Action-at-a-Distance Mutagenesis Induced by Oxidized Guanine in

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Action-at-a-Distance Mutagenesis Induced by Oxidized Guanine in Werner Syndrome Protein-Reduced Human Cells Hiroyuki Kamiya,*,†,‡,§,∥ Daiki Yamazaki,† Eri Nakamura,‡ Tetsuaki Makino,‡,∥ Miwako Kobayashi,§ Ichiro Matsuoka,§ and Hideyoshi Harashima† †

Faculty of Pharmaceutical Sciences, Hokkaido University, Kita-12, Nishi-6, Kita-ku, Sapporo 060-0812, Japan Graduate School of Science and Engineering, Ehime University, 2-5 Bunkyo-cho, Matsuyama 790-8577, Japan § College of Pharmaceutical Sciences, Matsuyama University, 4-2 Bunkyo-cho, Matsuyama 790-8578, Japan ∥ Graduate School of Biomedical and Health Sciences, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8553, Japan ‡

S Supporting Information *

ABSTRACT: 8-Oxo-7,8-dihydroguanine (GO, 8-hydroxyguanine) in DNA is one of the most important oxidatively damaged bases and causes G:C → T:A substitution mutations. The Werner syndrome protein (WRN) is a cancer-related RecQ DNA helicase and plays many roles in DNA replication and repair. To examine the relationships between GO-induced mutations and WRN, shuttle plasmid DNA containing a GO:C pair in the supF gene was transfected into human U2OS cells, in which WRN was knocked down. The plasmid DNA replicated in the knockdown cells was introduced into an Escherichia coli indicator strain. The knockdown of WRN increased the mutant frequency of the GO-plasmid DNA. Unexpectedly, however, the WRN knockdown only slightly enhanced the targeted G:C → T:A mutation. Instead, base-substitution mutations at various positions were more frequently detected, with statistical significance. The results obtained in this study suggested that the reduction of the cancerrelated WRN induced action-at-a-distance mutagenesis by the GO:C pair in human cells. In addition, the WRN knockdown decreased the GO:A-induced A:T → C:G mutations, suggesting that WRN may enhance the mutations caused by GO in the nucleotide pool.

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tively acts with DNA pol β by removing the mismatched primer terminus, and extracts of WRN-depleted cells lack long patch base excision repair (BER) activity.5 WRN interacts with 5′ flap endonuclease/5′-3′ exonuclease (FEN-1), replication protein A (RPA), XPG, and telomere-specific POT1.6−10 In addition, the WRN protein is reportedly involved in homologous recombination.11 Thus, the WRN protein functions to maintain genetic information in various ways. DNA and its related compounds are continuously oxidized by reactive oxygen species (ROS) that are produced endogenously (normal oxygen metabolism) and exogenously (environmental mutagens and carcinogens). The formation of oxidatively damaged bases in nucleic acids is considered to

INTRODUCTION

The Werner, Bloom, and Rothmund Thomson syndromes share cancer predisposition features and premature aging symptoms.1,2 Their responsible genes are three of the five members encoding DNA helicases homologous to Escherichia coli RecQ, and they are implicated in the maintenance of genome stability. Among them, the Werner syndrome protein (WRN) is one of the best-characterized human RecQ helicases. WRN possesses 3′ → 5′ helicase and 3′ → 5′ exonuclease activities, and functions in DNA replication, repair, and telomere maintenance. For example, replication fork progression is decreased in WRN-depleted cells treated with methylmethanesulfonate and hydroxyurea, and the cell cycle is delayed in these cells.3 WRN interacts with DNA polymerase (pol) β and stimulates strand displacement DNA synthesis via its helicase activity.4 The WRN exonuclease activity coopera© 2015 American Chemical Society

Received: October 15, 2014 Published: March 2, 2015 621

DOI: 10.1021/tx500418m Chem. Res. Toxicol. 2015, 28, 621−628

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Chemical Research in Toxicology

supplier’s recommendations. After 24 h, the plasmid DNAs containing GO (100 ng, 29 fmol) were transfected with Lipofectamine (Life Technologies). After 48 h of culture, the plasmid DNA amplified in the cells was recovered by the method of Stary and Sarasin.38 The recovered DNA was treated with Dpn I (New England Biolabs, Ipswich, Massachusetts, USA) to digest the unreplicated plasmid DNA. Determination of supF Mutant Frequency. The DNAs recovered from the cells were introduced into E. coli KS40/pOF105 by electroporation, using a Gene Pulser II transfection apparatus with a Pulse Controller II (Bio-Rad, Hercules, California, USA). The supF mutant frequency in the GO:C, G:C, and T:A experiments was determined 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), 5-bromo-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. The supF mutant frequency in the GO:A experiments was determined as the ratio of white and faint blue colonies to total colonies, on agar plates containing ampicillin, chloramphenicol, 5bromo-4-chloro-3-indolyl-β-D-galactopyranoside, and isopropyl-β-Dthiogalactopyranoside.31 Reverse Transcription (RT)-Quantitative PCR (qPCR) Analysis of mRNA. Total RNA was extracted from U2OS cells with an RNeasy Mini Kit (Qiagen, Hilden, Germany), combined with RNase-free DNase I (Takara, Otsu, Japan) for the degradation of genomic DNA in total RNA samples. First-strand cDNA synthesis was performed on 500 ng of total RNA, using an oligo dT-Adaptor primer and an RNA PCR Kit (AMV) (Takara), according to the manufacturer’s instructions. Each of the mRNA transcripts was measured by the qPCR method with an ABI 7500 Real Time PCR System and SYBRGreen chemistry (Life Technologies), using the following primer set: WRN upper, 5′-dTCAGGAAGTTGGGCTCCCTAA-3′; WRN lower, 5′-dCAACGATTGGAACCATTGGC-3′. Data are expressed as the ratio to the amount of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA, which was determined using the following primers: GAPDH upper, 5′-dAACTTTGGTATCGTGGAAGG-3′; GAPDH lower, 5′-dGTCTTCTGGGTGGCAGTGAT-3′. Western Blotting. The cells were extracted with radio immunoprecipitation assay buffer containing protease inhibitors. The whole cell extracts were fractionated on an 8% SDS−polyacrylamide gel and transferred to PVDF membranes. To detect WRN, the membranes were blocked in 100% Starting Block (Thermo Fisher Scientific, Waltham, Massachusetts, USA) containing 0.05% Tween 20 and probed with a rabbit anti-WRN antibody (catalogue no. sc-5629, Santa Cruz Biotechnology, Santa Cruz, California, USA) for 1 h in Can Get Signal solution 1 (Toyobo, Osaka, Japan) at 20 °C. For GAPDH, the membranes were blocked in 3% nonfat milk and probed with a rabbit anti-GAPDH antibody (catalogue no. G9545, Sigma-Aldrich, St. Louis, Missouri, USA) for 1 h in Tris-buffered saline (TBS), containing 0.1% Tween 20 and 3% nonfat milk, at 20 °C. After three washes of the membranes with TBS containing 0.1% Tween 20, they were incubated with horseradish peroxidase-conjugated antirabbit IgG (catalogue no. 7074S, Cell Signaling Technology, Danvers, Massachusetts, USA) for 1 h in TBS, containing 0.1% Tween 20 and 3% nonfat milk, at 20 °C. After three washes of the membranes with TBS containing 0.1% Tween 20, the proteins were then visualized using the Enhanced Chemiluminescence (ECL) System (GE Healthcare Bio-Sciences, Piscataway, New Jersey, USA) and detected with an LAS 3000 Luminescent Image Analyzer (Fujifilm, Tokyo, Japan). Statistical Analysis. Statistical significance was examined by the Student’s t-test. Levels of P < 0.05 were considered to be significant.

cause mutagenesis, carcinogenesis, aging, and neurodegeneration.12,13 8-Oxo-7,8-dihydroguanine (GO, also known as 8hydroxyguanine) is one of the major damaged nucleobases produced by ROS, and each day, 100−500 GO residues per cell are generated in DNA.14−16 This oxidized G base exhibits ambiguous coding potential and has the ability to form base pairs with A as well as C.17−20 GO is highly mutagenic and induces G:C → T:A and A:T → C:G transversion mutations when it is generated in the DNA and the nucleotide pool, respectively, in mammalian cells.21−27 Many cellular proteins are involved in the mutagenesis pathway of the damaged base. Some BER and nucleotide pool sanitization enzymes prevent the mutations caused by GO, and some specialized DNA pols play protective and enhancive roles in the process.27−32 WRN associates with the DNA glycosylase NEIL1 and stimulates its activities for GO in the bubble structure.33 Since the mutagenicity of GO is increased by the knockdown of NEIL1 in human cells,28 similar enhancement may also be observed when WRN is knocked down. Therefore, we surmised that the transfection of plasmid DNA containing a GO:C pair at a specific position would provide evidence for this hypothesis. In this study, double-stranded plasmid DNA containing the GO:C pair was transfected into human U2OS cells, in which WRN was knocked down. We found that the knockdown of WRN enhanced the frequency of mutants induced by GO. Unexpectedly, however, only a slight increase in the G:C → T:A transversion mutation at the GO site was observed in the WRN-knockdown cells. Instead, base-substitution mutations at untargeted positions were more frequently found, with statistical significance. These results suggested that the reduction of WRN induces action-at-a-distance mutagenesis by GO:C in human cells.

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MATERIALS AND METHODS

Materials. Oligodeoxyribonucleotides (ODNs) containing GO and their control ODNs were purchased from Nihon BioService (Asaka, Japan) and were purified by HPLC, as described previously.28,34 Other ODNs were obtained from Hokkaido System Science (Sapporo, Japan) and Sigma Genosys Japan (Ishikari, Japan) in purified forms. siRNAs (“stealth RNAi”, Life Technologies, Carlsbad, California, USA) were synthesized according to the BLOCK-iT RNAi Designer software, on the supplier’s Web site. The following siRNAs were used: WRN sense, 5′-AUCAGUGGCUGCAUACAGUUUCUGG-3′; WRN antisense, 5′-CCAGAAACUGUAUGCAGCCACUGAU-3′. Stealth RNAi Negative Control Medium GC duplex (% GC 48, Life Technologies) was used as the negative control. The E. coli strain KS40/pOF105 was provided by Professor Tatsuo Nunoshiba of International Christian University and was used as an indicator strain of the supF mutants.35 Construction of Plasmid DNAs Containing GO. The ODNs indicated below were synthesized and chemically phosphorylated at their 5′-end on the support during synthesis. G-122, 5′-dCGACTTCGAAGGTTCGAATCC-3′; GO-122, 5′-dCGACTTCGAAGGOTTCGAATC-3′; T-96, 5′-dGCAGACTCTAAATCTGCCGTCAT-3′; GO-96, 5′-dGCAGACTCGOAAATCTGCCGTCAT-3′. The double-stranded GO and control plasmid DNAs were constructed from the single-stranded forms of pZ189−107K/402E and the ODNs containing GO, G, and T bases, as described.36,37 G122, GO-122, T-96, and GO-96 were used for constructions of the G:C, GO:C, T:A, and GO:A plasmid DNAs, respectively. Mutagenesis Experiments for Shuttle Plasmids Containing GO. 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 (Life Technologies) and introduced into the cultured cells according to the

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RESULTS

WRN Knockdown Enhanced the Mutation Frequency of GO:C. We examined the effects of the knockdown of WRN by siRNA on the mutations induced by GO. First, the amounts of the WRN mRNA in human U2OS cells were measured by 622


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Chemical Research in Toxicology RT-qPCR (Figure 1A). The treatment of U2OS cells with siRNA reduced the amount of WRN mRNA. The knockdown efficiency was 85% at 24 h after siRNA introduction, the time point when the plasmid DNA was transfected. The knockdown at the same time point was confirmed by Western blotting, using an anti-WRN antibody (Figure 1B).

Figure 2. Effects of WRN knockdown in U2OS cells on the mutant frequency induced by GO:C. Open columns, control plasmid containing G:C at position 122; closed columns, plasmid containing GO:C at position 122. Transfection experiments were performed eight times. Data are expressed as the means + standard errors. *P < 0.05 vs control siRNA.

Figure 1. Knockdown of WRN by siRNA. (A) Amounts of WRN mRNA in U2OS cells treated with siRNA. The amount of mRNA was measured by RT-qPCR at 24 and 48 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 in duplicate (48 h) and triplicate (24 h). The data obtained in a single experiment and the mean values are represented by circles and bars, respectively. (B) The levels of WRN expression in U2OS cells at 24 h after siRNA introduction, detected by Western blot analysis.

WRN-knockdown cells (Figure 2). This result suggested that WRN suppresses the mutagenesis caused by GO. WRN Knockdown Induced Action-at-a-Distance Mutations. We expected that the enhanced mutant frequency in the knockdown cells was due to increased G:C → T:A mutations at the target site as some of the BER enzymes were knocked down.28 We then sequenced the supF genes from the colonies on the selection agar plates (Table 1). In accordance with the previous reports,28,30,31 the G:C → T:A transversion at position 122 was the mutation found most frequently in the colonies obtained from the GO-plasmid-transfected cells. Unexpectedly, however, the ratio of the targeted G:C → T:A mutation was lower for the knockdown cells than for the control cells. In the control siRNA-treated cells, the ratio of the targeted G:C → T:A mutation was 48% (Table 2). The ratio was reduced to 37% (Table 2) for the knockdown cells, although their total mutant frequency was 1.6-fold higher than the control cells. The total supF mutant frequencies were multiplied by the percentages of the targeted G:C → T:A transversion, to calculate its frequencies. The calculated G:C → T:A frequencies were 1.3 and 1.7 × 10−3 in the control and WRN experiments, respectively, and the targeted mutation frequency was increased slightly (by 0.4 × 10−3) by the WRN knockdown. Thus, the increase in the targeted mutation alone could not explain the 1.8 × 10−3 higher total mutant frequency. The spectra of the mutations found in the supF gene are summarized in Table 2. The distributions of the mutations, except for those at the target position, are shown in Supporting Information, Figure 1 for the GO experiments. Substitution mutations at G:C pairs were detected with enhanced frequency in the WRN-knockdown cells. The ratio of the untargeted substitutions at G:C sites (G:C → A:T, G:C → T:A, and G:C → C:G mutations at positions other than position 122) in the knockdown cells was 60% (34 mutations for the 57 colonies analyzed) and was higher than that of the targeted G:C → T:A mutation (Table 2). Meanwhile, the ratio of the substitutions at G:C sites in the control cells was only 32% (26 mutations for the 81 colonies analyzed). We then calculated the frequency of the untargeted substitution mutations at G:C pairs. The total supF mutant frequencies were multiplied by the percentages of the untargeted substitutions at G:C sites for each transfection experiment. The calculated frequencies were 0.4 (±0.2) and 2.5 (±0.6) × 10−3 in the control and WRN experiments, respectively (standard errors are shown in parentheses). These values were statistically different (P < 0.01), suggesting that the knockdown of WRN increased the substitution

We then transfected the plasmid DNAs containing a single G:C or GO:C pair at position 122 of the supF gene into the U2OS cells treated with the siRNA. The replicated DNA was recovered from the transfected cells and was then introduced into the indicator KS40/pOF105 E. coli strain, after the unreplicated DNA was removed.35 Typically, 100−150 supF mutant colonies were obtained on selection plates (10-fold dilution), and ∼300 total colonies were formed on titer plates (1,000-fold dilution), when the GO:C-derived plasmid DNA recovered from the transfected cells was electroporated into the KS40/pOF105 indicator strain. The mutant frequency was determined according to the numbers of mutant and total colonies, as described in the Materials and Methods section. The electroporation experiments were repeated several times in single transfection experiments, and the transfection into U2OS cells was independently conducted eight times. Judging by the numbers of colonies on the titer plates, which semiquantitatively reflected the amounts of plasmid DNA replicated in the cells, no obvious effects of the DNA helicase knockdown were observed. For example, three electroporation experiments were conducted in the eighth GO-transfection experiment, and total 1,210 (484, 362, and 364) and 1,031 (479, 248, and 304) colonies were formed on titer plates for the control and WRNknockdown cells, respectively (after 1,000-fold dilution). Thus, the WRN knockdown did not seem to have severe effects on replication efficiency. The supF mutant frequency for the cells treated with the siRNA against WRN was similar to that for the control siRNA in the experiments using the G:C plasmid (∼1 × 10−3, Figure 2, open columns), indicating that the reduction in the amount of the DNA helicase did not affect the background mutant frequency. The supF mutant frequency was 2.8 × 10−3 when the plasmid DNA with GO:C was transfected into U2OS cells treated with the control siRNA, indicating the mutagenicity of GO (Figure 2). Meanwhile, the mutant frequency was 4.6 × 10−3, representing a 1.6-fold (or 1.8 × 10−3) increase, in the 623


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Chemical Research in Toxicology Table 1. Mutations Detected in the supF Genea

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.41 We examined the effects of the knockdown of WRN on the mutations induced by GO:A, the intermediate in the dGOTP mutation pathway. We constructed plasmid DNAs containing a single T:A or GO:A pair at position 96 in the supF gene. This position was one of the A:T → C:G hot spots in the supF gene when dGOTP was introduced.26,27 The plasmid DNAs were transfected into the U2OS cells with knocked-down WRN. The treatment with the siRNA against WRN did not affect the supF mutant frequencies in the experiments using the T:Aplasmid (∼5 × 10−4, Figure 3A). The supF mutant frequency was quite high, 94 × 10−2 (94%), when the plasmid DNA with the GO:A pair at position 96 was introduced into U2OS cells treated with the control siRNA (Figure 3B). The removal of the A opposite GO by the MUTYH protein causes this high mutant frequency.28 The supF mutant frequency was decreased to 92 × 10−2 in the WRN-knockdown cells. Although the knockdown effect was small, it was statistically significant, suggesting that WRN may enhance the GO:A-induced mutations. We analyzed the sequences of the supF genes from the colonies on the selection agar plates. As expected, the A:T → C:G transversion was found in all progeny plasmid DNAs of the GO:A-plasmid (32 and 33 colonies for the control and knockdown experiments, respectively). Thus, the WRN protein was suggested to possibly contribute to the A:T → C:G mutations caused by GO:A.

G:C control 5G→C, 65G→A, 67G→A, 112G→A, 126G→C 65G→A, 67G→A, 112G→A, 126G→C 68T→G 70C→G, 71C→G 73G→T 74A→G 89C→A 91G→C 91G→A, 92A→T 91G→A, 112G→T 91G→A, 122G→T 121G→C 121G→C, 122G→T 130C→A 133T→△, 142A→C large deletion total colonies analyzed

si-WRN 2

5G→A, 91G→T

2

1

70C→T, 71C→T

1

2 1 1 1 2

71C→A 85G→A, 162G→A 91G→C 95C→G 91G→C, 92A→T, 118G→ A, 126G→C 118G→A 124, 125TC→TTC 130C→A 131C→T, 153C→T large deletion

2 1 2 6 1

1 1 1 1 1 1 1 1 6 24 total colonies analyzed GO:C

control

1 2 1 1 3

23

si-WRN

5G→A, 78G→A, 85G→A, 112G→C 51C→G, 95C→G, 147C→T 95C→G, 111C→A 101C→G 118G→A

2

5G→T, 67G→A, 84G→T

1

2 2 1 1

1 1 1 2

121G→T 122G→A 122G→T 130C→A large insertion large deletion

5 1 39 1 5 22

total colonies analyzed

81

5G→C, 86G→C 5G→C, 91G→T 27G→T, 66G→T, 73G→C 27G→A, 118G→A, 126G→T 66G→T, 126G→A 73G→T, 91G→C 88G→A, 101C→A 91G→C 95C→G 96T→G 96T→C 101C→T, 117C→G 118G→A 118G→C 121G→T 122G→T large insertion large deletion total colonies analyzed

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DISCUSSION In this study, we unexpectedly found that GO paired with C induced action-at-a-distance mutations in the WRN-knockdown cells (Table 2). As shown in Figure 2, the reduction of WRN significantly increased total supF mutant frequency when the plasmid DNA containing a GO:C pair in the supF gene was transfected. This result suggested that WRN suppresses the mutagenesis by GO in human cells. Since the oxidized base induces G:C → T:A transversion mutations, we had expected an increase in the transversion at the modified position (position 122), as in our previous studies in which BER enzymes and DNA pols η and ζ were knocked down.28,30,31 However, this type of mutation was minor in the WRNknockdown cells. Instead, substitution mutations at G:C sites other than position 122 were frequently found. The frequency of these untargeted mutations was significantly enhanced by the knockdown of WRN. Thus, WRN prevented the occurrence of the untargeted mutations induced by GO. The action-at-adistance mutations seemed to be broadly distributed in the supF gene, although the untargeted and background mutations could not be distinguished (Table 2 and Supporting Information, Figure 1B). We discuss possible mechanisms of the untargeted mutations in the following paragraphs: (i) reduced direct removal of mismatch, (ii) reduced mismatch repair (MMR) activity, (iii) involvement of error-prone TLS DNA pols, and (iv) secondary oxidation product(s) of GO. One possible explanation is the increased mismatch formation induced by GO plus WRN-reduction. WRN possesses 3′ → 5′ exonuclease activity, and it could prevent mismatch formation during replication. The WRN exonuclease activity excises 3′-terminal mismatches to enable primer extension by DNA pols β and δ.5,42 Moreover, the presence of damaged bases, including GO, reportedly inhibits or blocks the WRN exonuclease activity.43,44 The WRN protein was

1 1 1 2 2 2 1 1 2 1 3 21 1 12 57

a

Mutations detected in single colonies are represented. The sequence of the upper strand is shown. The numbers of colonies are shown on the right side.

mutations at untargeted G:C sites. When we focused on the DNA strand containing the GO base (the strand shown in Supporting Information, Figure 1), statistically significant untargeted substitution frequencies at G sites were found as well: 0.2 (±0.1) and 1.6 (±0.4) × 10−3 in the control and WRN experiments, respectively (P < 0.01). Thus, the GO base induced action-at-a-distance mutations at G or G:C sites in the WRN-knockdown cells. Effects of WRN Knockdown on GO:A-Induced Mutations. An oxidized form of dGTP, 8-oxo-7,8-dihydro-2′deoxyguanosine 5′-triphosphate (dGOTP), specifically induces A:T → C:G transversions in living cells.26,27,39,40 This type of 624


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Chemical Research in Toxicology Table 2. Spectra of Mutations Detected in the supF Genea GO:C

G:C control mutations at position 122 G:C → A:T G:C → T:A G:C → C:G mutations at other positions transition A:T → G:C G:C → A:T transversion A:T → T:A A:T → C:G G:C → T:A G:C → C:G small insertion (1−2 bp) large insertion (>2 bp) small deletion (1−2 bp) large deletion (>2 bp) others total mutations total colonies analyzed a

si-WRN

control

si-WRN

0(0) 2(8) 0(0)

0(0) 0(0) 0(0)

1 (1) 39 (48) 0(0)

0(0) 21 (37) 0(0)

1 (4) 12 (50)

0(0) 10 (43)

0(0) 9 (11)

1 (2) 10 (18)

1 (4) 3(13) 5(21) 10 (42) 0(0) 0(0) 1 (4) 6(25) 0(0) 41 24 (100)

1 (4) 0(0) 5(22) 10 (43) 2(9) 0(0) 0(0) 3(13) 0(0) 31 23 (100)

0(0) 0(0) 8 (10) 9 (11) 0(0) 5(6) 0(0) 22(27) 0(0) 93 81 (100)

0(0) 2(4) 13 (23) 11 (19) 0(0) 1 (2) 0(0) 12 (21) 0(0) 71 57 (100)

All data are represented as cases found (%).

increase in the mutant frequency was observed for the GOplasmid but not for the control plasmid (Figure 2). The WRN protein reportedly interacts with various DNA pols. WRN functionally interacts and forms a complex with DNA pol δ.42,47 WRN and DNA pol β physically interact, and the DNA pol β activity is stimulated via the WRN helicase activity.4 Moreover, the translesion synthesis (TLS) DNA pols η, ι, κ, and λ interact with WRN.48−50 We previously reported that DNA pols η, ζ, and κ are involved in TLS of the GO base.30,31 Thus, such TLS DNA pols might be abundant in/ near the replication complexes and relatively frequently loaded on the template DNA when the DNA regions near the GO base are replicated. Assuming that WRN plays a role in the recruiting and/or loading of high-fidelity DNA pol(s), the reduction of WRN could promote DNA synthesis by errorprone TLS DNA pols, and this might cause the untargeted mutations in the presence of GO. However, to our knowledge, mismatch formation by the TLS pols is more likely to occur at A:T sites rather than G:C sites. For example, human DNA pol η frequently misinserts dGTP opposite T and dATP plus dGTP opposite A.51 Likewise, the misincorporations induced by human DNA pols ι, κ, and λ are opposite T and A.52−56 Moreover, the mismatches opposite A and T are characteristic for yeast DNA pol ζ, and the mutational pattern of the mouse DNA pol ζ (Rev3 L2610F mutator) knock-in mice is a moderate increase in mutations at T bases.57,58 Thus, the TLS DNA pols, at least pols η, ι, κ, λ, and ζ, did not seem to be involved in the untargeted mutations observed in the present study. Pagano et al. examined (i) GO in leukocyte DNA, (ii) glutathione in whole blood, and (iii) glyoxal, methylglyoxal, uric acid, and ascorbic acid levels in plasma from Werner syndrome patients and concluded that the patients were in a prooxidant state.59 In addition, higher levels of serum and cardiac tissue ROS were detected in mice lacking part of the helicase domain of the murine WRN protein.60 These reports suggested that higher amounts of ROS were produced in the WRN-

Figure 3. Effects of WRN knockdown in U2OS cells on the mutant frequency 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. Transfection experiments were performed three (T:A) and four (GO:A) times. Data are expressed as the means + standard errors. *P < 0.05 vs control siRNA.

present in the knockdown cells, albeit to a lesser extent than the control cells (Figure 1). However, the function of the remaining WRN protein could be inhibited by the GO base. Thus, the untargeted mutations might have been formed when the GO-plasmid was introduced into the WRN-knockdown cells. WRN physically interacts with the heterodimer proteins involved in MMR, and nuclear extracts from cell lines derived from Werner syndrome patients were deficient in MMR.45,46 These studies suggested that WRN plays a role in MMR. Assuming that WRN stimulates MMR, the mismatches would remain in the DNA in the knockdown cells. However, this second explanation is apparently invalid since a significant 625


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knockdown cells than in the control cells. The GO base has redox activities, and its oxidation potential is lower than those of the four unmodified bases, suggesting that GO radicals may be formed more frequently in the knockdown cells.61−63 Moreover, the G bases around GO are vulnerable when DNA fragments are oxidized,64,65 and radical cations can migrate on DNA over distances longer than 55 bp.66,67 Thus, the action-ata-distance mutations induced by GO in the knockdown cells could be attributed to the formation of G damages distant from the modified position. In this case, the GO base at position 122 would be oxidized, and the G:C → T:A mutation observed might be induced by the secondary oxidation product(s) of GO. Further studies are necessary to examine this possibility. Phosphorylated H2AX (γH2AX) and p53 binding protein 1 nuclear foci, representing DNA damage responses, are induced after WRN-knockdown in primary human fibroblasts.68 In addition, the numbers of foci in the WRN-deficient cells are modestly higher immediately after exposure to H2O2, and several-fold higher after 28 h, than those in control cells. These observations suggested that the WRN-depleted cells are defective in oxidative DNA damage repair. This feature of the WRN-knockdown cells might be related to the untargeted mutations observed in the present study. Das et al. reported that WRN stimulates the NEIL1 DNA glycosylase for GO and that the number of GO lesions in DNA is higher in WRN-deficient cells.33 Moreover, the knockdown of NEIL1 promotes the G:C → T:A transversion mutations induced by GO.28 Thus, an increase in the targeted G:C → T:A transversion at position 122 had been expected, and this was our initial hypothesis. However, the results described in this study do not agree with this expectation since only a slight enhancement of this type of mutation was observed. The Bloom and RECQL4 proteins are also human homologues of the E. coli RecQ helicase and are responsible for the Bloom and Rothmund Thomson syndromes, respectively. When the plasmid DNAs containing GO were introduced into U2OS cells treated with siRNAs against these proteins, the frequencies of untargeted substitution mutations were also elevated, although the total supF mutant frequencies were similar to those of control cells (Yamazaki et al., unpublished results). Thus, the three human RecQ helicases contribute to high-fidelity DNA replication when GO is present at the replication fork. The frequency of the A:T → C:G transversion induced by the GO:A pair was slightly but significantly decreased by WRN knockdown (Figure 3B). Similar reductions were observed when DNA pols η and λ were knocked down.30,32 Since WRN interacts with these pols, the interactions might be related to the mutation pathway of the GO:A pair.48−50 Additional studies from various viewpoints will be needed to reveal role(s) of WRN on this issue. In conclusion, GO induced action-at-a-distance mutations at G:C sites when the WRN protein was knocked down. This work demonstrates again that experiments using DNA with site-directed modifications are useful in mutagenesis studies.69 The present results suggested that similar mutagenesis events occur in Werner syndrome patients and cause their cancer predispositions. Elucidation of the detailed mechanism(s) of the untargeted mutagenesis is quite important, and additional experiments are currently in progress.

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ASSOCIATED CONTENT

S Supporting Information *

Overall distribution of the substitution mutations detected in the supF gene. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Phone: +81-82-257-5300. Fax: +81-82-257-5334. 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 (20012001 and 25550032), and the Takeda Science Foundation to H.K. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Drs. Motoshi Suzuki, Shiro Koizume, Hideo Inoue, and Takemi Enomoto for discussions. ABBREVIATIONS BER, base excision repair; dGOTP, 8-oxo-7,8-dihydro-2′deoxyguanosine 5′-triphosphate; GO, 8-oxo-7,8-dihydroguanine; MMR, mismatch repair; ODN, oligodeoxyribonucleotide; pol, polymerase; qPCR, quantitative PCR; ROS, reactive oxygen species; RT, reverse transcription; TLS, translesion synthesis

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