiments to reveal their roles are currently in progress.

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REFERENCES

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AUTHOR INFORMATION

Corresponding Author

*Tel: +81-89-927-9609. Fax: +81-89-927-9590. E-mail: [email protected]. Funding

This work was supported in part by Grants-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan, and the Takeda Science Foundation. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Professor Satoru Suzuki and Ms. Mitsuko Ogo for assistance with the reverse transcription-PCR experiments.

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ABBREVIATIONS ROS, reactive oxygen species; GO, 8-oxo-7,8-dihydroguanine; pol, polymerase; dGOTP, 8-oxo-7,8-dihydro-dGTP; TLS, translesion synthesis 1775

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dx.doi.org/10.1021/tx300259x | Chem. Res. Toxicol. 2012, 25, 1771−1776