Functional Specialization of Chlamydomonas reinhardtii Cytosolic Thioredoxin h1 in the Response to Alkylation-Induced DNA Damage

Culture conditions, Ble-1 isolation, and genetic analysis.Unless noted otherwise, C. reinhardtii cells were grown under moderate light in Tris-acetate-phosphate (TAP) medium (28). To isolate insertional mutants hypersensitive to DNA-damaging agents, the wild-type strain CC-124 was transformed by the glass bead procedure with a plasmid containing a mutant form of protoporphyrinogen oxidase (rs-3 marker) conferring resistance to diphenyl ether herbicides (32). Herbicide-resistant transformants were tested for their ability to survive in the presence of 1.5 μg of bleomycin (Invitrogen)/ml or 2.5 mM MMS (Sigma). By using this approach, we recovered a mutant strain, Ble-1, very sensitive to genotoxic agents. For genetic analysis, Ble-1 was crossed to the wild-type strain of the opposite mating type (CC-125) and tetrads were dissected as previously described (27). The phenotype of the meiotic tetrad products was evaluated by spot tests on TAP medium containing 2.5 mM MMS (32). Five-microliter aliquots of appropriately diluted cells were pipetted onto the plates and incubated as previously reported (32).

Plasmid rescue, cosmid library screening, deletion mapping, and sequence analyses.The genomic sequence flanking one end of the integrated rs-3 marker in Ble-1 was recovered by plasmid rescue in E. coli (32). A 1.5-kb BamHI-NotI fragment from this flanking DNA was used as a probe to screen a Chlamydomonas genomic library (57, 78). Twelve hybridizing cosmid clones were isolated and mapped by restriction enzyme analysis. The longest one (cosmid 1) was cotransformed into Ble-1 together with plasmid pJK7, containing a genetically engineered acetolactate synthase gene conferring resistance to the herbicide sulfometuron methyl (36). However, none of the herbicide-resistant transformants showed complementation of Ble-1's hypersensitivity to genotoxic agents. Moreover, Southern hybridization with a 1.4-kb EcoRI-XhoI fragment from the 3′ end of cosmid 1 (distal to the cloned rs-3 flanking sequence) revealed a large chromosomal deletion in Ble-1. Thus, a combination of genome walking and Southern blot analyses (57, 78) was used to construct a contig of partly overlapping cosmid clones that spanned the deleted region. A subset of these clones, as well as several subclones, was used in complementation assays as described above. Some subclones were also partially sequenced, and putative transcriptional units were identified by searching the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov ) and the Chlamydomonas genome (http://genome.jgi-psf.org/chlre2/chlre2.home.html ) databases.

DNA and RNA analyses.Standard techniques were used to isolate nucleic acids from Chlamydomonas cells (32, 57). Electrophoretic fractionation of nucleic acids, transfer to nylon membranes, and hybridization with ³²P-labeled probes were carried out as previously described (32, 57).

Immunoblot analysis.Cells resuspended in sample buffer (30 mM Tris-HCl [pH 7.9], 1 mM phenylmethylsulfonyl fluoride) were lysed in a French press cell at 1,380 lb/in². After cellular debris was pelleted by consecutive centrifugations at 20,000 × g for 20 min and 45,000 × g for 45 min, soluble polypeptides were recovered from the supernatant. Samples were standardized for total protein concentration with the bicinchoninic acid assay (62). Next, 100-μg aliquots of proteins were boiled for 3 min in gel loading buffer (10% glycerol, 1.4% sodium dodecyl sulfate, 100 mM dithiothreitol, 30 mM Tris-HCl [pH 6.8]) and fractionated in sodium dodecyl sulfate-15% polyacrylamide gels (57). Electrophoretically separated samples were blotted onto nitrocellulose filters (Amersham) and blocked with Tris-buffered saline-Tween 20 (TBS-T) buffer (10 mM Tris-HCl [pH 7.4], 150 mM NaCl, 0.01% Tween 20) containing 5% nonfat dry milk. Further steps were performed in TBS-T buffer containing 1% milk. Membranes were incubated overnight at 4°C with primary antisera against Chlamydomonas Trxh1 and then reacted for 1 h at room temperature with anti-rabbit immunoglobulin G conjugated to horseradish peroxidase (Amersham). Signals were visualized by enhanced chemiluminescence (17).

Phenotypic characterization of Ble-1, complemented strains, and transgenic lines where Trxh1 expression was suppressed by RNAi.RNAi epi-mutants of Trxh1 were generated as previously described (56). Cell survival upon exposure to MMS or UVC irradiation was examined as already reported (32). To test for hypersensitivity to H₂O₂ or bleomycin, Chlamydomonas cells were grown to logarithmic phase, serially diluted, and spotted on TAP plates containing 1 mM H₂O₂ or 1.5 μg of bleomycin/ml. Cell growth was evaluated after 7 to 10 days of incubation under moderate light.

DNA damage repair analysis.Cells in logarithmic phase were collected by centrifugation, resuspended in TAP medium, and treated with 25 mM MMS for 30 min in the dark (8). Untreated control cells were incubated in TAP medium without MMS (8). Immediately afterwards, aliquots of control and MMS-treated cells were frozen for isolation of DNA corresponding to the zero time points. After three washes with TAP medium to remove unreacted MMS, the remaining cells were allowed to recover in the dark, with moderate shaking, for different time periods and aliquots were frozen as described before (8). Genomic DNA was isolated from the frozen samples, separated by denaturing gel electrophoresis (8), and hybridized with a ³²P-labeled probe corresponding to the TOC1 retroelement (32) present at about 15 to 20 copies per haploid nuclear genome. The distribution of radioactivity in each lane was quantified with a phosphorimager and Quantity One software (Amersham). For the analysis of DNA repair by PCR (33), cells were treated with 5 or 10 mM MMS and incubated as described above. Thirty nanograms of genomic DNA was used for the amplification of a 2.1-kb fragment corresponding to the Chlamydomonas Lsm5 (Like Sm 5) gene with primers Mut3-1 (5′-AGAGCTAGGGACCGTGGAGT-3′) and Mut3-2 (5′-TGTTCTCTGTTGCTTGTCTGACG-3′). The number of cycles showing a linear relationship between input DNA and the final product were determined in preliminary experiments. The PCR conditions consisted of 30 cycles at 93°C for 30 s, at 55°C for 30 s, and at 71°C for 180 s. Five-microliter aliquots of each PCR were resolved on 1% agarose gels and visualized by ethidium bromide staining, and signal intensities were quantified with Quantity One software (Bio-Rad). DNA lesion frequency was calculated, assuming a random Poisson distribution, as previously described (11, 33). Under the experimental conditions used (i.e., incubation of concentrated cells in the dark) and without MMS treatment, DNA replication associated with cell cycle progression in the asynchronous Chlamydomonas cultures increased the DNA content by less than 1.5-fold after 24 h (data not shown).

Subcellular localization of a GUS-Trxh1 fusion protein in transiently transformed onion epidermal cells.The coding sequence of Trxh1 was amplified by PCR from a full-length cDNA in plasmid CrTRXh1 (64) with primers Trx-cod-3′ (5′-CATGCCATGGCCGCGGCGGCGTGCTTGGC-3′, adding an NcoI site) and Trx-cod-5′ (5′-CATGCCATGGGCGGTTCTGTTATTGTG-3′, adding an NcoI site). The Trxh1 PCR fragment was then cloned in frame with the GUS start codon in plasmid pPTN134 (82). The transgenes GUS-Trxh1 and GUS alone were transformed into onion epidermal cells by microprojectile bombardment with DNA-coated tungsten particles (82). After bombardment, cells were allowed to recover for 20 h on regular Murashige and Skoog (MS) medium or on MS medium containing 1 mM MMS, 0.2 mM H₂O₂, or 0.5 μg of bleomycin/ml. Some epidermal peels, after 20 h of recovery on MS medium, were incubated for 1 h in liquid MS medium containing 8 mM MMS. GUS activity was detected by staining with X-glucuronide, whereas nuclei were identified with propidium iodide (PI) (82). Stained cells were observed by bright-field microscopy for distribution of GUS activity and by epifluorescent microscopy for PI labeling of the nucleus (71, 82).

Plasmid construction for the expression of Chlamydomonas Trxh1 in S. cerevisiae.EMY63 (MATaade2-1 ade3-100 his3-11 leu2-3 lys2-801 trp1-1 ura3-1 trx1::TRP1 trx2::LEU2), a yeast strain with both genes encoding cytosolic Trxs deleted, has been previously described (47). EMY63 cells were transformed by the lithium chloride method (24) with constructs for conditional expression of S. cerevisiae Trx1 or C. reinhardtii Trxh1 (CrTrxh1) in both its wild-type and redox-inactive C36S forms (25). Each thioredoxin sequence was amplified from the corresponding cDNA by PCR. The upstream primer allowed introduction of an MluI site and a start codon at the position corresponding to the N terminus of each protein, whereas the downstream primer allowed introduction of a BamHI restriction site after the stop codon. The PCR products were cloned under the control of the yeast Gal1 promoter into the centromere plasmid YCpGal2 containing the URA3 selectable marker (12). Sequence-verified recombinant plasmids were transformed into EMY63, and clones were selected and maintained on standard synthetic minimal medium (lacking uracil) supplemented with 2% glucose as the carbon source. For induction of recombinant Trx expression, 2% galactose was substituted for glucose.

Examination of tolerance of yeast strains to MMS or H₂O₂.Transformed EMY63 cells were grown to mid-log phase in glucose medium at 30°C and then diluted and transferred to inducing galactose medium. Growth was continued at 30°C, and cells in mid-log phase were used for tolerance tests. After dilution to an optical density at 600 nm of 0.2, cell growth and survival in the presence of genotoxic agents was evaluated by the halo assay (31). Cells were mixed with galactose top agar and spread on plates to obtain a uniform lawn. Disks containing 10 μl of MMS (1.36 M) or H₂O₂ (500 mM) were placed on the center of the yeast lawns, and cell growth was monitored after 3 to 5 days of incubation at 30°C.

Flow cytometry analysis.Yeast cells were grown in synthetic minimal medium (lacking uracil) supplemented with 2% galactose to an optical density at 600 nm of 0.5, centrifuged, and washed in 2 ml of 50 mM Tris-HCl (pH 8). Cells were then fixed in 70% ethanol for 1 h at room temperature, centrifuged, and resuspended in 1 ml of the Tris-HCl buffer containing 1 mg of RNase A/ml. After incubation for 2 h at 37°C, cells were pelleted (12,000 × g, 1 min), resuspended in 1 ml of Tris-HCl buffer containing PI (50 mg/ml), and allowed to stain in the dark at 4°C overnight under mild agitation. Analysis was performed on a fluorescence-activated cell sorter (Vantage; Becton Dickinson, Le pont de Claix, France). Nuclei were excited at 488 nm with an argon laser (Spectra-Physics, Mountain View, Calif.), and FL1-Height and FL1-Area were collected through a band-pass filter allowing light between 620 and 630 nm to reach the detector. Ten thousand nuclei were analyzed per sample. Data were collected with Cellquest software (Becton Dickinson, Mansfield, Mass.) and analyzed with MODFIT (Verity Software House, Inc., Topsham, Maine).

Isolation and genetic analysis of Ble-1.To identify C. reinhardtii genes involved in the cellular response to DNA damage, we carried out random insertional mutagenesis on the wild-type strain CC-124 (28). Cells from CC-124 were transformed with the rs-3 gene, which encodes a mutated form of protoporphyrinogen oxidase, conferring resistance to diphenyl ether herbicides (32). Herbicide-resistant transformants were then tested by replica plating for their ability to grow on media containing DNA-damaging agents. Since Chlamydomonas is haploid, nonlethal mutations in genes required for DNA damage repair and tolerance will result in reduced survival in the presence of genotoxic agents. By using this approach, we isolated a mutant strain (Ble-1) that is very sensitive to bleomycin and MMS. Ble-1 contained a single, although partly rearranged, copy of the rs-3 plasmid integrated into the nuclear genome (Fig. 1 and data not shown).

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FIG. 1.

Hypersensitivity to MMS cosegregates with the tagging rs-3 marker. (A) Growth and survival of cells on TAP medium or on TAP medium containing 2.5 mM MMS (TAP + MMS). The parental strains and the meiotic products of three tetrads from the cross between the wild-type (CC-125) and mutant Ble-1 strains are shown. Strains grown to logarithmic phase were diluted in TAP medium to 5 × 10⁴ cells per 5 μl, spotted on plates, and incubated as described in Materials and Methods. CTRL, control. (B) Southern blot analysis of the indicated strains. Genomic DNA was digested with HindIII and probed with the pBluescript sequence, the vector backbone of the rs-3 tagging plasmid.

To test whether the mutant phenotypes cosegregated with the rs-3 marker, Ble-1 was crossed with the wild-type strain of the opposite mating type, CC-125. The meiotic tetrad products were examined for survival on medium containing MMS or bleomycin (Fig. 1A and data not shown). Only tetrad products containing the rs-3 gene, as detected by Southern blot hybridization (Fig. 1B), were hypersensitive to MMS (Fig. 1A). In contrast, the tetrad products that did not carry the integrated mutagenic plasmid behaved like the wild type (Fig. 1). The analysis of 10 complete tetrads indicated that hypersensitivity to MMS and bleomycin segregated as a single Mendelian locus genetically linked (within five map units) to the integrated rs-3 marker.

Cloning and identification of the disrupted gene conferring MMS hypersensitivity on Ble-1.The chromosomal sequence flanking one end of the introduced rs-3 gene was obtained by plasmid rescue (66) and used as a probe to screen a Chlamydomonas genomic library. Although several partly overlapping cosmid clones were isolated, none complemented the Ble-1 mutant phenotype (Fig. 2A, e. g., Cosmid 1). These clones were mapped by restriction enzyme digestion, and the end of the longest one (distal to the cloned rs-3 flanking sequence) was used as a probe for Southern hybridization analysis of Ble-1 and CC-124. Lack of hybridization of this sequence to Ble-1 DNA revealed that integration of the rs-3 marker caused a large deletion in the nuclear genome of the mutant strain (Fig. 2A and data not shown). By using a genome walking approach, we next isolated an overlapping set of cosmids that encompassed the Ble-1 chromosomal deletion (Fig. 2A). The left end of this deletion was precisely defined by sequencing of the chromosome-plasmid junction. The right junction was not sequenced, but restriction enzyme mapping of the cosmid contig together with Southern blot analyses of the wild-type and mutant strains indicated that the deleted chromosomal region spans approximately 60 kb (Fig. 2A and data not shown).

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FIG. 2.

Genomic structure of the region affected by integration of the rs-3 marker in Ble-1 and phenotypic defects associated with a lack of cytosolic Trxh1. (A) The open-box diagram represents chromosomal DNA, whereas the dashed line immediately below indicates a deletion of ∼60 kb in Ble-1. The 3′ end of this deletion (dotted line) has not been precisely defined. Genes encoding a hybrid-cluster protein (HCP) and Trxh1 (Trxh1) are depicted by solid boxes. Cosmids and cosmid subclones used for complementation of Ble-1's hypersensitivity to MMS are also indicated. Southern blots of Ble-1 and the wild-type CC-124 strains are shown above the diagram. Genomic DNA was digested with the indicated enzymes and hybridized with different sequences (depicted as solid bars) to examine the deleted chromosomal region. Restriction enzyme sites: B, BamHI; E, EcoRI; P, PstI; X, XhoI. (B) Northern blot analysis of CC-124, Ble-1, and two independent transformants of Ble-1 complemented with a 3-kb PstI fragment containing Trxh1 [Ble-1(Trxh1)-10 and Ble-1(Trxh1)-33]. Total cell RNA was separated by denaturing agarose gel electrophoresis and sequentially probed with the coding sequence of Trxh1 (top panel) and with the coding sequence of the small subunit of the Rubisco gene (RbcS2, bottom panel) as a control for equal loadings in the lanes. (C) Immunoblot analysis of total soluble proteins, from the indicated strains, probed with polyclonal antibodies raised against Chlamydomonas Trxh1 (top panel) or the Rubisco holoenzyme (bottom panel). LS, large subunit of Rubisco. (D) RNA gel blot analysis of CC-124 and two independent strains where Trxh1 expression was suppressed by RNAi (Trxh1-IR-3 and Trxh1-IR-4). Total cell RNA was fractionated and sequentially hybridized with the Trxh1 probe (top panel), the coding sequence of the cytosolic thioredoxin h2 gene (Trxh2) (middle panel), and a fragment of the 25S rRNA gene (bottom panel) as a control for comparable sample loadings. (E) Growth and survival of the indicated strains on TAP medium or on TAP medium containing 2.5 mM MMS (TAP + MMS) or 1 mM hydrogen peroxide (TAP + H₂O₂).

To identify the gene(s) responsible for the hypersensitivity of Ble-1 to DNA-damaging agents, individual cosmid clones were tested for their ability to complement the mutant phenotypes. Ble-1 cells were cotransformed with each cosmid clone and with plasmid pJK7, encoding resistance to the herbicide sulfometuron methyl (36). Herbicide resistance transformants were then examined for their survival on medium containing MMS or bleomycin. This analysis showed that cosmid 4 complemented the hypersensitivity of Ble-1 to MMS but had only a partial effect on restoring bleomycin tolerance (Fig. 2A and 3). To define precisely the gene required for this phenotypic correction, several cosmid 4 subclones were cotransformed into Ble-1. In addition, partial sequence analysis of cosmid 4 revealed that it included the Trxh1 gene, which encodes cytosolic thioredoxin h1. Therefore, we also tested the complementation capability of a 3-kb PstI fragment exclusively containing Trxh1 (64). In all cases, transformation of Ble-1 with fragments that included a full-length Trxh1 gene reversed the MMS hypersensitivity, but the survival defect upon exposure to bleomycin was only partly corrected (Fig. 2A and 3).

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FIG. 3.

Effect of genotoxic agents on the survival of the mutant (Ble-1), the wild-type strain (CC-124), and transformants of Ble-1 containing the 3-kb Trxh1 fragment [Ble-1(Trxh1)-33] or all of cosmid 4 [Ble-1(Cos4)-20]. (A) The panels show the survival of cells grown on TAP medium containing increasing concentrations of MMS or exposed to increasing levels of UVC irradiation. Each graph point represents the mean (± standard deviation) of results of nine replicates (three independent experiments). Where the error bars are not visible, they are smaller than the symbols [□, CC-124; •, Ble-1; ▴, Ble-1(Cos4)-20; ⋄, Ble-1(Trxh1)-33]. (B) Growth and survival of the indicated strains on TAP medium with (TAP + Ble) or without (TAP) 1.5 μg of bleomycin/ml.

To evaluate further whether Trxh1 was required for tolerance to genotoxic agents, we examined its expression in the wild-type strain and the mutant Ble-1 strain, as well as two strains complemented with the 3-kb PstI fragment, Ble-1(Trxh1)-10 and Ble-1(Trxh1)-33. Northern blot analysis of total RNA revealed no detectable Trxh1 transcripts in Ble-1, whereas one complemented strain, Ble-1(Trxh1)-33, had RNA levels similar to those of the wild type (Fig. 2B). The other complemented strain, Ble-1(Trxh1)-10, had slightly reduced amounts of Trxh1 mRNA in comparison with CC-124 (Fig. 2B). Corresponding variations in Trxh1 protein levels were observed among these strains by immunoblot assay of total protein extracts probed with an anti-Trxh1 antibody (Fig. 2C). Interestingly, the Trxh1 expression level in independently complemented strains negatively correlated with their sensitivity to MMS (data not shown). Moreover, if Trxh1 is required for tolerance to MMS exposure, Trxh1 suppression by RNAi should result in a phenotype similar to that of Ble-1. To test this hypothesis, we transformed wild-type Chlamydomonas cells with inverted repeat constructs designed to produce double-stranded RNA homologous to Trxh1 (56). In several independent transformants, Trxh1 transcript levels were specifically down-regulated, whereas mRNA amounts for the closely related cytosolic thioredoxin h2 gene (Trxh2) (40) remained unperturbed (Fig. 2D, Trxh1-IR-3 and Trxh1-IR-4). Like Ble-1, these RNAi strains (Trxh1 epi-mutants) were hypersensitive to MMS treatment (Fig. 2E). However, they showed only mild survival defects when they were grown on bleomycin-containing medium (data not shown). These results, taken together, suggested a role for the cytosolic Trxh1 isoform in the cellular response to certain genotoxic agents such as MMS. Yet, Trxh1 did not fully complement the defect in the survival of Ble-1 when it was exposed to bleomycin or H₂O₂ (see below). Therefore, we hypothesize that disruption of another yet-to-be-identified gene(s) within the 60-kb deletion is responsible for the latter phenotypes.

Effect of genotoxic agents on cell survival and DNA damage repair in Ble-1, the Trxh1-complemented strains, and the Trxh1 RNAi-induced epi-mutants.To gain insight into the molecular role of Trxh1, we exposed cells to a variety of genotoxic agents causing different kinds of DNA lesions and requiring distinct pathways for their repair. In all cases, we compared the survival of the wild-type CC-124 strain, the Ble-1 mutant, a strain complemented with cosmid 4 [Ble-1(Cos4)-20], and a strain complemented with the Trxh1-containing 3-kb PstI fragment [Ble-1(Trxh1)-33]. All strains behaved similarly to the wild type when they were irradiated with UVC light and allowed to recover under nonphotoreactivating conditions (Fig. 3A). In contrast, Ble-1 was very sensitive to treatment with MMS and this phenotype was nearly fully complemented by ectopic expression of Trxh1 (Fig. 3A). In addition, as already discussed, strains where Trxh1 was suppressed by RNAi displayed hypersensitivity to MMS (Fig. 2E). Ble-1 survival was also compromised by exposure to H₂O₂ or bleomycin (Fig. 2E and 3B). However, bleomycin sensitivity was only partly reversed by transformation with a wild-type copy of Trxh1 (Fig. 3B), whereas the defect in H₂O₂ tolerance was not corrected (Fig. 2E). Conversely, down-regulation of Trxh1 expression by RNAi caused only mild hypersensitivity to bleomycin (data not shown) and did not affect survival in the presence of H₂O₂ (Fig. 2E). Somewhat surprisingly, given the known role of thioredoxins in the oxidative-stress response (14, 35, 38), these findings indicated that Trxh1 plays a key role in cellular protection against MMS and, to some extent, bleomycin but has no apparent effect on the tolerance to H₂O₂.

To test whether Trxh1 is required for the repair of MMS-induced DNA damage, cells were briefly treated with this chemical and then allowed to recover for different periods of time. The extent of induced or residual DNA damage was evaluated by alkaline gel electrophoresis (8). Under these conditions, both SSBs and DSBs as well as alkali-labile sites (i.e., abasic sites) arising from BER (21) are detectable by the enhanced electrophoretic mobility of fragmented DNA. Examination of DNA isolated immediately after the treatment revealed similar extents of MMS-induced nuclear DNA damage in the wild-type, Ble-1, and Ble-1(Trxh1)-33 strains (Fig. 4). However, after incubation in drug-free medium, Ble-1 cells repaired DNA strand breaks very slowly in comparison with the wild type, as indicated by the smaller average molecular mass of DNA molecules at each time point (Fig. 4). The complemented strain, Ble-1(Trxh1)-33, showed an intermediate phenotype (Fig. 4). This partial correction of the DNA repair capacity differs from the nearly full reversal of the survival defect in the same strain when it is exposed to MMS (Fig. 3A).

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FIG. 4.

Analysis of nuclear DNA repair after MMS-induced damage in the wild-type, Ble-1, and Ble-1(Trxh1)-33 strains. (A) Southern blot showing DNA repair after exposure to 25 mM MMS for 30 min. Genomic DNA was isolated from untreated control cells (CTRL) and MMS-treated cells either immediately after the treatment (0 h) or after recovery in the absence of MMS for 6, 12, or 24 h. The DNA was separated by alkaline gel electrophoresis and hybridized with a sequence corresponding to the TOC1 transposable element. (B) In the same kind of experiment described above, the relative distribution of radioactivity in each lane (indicative of the DNA mass distribution) was analyzed with a phosphorimager and plotted as a function of migration distance. Graphs represent the average of results of two independent experiments. CC-124 is indicated by dotted lines, Ble-1 is indicated by solid lines, and Ble-1(Trxh1)-33 is indicated by dashed lines. The vertical solid line indicates the average molecular mass of damaged DNA (similar in all strains) immediately after MMS treatment.

MMS induces high levels of N-methylpurines and secondary lesions resulting from DNA damage processing and replication, such as SSBs and DSBs (21). We speculate that, at the higher MMS concentration (25 mM) used to cause DNA damage in the repair experiments (in comparison with the concentrations employed to test for cell survival), a greater proportion of MMS-induced lesions corresponds to secondary DSBs. Indeed, at relatively high concentrations, MMS behaves as a radiomimetic agent in a manner similar to that of DSB-inducing bleomycin (43). Hence, considering also that Ble-1 shows hypersensitivity to bleomycin and that this phenotype is only partly corrected by the expression of Trxh1, Ble-1 is likely defective in the repair of DSBs, but this deficiency is not a consequence of the lack of Trxh1.

We also tested the role of Trxh1 in the repair of DNA lesions induced by a lower concentration of MMS, one comparable to that employed in the survival experiments (see Material and Methods), by using a semiquantitative PCR approach (11, 33). The ability of a DNA fragment to support PCR amplification is an indicator of its in vivo intactness, since DNA sequences containing DNA polymerase-blocking or -terminating lesions will not be amplified in this assay (11, 33). However, removal of DNA lesions through DNA repair will enhance the template integrity of genomic DNA, enabling more PCR amplification. The MMS treatment generated similar levels of DNA lesions in the wild-type and Trxh1 RNAi epi-mutants (Fig. 5), suggesting that Trxh1 does not play a major role in the detoxification of MMS and prevention of alkylation DNA damage. In contrast, the strains where Trxh1 expression was suppressed by RNAi showed a lower rate of DNA repair (measured as the recovery of amplification signal at 6 and 12 h after treatment) in comparison with the wild type (Fig. 5). It is unlikely that the increase in DNA amplification over time results predominantly from the replication of intact DNA molecules since, under the conditions used, even in the absence of treatment with genotoxic agents, measured DNA replication was less than 1.5-fold after 24 h (data not shown). Therefore, our results suggest that Chlamydomonas Trxh1 is required, either directly or indirectly (see discussion), for the repair of alkali-labile abasic sites and/or SSBs induced by treatment with alkylating agents.

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FIG. 5.

Repair of MMS-induced DNA lesions in the wild type (CC-124) and a Trxh1 RNAi epi-mutant (Trxh1-IR-3) as examined by semiquantitative PCR. (A) Amplification of a 2.1-kb genomic DNA fragment (Lsm5) by using, as the template, DNA isolated from cells immediately after treatment with 10 mM MMS (0 h) or after allowing the cells to recover for 6 or 12 h in the absence of MMS. Untreated control cells (CTRL) were also analyzed. The amplified products were resolved by agarose gel electrophoresis and stained with ethidium bromide. (B) Relative amplification of the 2.1-kb fragment calculated by dividing the amount of amplification from damaged samples (A_D) by the amount of amplification from nondamaged controls (A_C). Each graph point represents the mean (± standard error) of results of three independent experiments. Symbols: ▴, CC-124; ▪, Trxh1-IR-3.

Relocalization of a Trxh1-GUS fusion protein to the nucleus in cells exposed to genotoxic agents.The Chlamydomonas Trxh1 isoform does not contain a canonical nuclear localization signal, and it has been assumed to localize to the cytosol (64). However, if Trxh1 were directly involved in DNA repair, it would be expected to localize to the nucleus and/or to redistribute to the nucleus in response to treatment with genotoxic agents. To test this hypothesis, we examined the subcellular partitioning of a fusion polypeptide consisting of the Trxh1 coding sequence linked to the N terminus of the E. coli GUS protein. A transgene expressing GUS alone was used as a control, since this 68-kDa polypeptide is largely excluded from the nucleus (71). Both constructs were placed under the control of the Cauliflower mosaic virus 35S promoter and introduced into onion epidermal cells by particle bombardment. Onion epidermal peels were used as a transient gene expression system because the large, transparent (chlorophyll-less) cells facilitate the imaging of subcellular structures (71). In addition, we and others have previously demonstrated the correct subcellular localization of Chlamydomonas fusion proteins in this system (79, 82).

After particle bombardment, the onion peels were incubated for 18 to 20 h on MS medium with or without MMS, bleomycin, or H₂O₂. In other cases, epidermal peels were incubated overnight on MS and then transferred for a short period to medium containing genotoxic agents. The subcellular distribution of the expressed proteins was determined histochemically by the X-glucuronide assay (71). Regardless of treatment, the GUS polypeptide was predominantly localized in the cytoplasm of onion cells (Fig. 6B and D). Likewise, in the absence of genotoxic agents or upon treatment with H₂O₂, the Trxh1-GUS protein was largely found in the cytosol (Fig. 6A and C). In contrast, after treatment with MMS or bleomycin, the fusion polypeptide showed dual localization, in both the nucleus and the cytoplasm, in the majority of the examined cells (Fig. 6A and C and data not shown). Thus, consistent with a role of Trxh1 in DNA repair, our data indicated that the protein relocalizes to the nucleus in response to certain DNA-damaging agents.

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FIG. 6.

Subcellular distribution of GUS and GUS-Trxh1 fusion proteins in transiently transformed onion epidermal cells. Polypeptides were localized histochemically by the X-glucuronide assay. (A) Representative cellular staining patterns corresponding to GUS-Trxh1. Onion epidermal peels bombarded with DNA-coated tungsten particles were incubated on MS medium alone (No treatment) or with the indicated concentrations of MMS, bleomycin (Ble), or hydrogen peroxide (H₂O₂). Tissues were simultaneously analyzed by X-glucuronide staining (left panels, blue color) and nucleus-specific PI staining (right panels, orange color). Nuclei are indicated with arrowheads. (B) Representative staining patterns in onion epidermal cells transiently transformed with GUS and treated as described for panel A. Panels stained with X-glucuronide (left) and PI (right) are shown. (C) Frequency analysis of GUS-Trxh1 subcellular distribution under the indicated treatments. Transformed cells were classified as showing exclusive cytoplasmic localization (black bars) or dual, nuclear, and cytoplasmic localization (red bars) of GUS activity. The results show the averages (± standard deviations) of results of three independent experiments (300 cells analyzed per treatment). (D) Frequency analysis of GUS subcellular localization under the indicated treatments as described for panel C.

Functional complementation of a S. cerevisiae trx1 trx2 double mutant by expression of C. reinhardtii Trxh1.Budding yeast contains two genes encoding cytoplasmic thioredoxins (Trx1 and Trx2), which are dispensable during normal growth conditions (47). However, deletion of both Trx genes results in hypersensitivity to oxidative agents and defects in the cell cycle, particularly a prolonged S phase (47, 69). Trx functions as a reductant for ribonucleotide reductase, an essential enzyme in deoxyribonucleotide biosynthesis (69), but the alterations in the cell cycle do not result from reduced levels of deoxyribonucleotides (46). In fact, glutaredoxin can also function as an electron donor for ribonucleotide reductase (54) and the thioredoxin and glutathione-glutaredoxin systems appear to have overlapping functions, since only one is required for S. cerevisiae viability (16, 68). The actual role of Trxs in the yeast cell cycle has remained elusive, but we hypothesize that lack of cytosolic Trxs may result in increased DNA damage by endogenously produced ROS and/or a slower repair of spontaneous DNA lesions that activate the S-phase DNA damage checkpoint, preventing entry into mitosis (42, 67).

Little is known about a potential function of yeast Trxs in response to monofunctional alkylating agents, although Trx2 and the thioredoxin reductase 1 gene are transcriptionally activated by MMS treatments (10, 35). Therefore, we examined whether the S. cerevisiae trx1 trx2 double mutant was hypersensitive to a variety of genotoxic agents and whether Chlamydomonas Trxh1 could complement the phenotypic deficiencies, suggestive of evolutionary conservation of function. S. cerevisiae cells with both Trx1 and Trx2 deleted showed defects in survival (although to different degrees) when they were exposed to MMS or H₂O₂ (Fig. 7A). Expression of Chlamydomonas Trxh1 from a yeast-replicating vector, under the control of a galactose-inducible promoter, resulted only in reversion of MMS hypersensitivity. However, a mutant form of Chlamydomonas Trxh1 where a cysteine residue in the redox-active site was replaced by serine (C36S) (25) failed to complement this phenotype (Fig. 7A). In flow cytometry analysis of asynchronous cultures, the yeast trx1 trx2 mutant also displays a considerably lengthened S phase that becomes apparent by a marked decrease in the proportion of cells having a G₁ (1N) or, to a lower degree, G₂ (2N) DNA content (47). Expression of Chlamydomonas Trxh1 partly corrected this deficiency in cell cycle progression, provided that the protein contained the wild-type cysteine residues in its catalytic site (Fig. 7B). Thus, these results suggest functional conservation between budding yeast and Chlamydomonas cytosolic thioredoxins as key components in the cellular response to alkylating DNA-damaging agents. Moreover, a redox-active Cys-X-X-Cys site is necessary for this function.

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FIG. 7.

Phenotypic complementation of an S. cerevisiae trx1 trx2 double mutant (EMY63) by expression of Chlamydomonas Trxh1. (A) Expression of Trxh1, under the control of a galactose-inducible promoter in a centromeric plasmid restored the tolerance of the mutant to MMS to the same extent that transformation with S. cerevisiae Trx1 did (left panel). In contrast, Trxh1 did not complement the hypersensitivity to H₂O₂ (right panel). Yeast cells were transformed with the empty plasmid (YCpGal2), with S. cerevisiae Trx1 (Yeast Trx1), with Chlamydomonas Trxh1 (CrTrxh1), or with a mutant form of Trxh1 lacking a catalytically active cysteine (CrTrxh1 C36S). Cells were plated, forming a lawn, and exposed to a gradient of MMS or H₂O₂ concentrations, established by diffusion from a centrally placed disk containing 1.36 M MMS or 500 mM H₂O₂. The size of the halo where cell growth was suppressed was used as an estimation of the sensitivity to genotoxic agents. The results show the averages (± standard deviations) of three to five independent experiments. (B) Relative DNA content in asynchronous cultures of transformed trx1 trx2 mutant cells. Yeast cells of the indicated transformants were grown to logarithmic phase, stained with PI, and analyzed by flow cytometry. Relative fluorescence intensity (DNA content) is plotted against the number of counted events (Nuclei counts). Relative DNA contents corresponding to 1N (G₁) and 2N (G₂) are indicated.