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[Cancer Research 66, 748-754, January 15, 2006]
© 2006 American Association for Cancer Research

Molecular Biology, Pathobiology, and Genetics

Critical Roles for Polymerase {zeta} in Cellular Tolerance to Nitric Oxide–Induced DNA Damage

Xiaohua Wu1, Katsuya Takenaka1,2, Eiichiro Sonoda1,2, Helfrid Hochegger1,2, Shosuke Kawanishi3, Takuo Kawamoto1,2, Shunichi Takeda1,2 and Mitsuyoshi Yamazoe1,2

1 Department of Radiation Genetics, Graduate School of Medicine, Kyoto University, Yoshida Konoe, Sakyo-ku, Kyoto, Japan; 2 Core Research for Evolutional Science and Technology, Japan Science and Technology, Saitama, Japan; and 3 Department of Environmental and Molecular Medicine, Mie University School of Medicine, Edobashi, Tsu, Mie, Japan

Requests for reprints: Mitsuyoshi Yamazoe, Department of Radiation Genetics, Faculty of Medicine, Kyoto University, Konoe Yoshida, Sakyo-ku, Kyoto 606-8501, Japan. Phone: 81-75-753-4410; Fax: 81-75-753-4419; E-mail: yamazoe{at}rg.med.kyoto-u.ac.jp.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Nitric oxide (NO), a signal transmitter involved in inflammation and regulation of smooth muscle and neurons, seems to cause mutagenesis, but its mechanisms have remained elusive. To gain an insight into NO-induced genotoxicity, we analyzed the effect of NO on a panel of chicken DT40 clones deficient in DNA repair pathways, including base and nucleotide excision repair, double-strand break repair, and translesion DNA synthesis (TLS). Our results show that cells deficient in Rev1 and Rev3, a subunit essential for DNA polymerase {zeta} (Pol{zeta}), are hypersensitive to killing by two chemical NO donors, spermine NONOate and S-nitroso-N-acetyl-penicillamine. Mitotic chromosomal analysis indicates that the hypersensitivity is caused by a significant increase in the level of induced chromosomal breaks. The data reveal the critical role of TLS polymerases in cellular tolerance to NO-induced DNA damage and suggest the contribution of these error-prone polymerases to accumulation of single base substitutions. (Cancer Res 2006; 66(2): 748-54)


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Nitric oxide (NO) is generated by a variety of cells to regulate numerous biological processes, including signal transduction and macrophage-mediated immunity. However, excessive or prolonged exposure to NO may lead to a number of pathophysiologic processes including carcinogenesis (1, 2). The carcinogenesis seems to be caused by accumulation of mutations rather than by promotion of tumorigenesis as previous studies have shown that NO induces mutagenesis both in vivo as well as in vitro (3–7). In vitro studies indicate that NO itself is not very reactive with purified DNA. It can react in vivo with oxygen and superoxide to create potent DNA-damaging agents, reactive nitrogen species such as N2O3 and peroxynitrite. NO-mediated oxidation products may cause direct deamination, oxidation, or nitration of bases, strand breaks, cross-links, and possibly inhibition of some repair enzymes (8–12). It is believed that among these lesions, base damage and strand break seem to be the major lesion induced by NO (13, 14). In fact, apurinic/apyrimidinic sites, products following base damage, are the earliest detectable lesion induced by NO in Chinese hamster ovary cells (4). However, the mechanisms underlying the NO-induced mutagenesis have hardly been investigated because the vast majority of NO-induced base damage is quickly and accurately repaired by base excision repair. Furthermore, the genotoxic effect of NO could not be specifically addressed in in vivo experiments because the exposure of cells to high doses of NO provokes a wide variety of cellular responses involving signal transduction.

Large numbers of base damage and single-strand breaks arise daily as the genomic DNA is constantly assaulted by environment mutagens and cellular activities. Resulting DNA lesions are repaired by excision repair pathways including base excision repair, nucleotide excision repair, and single-strand break repair pathways (reviewed in ref. 15). DNA damage, if left unrepaired, may arrest DNA replication, leading to double-strand breaks and single-strand gaps on the relevant sister chromatids. These lesions are processed by two major postreplication repair pathways: homologous recombination repair and translesion DNA synthesis (TLS; ref. 16). Homologous recombination releases the replication block by facilitating DNA synthesis using the other intact sister as a template strand whereas TLS fills a daughter strand gap, employing a number of specialized error-prone DNA polymerases, including Pol{eta} (Rad30), Pol{kappa}, Pol{theta}, Pol{zeta}, and a regulatory protein, Rad18 (reviewed in ref. 17). A defect in homologous recombination or TLS results in an increase in the level of double-strand breaks, which can be evaluated by counting chromosomal breaks in mitotic cells. Double-strand breaks that arise during replication are preferentially repaired by homologous recombination whereas ionizing radiation–induced double-strand breaks are mainly repaired by nonhomologous end-joining in mammalian cells (reviewed in ref. 18).

Pol{zeta} seems to play an important role in TLS because DT40 cells deficient in Pol{zeta} display hypersensitivity to a variety of DNA damaging agents, including UV, X-rays, H2O2, monofunctional alkylating agent (methyl methanesulfonate), cross-linking agents such as cisplatin and nitrogen mustard, 4OH-estradiol, and tamoxifen, an antiestrogen agent (19). Pol{zeta} is composed of two subunits, Rev3, the catalytic subunit, and Rev7. In addition, genetic studies with yeast and DT40 cells have shown that Pol{zeta} may intimately collaborate with Rev1 to perform TLS (ref. 20; reviewed in ref. 21). Rev3 plays an essential role in genome maintenance in mammals as the disruption of the Rev3 gene in mice results in early embryonic lethality (22–24). In yeast, Pol{zeta} is the major source of base substitutions and frameshifs not only after exogenous DNA damage but also during physiologic cell cycle (ref. 25; reviewed in ref. 21). Although the contribution of mammalian Pol{zeta} to spontaneous mutagenesis has remained elusive, the critical role for Pol{zeta} in genome maintenance as well as its error-prone character suggests that Pol{zeta} may play a dominant role in spontaneous mutagenesis.

We here examined the toxicity of NO toward cells using a panel of gene-disrupted DT40 clones deficient in DNA repair pathways. DT40 cells possess a number of advantages as a tool for studying the DNA repair pathway that is coupled with DNA replication (reviewed in ref. 26). First, because ~60% of cycling cells are in S phase, exogenous DNA damage may have a direct effect on DNA replication. Second, the absence of functional p53, which induces apoptosis on DNA damage, seems to contribute to a sensitive evaluation of genome instability by measuring chromosomal breaks in mitotic cells because cells carrying double-strand breaks can enter the M phase without stimulating the apoptosis pathway. Thus, DT40 cells allow us to reliably identify the environmental mutagens that cause replication block by simply doing chromosome analysis, as we previously showed the genotoxic potential of tamoxifen (19). Our present study proves that Pol{zeta} is a critical factor for tolerating NO toxicity and prevents chromosomal breaks probably by releasing replication block at NO-damaged template strands.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Chemicals. Spermine NONOate (SPER/NO) was obtained from Sigma (St. Louis, MO). Spermine and S-nitroso-N-acetyl-penicillamine (SNAP) were purchased from Wako (Osaka, Japan). Stock solutions of SNAP (1 mol/L) and SPER/NO (1 mol/L) were prepared freshly every time before use in DMSO and 0.1 mol/L NaOH, respectively. Stock solution of spermine (1 mol/L) was prepared in PBS and stored at –20°C.

Cell lines and cell culture. Cell lines used in this study are listed in Table 1. Some of these phenotypes have been previously described (27–32). Cells were cultured as described before (28).


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  		Table 1. DNA repair and checkpoint genes mutated in the analyzed DT40 clones

 
Colony formation assay. The colony formation assay was done as previously described (28). Cells (1 x 105) were incubated at 39.5°C in 1 mL of complete medium containing the NO donor for 1 hour. The cells were also treated with 1% DMSO or 1% 0.1 mol/L NaOH as control.

Measurement of cell cycle distribution. Cells (5 x 105/mL) were treated with 2 mmol/L SPER/NO or 4 mmol/L SNAP in the complete medium for 1 hour, and then washed with PBS twice. Cells were labeled for 10 minutes with 20 µmol/L 5-bromo-2'-deoxyuridine (BrdUrd) and subsequently harvested every 3 hours. Harvested cells were fixed and analyzed as previously described (33).

Measurement of the length of cell cycle using BrdUrd pulse-chase labeling. Cells (5 x 105/mL) were labeled for 10 minutes with 20 µmol/L BrdUrd. Cells were washed with PBS once and treated with 2 mmol/L SPER/NO in the complete medium for 1 hour, followed by washing with PBS twice. Cells were harvested every 2 hours. Harvested cells were fixed at 4°C overnight with 70% ethanol and analyzed as previously described (28).

Analysis of chromosomal aberrations. Karyotype analysis was done as previously described (33). Briefly, 5 x 105 cells/mL were treated with 2 mmol/L SPER/NO in the complete medium for 1 hour and harvested every 3 hours. To enrich mitotic cells, cells were treated with 0.1 µg/mL of colcemid (Invitrogen, Carlsband, CA) during the final 3 hours before harvest. A portion of the fixed cells was dropped onto a wet (with 50% ethanol) glass slide and immediately flame dried. The slides were stained with 3% Giemsa solution at pH 6.4 for 20 minutes. Chromosomal aberrations were assessed by microscope.

Measurement of NO-induced sister chromatid exchange levels. Measurement of sister chromatid exchange (SCE) levels was done as previously described (34). Briefly, cells were treated with 25 or 50 µmol/L of SPER/NO for the last 8 hours of incubation with 10 µmol/L BrdUrd for 18 hours. The cells were also treated with 0.1 µg/mL of colcemid for the final 2 hours of the incubation to enrich mitotic cells.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
rev3 cells are sensitive to NO. To comprehensively investigate NO-induced damage response, we exposed a panel of gene-disrupted clones (Table 1) to NO and evaluated cellular response using a colony survival assay. Figure 1A shows a representative of this assay wherein REV3–/– (hereafter called rev3) and wild-type cells were treated for 1 hour with different concentrations of SPER/NO, which has been widely used as a NO donor. As a control, the cells were treated with spermine, which does not release NO (Fig. 1B). Interestingly, rev3 cells exhibited marked increase in sensitivity to 1 to 3 mmol/L SPER/NO when compared with wild-type cells (Fig. 1A) whereas neither rev3 nor wild-type cells showed elevated sensitivity to the spermine control (Fig. 1B). Thus, NO may be responsible for the observed hypersensitivity of the rev3 mutant. To confirm this conclusion, the cells were exposed to another NO donor, SNAP, and rev3 cells also exhibited significant hypersensitivity (Fig. 1C). These observations support the idea that Rev3 plays a key role in tolerance to genotoxicity caused by NO.


Figure 1
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  		Figure 1. Sensitivity of wild-type (WT) and rev3 cells to SPER/NO, spermine, and SNAP. A to C, the fractions of surviving colonies after the indicated treatment compared with untreated controls of the same genotype are shown on the y axis with a logarithmic scale. A, SPER/NO. B, spermine. C, SNAP. D, sensitivity of wild-type and rev3 cells to 0.5 mmol/L SPER/NO with different exposure time. 1% DMSO or 0.1 mol/L NaOH did not show any significant effects on cell survival. Representative results from at least six independent experiments for SPER/NO and SNAP. Points, mean; bars, SD.

 
To further evaluate the effect of NO, we exposed cells to a lower concentration of SPER/NO for different periods, ranging from 45 to 135 minutes, and subsequently measured their plating efficiency in NO-free medium. It is known that the release of NO from SPER/NO declines to half every 39 minutes on addition of the agent to the culture medium whereas the half-life of released NO is around a few seconds in the medium. The colony survival of rev3 cells, but not of wild-type cells, was reduced nearly exponentially as incubation time with NO was linearly increased (Fig. 1D). The data are consistent with the idea that released NO causes DNA damage that may be processed by Rev3-dependent mechanisms.

Using the colony formation assay, the sensitivity of other genotypes to NO was measured. The relative sensitivity of each genotype is shown by calculating the LD50 (i.e., the dose of NO that reduces the cellular viability to 50% in the colony formation assay; Fig. 2A and B). As expected, rev1 cells exhibited NO sensitivity very similar to that of rev3 cells, which is in agreement with previous results (20). Among the TLS-deficient gene-disrupted clones, neither pol{eta} nor pol{kappa} clones were sensitive to NO. To assess the role of base excision repair in cellular response to NO, we examined fen1, polß, and parp1 cells, which are all partially defective in base excision repair (32).4 fen1 cells, but not polß or parp1 cells, showed a slight increase in NO sensitivity. We may have failed to identify the important contribution of base excision repair to cellular tolerance to NO presumably because complete depletion of base excision repair seems to be lethal to the cells (35). To investigate the roles of two major double-strand break repair pathways, homologous recombination–deficient rad54 and xrcc3 cells as well as nonhomologous end-joining–deficient ku70 and DNA-pkcs cells were analyzed. These mutants did not show hypersensitivity to NO, which is apparently different from the high sensitivity of each pol{zeta}, ku70, and rad54 mutant to tamoxifen (19). Likewise, atm cells, which are defective of double-strand break–induced damage checkpoint (36, 37) and susceptible to oxidative stress (38), did not display hypersensitivity to NO treatment. The fancd2 mutant exhibited only little sensitivity, if any, although previous reports indicate generation of interstrand cross-link by NO (11) and that FancD2 is involved in cellular tolerance to cross-linking agents (39). In summary, significant increase in NO sensitivity is detected specifically in the rev1 and rev3 mutants.


Figure 2
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  		Figure 2. Toxicity profile of SPER/NO and SNAP on a panel of the repair-deficient DT40 mutants. The relative LD50 dose was calculated from two to three independent experiments and normalized according to the LD50 values of parental wild-type cells. A, SPER/NO. B, SNAP. Columns, mean; bars, SD.

 
NO treatment induces extensive G2 phase arrest. To understand the causal relationship between the rev mutation and NO hypersensitivity, we first studied the effect of NO treatment on cell cycle and cell death. To this end, cells were treated with either 2 mmol/L SPER/NO or 4 mmol/L SNAP for 1 hour, wherein the colony survival was reduced to 40% to 50% in wild-type and to 5% to 6% in rev3 cells (Fig. 1A and C), and their cell cycle progression was followed every 3 hours up to 32 hours using BrdUrd pulse labeling. Figure 3A shows representative cell cycle distribution of wild-type and rev3 cells at 18 and 32 hours after SPER/NO or SNAP exposure. Transient cell cycle arrest in the G2 phase is a common checkpoint response to DNA damage in DT40 cells as ionizing radiation causes G2 arrest for up to 2 hours (36, 37). To our surprise, in marked contrast with only a 2-hour delay in G2 following ionizing radiation, cell cycle delay in the G2 phase persisted for a more extended time after both SPER/NO and SNAP treatment (Fig. 3B and C). Cell accumulation in G2 reached the maximum at 12 hours and remained up to 18 hours after NO donor treatments in both wild-type and rev3 cells.


Figure 3
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  		Figure 3. Cell cycle analysis of wild-type and rev3 cells in response to NO treatment. Wild-type and rev3 cells were exposed to 2 mmol/L SPER/NO or 4 mmol/L SNAP for 1 hour. A, representative cell cycle distribution of wild-type and rev3 cells at 18 and 32 hours after SPER/NO or SNAP exposure. Cells were pulse-labeled for 10 minutes and subsequently stained with FITC-anti-BrdUrd to detect BrdUrd incorporation (y axis, log scale) and with propidium iodide to detect total DNA (x axis, linear scale). The lower-left gate identifies G1 cells, the upper gate identifies cells incorporating BrdUrd (~S phase), and the lower-right gate identifies G2-M cells. Sub-G1 cells indicated on the most left gate reflect dead cells. Numbers show the percentages of cells decreasing in each gate. B and C, kinetic change of cell population in G2 phase following SPER/NO (B) or SNAP (C) exposure. The percentages of G2 phase cells were calculated by fluorescence-activated cell sorting as indicated in (A) (lower-right box). The x axis indicates time after NO donor treatment. –, experiment without NO donor treatment. Columns, mean of two experiments; bars, SD. D, the calculated relative percentages of wild-type and rev3 cells in mid S phase with or without SPER/NO exposure are plotted with time. The progression of BrdUrd-labeled (i.e., S phase) cells out of S phase and into first G2-M (4n DNA content), then G1 (2n DNA content), and back into S phase with time is indicated. X axis, period of chase after treatment with 2 mmol/L of SPER/NO for 1 hour.

 
We also evaluated the effect of NO on the cell cycle by a pulse-chase experiment (Fig. 3D). Following exposure to SPER/NO, both BrdUrd-labeled wild-type and rev3 cells began to enter the next mid S phase at 10 hour, which is 2 hours longer than for the normal cell cycle time for both types of cells. Thus, cell cycle delay at the G2 phase seems to vary from 2 to 24 hours among individual cells.

In wild-type cells, the cell cycle had almost fully recovered by 32 hours whereas increased cell death (sub-G1) was observed in rev3 cells (Fig. 3A). These observations support the idea that NO may cause DNA lesions that are left unrepaired in rev3 cells, resulting in activation of G2 checkpoint. This accumulation in G2 eventually leads to cell death correlating well with the reduced plating efficiencies that we observed in the colony formation assays (Fig. 1A and C).

Pol{zeta}-deficient cells exhibit significant increase in the level of NO-induced chromosomal aberrations. To understand the cause of cell death, we measured cytologically detectable chromosomal breaks in mitotic cells. To this end, asynchronous populations of wild-type and rev3 cells were exposed to 2 mmol/L SPER/NO for 1 hour and mitotic cells were subsequently harvested every 3 hours. Cells that were treated with SPER/NO in the G2 phase can enter the M phase within 3 hours. Interestingly, wild-type cells showed a biphasic pattern of induced chromosomal breaks; two peaks were detectable at 0 to 3 and 6 to 9 hours (Fig. 4), indicating that exposure of cells to NO in G2 and S phases may significantly induce chromosomal breaks, which are repaired with time. Likewise, rev3 cells also exhibited biphasic pattern with the latter peak appearing at 12 to 15 hours after SPER/NO exposure. Remarkably, in rev3 cells, the vast majority of NO-induced chromosomal aberrations were of isochromatid type wherein two sister strands were broken at the same sites. Furthermore, the level of chromosomal aberrations at the second peak was significantly higher in rev3 cells than in wild-type cells. In summary, Pol{zeta} can prevent chromosomal breaks induced by NO in cycling cells.


Figure 4
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  		Figure 4. NO-induced chromosome aberrations (CA) and SCE in wild-type and rev3 cells. A, NO-induced chromosome aberrations of wild-type and rev3 cells. Cells were exposed to 2 mmol/L SPER/NO for 1 hour. B, SCE events in wild-type and rev3 cells with or without SPER/NO treatment. Cells were treated with 25 or 50 µmol/L of SPER/NO for 18 hours. Columns, mean; bars, SE.

 
Replication block can be released by homologous recombination and TLS, which seem to function complementarily to each other (27, 40). In fact, TLS-deficient cells, such as rad18 and pol{zeta} cells, consistently show increased levels of spontaneously arising SCE (27, 40), which may reflect homologous recombination associated with crossover (34). Following SPER/NO treatment, marked induction of SCE events was found in wild-type cells whereas only a few SCEs were induced in rev3 cells (Fig. 4B). These observations may shed light on the complex actions of Pol{zeta} in cellular tolerance to NO-induced DNA damage.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Here we found that the gene-disrupted, TLS mutant pol{zeta} cells are hypersensitive to two NO donors, exhibiting marked increase in chromosomal breaks. NO concentration in the medium in the present experiments is 20 to 30 µmol/L, which is ~10 times higher than the local NO concentration wherein macrophages are stimulated at the site of inflammation (41, 42). Thus, prolonged in vivo exposure to this level of NO generated from macrophages is expected to be well compatible with 1-hour exposure to the NO concentration used in this study. We therefore conclude that substantial quantities of NO generated by chronic inflammation may in turn lead to unwanted collateral damage to normal neighboring tissues, including DNA damage and resulting replication block, which occasionally leads to chromosomal breaks as shown in this study.

NO-induced DNA damage leads to prolonged G2 arrest. The data from the pulse BrdUrd labeling and pulse-chase experiments indicate that the effect of NO treatment on each cell varied remarkably, with some cells restarting the cell cycle after only 2 hours of delay (Fig. 3D) and the majority of the cells staying in the G2 phase for >12 hours (Fig. 3B and C). Furthermore, whereas recovery from the G2 arrest began at a similar time in both genotypes, the recovery tended to take longer in rev3 cells (18 hours) than in wild-type cells (12 hours). Such marked divergence in the extent of G2 arrest among individual cells was not found following other genotoxic stressors such as ionizing radiation (36). Another difference between the NO treatment and ionizing radiation is the extent of cell killing relative to that of transient cell cycle arrest. Despite the marked cell cycle delay in the G2 phase following exposure to the NO donors, substantial fractions of wild-type cells appeared to restart cell cycling as 1-hour exposure of wild-type cells to 2 mmol/L SPER/NO reduced the colony survival to only 40% to 50%. This is in contrast with ionizing radiation–induced G2 arrest wherein LD40 to 50% dose of ionizing radiation (i.e., 2 Gy for wild-type cells) results in modest G2 delay only up to 2 hours in DT40 cells (36, 37). Presumably, the prolonged G2 arrest may be attributed to altered protein function and gene expression involved in cell cycle progression (43), DNA repair (12), or replication enzymes (44). Alternatively, compared with ionizing radiation, NO may induce a larger number of less toxic DNA damage that blocks DNA replication. In this scenario, NO-treated cells would stay for a longer time in the G2 phase to complete DNA replication. The longer G2 arrest in the absence of Rev3 is in agreement with the notion that Pol{zeta} contributes to DNA replication as a TLS polymerase.

Rev3 releases NO-induced replication block. NO-induced chromosomal aberrations exhibit a couple of unique characteristics. Mitotic cells displayed a biphasic pattern of chromosomal aberrations after an asynchronous population of wild-type cells were transiently exposed to the NO donor, with the first and second peaks appearing at 3 and 9 hours. Because a majority of the mitotic cells that enter within 3 hours should have been exposed to NO in the G2 phase (27), an increase in the level of chromatid breaks at 3 hours suggests that NO may directly induce double-strand breaks. Wild-type and rev3 cells showed similar levels of chromatid breaks, indicating that Rev3 is not involved in double-strand break repair. This observation is in marked contrast with the significant increase in ionizing radiation–induced chromosomal breaks in rev1 and rev3 cells compared with wild-type cells (20, 27). The second peak likely reflects double-strand breaks caused by replication block at NO-induced lesions. The second peak observed in pol{zeta} cells differs from that in wild-type cells in the following two points. First, a higher peak appeared at a later time, 15 hours post-NO treatment. This delayed appearance of the second peak is consistent with longer retention of cells in the G2 phase in rev3 cells compared with wild-type cells. Second, a majority of the chromosomal aberrations at 15 hours in rev3 cells were isochromatid-type breaks. This observation is in contrast with the data that cisplatin mainly induces chromatid-type breaks (45, 46). There are three possible explanations for the occurrence of isochromatid-type breaks: (a) double-strand breaks or interstrand cross-link at template DNA for replication; (b) defective resolution during homologous recombination between sister chromatids (47); and (c) replication collapse at DNA replication block. Because DT40 mutants defective in double-strand break repair, such as rad54, ku70, and DNA-pk, or mutants defective in interstrand cross-link repair, such as fancD2, did not show significant sensitivity to NO, the first explanation is unlikely. The second explanation assumes the involvement of Pol{zeta} in the resolution of homologous recombination although this notion is not supported by any biochemical study on yeast Pol{zeta}. Finally, supposing that NO, but not cisplatin, may inhibit DNA repair or replication enzymes, replication machinery would be more susceptible to breakdown especially in the absence of TLS polymerase. This notion may explain that different types of chromosomal breaks are induced by NO and cisplatin. Obviously, further study is required to understand the inhibitory effect of NO on enzymes involved in DNA metabolism.

We found a significant increase in the level of SCE induced by NO in wild-type cells. This indicates that NO treatment may increase the number of DNA lesions that are left unrepaired before replication. These unrepaired lesions result in the stalling and collapsing of replication forks, which have to be restarted by homologous recombination using the other intact sister-chromatid as a template. rev3 cells showed fewer SCE compared with wild-type cells after NO treatment. This can be explained by reduced homologous recombination functions because Rev3 as well as Pol{eta} plays a role in some homologous recombination reactions (27, 48).

Pol{zeta} is involved in cellular tolerance to NO-induced DNA damage and accumulation of mutations. NO and its oxidative products induce deamination, nitration, and oxidation of guanine, producing xanthine, 8-nitro-guanine, and 8-oxo-guanine, respectively. In particular, xanthine and 8-nitro-guanine are unstable in DNA and can be quickly converted to apurinic/apyrimidinic sites through depurination (9). Apurinic/apyrimidinic sites might be the major lesion that requires Pol{zeta} for efficient TLS although the exact role of mammalian Pol{zeta} in TLS remains to be elucidated because no biochemical study has successfully reconstituted its polymerase activity. Studies in yeast have shown that Pol{zeta} can efficiently bypass abasic sites by extending from nucleotides inserted opposite the lesion by other TLS DNA polymerases (49). Likewise, Pol{zeta} may be involved in the extension step in higher eukaryotic cells. Pol{zeta}-dependent TLS past apurinic/apyrimidinic sites likely results in extensive accumulation of point mutations. Indeed, G:C to T:A transversion is the most common form of base substitution induced by peroxynitrite (50) and most frequently seen in p53 gene from liver and lung cancers (51). Future biochemical study on Pol{zeta} may allow for evaluation of the contribution of Pol{zeta} to each type of single base substitutions.

NO seems to be associated with progression of Alzheimer's disease (52). The data imply that NO damages neurons not only by affecting signal transduction but also by increasing base damages in a Pol{zeta}-dependent manner, although the role of Pol{zeta} in resting cells remains to be defined. In addition to the pathologic role, NO recently has been considered as a potential therapeutic agent for cancer (53). To screen for better agents that have less mutagenic potential, the Pol{zeta}-deficient DT40 would be useful because of rapid proliferation and relatively stable phenotype even in the absence of Pol{zeta}.


   Acknowledgments
 
Grant support: Core Research for Evolutional Science and Technology, Japan Science and Technology (Saitama, Japan); the Center of Excellence grant for Scientific Research from the Ministry of Education, Culture, Sports and Technology; the Uehara Memorial Foundation and the Naito Foundation; and the Virtual Research Institute of Aging of Nippon Boehringer Ingelheim.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   Footnotes
 
4 Tano et al., in preparation; Hochegger et al., submitted. Back

Received 8/15/05. Revised 10/12/05. Accepted 11/11/05.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Tamir S, Tannenbaum SR. The role of nitric oxide (NO.) in the carcinogenic process. Biochim Biophys Acta 1996;1288:F31–6.[Medline]
  2. Wink DA, Vodovotz Y, Laval J, Laval F, Dewhirst MW, Mitchell JB. The multifaceted roles of nitric oxide in cancer. Carcinogenesis 1998;19:711–21.[Abstract/Free Full Text]
  3. Nguyen T, Brunson D, Crespi CL, Penman BW, Wishnok JS, Tannenbaum SR. DNA damage and mutation in human cells exposed to nitric oxide in vitro. Proc Natl Acad Sci U S A 1992;89:3030–4.[Abstract/Free Full Text]
  4. Tamir S, Burney S, Tannenbaum SR. DNA damage by nitric oxide. Chem Res Toxicol 1996;9:821–7.[CrossRef][Medline]
  5. Burney S, Tamir S, Gal A, Tannenbaum SR. A mechanistic analysis of nitric oxide-induced cellular toxicity. Nitric Oxide 1997;1:130–44.[CrossRef][Medline]
  6. Zhuang JC, Lin C, Lin D, Wogan GN. Mutagenesis associated with nitric oxide production in macrophages. Proc Natl Acad Sci U S A 1998;95:8286–91.[Abstract/Free Full Text]
  7. Routledge MN. Mutations induced by reactive nitrogen oxide species in the supF forward mutation assay. Mutat Res 2000;450:95–105.[Medline]
  8. Kennedy LJ, Moore KJ, Caulfield JL, Tannenbaum SR, Dedon PC. Quantitation of 8-oxoguanine and strand breaks produced by four oxidizing agents. Chem Res Toxicol 1997;10:386–92.[CrossRef][Medline]
  9. Yermilov V, Rubio J, Ohshima H. Formation of 8-nitroguanine in DNA treated with peroxynitrite in vitro and its rapid removal from DNA by depurination. FEBS Lett 1995;376:207–10.[CrossRef][Medline]
  10. Horiike S, Kawanishi S, Kaito M, et al. Accumulation of 8-nitroguanine in the liver of patients with chronic hepatitis C. J Hepatol 2005;43:403–10.[CrossRef][Medline]
  11. Caulfield JL, Wishnok JS, Tannenbaum SR. Nitric oxide-induced interstrand cross-links in DNA. Chem Res Toxicol 2003;16:571–4.[Medline]
  12. Liu L, Xu-Welliver M, Kanugula S, Pegg AE. Inactivation and degradation of O(6)-alkylguanine-DNA alkyltransferase after reaction with nitric oxide. Cancer Res 2002;62:3037–43.[Abstract/Free Full Text]
  13. Phoa N, Epe B. Influence of nitric oxide on the generation and repair of oxidative DNA damage in mammalian cells. Carcinogenesis 2002;23:469–75.[Abstract/Free Full Text]
  14. Zhou X, Liberman RG, Skipper PL, Margolin Y, Tannenbaum SR, Dedon PC. Quantification of DNA strand breaks and abasic sites by oxime derivatization and accelerator mass spectrometry: Application to {gamma}-radiation and peroxynitrite. Anal Biochem 2005;343:84–92.[CrossRef][Medline]
  15. Barnes DE, Lindahl T. Repair and genetic consequences of endogenous DNA base damage in mammalian cells. Annu Rev Genet 2004;38:445–76.[CrossRef][Medline]
  16. Hochegger H, Sonoda E, Takeda S. Post-replication repair in DT40 cells: translesion polymerases versus recombinases. Bioessays 2004;26:151–8.[CrossRef][Medline]
  17. Friedberg EC, Lehmann AR, Fuchs RP. Trading places: how do DNA polymerases switch during translesion DNA synthesis? Mol Cell 2005;18:499–505.[CrossRef][Medline]
  18. van Gent DC, Hoeijmakers JH, Kanaar R. Chromosomal stability and the DNA double-stranded break connection. Nat Rev Genet 2001;2:196–206.[CrossRef][Medline]
  19. Mizutani A, Okada T, Shibutani S, et al. Extensive chromosomal breaks are induced by tamoxifen and estrogen in DNA repair-deficient cells. Cancer Res 2004;64:3144–7.[Abstract/Free Full Text]
  20. Okada T, Sonoda E, Yoshimura M, et al. Multiple roles of vertebrate REV genes in DNA repair and recombination. Mol Cell Biol 2005;25:6103–11.[Abstract/Free Full Text]
  21. Lawrence CW, Maher VM. Eukaryotic mutagenesis and translesion replication dependent on DNA polymerase {zeta} and Rev1 protein. Biochem Soc Trans 2001;29:187–91.[CrossRef][Medline]
  22. Bemark M, Khamlichi AA, Davies SL, Neuberger MS. Disruption of mouse polymerase {zeta} (Rev3) leads to embryonic lethality and impairs blastocyst development in vitro. Curr Biol 2000;10:1213–6.[CrossRef][Medline]
  23. Esposito G, Godindagger I, Klein U, Yaspo ML, Cumano A, Rajewsky K. Disruption of the Rev3l-encoded catalytic subunit of polymerase {zeta} in mice results in early embryonic lethality. Curr Biol 2000;10:1221–4.[CrossRef][Medline]
  24. Wittschieben J, Shivji MK, Lalani E, et al. Disruption of the developmentally regulated Rev3l gene causes embryonic lethality. Curr Biol 2000;10:1217–20.[CrossRef][Medline]
  25. Harfe BD, Jinks-Robertson S. DNA polymerase {zeta} introduces multiple mutations when bypassing spontaneous DNA damage in Saccharomyces cerevisiae. Mol Cell 2000;6:1491–9.[CrossRef][Medline]
  26. Yamazoe M, Sonoda E, Hochegger H, Takeda S. Reverse genetic studies of the DNA damage response in the chicken B lymphocyte line DT40. DNA Repair (Amst) 2004;3:1175–85.[Medline]
  27. Sonoda E, Okada T, Zhao G, et al. Multiple roles of Rev3, the catalytic subunit of pol{zeta} in maintaining genome stability in vertebrates. EMBO J 2003;22:3188–97.[CrossRef][Medline]
  28. Takata M, Sasaki MS, Sonoda E, et al. Homologous recombination and non-homologous end-joining pathways of DNA double-strand break repair have overlapping roles in the maintenance of chromosomal integrity in vertebrate cells. EMBO J 1998;17:5497–508.[CrossRef][Medline]
  29. Bezzubova O, Silbergleit A, Yamaguchi-Iwai Y, Takeda S, Buerstedde JM. Reduced X-ray resistance and homologous recombination frequencies in a RAD54–/– mutant of the chicken DT40 cell line. Cell 1997;89:185–93.[CrossRef][Medline]
  30. Fukushima T, Takata M, Morrison C, et al. Genetic analysis of the DNA-dependent protein kinase reveals an inhibitory role of Ku in late S-G2 phase DNA double-strand break repair. J Biol Chem 2001;276:44413–8.[Abstract/Free Full Text]
  31. Okada T, Sonoda E, Yamashita YM, et al. Involvement of vertebrate pol{kappa} in Rad18-independent postreplication repair of UV damage. J Biol Chem 2002;277:48690–5.[Abstract/Free Full Text]
  32. Matsuzaki Y, Adachi N, Koyama H. Vertebrate cells lacking FEN-1 endonuclease are viable but hypersensitive to methylating agents and H2O2. Nucleic Acids Res 2002;30:3273–7.[Abstract/Free Full Text]
  33. Sonoda E, Sasaki MS, Buerstedde JM, et al. Rad51-deficient vertebrate cells accumulate chromosomal breaks prior to cell death. EMBO J 1998;17:598–608.[CrossRef][Medline]
  34. Sonoda E, Sasaki MS, Morrison C, Yamaguchi-Iwai Y, Takata M, Takeda S. Sister chromatid exchanges are mediated by homologous recombination in vertebrate cells. Mol Cell Biol 1999;19:5166–9.[Abstract/Free Full Text]
  35. Izumi T, Brown DB, Naidu C, et al. Two essential but distinct functions of the mammalian abasic endonuclease. Proc Natl Acad Sci U S A 2005;102:5739–43.[Abstract/Free Full Text]
  36. Morrison C, Sonoda E, Takao N, Shinohara A, Yamamoto K, Takeda S. The controlling role of ATM in homologous recombinational repair of DNA damage. EMBO J 2000;19:463–71.[CrossRef][Medline]
  37. Takao N, Kato H, Mori R, et al. Disruption of ATM in p53-null cells causes multiple functional abnormalities in cellular response to ionizing radiation. Oncogene 1999;18:7002–9.[CrossRef][Medline]
  38. Ito K, Hirao A, Arai F, et al. Regulation of oxidative stress by ATM is required for self-renewal of haematopoietic stem cells. Nature 2004;431:997–1002.[CrossRef][Medline]
  39. Yamamoto K, Hirano S, Ishiai M, et al. Fanconi anemia protein FANCD2 promotes immunoglobulin gene conversion and DNA repair through a mechanism related to homologous recombination. Mol Cell Biol 2005;25:34–43.[Abstract/Free Full Text]
  40. Yamashita YM, Okada T, Matsusaka T, et al. RAD18 and RAD54 cooperatively contribute to maintenance of genomic stability in vertebrate cells. EMBO J 2002;21:5558–66.[CrossRef][Medline]
  41. Ioannidis I, Batz M, Paul T, Korth HG, Sustmann R, De Groot H. Enhanced release of nitric oxide causes increased cytotoxicity of S-nitroso-N-acetyl-DL-penicillamine and sodium nitroprusside under hypoxic conditions. Biochem J 1996;318:789–95.[Medline]
  42. Lewis RS, Tamir S, Tannenbaum SR, Deen WM. Kinetic analysis of the fate of nitric oxide synthesized by macrophages in vitro. J Biol Chem 1995;270:29350–5.[Abstract/Free Full Text]
  43. Pervin S, Singh R, Chaudhuri G. Nitric oxide-induced cytostasis and cell cycle arrest of a human breast cancer cell line (MDA-MB-231): potential role of cyclin D1. Proc Natl Acad Sci U S A 2001;98:3583–8.[Abstract/Free Full Text]
  44. Lepoivre M, Flaman JM, Bobe P, Lemaire G, Henry Y. Quenching of the tyrosyl free radical of ribonucleotide reductase by nitric oxide. Relationship to cytostasis induced in tumor cells by cytotoxic macrophages. J Biol Chem 1994;269:21891–7.[Abstract/Free Full Text]
  45. Yonetani Y, Hochegger H, Sonada E, et al. Differential and collaborative actions of Rad51 paralog proteins in celluloar response to DNA damage. Nucleic Acids Res 2005;33:4544–52.[Abstract/Free Full Text]
  46. Nojima K, Hochegger H, Saberi A, et al. Multiple repair pathways mediate tolerance to chemotherapeutic cross-linking agents in vertebrate cells. Cancer Res 2005;65:11704–11.
  47. French CA, Masson JY, Griffin CS, O'Regan P, West SC, Thacker J. Role of mammalian RAD51L2 (RAD51C) in recombination and genetic stability. J Biol Chem 2002;277:19322–30.[Abstract/Free Full Text]
  48. Kawamoto T, Araki K, Sonada E. Dual roles for DNA polymerase h in homologous DNA recombination and translesion dna synthesis. Mol Cell. In press 2005.
  49. Haracska L, Unk I, Johnson RE, et al. Roles of yeast DNA polymerases {delta} and {zeta} and of Rev1 in the bypass of abasic sites. Genes Dev 2001;15:945–54.[Abstract/Free Full Text]
  50. Jeong JK, Juedes MJ, Wogan GN. Mutations induced in the supF gene of pSP189 by hydroxyl radical and singlet oxygen: relevance to peroxynitrite mutagenesis. Chem Res Toxicol 1998;11:550–6.[CrossRef][Medline]
  51. Loeb LA, Preston BD. Mutagenesis by apurinic/apyrimidinic sites. Annu Rev Genet 1986;20:201–30.[CrossRef][Medline]
  52. Zhang J, Dawson VL, Dawson TM, Snyder SH. Nitric oxide activation of poly(ADP-ribose) synthetase in neurotoxicity. Science 1994;263:687–9.[Abstract/Free Full Text]
  53. Zanon M, Piris A, Bersani I, et al. Apoptosis protease activator protein-1 expression is dispensable for response of human melanoma cells to distinct proapoptotic agents. Cancer Res 2004;64:7386–94.[Abstract/Free Full Text]
  54. Simpson LJ, Sale JE. Rev1 is essential for DNA damage tolerance and non-templated immunoglobulin gene mutation in a vertebrate cell line. EMBO J 2003;22:1654–64.[CrossRef][Medline]
  55. Takata M, Sasaki MS, Tachiiri S, et al. Chromosome instability and defective recombinational repair in knockout mutants of the five Rad51 paralogs. Mol Cell Biol 2001;21:2858–66.[Abstract/Free Full Text]



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