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. 2014 Jan 16;29(2):107–114. doi: 10.1093/mutage/get067

An increase of oxidised nucleotides activates DNA damage checkpoint pathway that regulates post-embryonic development in Caenorhabditis elegans

Yu Sanada 1, Qiu-Mei Zhang-Akiyama 1,*
PMCID: PMC3924892  PMID: 24435662

Abstract

8-Oxo-dGTP, an oxidised form of dGTP generated in the nucleotide pool, can be incorporated opposite adenine or cytosine in template DNA, which can in turn induce mutations. In this study, we identified a novel MutT homolog (NDX-2) of Caenorhabditis elegans that hydrolyzes 8-oxo-dGDP to 8-oxo-dGMP. In addition, we found that NDX-1, NDX-2 and NDX-4 proteins have 8-oxo-GTPase or 8-oxo-GDPase activity. The sensitivity of ndx-2 knockdown C. elegans worms to methyl viologen and menadione bisulphite was increased compared with that of control worms. This sensitivity was rescued by depletion of chk-2 and clk-2, suggesting that growth of the worms is regulated by the checkpoint pathway in response to the accumulation of oxidised nucleotides. Moreover, we found that the sensitivity to menadione bisulphite of ndx-1 and ndx-2-double knockdown worms was enhanced by elimination of XPA-1, a factor involved in nucleotide excision repair. The rescue effect by depletion of chk-2 and clk-2 was limited in the xpa-1 mutant, suggesting that the chk-2 and clk-2 checkpoint pathway is partially linked to the function of XPA-1.

Introduction

Oxidative damage to bases occurs through normal cellular metabolism and through exposure to ionizing radiation and various chemical oxidising agents (1). Among such damaged bases, an oxidised form of guanine, 8-oxo-7,8-dihydroguanine (8-oxoG), can alter genetic information since it pairs with adenine and cytosine (2,3). 8-oxo-dGTP, an oxidised form of dGTP generated in the nucleotide pool, is incorporated opposite adenine or cytosine of template DNA, causing A:T to C:G or G:C to T:A transversions (4,5).

To prevent such deleterious effects, Escherichia coli MutT degrades 8-oxo-dGTP and 8-oxo-dGDP to 8-oxo-dGMP, and thereby prevents incorporation of the 8-oxoG-containing nucleotide (1,6). Mammalian cells also possess enzymes that eliminate 8-oxoG-containing nucleotides from the nucleotide pool. These include MTH1 (5,7), MTH2 (NUDT15) (8), MTH3 (NUDT18) (9) and NUDT5 (10). MTH1 and MTH2 preferentially degrade 8-oxodGTP, whereas NUDT5 and MTH3 hydrolyse 8-oxo-dGDP but show little activity towards 8-oxo-dGTP. In Caenorhabditis elegans, two MutT homologs, NDX-1 and NDX 4, have been identified (11,12). NDX-1 cleaves 8-oxo-dGDP but not 8-oxo-dGTP, while NDX-4 cleaves 8-oxo-dGTP but not 8-oxo-dGDP. In this study, we searched for another MutT homolog of C. elegans and found a novel hydrolase for 8-oxo-dGDP encoded by ndx-2.

Furthermore, it has been shown that 8-oxoG can be incorporated into RNA by RNA polymerase (13). 8-oxo-GTP is incorporated opposite adenine in the DNA template during transcription and the altered transcripts could lead to mistranslated proteins. E. coli MutT protein has the ability to degrade both 8-oxo-GDP and 8-oxo-GTP in the nucleotide pool (14,15). Many MutT homologs have hydrolytic activity towards 8-oxo-GTP or 8-oxo-GDP (9,16). Thus, we investigated whether the NDX proteins act on 8-oxoG-containing ribonucleotides.

Eukaryotes respond to DNA damage by activating the DNA damage response (DDR) pathway (17). The DDR includes DNA repair processes and checkpoint pathways that regulate cell cycle progression and affect development, growth rate, lifespan and sensitivity to DNA damage (18). In C. elegans, checkpoint pathways are conserved and DNA damage checkpoint genes such as chk-2 and clk-2 have been described (19). The CLK-2 checkpoint protein has been found to be involved in the activation of cell cycle arrest and apoptosis in response to misincorporation of uracil (20). Interaction between the base excision repair (BER) pathway and activation of the DDR signalling pathway through CLK-2 has been described (20). We examined whether the DDR pathway is activated in response to an increased amount of oxidised nucleotides or their misincorporation. Here, we report that CHK-2 and CLK-2 checkpoint proteins function in post-embryonic development and growth control.

If incorporated into DNA, the 8-oxoG lesion should be repaired. Bacteria and eukaryotes are equipped with mechanisms to prevent mutations by 8-oxoG (21). One such mechanism is BER for oxidatively damaged bases in DNA. In E. coli, MutM removes 8-oxoG paired with cytosine in DNA from DNA (21,22). In yeast and mammalian cells, 18-Oxoguanine glycosylase (OGG1) has been shown to remove 8-oxoG to initiate BER (23,24). In contrast, little is known about repair pathway(s) processing 8-oxoG in DNA in C. elegans. C. elegans lacks homologs of E. coli MutM and mammalian OGG1. We carried out a survey of repair pathways that would act on 8-oxoG-containing nucleotides. Here, we report that XPA-1, which is a factor acting in nucleotide excision repair (NER), seems to be involved in repair of incorporated 8-oxoG, and that activation of the checkpoint pathway is partially linked to the function of XPA-1.

Materials and Methods

Assay for the sensitivity of E. coli to oxidative stress

Plasmids used in this experiment were previously documented (11). E. coli CC101-harboring pGEX4T-1, CC101 mutT-harboring pGEX4T-1 or CC101 mutT-expressing NDX-2 was grown to stationary phase in 5ml of Luria-Bertani (LB) medium containing 100 μg/ml ampicillin at 37°C. Appropriate dilutions of each culture were plated on LB plates containing methyl viologen (MV). After incubation at 37°C for 20h, the number of colonies was counted to estimate survival.

Assay for enzymatic activity

C. elegans NDX-1, NDX-2 and NDX-4 were purified as previously described (11). 8-oxo-dGTP was purchased from TriLink Biotechnologies (San Diego, CA, USA) and 8-oxo-dGDP and 8-oxo-GTP were purchased from Cosmo Bio (Tokyo, Japan). 8-oxo-GDP was prepared as the product of hydrolysis of 8-oxo-GTP by NDX-4. Reaction mixtures (25 μl) containing 20mM Tris-HCl (pH 8.0), 4mM MgCl2, 40mM NaCl, 8mM dithiothreitol, 5% glycerol and each substrate at 20 μM were incubated at 37°C for 30min with purified NDX protein. The reaction was terminated by adding 25 μl of 5mM EDTA. The mixture was subjected to high-performance liquid chromatography (HPLC) using a TSK-GEL DEAE-2SW column (Tosoh, Tokyo, Japan) at a flow rate of 1.0ml/min for the mobile phase buffer (75mM sodium phosphate, pH 6.0, 1mM EDTA and 20% acetonitrile). The substrates and reaction products were detected by measuring ultraviolet absorbance at 254nm for dGMP, dGDP, dGTP, GMP, GDP and GTP, or 293nm for 8-oxo-dGMP, 8-oxo-dGDP, 8-oxo-dGTP, 8-oxo-GMP, 8-oxo-GDP and 8-oxo-GTP.

C. elegans strains and culture conditions

C. elegans N2, RB1054 ndx-4 (ok1003), RB1572 mlh-1 (ok1917), RB877 nth-1 (ok724) and RB864 xpa-1 (ok698) were obtained from the Caenorhabditis Genetics Center (Minneapolis, MN, USA). C. elegans worms were cultured at 20°C on NGM agar plates (0.3% NaCl, 0.25% polypeptone, 0.005% cholesterol, 1mM MgSO4, 1mM CaCl2, 25mM potassium phosphate, pH 6.0 and 0.17% agar) with a lawn of E. coli OP50.

Bacteria-mediated RNA interference

For knockdown experiments, we used the well-established RNA interference (RNAi) method (25,26). C. elegans ndx-1, ndx-2, chk-2 (Y60A3A.12) and clk-2 (C07H6.6) complementary DNA (cDNA) clones were amplified by PCR from a cDNA library using the following primers: for ndx-1, 5′-CCAAGCAAGCTTCCACTTGGAAAGTTG-3′ (forward with a HindIII site) and 5′-AGGCTCGAGTTAAAGCATATGAAGTGACGG-3′ (reverse with an XhoI site); for ndx-2, 5′-AGGGAAAAAAGCGGCCGCACGT CATCGGCCACA-3′ (forward with a NotI site) and 5′-CTATCTGC AGCTAGATCGTGGCGAATT-3′ (reverse with a PstI site); for chk-2, 5′-TTAAGCGCGGCCGCATGGTTCGCGGGACA-3′ (forward with a NoII site) and 5′-TTAAGCCTGCAGTCACATTTTTGCCTTTTTCACAG-3′ (reverse with a PsII site) and for clk-2, 5′-TTAAGCAAGCTTATGAATTTAC GAAGTCGCCT-3′ (forward with a HindIII site) and 5′-TTAAGCCTCGAGTG CGTAATTGAGATCACTC-3′ (reverse with an XhoI site). The amplified PCR products were subcloned into plasmid L4440 for bacteria-mediated feeding RNAi (RNAi plasmid). Double RNAi experiments were performed by mixing equal amounts of overnight cultures of E. coli HT115 (DE3) that had been transformed with the respective RNAi plasmids and then plating the mixture on NGM plates containing 1mM IPTG (RNAi plates). The transformant-harboring L4440 was used for a negative control of RNAi and for single knockdown experiments. To confirm the effect of RNAi, reverse transcription–polymerase chain reaction was carried out with total RNA prepared from RNAi and control worms (supplementary Figure 1, available at Mutagenesis Online).

Assay for survival of C. elegans in adulthood under oxidative conditions

To assay survival of C. elegans in adulthood, young adult worms were transferred to plates containing 5mM menadione bisulphite and survival was monitored daily. This protocol was similar to the assay for lifespan (11), except that the plates contained 5mM menadione bisulphite.

Assay for the sensitivity of C. elegans to oxidative stress

To assay sensitivity to menadione bisulphite during development, eggs were placed on RNAi plates containing MV or menadione bisulphite. After incubation at 20°C for 4 days, L4 and adult worms were counted and the number of L4 and adults/the number of eggs was determined. Smaller larvae were also counted on the previous day and the number of L1 to L3 larvae/the number of eggs was determined. Then, to determine growth in the first filial generation (F1) progeny animals, eggs were collected from the ndx-4 mutants that developed to adulthood on RNAi plates containing 0mM (untreated) or 0.5mM menadione bisulphite and placed on NGM plates in the absence of menadione bisulphite. After incubation for four additional days, L4 and adult worms were counted and the number of (F1 L4 and adults)/the number of F1 eggs placed on NGM plates was determined.

Results

Complementation of sensitivity to MV of E. coli mutT by NDX-2

Previous studies revealed that there are at least two MutT homologs, NDX-1 and NDX 4, in C. elegans (11,12). We first examined whether C. elegans possess additional proteins with pyrophosphatase activity towards 8-oxo-dGTP and/or 8-oxo-dGDP. C. elegans NDX-2 is predicted to be an ADP sugar diphosphatase orthologous to human NUDT5. NUDT5 also has 8-oxo-dGDPase activity and plays an important role in preventing mutations. Expression of NDX-2 was able to reduce the mutation frequency in E. coli mutT, as previously described (11,12). To examine whether NDX-2 complements the sensitivity of E. coli mutT mutants to MV, we compared the survival of E. coli CC101 mutT expressing NDX-2 with that of wild-type CC101 and CC101 mutT. We found that E. coli CC101 mutT-expressing NDX-2 was more resistant to MV compared with CC101 mutT bearing the vector alone (supplementary Figure 2, available at Mutagenesis Online). Therefore, in this study, we first focussed on the properties of NDX-2 of C. elegans as an E. coli MutT homolog.

Purification and enzymatic activity of C. elegans NDX-2

C. elegans NDX-2 was expressed as a glutathione S-transferase (GST)-fused protein in E. coli CC101 mutT, the GST-NDX-2 fusion protein was purified by glutathione-Sepharose 4B column chromatography, and the GST-tag was removed with thrombin (Figure 1A). Assays for enzyme activities were carried out using 20 μM 8-oxo-dGDP or 8-oxo-dGTP as substrate, and the products were analyzed by HPLC. As shown in Figure 1B, NDX-2 degraded 8-oxo-dGDP to its monophosphate form, and hydrolysis of 8-oxo-dGTP was hardly detected. The substrate specificity of NDX-2 is shown in Table I.

Fig. 1.

Fig. 1.

Fig. 1.

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Cleavage activity of C. elegans NDX proteins against 8-oxoG-containing nucleotides. (A) Purification of NDX-2 and NDX-4. NDX-2 and NDX-4 were obtained by the cleavage of the GST-fused protein with thrombin. Proteins were analysed by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (12% polyacrylamide) and stained with Coomassie Brilliant blue R 250. Lane 1, molecular weight markers; Lane 2, purified NDX-2 or NDX-4 protein. The arrows indicate purified NDX-2 and NDX-4. (B) Substrate specificity of NDX-2. 8-oxo-dGDP or 8-oxo-dGTP after incubation at 37°C for 30min without (top) or with (bottom) 5 μM purified NDX-2. 8-Oxo-dGMP, 8-oxo-dGDP and 8-oxo-dGTP are indicated by (1), (2) and (3), respectively. (C) Substrate specificity of NDX proteins. 8-Oxo-GDP or 8-Oxo-GTP after incubation at 37°C for 30min without or with 0.73 μM purified NDX-1, 5 μM NDX-2 or 1.51 μM NDX-4. 8-Oxo-GMP, 8-oxo-GDP and 8-oxo-GTP are indicated by (1), (2) and (3), respectively.

Table I.

Hydrolysis of oxidised and normal nucleotides by C. elegans NDX-1, NDX-2 and NDX-4

NDX-1 NDX-2 NDX-4
dGDP – 66.4% –
dGTP – – 24.0%
8-oxo-dGDP 62.7% 54.4% –
8-oxo-dGTP – – 59.5%
GDP –   5.40% –
GTP – – 33.7%
8-oxo-GDP 63.2% 25.2% –
8-oxo-GTP 10.5% – 34.3%
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Each substrate at 20 μM was incubated with purified NDX-1 (0.73 μM), NDX-2 (5 μM) or NDX-4 (1.51 μM) at 37°C for 30min. The hydrolysis percentage was measured by HPLC. Dashes indicate <5% hydrolysis.

E. coli MutT protein and many MutT homologs also have activity towards the oxidised ribonucleotide, as reported previously (9,14–16). We examined whether NDX-1, NDX-2 and NDX-4 also hydrolyze 8-oxo-GTP and/or 8-oxo-GDP. As shown in Figure 1C and Table I, NDX-1 and NDX-2 showed 8-oxo-GDPase activity. Under the same conditions, hydrolysis of 8-oxo-GTP by NDX-2 was hardly detected. NDX-4 showed 8-oxo-GTPase activity, while the hydrolysis of 8-oxo-GDP was not detected.

The effect of superoxide generators on survival in C. elegans treated in the adult stages

Previous studies showed that the lifespan of C. elegans is not affected by the knockdown of ndx-1 or knockout of ndx-4 (11,12). The lifespan of ndx-2-RNAi worms was also similar to that of control worms (data not shown). We next examined whether oxidative stress affects the survival of ndx-1- and ndx-2-RNAi worms and ndx-4 mutants. To test this, young adult worms were incubated on plates containing 5mM menadione bisulphite, a superoxide generator. The survival was assessed by the percentage of live worms. While ndx-1-RNAi and ndx-2-RNAi worms showed similar survival to control worms, mutation of ndx-4 decreased the survival of worms (Figure 2). Furthermore, at Day 7, 62% of ndx-4 mutants were dead and 95% of ndx-1 and ndx-2 double knockdown ndx-4 mutants were dead (P < 0.01, log-rank test). Double knockdown of ndx-1 and ndx-2 in ndx-4 mutants resulted in significantly lower survival, suggesting that NDX-1, NDX-2 and NDX-4 co-ordinately participate in sanitisation of the nucleotide pool.

Fig. 2.

Fig. 2.

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The survival of ndx-4 (ok1003) mutants in adulthood under oxidative conditions. ndx-4 (ok1003) and ndx-1; ndx-2 (double RNAi)/ndx-4 (ok1003) worms showed decreased survival on 5mM menadione bisulphite plates compared to control worms, The surviving population was counted daily from Day 3. Closed diamonds, control worms; closed squares, ndx-4 (ok1003) worms; closed triangles, ndx-1; ndx-2 (double RNAi)/ndx-4 (ok1003) worms.

Increased sensitivity to oxidative stress during post-embryonic development in C. elegans by knockdown and knockout of the ndx genes

Previous studies showed that ndx-4 mutants are slightly sensitive to paraquat (MV) (12). ndx-1-RNAi worms also show higher sensitivity to MV and menadione bisulphite compared with control worms (11). We compared the sensitivity of ndx-2-RNAi and control worms to MV or menadione bisulphite by assessing the decrease in the growth rate of the worms. The eggs of the worms were exposed to the agents on plates for 4 days. The percent of eggs that developed to adults was determined as the growth rate. As shown in Figure 3A, like ndx-1-RNAi worms, ndx-2-RNAi worms showed higher sensitivity to both MV and menadione bisulphite than the control worms.

Fig. 3.

Fig. 3.

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The sensitivity of ndx-1 (RNAi) and ndx-2 (RNAi) worms and ndx-4 (ok1003) mutants to MV and menadione bisulphite. (A) ndx-2 (RNAi) worms showed decreased development on MV and menadione bisulphite plates compared to control worms. Closed diamonds, control worms; closed triangles, ndx-2 (RNAi) worms. (B) ndx-4 (ok1003) worms similarly exhibited sensitivity to MV and menadione bisulphite. Co-application of ndx-1 (RNAi) and ndx-2 (RNAi) did not enhance the sensitivity of the ndx-4 mutant. Closed diamonds, control worms; closed squares, ndx-4 (ok1003) worms; closed triangles, ndx-1 (RNAi)/ndx-4 (ok1003) worms; closed circles, ndx-2 (RNAi)/ndx-4 (ok1003) worms; ×, ndx-1; ndx-2 (double RNAi)/ndx-4 (ok1003) worms. The ratio of the number of viable L4 and adults to the number of eggs was determined and normalised to the untreated control. The values represent the mean ± SD (n = 3).

It is likely that NDX-1, NDX-2 and NDX-4 co-ordinately degrade 8-oxoG-containing nucleotides. To test this, we examined whether the knockdown or knockout of multiple ndx genes increased the sensitivity to MV and menadione bisulphite. In this study, we used the feeding RNAi method for the knockdown of ndx-1 and ndx-2, and the ndx-4 mutant for the knockout of ndx-4. As shown in Figure 3B, unlike the case of survival in adulthood, application of ndx-1- and/or ndx-2-RNAi did not increase the sensitivity of the ndx-4 mutant.

The effect of knockdown of chk-2 and clk-2 on the sensitivity to oxidative stress

Although the number of L4-adult stage worms was decreased in the plates containing menadione bisulphite, a few small larvae were observed in these plates (Figure 4A). This suggested that some of the worms that did not reach the adult stage underwent retarded development or arrest at the larval stage rather than death under the oxidative condition. We hypothesised that activation of checkpoint pathways caused this arrest. In C. elegans, various checkpoint pathways are conserved and DNA damage checkpoint genes such as chk-2 and clk-2 have been described (19). It is known that cell cycle arrest and apoptosis are activated in response to 2′-deoxyuridine, 5′-triphosphate (dUTP) misincorporation into DNA in C. elegans (20). To assess whether the growth arrest involved CHK-2- and/or CLK2-mediated checkpoint pathways, we examined the effect of knockdown of chk-2 and clk-2 on the growth rate of ndx-1- or ndx-2-RNAi worms or ndx-4 mutants. While ndx-1- or ndx-2-RNAi worms or ndx-4 mutants showed sensitivity to menadione bisulphite, as shown in Figure 3B, the number of worms that were able to develop to adulthood was restored to the control level by the knockdown of chk-2 and clk-2 (Figure 4A). That is, the sensitivity was rescued by the knockdown of chk-2 and clk-2.

Fig. 4.

Fig. 4.

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Activation of checkpoint pathways in response to an increased amount of oxidised nucleotides. (A) Effect of knockdown of chk-2 and clk-2 on the sensitivity of ndx-1 (RNAi) and ndx-2 (RNAi) worms and ndx-4 mutants to 1mM bisulphite. The values indicate the number of viable L1–L3/the number of eggs (white bar) and viable L4 and adults/the number of eggs (gray bar). The values represent the mean ± SD (n = 3). *P < 0.05, **P < 0.01; t-test. (B) Comparison of growth under normal conditions of the first filial generation (F1) progeny animals of the ndx-4 (ok1003) mutants that were able to develop to adulthood under versus not under the oxidative condition caused by menadione bisulphite (0.5mM). Left panel shows a schematic depiction of the method. The ratio of the number of F1 L4-adults worms to the number of F1 eggs from P0 adult worms was determined and normalised by the value for the untreated control. The values represent the mean ± SD (n = 3). *P < 0.05, **P < 0.01; t-test versus ndx-4 mutant.

Despite the rescue of growth rate by the knockdown of chk-2 and clk-2, DNA damage seemed not to be repaired, which could result in serious physiological problems. We therefore examined the influence on the reproduction of the ndx-4 mutants that had evaded growth arrest at the larval stage and their progeny. All the adult worms in the chk-2- and clk-2-RNAi plates could lay eggs. We did not observe any obvious differences in the hatching rate compared to the control. We next examined the growth of the first filial generation (F1) progeny of the chk-2- and clk-2-RNAi worms. As shown in Figure 4B, the progeny of the chk-2- and clk-2-RNAi worms exhibited a lower growth rate than the progeny of the control RNAi worms.

The effect of elimination of xpa-1 on the sensitivity to oxidative stress

Once the abnormal nucleotides are incorporated, they should be removed and then the DNA should be repaired properly. Pathways including mismatch repair (MMR), BER and NER are conserved mechanisms for DNA repair in C. elegans. However, little is known about pathways for the repair of incorporated 8-oxoG in C. elegans. Multiple repair genes are conserved in C. elegans, including mlh-1 (MMR), nth-1 (BER) and xpa-1 (NER), and their knockout mutants, mlh-1 (ok1917), nth-1 (ok724) and xpa-1 (ok698), are available. We examined whether the knockout of the mlh-1, nth-1 or xpa-1 repair gene affects the sensitivity to menadione bisulphite in ndx-1- and ndx-2-double RNAi worms. As shown in Figure 5A, the knockout of mlh-1 or nth-1 did not affect the sensitivity. The knockout of xpa-1 slightly increased the sensitivity of the ndx-1-RNAi worms but did not significantly increase the sensitivity of the ndx-2-RNAi worms (Figure 5B). On the other hand, the knockout of xpa-1 increased the sensitivity of the ndx-1- and ndx-2-double RNAi worms (Figure 5A).

Fig. 5.

Fig. 5.

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A survey of DNA repair pathways that process the incorporated oxidised nucleotides. (A) Effect of knockout of DNA repair genes on the sensitivity of ndx-1 and ndx-2 (double RNAi) worms. (B) Effect of knockout of xpa-1 on the sensitivity of ndx-1 (single RNAi) and ndx-2 (single RNAi) worms. (C) Effect of knockdown of chk-2 and clk-2 on the increased sensitivity of ndx-1 (RNAi)/xpa-1 (ok698) and ndx-2 (RNAi)/xpa-1 (ok698) knockdown worms to menadione bisulphite. The values are shown as the number of viable L4 and adults/the number of eggs. The values represent the mean ± SD (n = 3). *P < 0.05; t-test.

As described above, knockdown of chk-2 and clk-2 rescued the sensitivity of ndx-1- and ndx-2-RNAi worms. We next examined whether knockdown of chk-2 and clk-2 rescues the sensitivity of ndx-1- and ndx-2-RNAi xpa-1 mutant. As shown in Figure 5C, the sensitivity was only slightly (not significantly) rescued by the knockdown of chk-2 and clk-2.

Discussion

MutT homologs that eliminate oxidised nucleotides have been identified in various organisms. In C. elegans, NDX-1 and NDX-4 selectively degrade 8-oxo-dGDP and 8-oxo-dGTP, respectively (11,12). We first focussed on the properties of NDX-2 of C. elegans as an E. coli MutT homolog. To examine the enzymatic activity of C. elegans NDX-2, the NDX-2 protein was purified (Figure 1A). Biochemical studies revealed that NDX-2, like NDX-1, hydrolyzes 8-oxo-dGDP, but not 8-oxo-dGTP, to the monophosphate form (Figure 1B). NDX-2 can hydrolyze normal dGDP as efficiently as 8-oxo-dGDP (Table I). This property is similar to that of NDX-4 in that these enzymes hydrolyse normal nucleotides (12). The 8-oxo-dGDPase activity of NDX-2 was significantly lower than that of NDX-1. This fact might explain why the suppression of mutagenesis by the expression of NDX-2 was not as efficient as that by NDX-1 or NDX-4 (11,12). However, NDX-1 and NDX-2 provide mutual backup in sanitisation of the nucleotide pool. As reported previously, 8-oxo-dGDP strongly inhibits 8-oxo-dGTPase activity of MTH1 (27). Likewise, the presence of 8-oxo-dGDP or dGDP reduces NDX-4’s 8-oxo-dGTPase activity (supplementary Figure 3, available at Mutagenesis Online). The enzymatic activity of NDX-2 towards both 8-oxo-dGDP and dGDP might be necessary to avoid a reduction of the activity of NDX-4. 8-oxoG incorporation into RNA is considered to cause translational errors resulting in the synthesis of abnormal proteins (13). Although 8-oxoG in DNA is removed by DNA glycosylases such as MutM, OGG1 and NEIL1 to initiate BER and repair using the other strand (22–24,28), 8-oxoG formed in RNA cannot be similarly removed. Therefore, organisms must be equipped with mechanisms to prevent transcriptional and translational errors caused by oxidative damage. MutT and many homologs of MutT also hydrolyse 8-oxo-GTP and/or 8-oxo-GDP (9,14,16). In this study, we examined the enzymatic activity of NDX-1, NDX-2 and NDX-4 towards oxidised ribonucleotides and found that NDX-1 and NDX-2 had 8-oxo-GDPase activity, and NDX-4 had 8-oxo-GTPase activity. These facts suggest that NDX-1, NDX-2 and NDX-4 are also involved in sanitisation of the ribonucleotide pool and play a critical role in preventing the synthesis of abnormal proteins. As shown in Figure 2, ndx-1, ndx-2 double RNAi ndx-4 mutants displayed lower survival when placed on plates containing menadione bisulphite in adulthood. In C. elegans, the germline is the only tissue in which cell divisions occur into adulthood (29). Therefore, the lower survival seems to have little to do with oxidatively damaged deoxyribonucleotides such as 8-oxo-dGTP. The lower survival may instead be explained by NDX’s inability to sanitise the ribonucleotide pool.

We examined the effect of MV and menadione bisulphite on growth rate to assess the sensitivity during development. As shown in Figure 3A, knockdown of ndx-2 caused sensitivity to both MV and menadione bisulphite. Although NDX-1, NDX-2 and NDX-4 may collaborate to prevent the misincorporation of 8-oxoG, knockdown of ndx-1 or ndx-2 did not enhance the sensitivity of the ndx-4 mutant (Figure 3B). We have, up to this point, determined the ratio of the number of viable L4 and adults, but a few small larvae were also observed in the plates containing the oxidizing agents. We hypothesised that some of the worms were arrested at the larval stage because the checkpoint pathways that regulate development are activated in response to an increase in the amount of oxidised nucleotides such as 8-oxo-dGTP (Figure 4A). To test this possibility, we examined the effect of the knockdown of chk-2 and clk-2, DNA damage checkpoint genes in C. elegans, on the sensitivity of ndx knockdown or knockout worms. As shown in Figure 4A, the sensitivity was rescued by the knockdown of chk-2 or clk-2. These results suggest that development is regulated by the function of CHK-2 and CLK-2. Checkpoint pathways are activated in response to dUTP misincorporation into DNA (20), and we speculate that the checkpoint pathways may also be activated in response to 8-oxo-dGTP misincorporation into DNA, although it is possible that the checkpoint pathways respond independently of misincorporation of 8-oxo-dGTP.

We next asked if adult worms that had evaded the growth arrest had some potential problem. To do this, we examined the effects on the reproduction of these adult worms and their progeny using the ndx-4 mutant. Although there were no abnormalities in the hatching rate of eggs from the adult worms, the growth of the first filial generation (F1) progeny of the chk-2- and clk-2-RNAi worms was retarded compared to that of control RNAi worms (Figure 4B). This result suggests that the checkpoint pathway is probably important to make sufficient time available for repair and to prevent transgenerational accumulation of DNA damage that would impair growth.

8-oxoG is generated in DNA by misincorporation of 8-oxo-dGTP. Therefore, it is also important to examine DNA repair pathways for 8-oxoG. The BER pathway is the main mechanism for removal of 8-oxoG in DNA (30). Furthermore, MMR plays a role in the prevention of the mutagenic effect of 8-oxoG (31,32). We examined whether the knockout of nth-1 and mlh-1 affects the sensitivity of ndx-1, ndx-2 double RNAi worms to menadione bisulphite and found there were no changes in the sensitivity. C. elegans lacks homologues of E. coli MutM and mammalian OGG1 that remove 8-oxoG from DNA. Although C. elegans NTH-1, a homologue of endonuclease III, was reported to have activity towards 8-oxoG paired with G, this activity is very weak (33). Therefore, we tested the possibility that another pathway is primarily involved in the repair of 8-oxoG in C. elegans. In yeast, NER also contributes to the release of 8-oxoG in damaged DNA (32). It has been proposed that oxidative lesions relevant to aging are normally repaired by NER in C. elegans (34). Thus, we examined whether the knockout of xpa-1 affected the sensitivity of ndx-1, ndx-2 double RNAi worms to menadione bisulphite and found that knockout of xpa-1 greatly increased the sensitivity (Figure 5A). XPA-1 is an orthologue of the human NER gene XPA. It is generally reported that short-patch BER accounts for the majority of 8-oxoG repair and that NER may function in the repair of 8-oxoG. Reduced repair of oxidative DNA damage in XP-A cell extracts has previously been reported (35,36). In C. elegans, the xpa-1 (ok698) mutant is not hypersensitive to MV in adulthood and shows mild sensitivity to exposure to juglone, a superoxide generating agent (34,37). The xpa-1 (ok698) mutant was not sensitive to menadione bisulphite during development (Figure 5A). The ndx-1- and ndx-2-single knockdown xpa-1 mutants were slightly sensitive and not sensitive, respectively (Figure 5B). In contrast, the ndx-1- and ndx-2-double knockdown xpa-1 mutant was hypersensitive to menadione bisulphite (Figure 5A). The result suggests that XPA-1 plays an important role in defence against oxidative stress during development in C. elegans. The oxidising compounds used in this study, MV and menadione bisulphite, produce superoxide. It was reported that superoxide may participate in reactions to generate hydroxyradical, one of the most reactive free radicals (38), and the oxidation of DNA by a hydroxyradical has been reported to generate 8-oxoG (39). The oxidising agents are thought to induce the production of oxidised nucleotides, such as 8-oxo-dGTP or 8-oxo-dGDP. In addition, they should also induce oxidisation of bases existing in DNA strands. However, hypersensitivity to menadione bisulphite was observed when ndx genes were knocked down. We think therefore that XPA-1 may be involved in repair of 8-oxoG and the function of XPA-1 is linked to the oxidised nucleotides.

Moreover, we assessed whether the increased sensitivity of the xpa-1 mutant was rescued by knockdown of chk-2 or clk-2. The sensitivity was slightly, but not significantly, rescued by the knockdown of chk-2 or clk-2 (Figure 5C). This result suggests that the chk-2 and clk-2 checkpoint pathway is at least partially linked to the function of XPA-1.

In this study, we investigated the physiological roles of NDX proteins and the response of C. elegans to an increased amount of oxidised nucleotides in the nucleotide pool. We identified factors that participate in the response to the oxidative DNA lesions. However, how they are functioning remains unclear and the molecular mechanisms should be studied in detail.

Supplementary data

Supplementary Figures 1–3 are available at Mutagenesis Online.

Funding

This work was supported by Grants-in-Aid for Scientific Research (#24510071) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan (to Q.-M.Z.-A.), the Global Center of Excellence Program ‘Formation of a Strategic Base for Biodiversity and Evolutionary Research (A06): from Genome to Ecosystem’ and the Grants for Excellent Graduate Schools program (MEXT). We are also grateful to the Shiseido Female Research Science Grant for supporting Q.-M.Z.-A. Some strains were provided by the CGC, which is funded by the NIH Office of Research Infrastructure Programs (P40 OD010440).

Supplementary Material

Supplementary Data
supp_29_2_107__index.html (1.1KB, html)

Acknowledgements

The authors thank Dr Elizabeth Nakajima for critically reading the manuscript. The authors thank Dr Kazunari Hashiguchi for kindly providing plasmids used in this work.

Conflict of interest statement: None declared.

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