Mol Genet Genomics (2001) 265: 1087±1096 DOI 10.1007/s004380100507
O R I GI N A L P A P E R
L. Gellon á R. Barbey á P. Auret van der Kemp D. Thomas á S. Boiteux
Synergism between base excision repair, mediated by the DNA glycosylases Ntg1 and Ntg2, and nucleotide excision repair in the removal of oxidatively damaged DNA bases in Saccharomyces cerevisiae Received: 9 February 2001 / Accepted: 19 April 2001 / Published online: 6 June 2001 Ó Springer-Verlag 2001
Abstract In Saccharomyces cerevisiae, inactivation of the two DNA N-glycosylases Ntg1p and Ntg2p does not result in a spontaneous mutator phenotype, whereas simultaneous inactivation of Ntg1p, Ntg2p and Rad1p or Rad14p, both of which are involved in nucleotide excision repair (NER), does. The triple mutants rad1 ntg1 ntg2 and rad14 ntg1 ntg2 show 15- and 22-fold increases, respectively, in spontaneous forward mutation to canavanine resistance (CanR) relative to the wild-type strain (WT). In contrast, neither of these triple mutants shows an increase in the incidence of Lys+ revertants of the lys1-1 ochre allele. Furthermore, the rad1 ntg1 ntg2 mutant is hypersensitive to the lethal eect of H2O2 relative to WT, rad1 and ntg1 ntg2 mutant strains. Moreover, the rad1 ntg1 ntg2 strain is hypermutable (CanR and Lys+) upon exposure to H2O2, relative to WT, rad1 and ntg1 ntg2 strains. Mutagen sensitivity and enhanced mutagenesis in the rad1 ntg1 ntg2 triple mutant, relative to the other strains tested, were also observed upon exposure to oxidizing agents such as tertbutylhydroperoxide and menadione. In contrast, the sensitivity of the rad1 ntg1 ntg2 triple mutant to c-irradiation does not dier from that of the WT. However, the triple mutant shows an increase in the frequency of Lys+ revertants recovered after c-irradiation. The results reported in this study demonstrate that base excision repair (BER) mediated by Ntg1p and Ntg2p acts synergistically with NER to repair endogenous or inCommunicated by R. Devoret L. Gellon á P. Auret van der Kemp á S. Boiteux (&) CEA, DeÂpartement de Radiobiologie et Radiopathologie, UMR217 CNRS-CEA ``Radiobiologie MoleÂculaire et Cellulaire'', 92265 Fontenay aux Roses, France E-mail:
[email protected] Tel.: +33-1-46548858 Fax: +33-1-46548859 R. Barbey á D. Thomas Centre de GeÂneÂtique MoleÂculaire, UPR2167 CNRS, 91190-Gif sur Yvette, France
duced lethal and mutagenic oxidative DNA damage in yeast. The substrate speci®city of Ntg1p and Ntg2p, and the spectrum of lesions induced by the DNA-damaging agents used, strongly suggest that oxidized DNA bases, presumably oxidized pyrimidines, represent the major targets of this repair pathway. Keywords Oxidative damage á DNA repair and mutagenesis á NTG1/NTG2/RAD1/ RAD14 á Saccharomyces cerevisiae
Introduction Oxidative DNA damage is unavoidable, and in the course of evolution organisms have developed a spectrum of DNA repair mechanisms that are critical for cell viability and genome stability (Friedberg et al. 1995). Reactive oxygen species (ROS) generated by cellular metabolism or exogenous agents can induce several types of lesions, such as oxidized bases, apurinic/ apyrimidinic (AP) sites and DNA strand breaks (Dizdaroglu 1991; Cadet et al. 1997). Oxidative DNA damage has been implicated in several pathologies in mammals, such as cancer or neurodegenerative diseases (Feig et al. 1994; Boiteux and Radicella 2000; Le Page et al. 2000). Oxidized DNA bases are substrates for DNA N-glycosylases, which initiate the base excision repair (BER) pathway (Krokan et al. 1997; Lindahl and Wood 1999; Memisoglu and Samson 2000). In Escherichia coli, three DNA glycosylases are known, which are able to recognise and excise modi®ed bases in DNA that has been exposed to chemical oxidants or ionizing radiation: Nth (endonuclease III), Nei (endonuclease VIII) and Fpg (MutM) (Krokan et al. 1997). These enzymes are DNA N-glycosylases/AP lyases, catalysing both cleavage of the glycosylic bond and incision of the phosphodiester backbone at the resulting AP site via b- or b- and d-elimination reactions (Boiteux 1993; McCullough et al. 1999). Nth and Nei remove pyrimidine-derived lesions,
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whereas Fpg primarily acts on purine-derived lesions (Boiteux et al. 1992; Dizdaroglu et al. 1993; Jiang et al. 1997b). In E. coli, inactivation of both nth and nei genes results in hypersensitivity to X-rays and H2O2 and generates a spontaneous mutator phenotype (Jiang et al. 1997a; Saito et al. 1997). In E. coli, the nucleotide excision repair (NER) pathway, which is initiated by the UvrABC endonuclease, is also involved in the repair of oxidized DNA bases. Both in vitro and in vivo, the UvrABC endonuclease from E. coli recognizes and excises thymine glycol residues from DNA (Lin and Sancar 1989; Kow et al. 1990). In Saccharomyces cerevisiae, three DNA N-glycosylases/AP lyases are also involved in the repair of oxidatively damaged DNA bases; Ntg1p, Ntg2p and Ogg1p (Girard and Boiteux 1997). Ntg1p and Ntg2p are closely related to each other and to E. coli Nth (Eide et al. 1996; Augeri et al. 1997; Senturker et al. 1998; You et al. 1998,1999; Alseth et al. 1999). However, Ntg2p, but not Ntg1p, possesses the consensus sequence for an iron-sulfur center found in most of the Nth homologues (Eide et al. 1996; Augeri et al. 1997; Senturker et al. 1998; You et al. 1998, 1999; Alseth et al. 1999). Analysis of their subcellular localisation indicates that Ntg2p is exclusively localised in the nucleus, whereas Ntg1p is present in both the nucleus and the mitochondria (Alseth et al. 1999; You et al. 1999). Furthermore, Ntg1p is damage inducible, whereas Ntg2p is constitutively expressed (Eide et al. 1996; Augeri etal. 1997; Alseth et al. 1999). Ogg1p, on the other hand, does not show signi®cant sequence homology to Ntg1p and Ntg2p, except for the Helix-hairpin-Helix-GPD/K active-site domain (Nash et al. 1996; van der Kemp et al. 1996; Guibourt et al. 2000). Ntg1p and Ntg2p display a broad substrate speci®city, releasing oxidized pyrimidines such as 5,6dihydrothymine, 5,6-dihydrouracil, 5-hydroxy-5-methylhydantoin, 5-hydroxyuracil, 5-hydroxycytosine or thymine glycol (Eide et al. 1996; Augeri et al. 1997; Senturker et al. 1998; You et al. 1998, 1999; Alseth et al. 1999). Ntg1p and Ntg2p also release purine-derived lesions, 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyG) and 4,6-diamino-5-formamidopyrimidine (FapyA), from c-irradiated DNA (Senturker et al. 1998). Ntg1p and Ntg2p have very similar, but not identical, substrate speci®cities. Ntg1p cleaves UV-irradiated DNA more eciently than Ntg2p does (Alseth et al. 1999)and excises 8-OxoG when mispaired with a guanine (Senturker et al. 1998). On the other hand, Ogg1p exhibits a narrow substrate speci®city, catalysing the removal of 8-OxoG and FapyG from DNA exposed to c-irradiation or H2O2 (Karahalil et al. 1998). Finally, Ntg1p, Ntg2p and Ogg1p incise DNA at AP sites via a b-elimination reaction (Karahalil et al. 1998; Alseth et al. 1999). The biological functions of the DNA N-glycosylases Ntg1p, Ntg2p and Ogg1p in S. cerevisiae have been investigated by analysing the phenotypes of mutant strains. In vitro, cell-free protein extracts of the ntg1 ntg2 double mutant do not show detectable activity on substrates containing a single 5,6-dihydrouracil, indi-
cating that these two activities are probably the major DNA N-glycosylases capable of removing oxidized pyrimidines in yeast (You et al. 1998, 1999). In vivo, a mutator phenotype has been reported for ntg1 and ntg2 mutant strains (Alseth et al. 1999), but this was not observed in another study (Swanson et al. 1999). Ntg1p and Ntg2p have also been implicated in the repair of AP sites induced by an alkylating agent (You et al. 1999). On the other hand, inactivation of the Ogg1p results in a spontaneous GC to TA mutator phenotype which is probably due to the accumulation of 8-OxoG in DNA (Thomas et al. 1997; Scott et al. 1999). The aim of the work presented here was to assess the relative contribution of BER, mediated by the DNA N-glycosylases Ntg1p and Ntg2p, and NER in the repair of oxidized DNA bases in S.cerevisiae. We therefore constructed a series of yeast strains de®cient in Ntg1p and Ntg2p and Rad1p or Rad14p. These strains were analysed for spontaneous mutagenesis, survival and mutagenesis following exposure to DNA-damaging agents that generate an oxidative stress, such as H2O2, tert-butylhydroperoxide, menadione and c-irradiation.
Materials and methods Media and growth conditions Yeast strains were grown in YEPD medium (1% yeast extract, 2% Bacto-peptone, 2% glucose, with 2.5% agar for plates) or YNBD medium (2% glucose-0.17% yeast nitrogen base without amino acids and with 0.5% ammonium sulfate-2% agar for plates) supplemented with the required amino acids and bases at recommended concentrations. Supplemented YNBD medium lacking lysine was used for selective growth of Lys+ revertants. Supplemented YNBD medium lacking arginine but containing canavanine (Sigma) at 60 mg/l was used for the selective growth of canavanine-resistant (CanR) mutants. All products used for growth media, including agar, were purchased from Difco. Amino acids and uracil were from Sigma. Strains and microbiological methods All strains used in this study are derivatives of FF18733 and their relevant genotypes and origins are listed in Table 1. S. cerevisiae was transformed after acetate lithium treatment as described by Gietz et al. (1992). Recombinant DNA methods The ogg1::TRP1 disruption mutant was previously described (Thomas et al. 1997). In order to disrupt NTG1 an EcoRI-BamHI fragment of yeast Chromosome I containing the NTG1 gene was ®rst cloned into plasmid pUC19, yielding plasmid pNTG1-1. To disrupt NTG1 with the URA3 marker, pNTG1-1 was digested with BglII, treated with Klenow fragment to create blunt ends, dephosphorylated and ligated with a blunt-ended BglII fragment of pFL44 containing the URA3 gene (Bonneaud et al. 1991). The resulting plasmid was then digested with EcoRI and BamHI, treated with the Bal31 nuclease for 5 min, and the product of the digestion was used to transform a wild-type strain. Transformants were selected for uracil prototrophy. Correct disruption events were veri®ed by Southern analysis, performed with genomic DNA prepared as described by Homan and Winston (1987). Probes
1089 Table 1 Saccharomyces cerevisiae strains used in this study
Strain
Genotype
Source/reference
FF18733 FF181481 BG300 CD169 CD183 FF181134 CD182 CC892-1A CC892-2A CC892 BG310 BG311
MATa, his7, leu2, lys1, trp1, ura3 FF18733 with rad1D::LEU2 FF18733 with rad14D::URA3 FF18733 with ntg2D::TRP1 FF18733 with ntg1D::URA3 FF18733 with rev3D::URA3 FF18733 with ntg1D::URA3, ntg2D::TRP1 FF18733 with rad1D::LEU2, ntg1D::URA3 FF18733 with rad1D::LEU2, ntg2D::TRP1 FF18733 with rad1D::LEU2, ntg1D::URA3, ntg2D::TRP1 FF18733 with ntg1D::URA3, ntg2D::TRP1, rad14D::kanMX6 FF18733 with rad1D::LEU2, ntg1D::URA3, ntg2D::TRP1, rev3D::kanMX6 FF18733 with ogg1D::TRP1
F. Fabre F. Fabrea This studyb This study This study F. Fabrec This study This study This study This study This study This study
CD138
Thomas et al. (1997)
a The plasmid used to disrupt RAD1 was pWJ153, originally obtained from R. Rothstein (Coic et al. 2000) b The plasmid used to disrupt RAD14 was pBM189, described by Scott et al. (1999) c The plasmid used to disrupt the REV3 gene was originally obtained from R. D. Gietz
were radioactively labelled by the random-priming method as described by Hodgson and Fisk (1987). To disrupt NTG2, an Asp718-PstI fragment of yeast Chromosome XV containing the NTG2 gene was ampli®ed by PCR and cloned into the pUC19 vector, yielding plasmid pNTG2-1. To disrupt NTG2 with the TRP1 marker, pNTG2-1 was digested with BsmFI and EcoRI, treated with Klenow fragment to create blunt ends, dephosphorylated and ligated with a blunt-ended BglII fragment of pFL39 containing the TRP1 gene (Bonneaud et al. 1991). The resulting plasmids were then digested with Asp718 and PstI, and the products were used to transform a wild-type strain. Transformants were selected for tryptophan prototrophy. Correct disruption events were veri®ed by Southern analyses. To create REV3 and RAD14 disruptions, the PCR disruption technique with the kanMX module was used to delete the entire open reading frame of the REV3 and RAD14 genes in a rad1Dntg1Dntg2D and ntg1Dntg2D background, respectively. For REV3 disruption, the primers 5¢-GTCAATACAAAACTACAAGTTGTGGCGAAATAAAATGTTTGGAAc ggatccccgggttaattaa-3¢ (forward) and 5¢-GAAACAAATAACTACTCATCATTTTGCGAGACATATCTGTGTCTAGAgaattcgagctcgtttaaac-3¢ (reverse) were used to amplify a 2.4-kb disruption fragment using pFA6a-13Myc-kanMX6 (Longtine et al. 1998) as a template. For RAD14 disruption, the primers 5¢-CATAAGGAAACAAGATTACATTGAGTACGATTTTGCCACCcggatccccgggttaattaa3¢ (forward) and 5¢-CCTTATTATGACTTTCTT GTTATATTCTTATATACATAACCAACATgaattcgagctcgtttaaa c-3¢ (reverse) were used. The lower case letters indicate nucleotide sequences that belong to the kanMX cassette, sequences corresponding to the speci®c gene are given in uppercase. The PCR product was used directly for transformation (Baudin et al. 1993). Following transformation, cells were grown for 3 h in 3 ml of YEPD before selective plating on YEPD containing geneticin (Sigma) at 200 mg/l. After 2 days at 30°C, the colonies were replica-plated onto fresh geneticin-containing medium. Gene disruption was con®rmed by PCR analysis of genomic DNA and by measuring the UV sensitivity of mutants.
centrations for 20 min at 30°C with shaking. In the case of cirradiation, cells were irradiated with a 137Cs source at a dose rate of 52.4 Gy/min. For all experiments, untreated and treated cells were immediately diluted and plated on solid YEPD medium, and colonies were scored after 2±3 days of growth at 30°C. All experiments were independently carried out in triplicate. Spontaneous mutation frequencies To measure spontaneous mutation frequencies, cells were grown to saturation in YEPD medium. Then, about 103 cells were inoculated into ®ve separate 3-ml portions of YEPD medium. These cultures were grown to saturation at 30°C. Cell density was measured by plating dilutions on YEPD agar plates and counting colonies after 2±3 days at 30°C. To screen for canavanine-resistant mutants (CanR) and Lys+ revertants, appropriate dilutions were plated on selective medium and colonies counted after 3±4 days at 30°C. All experiments were carried out independently at least three times, and mutation frequencies were determined by the method of the median (Lea and Coulson 1949). Induced mutation frequencies Cells were treated with the dierent DNA-damaging agents as described above. After treatment, cells were harvested by centrifugation, washed twice with sterile water and resuspended in 2 ml of YEPD medium. Untreated and treated cultures were grown for 24 h at 30°C without selection. Cell density was measured by plating dilutions on YEPD agar plates and counting colonies after 2±3 days at 30°C. To screen for canavanine-resistant mutants (CanR) and Lys+ revertants, appropriate dilutions were plated on selective medium and colonies counted after 3±4 days at 30°C. All experiments were carried out in triplicate and mutation frequencies were determined and expressed as averages (SD).
Sensitivity of strains to DNA-damaging agents Yeast strains were grown in YEPD medium at 30°C with agitation, to a cell density of 107 cells/ml. Cells were pelleted and resuspended in the same volume of sterile water. Aliquots of cells were then exposed to various concentrations of DNA-damaging agent. Cells were exposed to increasing concentrations of H2O2 (Sigma), tert-butylhydroxyperoxide (Sigma) or menadione (Sigma) for 30 min at 30°C with shaking. In the case of methyl methanesulfonate (Sigma) treatment, cells were treated with increasing con-
Results Simultaneous inactivation of Ntg1p, Ntg2p and NER generates a spontaneous mutator phenotype in yeast To investigate the mutagenic potential of endogenous oxidative DNA damage, we measured spontaneous
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mutation frequencies in isogenic yeast strains de®cient in Ntg1p and Ntg2p. In vitro studies have shown that Ntg1p and Ntg2p are DNA N-glycosylases that are capable of releasing a variety of oxidatively damaged pyrimidines and purines from damaged DNA (Eide et al. 1996; Augeri et al. 1997; Senturker et al. 1998; You et al. 1998, 1999; Alseth etal. 1999). Spontaneous mutagenesis was monitored using two systems: forward mutation leading to inactivation of the arginine permease gene, CAN1, to yield canavanine-resistant (CanR) mutants and reversion of the lys1-1 ochre allele to yield Lys+ revertants (Cassier-Chauvat and Fabre 1991). Spontaneous mutation frequencies (CanR and Lys+) are no higher in single ntg1 or ntg2 or double ntg1 ntg2 mutant strains than in the WT (Table 2). The lack of a mutator phenotype of the ntg1 ntg2 double mutant could be due to the action of an alternative repair pathway such as NER (Swanson et al. 1999; Memisoglu and Samson 2000). Therefore, we investigated spontaneous mutagenesis in strains that were defective in Ntg1p, Ntg2p and a NER protein (Rad1p or Rad14p). The spontaneous CanR mutant frequency is moderately stimulated over the wild-type level in rad1 and rad14 strains (4.7fold and 2-fold, respectively) (Table 2). In contrast, simultaneous inactivation of Ntg1p, Ntg2p and the NER pathway in rad1 ntg1 ntg2 or rad14 ntg1 ntg2 triple mutants resulted in a synergistic, i.e., greater than additive, 15-fold and 22-fold increase in CanR mutation frequency relative to the WT (Table 2). Interestingly, the frequency of Lys+ revertants is not enhanced in the rad1 ntg1 ntg2 or rad14 ntg1 ntg2 triple mutant, relative to the WT (Table 2). On the other hand, both CanR and Lys+ events are enhanced in the ogg1 single mutant (Table 2). The results also show that Ntg1p and Ntg2p can substitute for each other in the removal of endogenous mutagenic DNA damage, since rad1 ntg1 and rad1 ntg2 double mutant strains exhibit CanR mutation frequencies identical to those of the rad1 mutant alone. Furthermore, inactivation of the mutagenic polymerase f,
Table 2 Spontaneous mutation frequencies in yeast strains de®cient in BER and NER pathways
Relevant genotype
WT ntg1 ntg2 ntg1 ntg2 rad1 rad14 rad1 ntg1 rad1 ntg2 rad1 ntg1 ntg2 rad14 ntg1 ntg2 rev3 rev3 rad1 ntg1 ntg2 ogg1 a
encoded by the REV3 gene, to yield the rad1 ntg1 ntg2 rev3 quadruple mutant, results in a decrease in the frequency of spontaneous CanR mutations, indicating that CanR mutants mostly result from translesion synthesis at sites of DNA damage by Rev3p (Table 2). Sensitivity and mutability of Ntg1p-, Ntg2pand NER-de®cient yeast cells following exposure to hydrogen peroxide If Ntg1p and Ntg2p act synergistically with the NER pathway to remove endogenous oxidative damage, the same DNA repair pathways could prevent lethal and mutagenic eects of oxidizing agent such as H2O2 in yeast. Figure 1 shows that ntg1, ntg2, rad1 or rad14 single, and ntg1 ntg2, rad1 ntg1 or rad1 ntg2 double mutants, do not show an increase in sensitivity to H2O2 over the WT. In contrast, the triple mutants rad1 ntg1 ntg2 and rad14 ntg1 ntg2 are more sensitive to H2O2 than the WT (Fig. 1). The shape of the survival curves indicates a drop in viability at low concentration of H2O2 (0.5 mM), followed by resistance up to 2.0 mM, and a second phase of sensitivity at higher doses. The bi-phasic shape of the survival curves is more pronounced for the rad1 ntg1 ntg2 and rad14 ntg1 ntg2 triple mutants (Fig. 1). Therefore, BER mediated by Ntg1p and Ntg2p, and NER mediated by Rad1p or Rad14p, act synergistically to remove lethal oxidative DNA damage induced by H2O2 in yeast. Upon exposure to H2O2, CanR and Lys+ mutant frequencies are enhanced at the doses used (0.2±6 mM) (Fig. 2). The H2O2-induced CanR and Lys+ mutant frequency curves are similar for wild type, ntg1, ntg2, rad1 and rad14 single mutants (Fig. 2A, C, D). We observed a moderate increase in H2O2-induced CanR and Lys+ mutant frequencies for the ntg1 ntg2 double mutant strain relative to the WT (Fig. 2A, D). In contrast, H2O2-induced CanR and Lys+ mutant fre-
Spontaneous mutation frequenciesa CanR mutants (per 107 cells)
Lys+ revertants (per 108 cells)
9 8 6 8 42 18 27 31 134 199 2 41 56
7 (1.0) 8 (1.1) 7 (1.0) 9 (1.3) 11 (1.6) 5 (0.7) 6 (0.9) 8 (1.1) 9 (1.3) 6 (0.9) 0.5 (0.07) 0.6 (0.09) 150 (21.4)
(1.0) (0.9) (0.7) (0.9) (4.7) (2.0) (3.0) (3.4) (14.9) (22.1) (0.2) (4.6) (6.2)
Mutation frequencies were calculated by the method of the median from experiments using at least 15 independent cultures (see Materials and methods). The numbers in parentheses indicate the relative increase over WT values
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quencies are greatly increased in the rad1 ntg1 ntg2 or rad14 ntg1 ntg2 triple mutant relative to the other strains tested (Fig. 2A, C, D). It should be noted that the curves for H2O2-induced mutagenesis are also clearly bi-phasic for the rad1 ntg1 ntg2 and rad14 ntg1 ntg2 triple mutants, showing two peaks at 0.5 mM and 6 mM, respectively (Fig. 2A, C, D). Therefore, H2O2induced survival and mutagenesis curves present the same pro®le, suggesting that the two phenomena are correlated. To investigate the respective roles of Ntg1p and Ntg2p in a NER-de®cient background, we measured H2O2-induced mutagenesis in rad1 ntg1 or rad1 ntg2 double mutants. Figure 2B shows a signi®cant increase in CanR mutant frequency in the rad1 ntg1 mutant compared to the WT, whereas this is not the case for the rad1 ntg2 mutant. Fig. 1 Sensitivity of yeast mutant strains to H2O2. Exponentially growing cells were exposed to increasing concentrations of H2O2 (0.1±6 mM) for 30 min at 30°C and plated on non-selective medium. The relevant genotypes of yeast strains are indicated (see Table 1). The data shown are from a representative experiment, similar results were obtained from three independent experiments
Fig. 2A±D H2O2-induced mutagenesis of yeast mutant strains at the CAN1 and LYS1 locus. Exponentially growing cells were treated with H2O2 at the indicated concentration for 30 min at 30°C, washed and allowed to grow for an additional 24 h at 30°C. Cell density was determined after plating on non-selective medium. CanR mutants and Lys+ revertants were scored on selective plates as described in Materials and methods. The data for each curve represent the average values (SE) of at least three experiments for each strain. Values reported in the plots represent H2O2-induced mutation frequencies per 107 surviving cells: f[H2O2-treated] ± f[Untreated]. A WT, rad1, ntg1 ntg2, rad1 ntg1 ntg2. B rad1 ntg1 and rad1 ntg2. C, D WT, rad14, ntg1 ntg2, rad14 ntg1 ntg2. The strains are indicated as follows: ®lled squares, WT; ®lled triangles, rad1; ®lled circles, rad14, open diamonds, rad1 ntg1; ®lled diamonds, rad1 ntg2; open squares, ntg1 ntg2; open circles, rad1 ntg1 ntg2; open triangles, rad14 ntg1 ntg2
Sensitivity and mutability of Ntg1p-, Ntg2p-, and NER-de®cient yeast cells upon exposure to t-butylhydroperoxide The eects of the organic peroxide tert-butylhydroperoxide (t-BH), on survival and mutagenesis of WT, rad1, ntg1 ntg2 and rad1 ntg1 ntg2 mutant strains, were also
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Fig. 3A±C Sensitivity and mutability of yeast mutant strains on exposure to t-butylhydroperoxide. A Sensitivity of yeast mutant strains to t-BH (®lled squares, WT; ®lled triangles, rad1; open squares, ntg1 ntg2; open circles, rad1 ntg1 ntg2) B, C t-BH-induced mutagenesis was measured as described in Materials and methods and in the legend of Fig. 1
investigated. Figure 3A shows that the rad1 single mutant does not exhibit a signi®cant increase in sensitivity to t-BH. On the other hand, the ntg1 ntg2 double mutant exhibits an intermediate sensitivity to t-BH, between those of the WT and the rad1 ntg1 ntg2 triple mutant ± which is the most sensitive strain. Upon exposure to t-BH, CanR and Lys+ mutant frequencies are enhanced over the spontaneous level (Fig. 3B, C). The results show that CanR and Lys+ mutant frequencies for a given dose of t-BH are very similar in wild type, rad1 and ntg1 ntg2 double mutant strains (Fig. 3B, C). In contrast, t-BH-induced CanR Fig. 4A±C Sensitivity and mutability of yeast mutant strains on exposure to menadione. A Sensitivity of yeast mutant strains to MD (®lled squares, WT; ®lled triangles, rad1; open squares, ntg1 ntg2; open circles, rad1 ntg1 ntg2). B, C MD-induced mutagenesis was assayed as described in Materials and methods and in the legend of Fig. 1
and Lys+ mutant frequencies are greatly increased in the rad1 ntg1 ntg2 triple mutant relative to the three other strains (Fig. 3B, C). These results con®rm that Ntg1p, Ntg2p and NER act synergistically to remove mutagenic lesions induced by chemical oxidants in yeast. Sensitivity and mutability of Ntg1p-, Ntg2p-, and NERde®cient yeast cells upon exposure to menadione The eects of the redox cycling agent menadione (MD) on the survival and mutagenesis of WT, rad1, ntg1 ntg2 and rad1 ntg1 ntg2 mutant strains, were also investigated. The results show that the ntg1 ntg2 double and rad1 ntg1 ntg2 triple mutants are signi®cantly more sensitive to MD than the rad1 or the WT strains (Fig. 4A). However, the rad1 ntg1 ntg2 triple mutant is reproducibly more sensitive than the ntg1 ntg2 double mutant. After exposure to MD, CanR and Lys+ mutant frequencies are signi®cantly higher in the ntg1 ntg2 double mutant relative to rad1 and WT (Fig. 4B, C). However, the rad1 ntg1 ntg2 triple mutant shows much higher CanR and Lys+ mutant frequencies than the ntg1 ntg2 double mutant (Fig. 4B, C).
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Sensitivity and mutability of Ntg1p-, Ntg2p-, and NER-de®cient yeast cells upon exposure to c-irradiation Oxidative DNA damage can also be induced by c-irradiation. Figure 5A shows that WT, rad1, ntg1 ntg2 and rad1 ntg1 ntg2 mutant strains exhibit the same sensitivity to c-irradiation. Upon exposure to c-irradiation, CanR and Lys+ mutant frequencies are enhanced in a dosedependent manner (Fig. 5B, C). Our results also show that c-irradiation stimulates the appearance of CanR mutants to the similar levels in WT, rad1, ntg1 ntg2, and rad1 ntg1 ntg2 mutant strains (Fig. 5B). In contrast, Lys+ mutant frequency is considerably increased in the rad1 ntg1 ntg2 triple mutant relative to the WT, rad1 and ntg1 ntg2 strains (Fig. 5C). Therefore, synergism between Ntg1p, Ntg2p and NER after c-irradiation is only observed for Lys+ revertants. Sensitivity and mutability of Ntg1p-, Ntg2p-, and NER-de®cient yeast cells upon exposure to methyl methanesulfonate Oxidative DNA damage also produces AP sites which are primarily repaired by Apn1p, Apn2p and NER (Torres-Ramos et al. 2000). Since, Ntg1p and Ntg2p are AP lyases, it was critical to measure survival and mutagenesis of the rad1 ntg1 ntg2 triple mutant following to an alkylating agent, such as methyl methanesulfonate (MMS), that generates AP sites upon chemical or enzymatic excision of methylated bases in DNA. The results show that rad1 and rad1 ntg1 ntg2 mutant strains are moderately sensitive to MMS relative to the WT (Fig. 6A). Upon exposure to MMS, CanR and Lys+ Fig. 5A±C Sensitivity and mutability of yeast mutant strains on exposure to c-irradiation. A: Sensitivity of yeast mutant strains to c-irradiation (®lled squares, WT; ®lled triangles, rad1; open squares, ntg1 ntg2; open circles, rad1 ntg1 ntg2). B, C Gammairradiation -induced mutagenesis was measured as described in Materials and Methods and in the legend of Fig. 1
mutant frequencies are similar in the rad1 ntg1 ntg2 triple mutant and in the WT (Fig. 6B, C). Therefore, synergism between Ntg1p, Ntg2p and NER is not observed in the repair of AP sites generated by exposure to MMS.
Discussion In S. cerevisiae, three DNA glycosylases, Ntg1p, Ntg2p and Ogg1p, excise a wide spectrum of oxidatively damaged DNA bases and nick DNA at AP sites. The Ogg1p has a narrow substrate speci®city, releasing 8-OxoG andFapyG (Karahalil et al. 1998). In contrast, Ntg1p and Ntg2p recognise a variety of oxidatively damaged pyrimidines and purines (Eide et al. 1996; Augeri et al. 1997; Senturker et al. 1998; You et al. 1998, 1999; Alseth et al. 1999). The aim of this work was to assess the biological role of Ntg1p and Ntg2p and the impact of oxidized DNAbases on genetic stability. Our results show that single (ntg1 or ntg2) and double (ntg1 ntg2) mutant strains do not exhibit a spontaneous mutator phenotype. However, a spontaneous mutator phenotype is revealed by the simultaneous inactivation of Ntg1p, Ntg2p and a protein required for NER (Rad1p or Rad14p), indicating that Ntg1p, Ntg2p and NER act synergistically to remove endogenous DNA damage that can result in CanR mutations in yeast. Since rad1 ntg1 ntg2 and rad14 ntg1 ntg2 triple mutants behave similarly, we conclude that the mutator phenotype is due to the inactivation of the NER pathway and not any other repair pathway requiring Rad1p (Fishman-Lobell and Haber 1992; Kirkpatrick and Petes 1997). Interestingly, rad1 ntg1 ntg2 and rad14 ntg1 ntg2 triple mutants do not show enhanced Lys+ mutant frequency relative to the WT. This last observation suggests that spontaneous CanR mutations are not base substitutions or, alternatively, are base substitutions that cannot generate a Lys+ revertant (Cassier-Chauvat and Fabre 1991). Although they do not allow determination of the chemical nature of endogenous mutagenic DNA damage, these data provide some clues. The endogenous lesions prob-
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Fig. 6A±C Sensitivity and mutability of yeast mutant strains on exposure to methyl methanesulfonate. A: Sensitivity of yeast mutant strains to MMS. (®lled squares, WT; ®lled triangles, rad1; open squares, ntg1 ntg2; open circles, rad1 ntg1 ntg2). B, C: MMSinduced mutagenesis was measured as described in Materials and Methods and in the legend of Fig. 1
ably represent oxidative DNA damage, because they are repaired by a pathway involving both Ntg1p and Ntg2p. They are not AP sites because the major AP endonucleases, Apn1p and Apn2p, are functional in these strains. They are not 8-OxoG nor FapyG, which are repaired by the Ogg1p. Some of them are DNA replication-blocking lesions which require Rev3p for translesion synthesis and mutagenesis (Paulovich et al. 1998). This pro®le suggests oxidized pyrimidines, such as thymine glycol or 5-hydroxycytosine (5-OH-C), as the major class of putative endogenous mutagenic damage in the rad1 ntg1 ntg2 triple mutant. In E. coli, thymine glycol is substrate for both the Nth protein and NER, and causes a T to C transition (Basu et al. 1989; Lin and Sancar 1989; Dizdaroglu et al. 1993) whereas 5-OH-C causes a C to T transition (Feig et al. 1994). Sequence analysis of spontaneous CanR mutants in the WT and the rad1 ntg1 ntg2 triple mutant will provide more information on the identity of the endogenous mutagenic DNA base damage in yeast. Synergism between Ntg1p, Ntg2p and NER was further demonstrated upon exposure of yeast cells to DNA-damaging agents. The results show that rad1 ntg1 ntg2 or rad14 ntg1 ntg2 triple mutants are hypersensitive to H2O2, relative to WT, or ntg1 ntg2, rad1 ntg1 and rad1 ntg2 double mutants. Furthermore, H2O2-induced mutagenesis (CanR and Lys+) is strongly enhanced in the rad1 ntg1 ntg2 and rad14 ntg1 ntg2 triple mutants relative to the other strains tested. The stimulation of lys1-1 reversion indicates that H2O2 induces a spectrum of lesions that diers from that induced by endogenous stress. Although maximal stimulation of CanR mutagenesis is obtained for the rad1 ntg1 ntg2 triple mutant, a signi®cant increase is also observed in the rad1 ntg1 double mutant, indicating a critical role for the Ntg1p.
This may re¯ect the broader substrate speci®city of Ntg1p or the presence of higher amounts of Ntg1p over Ntg2p or both. Indeed, Ntg1p has been shown to be more ecient than Ntg2p in the recognition of UVinduced DNA damage (Alseth et al. 1999). In addition, Ntg1p is inducible upon exposure to H2O2, which may result in higher levels of Ntg1p relative to Ntg2p (Eide et al. 1996; You et al. 1998; Alseth et al. 1999). The fact that inactivation of Ntg1p stongly aects mutagenesis atthe nuclear gene CAN1 demonstrates that the protein is indeed present in the nucleus as well as in mitochondria (Alseth et al. 1999; You et al. 1999). Our results also show that H2O2-induced mutagenesis in the rad1 ntg1 ntg2 triple mutant is bi-phasic, suggesting the induction of repair systems that protect against oxidative DNA damage. The ®rst peak at 0.5 mM H2O2 possibly re¯ects DNA damage that occurs without eciently triggering the adaptive response. The survival curves suggest that adaptation is induced at the same concentration in the WT and the rad1 ntg1 ntg2 strain. Similarly, adaptation to H2O2 occurs at the same concentration in WT and the H2O2-sensitive rad9 mutant (Flattery-O'Brien and Dawes 1998). The synergism between Ntg1p, Ntg2p and NER was also observed upon exposure to other chemical oxidants, t-BH and MD. However, the biological response to MD is clearly dierent from that to H2O2 and t-BH. Upon exposure to MD, the ntg1 ntg2 double mutant is signi®cantly more sensitive and mutable than the WT. Dierent responses to H2O2 and MD were previously reported in yeast. For example, the rad9 mutant strain is hypersensitive to H2O2 but not to MD (Flattery-O'Brien andDawes 1998). Furthermore, H2O2 causes G2 arrest, whereas MD-treated cells arrested in G1 (FlatteryO'Brien and Dawes 1998). This may be due to the types of lesion produced in DNA or to the signals induced by H2O2 and MD, respectively. In E. coli, H2O2 primarily induces the OxyR regulon, whereas superoxide-generating systems such as MD induce the SoxRS regulon (Demple 1991). Although ionising radiation induces DNA base damage similar to that caused by H2O2, it also induces other lesions in DNA, such as double
1095
strand breaks (Dizdaroglu 1991; Cadet et al. 1997). Indeed, we do not observe hypersensitivity of the ntg1 ntg2 rad1 triple mutant relative to the WT, indicating that oxidized DNA bases are not a major cause of lethality after c-irradiation. Upon exposure to c-irradiation, the rate of CanR forward mutagenesis does not increase in the ntg1 ntg2 rad1 triple mutant relative to the WT, whereas that of Lys+ mutagenesis does. This is probably due to the fact that c-irradiation mostly induces CanR mutants that are not base substitutions. In contrast, reversion of the lys1-1 ochre allele speci®cally selects for base substitution events resulting from DNA damage induced by c-irradiation (Kuipers et al. 1999). These results further suggest that Ntg1p, Ntg2p and NER act synergistically to remove lethal and mutagenic oxidized DNA bases induced by chemical oxidants and c-irradiation. The substrate speci®city of Ntg1p and Ntg2p also includes AP sites which may be at the origin of CanR and Lys+ mutants observed in the rad1 ntg1 ntg2 triple mutant upon exposure to oxidizing agents. To avoid a major contribution of AP sites to the phenotypes analysed, the yeast strains used in this study have functional Apn1p and Apn2p (Ramotar et al. 1991; Johnson et al. 1998; Bennett 1999). Our results also show that the induction of an excess of AP sites in yeast cells exposed to MMS does not result in enhanced mutagenesis in the rad1 ntg1 ntg2 triple mutant relative to the WT. Taken together, the results reported in this study strongly suggest that oxidized DNA bases, probably oxidized pyrimidines, that arise due to endogenous or environmental oxidative stress, are mutagenic lesions in yeast. Therefore, the biological role of Ntg1p and Ntg2p is to protect the genetic material from such damage. The NER pathway acts as a very ecient backup system for the removal of some oxidized DNA bases, since inactivationof NER is required to reveal sensitivity or mutability of yeast cells that are de®cient in Ntg1p and Ntg2p. This is in contrast with the role of NER in the repair of alkylation DNA damage, where BER mutants, apn1, apn1 apn2 or mag1, are already hypersensitive to MMS (Ramotar et al. 1991; Xiao and Chow 1998; Torres-Ramos et al. 2000). These results further demonstrate that organisms have developed overlapping DNA repair pathways to protect DNA from unavoidable damage. Acknowledgements The authors thank Drs F.Fabre, M.C.Marsolier and J.P. Radicella and for yeast strains, plasmids, discussion and critical reading of the manuscript. This work was supported by the Centre National de la Recherche Scienti®que (CNRS), the Commissariat aÁ l'Energie Atomique (CEA) and Electricite de France (EDF).
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