The 3'->5' exonuclease of Apn1 provides an alternative pathway to repair 7,8-dihydro-8-oxodeoxyguanosine in Saccharomyces cerevisiae

The 8-oxo-7,8-dihydrodeoxyguanosine (8oxoG), a major mutagenic DNA lesion, results either from direct oxidation of guanines or misincorporation of 8oxodGTP by DNA polymerases. At present, little is known about the mechanisms preventing the mutagenic action of 8oxodGTP in Saccharomyces cerevisiae. Herein, we report for the first time the identification of an alternative repair pathway for 8oxoG residues initiated by S. cerevisiae AP endonuclease Apn1, which is endowed with a robust progressive 3'-->5' exonuclease activity towards duplex DNA. We show that yeast cell extracts, as well as purified Apn1, excise misincorporated 8oxoG, providing a damage-cleansing function to DNA synthesis. Consistent with these results, deletion of both OGG1 encoding 8oxoG-DNA glycosylase and APN1 causes nearly 46-fold synergistic increase in the spontaneous mutation rate, and this enhanced mutagenesis is primarily due to G . C to T . A transversions. Expression of the bacterial 8oxodGTP triphosphotase MutT in the apn1Delta ogg1Delta mutant reduces the mutagenesis. Taken together, our results indicate that Apn1 is involved in an S. cerevisiae 8-oxoguanine-DNA glycosylase (Ogg1)-independent repair pathway for 8oxoG residues. Interestingly, the human major AP endonuclease, Ape1, also exhibits similar exonuclease activity towards 8oxoG residues, raising the possibility that this enzyme could participate in the prevention of mutations that would otherwise result from the incorporation of 8oxodGTP.     

Abasic sites linked to dUTP incorporation in DNA are a major cause of spontaneous mutations in absence of base excision repair and Rad17-Mec3-Ddc1 (9-1-1) DNA damage checkpoint clamp in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, inactivation of base excision repair (BER) AP endonucleases (Apn1p and Apn2p) results in constitutive phosphorylation of Rad53p and delay in cell cycle progression at the G2/M transition. These data led us to investigate genetic interactions between Apn1p, Apn2p and DNA damage checkpoint proteins. The results show that mec1 sml1, rad53 sml1 and rad9 is synthetic lethal with apn1 apn2. In contrast, apn1 apn2 rad17, apn1 apn2 ddc1 and apn1 apn2 rad24 triple mutants are viable, although they exhibit a strong Can(R) spontaneous mutator phenotype. In these strains, high Can(R) mutation rate is dependent upon functional uracil DNA N-glycosylase (Ung1p) and mutation spectra are dominated by AT to CG events. The results point to a role for Rad17-Mec3-Ddc1 (9-1-1) checkpoint clamp in the prevention of mutations caused by abasic (AP) sites linked to incorporation of dUTP into DNA followed by the excision of uracil by Ung1p. The antimutator role of the (9-1-1) clamp can either rely on its essential function in the induction of the DNA damage checkpoint or to another function that specifically impacts DNA repair and/or mutagenesis at AP sites. Here, we show that the abrogation of the DNA damage checkpoint is not sufficient to enhance spontaneous mutagenesis in the apn1 apn2 rad9 sml1 quadruple mutant. Spontaneous mutagenesis was also explored in strains deficient in the two major DNA N-glycosylases/AP-lyases (Ntg1p and Ntg2p). Indeed, apn1 apn2 ntg1 ntg2 exhibits a strong Ung1p-dependent Can(R) mutator phenotype with a spectrum enriched in AT to CG, like apn1 apn2 rad17. However, genetic analysis reveals that ntg1 ntg2 and rad17 are not epistatic for spontaneous mutagenesis in apn1 apn2. We conclude that under normal growth conditions, dUTP incorporation into DNA is a major source of AP sites that cause high genetic instability in the absence of BER factors (Apn1p, Apn2p, Ntg1p and Ntg2p) and Rad17-Mec3-Ddc1 (9-1-1) checkpoint clamp in yeast.     

Cellular role of yeast Apn1 apurinic endonuclease/3''-diesterase: repair of oxidative and alkylation DNA damage and control of spontaneous mutation.

The APN1 gene of Saccharomyces cerevisiae encodes the major apurinic/apyrimidinic endonuclease and 3'-repair DNA diesterase in yeast cell extracts. The Apn1 protein is a homolog of Escherichia coli endonuclease IV, which functions in the repair of some oxidative and alkylation damages in that organism. We show here that yeast strains lacking Apn1 (generated by targeted gene disruption or deletion-replacement) are hypersensitive to both oxidative (hydrogen peroxide and t-butylhydroperoxide) and alkylating (methyl- and ethylmethane sulfonate) agents that damage DNA. These cellular hypersensitivities are correlated with the accumulation of unrepaired damages in the chromosomal DNA of apn1 mutant yeast cells. Hydrogen peroxide-treated APN1+ but not apn1 mutant cells regenerate high-molecular-weight DNA efficiently after the treatment. The DNA strand breaks that accumulate in the Apn1-deficient mutant contain lesions that block the action of DNA polymerase but can be removed in vitro by purified Apn1. An analogous result with DNA from methylmethane sulfonate-treated cells corresponded to the accumulation of unrepaired DNA apurinic sites in the apn1 mutant cells. The rate of spontaneous mutation in apn1 mutant S. cerevisiae was 6- to 12-fold higher than that measured for wild-type yeast cells. This increase indicates that under normal growth conditions, the production of DNA damages that are targets for Apn1 is substantial and that such lesions can be mutagenic when left unrepaired.     

Endogenous DNA abasic sites cause cell death in the absence of Apn1, Apn2 and Rad1/Rad10 in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, mutations in APN1, APN2 and either RAD1 or RAD10 genes are synthetic lethal. In fact, apn1 apn2 rad1 triple mutants can form microcolonies of approximately 300 cells. Expression of Nfo, the bacterial homologue of Apn1, suppresses the lethality. Turning off the expression of Nfo induces G(2)/M cell cycle arrest in an apn1 apn2 rad1 triple mutant. The activation of this checkpoint is RAD9 dependent and allows residual DNA repair. The Mus81/Mms4 complex was identified as one of these back-up repair activities. Furthermore, inactivation of Ntg1, Ntg2 and Ogg1 DNA N-glycosylase/AP lyases in the apn1 apn2 rad1 background delayed lethality, allowing the formation of minicolonies of approximately 10(5) cells. These results demonstrate that, under physiological conditions, endogenous DNA damage causes death in cells deficient in Apn1, Apn2 and Rad1/Rad10 proteins. We propose a model in which endogenous DNA abasic sites are converted into 3'-blocked single-strand breaks (SSBs) by DNA N-glycosylases/AP lyases. Therefore, we suggest that the essential and overlapping function of Apn1, Apn2, Rad1/Rad10 and Mus81/Mms4 is to repair 3'-blocked SSBs using their 3'-phosphodiesterase activity or their 3'-flap endonuclease activity, respectively.     

Lack of contribution of covalent benzo[a]pyrene-7,8-quinone–DNA adducts in benzo[a]pyrene-induced mouse lung tumorigenesis

Benzo[a]pyrene (B[a]P) is a potent human and rodent lung carcinogen. This activity has been ascribed in part to the formation of anti-trans-7,8-dihydroxy-7,8-dihydroB[a]P-9,10-epoxide (BPDE)-DNA adducts. Other carcinogenic mechanisms have been proposed: (1) the induction of apurinic sites from radical cation processes, and (2) the metabolic formation of B[a]P-7,8-quinone (BPQ) that can form covalent DNA adducts or reactive oxygen species which can damage DNA. The studies presented here sought to examine the role of stable BPQ-DNA adducts in B[a]P-induced mouse lung tumorigenesis. Male strain A/J mice were injected intraperitoneally once with BPQ or trans-7,8-dihydroxy-7,8-dihydroB[a]P (BP-7,8-diol) at 30, 10, 3, or 0 mg/kg. Lungs and livers were harvested after 24 h, the DNA extracted and subjected to ³²P-postlabeling analysis. Additional groups of mice were dosed once with BPQ or BP-7,8-diol each at 30 mg/kg and tissues harvested 48 and 72 h later, or with B[a]P (50 mg/kg, a tumorigenic dose) and tissues harvested 72 h later. No BPQ or any other DNA adducts were observed in lung or liver tissues 24, 48, or 72 h after the treatment with 30 mg/kg BPQ. BP-7,8-diol gave BPDE-DNA adducts at all time points in both tissues and B[a]P treatment gave BPDE-DNA adducts in the lung. In each case, no BPQ-DNA adducts were detected. Mouse body weights significantly decreased over time after BPQ or BP-7,8-diol treatments suggesting that systemic toxicity was induced by both agents. Model studies with BPQ and N-acetylcysteine suggested that BPQ is rapidly inactivated by sulfhydryl-containing compounds and not available for DNA adduction. We conclude that under these treatment conditions BPQ does not form stable covalent DNA adducts in the lungs or livers of strain A/J mice, suggesting that stable BPQ-covalent adducts are not a part of the complex of mechanisms involved in B[a]P-induced mouse lung tumorigenesis.     

A general role of the DNA glycosylase Nth1 in the abasic sites cleavage step of base excision repair in Schizosaccharomyces pombe

One of the most frequent lesions formed in cellular DNA are abasic (apurinic/apyrimidinic, AP) sites that are both cytotoxic and mutagenic, and must be removed efficiently to maintain genetic stability. It is generally believed that the repair of AP sites is initiated by the AP endonucleases; however, an alternative pathway seems to prevail in Schizosaccharomyces pombe. A mutant lacking the DNA glycosylase/AP lyase Nth1 is very sensitive to the alkylating agent methyl methanesulfonate (MMS), suggesting a role for Nth1 in base excision repair (BER) of alkylation damage. Here, we have further evaluated the role of Nth1 and the second putative S.pombe AP endonuclease Apn2, in abasic site repair. The deletion of the apn2 open reading frame dramatically increased the sensitivity of the yeast cells to MMS, also demonstrating that the Apn2 has an important function in the BER pathway. The deletion of nth1 in the apn2 mutant strain partially relieves the MMS sensitivity of the apn2 single mutant, indicating that the Apn2 and Nth1 act in the same pathway for the repair of abasic sites. Analysis of the AP site cleavage in whole cell extracts of wild-type and mutant strains showed that the AP lyase activity of Nth1 represents the major AP site incision activity in vitro. Assays with DNA substrates containing base lesions removed by monofunctional DNA glycosylases Udg and MutY showed that Nth1 will also cleave the abasic sites formed by these enzymes and thus act downstream of these enzymes in the BER pathway. We suggest that the main function of Apn2 in BER is to remove the resulting 3′-blocking termini following AP lyase cleavage by Nth1.     

The role of Schizosaccharomyces pombe DNA repair enzymes Apn1p and Uve1p in the base excision repair of apurinic/apyrimidinic sites

In Schizosaccharomyces pombe the repair of apurinic/apyrimidinic (AP) sites is mainly initiated by AP lyase activity of DNA glycosylase Nth1p. In contrast, the major AP endonuclease Apn2p functions by removing 3'-alpha,beta-unsaturated aldehyde ends induced by Nth1p, rather than by incising the AP sites. S. pombe possesses other minor AP endonuclease activities derived from Apn1p and Uve1p. In this study, we investigated the function of these two enzymes in base excision repair (BER) for methyl methanesulfonate (MMS) damage using the nth1 and apn2 mutants. Deletion of apn1 or uve1 from nth1Delta cells did not affect sensitivity to MMS. Exogenous expression of Apn1p failed to suppress the MMS sensitivity of nth1Delta cells. Although Apn1p and Uve1p incised the oligonucleotide containing an AP site analogue, these enzymes could not initiate repair of the AP sites in vivo. Despite this, expression of Apn1p partially restored the MMS sensitivity of apn2Delta cells, indicating that the enzyme functions as a 3'-phosphodiesterase to remove 3'-blocked ends. Localization of Apn1p in the nucleus and cytoplasm hints at an additional function of the enzyme other than nuclear DNA repair. Heterologous expression of Saccharomyces cerevisiae homologue of Apn1p completely restored the MMS resistance of the nth1Delta and apn2Delta cells. This result confirms a difference in the major pathway for processing the AP site between S. pombe and S. cerevisiae cells.     

The major role of human AP-endonuclease homolog Apn2 in repair of abasic sites in Schizosaccharomyces pombe

The abasic (AP) sites, the major mutagenic and cytotoxic genomic lesions, induced directly by oxidative stress and indirectly after excision of damaged bases by DNA glycosylases, are repaired by AP-endonucleases (APEs). Among two APEs in Saccharomyces cerevisiae, Apn1 provides the major APE activity, and Apn2, the ortholog of the mammalian APE, provides back-up activity. We have cloned apn1 and apn2 genes of Schizosaccharomyces pombe, and have shown that inactivation of Apn2 and not Apn1 sensitizes this fission yeast to alkylation and oxidative damage-inducing agents, which is further enhanced by Apn1 inactivation. We also show that Uve1, present in S.pombe but not in S.cerevisiae, provides the back-up APE activity together with Apn1. We confirmed the presence of APE activity in recombinant Apn2 and in crude cell extracts. Thus S.pombe is distinct from S.cerevisiae, and is similar to mammalian cells in having Apn2 as the major APE.     

Abrogation of the Chk1-Pds1 checkpoint leads to tolerance of persistent single-strand breaks in Saccharomyces cerevisiae

In budding yeast, Apn1, Apn2, Tpp1, and Rad1/Rad10 are important enzymes in the removal of spontaneous DNA lesions. apn1 apn2 rad1 yeast are inviable due to accumulation of abasic sites and strand breaks with 3' blocking lesions. We found that tpp1 apn1 rad1 yeast exhibited slow growth but frequently gave rise to spontaneous slow growth suppressors that segregated as single-gene mutations. Using a candidate gene approach, we identified several tpp1 apn1 rad1 suppressors. Deleting uracil glycosylase suppressed both tpp1 apn1 rad1 and apn1 apn2 rad1 growth defects by reducing the abasic site burden. Mutants affecting the Chk1-Pds1 metaphase-anaphase checkpoint only suppressed tpp1 apn1 rad1 slow growth. In contrast, most S-phase checkpoint mutants were synthetically lethal in a tpp1 apn1 rad1 background. Epistasis analyses showed an additive effect between chk1 and ung1, indicating different mechanisms of suppression. Loss of Chk1 partially restored cell-growth parameters in tpp1 apn1 rad1 yeast, but at the same time exacerbated chromosome instability. We propose a model in which recombinational repair during S phase coupled with failure of the metaphase-anaphase checkpoint allows for tolerance of persistent single-strand breaks at the expense of genome stability.     

Msh1p counteracts oxidative lesion-induced instability of mtDNA and stimulates mitochondrial recombination in Saccharomyces cerevisiae

The proximity of the mitochondrial genome to the respiratory chain, a major source of ROS (radical oxygen species), makes mtDNA more vulnerable to oxidative damage than nuclear DNA. Mitochondrial BER (base excision repair) is generally considered to be the main pathway involved in the prevention of oxidative lesion-induced mutations in mtDNA. However, we previously demonstrated that the increased frequency of mitochondrial Oli^r mutants in an ogg1Δ strain, lacking the activity of a crucial mtBER glycosylase, is reduced in the presence of plasmids encoding Msh1p, the mitochondrial homologue of the bacterial mismatch protein MutS. This finding suggested that Msh1p might be involved in the prevention of mitochondrial mutagenesis induced by oxidative stress. Here we show that a double mutant carrying the msh1-R813W allele, encoding a variant of the protein defective in the ATP hydrolysis activity, combined with deletion of SOD2, encoding the mitochondrial superoxide dismutase, displays a synergistic effect on the frequency of Oli^r mutants, indicating that Msh1p prevents generation of oxidative lesion-induced mitochondrial mutations. We also show that double mutants carrying the msh1-R813W allele, combined with deletion of either OGG1 or APN1, the latter resulting in deficiency of the Apn1 endonuclease, exhibit a synergistic effect on the frequency of respiration-defective mutants having gross rearrangements of the mitochondrial genome. This suggests that Msh1p, Ogg1p and Apn1p play overlapping functions in maintaining the stability of mtDNA. In addition, we demonstrate, using a novel ARG8^m recombination assay, that a surplus of Msh1p results in enhanced mitochondrial recombination. Interestingly, the mutant forms of the protein, msh1p-R813W and msh1p-G776D, fail to stimulate recombination. We postulate that the Msh1p-enhanced homologous recombination may play an important role in the prevention of oxidative lesion-induced rearrangements of the mitochondrial genome.     

Pir1p mediates translocation of the yeast Apn1p endonuclease into the mitochondria to maintain genomic stability

The mitochondrial genome is continuously subject to attack by reactive oxygen species generated through aerobic metabolism. This leads to the formation of a variety of highly genotoxic DNA lesions, including abasic sites. Yeast Apn1p is localized to the nucleus, where it functions to cleave abasic sites, and apn1 Delta mutants are hypersensitive to agents such as methyl methanesulfonate (MMS) that induce abasic sites. Here we demonstrate for the first time that yeast Apn1p is also localized to the mitochondria. We found that Pir1p, initially isolated as a cell wall constituent of unknown function, interacts with the C-terminal end of Apn1p, which bears a bipartite nuclear localization signal. Further analysis revealed that Pir1p is required to cause Apn1p mitochondrial localization, presumably by competing with the nuclear transport machinery. pir1 Delta mutants displayed a striking (approximately 3-fold) increase of Apn1p in the nucleus, which coincided with drastically reduced levels in the mitochondria. To explore the functional consequences of the Apn1p-Pir1p interaction, we measured the rate of mitochondrial mutations in the wild type and pir1 Delta and apn1 Delta mutants. pir1 Delta and apn1 Delta mutants exposed to MMS exhibited 3.6- and 5.8-fold increases, respectively, in the rate of mitochondrial mutations, underscoring the importance of Apn1p in repair of the mitochondrial genome. We conclude that Pir1p interacts with Apn1p, at the level of either the cytoplasm or nucleus, and facilitates Apn1p transport into the mitochondria to repair damaged DNA.     

Base excision repair activities required for yeast to attain a full chronological life span

The chronological life span of yeast, the survival of stationary (G0) cells over time, provides a model for investigating certain of the factors that may influence the aging of non-dividing cells and tissues in higher organisms. This study measured the effects of defined defects in the base excision repair (BER) system for DNA repair on this life span. Stationary yeast survives longer when it is pre-grown on respiratory, as compared to fermentative (glucose), media. It is also less susceptible to viability loss as the result of defects in DNA glycosylase/AP lyases (Ogg1p, Ntg1p, Ntg2p), apurinic/apyrimidinic (AP) endonucleases (Apn1p, Apn2p) and monofunctional DNA glycosylase (Mag1p). Whereas single BER glycosylase/AP lyase defects exerted little influence over such optimized G0 survival, this survival was severely shortened with the loss of two or more such enzymes. Equally, the apn1delta and apn2delta single gene deletes survived as well as the wild type, whereas a apn1delta apn2delta double mutant totally lacking in any AP endonuclease activity survived poorly. Both this shortened G0 survival and the enhanced mutagenicity of apn1delta apn2delta cells were however rescued by the over-expression of either Apn1p or Apn2p. The results highlight the vital importance of BER in the prevention of mutation accumulation and the attainment of the full yeast chronological life span. They also reveal an appreciable overlap in the G0 maintenance functions of the different BER DNA glycosylases and AP endonucleases.     

The Saccharomyces cerevisiae ETH1 gene, an inducible homolog of exonuclease III that provides resistance to DNA-damaging agents and limits spontaneous mutagenesis

The recently sequenced Saccharomyces cerevisiae genome was searched for a gene with homology to the gene encoding the major human AP endonuclease, a component of the highly conserved DNA base excision repair pathway. An open reading frame was found to encode a putative protein (34% identical to the Schizosaccharomyces pombe eth1(+) [open reading frame SPBC3D6.10] gene product) with a 347-residue segment homologous to the exonuclease III family of AP endonucleases. Synthesis of mRNA from ETH1 in wild-type cells was induced sixfold relative to that in untreated cells after exposure to the alkylating agent methyl methanesulfonate (MMS). To investigate the function of ETH1, deletions of the open reading frame were made in a wild-type strain and a strain deficient in the known yeast AP endonuclease encoded by APN1. eth1 strains were not more sensitive to killing by MMS, hydrogen peroxide, or phleomycin D1, whereas apn1 strains were approximately 3-fold more sensitive to MMS and approximately 10-fold more sensitive to hydrogen peroxide than was the wild type. Double-mutant strains (apn1 eth1) were approximately 15-fold more sensitive to MMS and approximately 2- to 3-fold more sensitive to hydrogen peroxide and phleomycin D1 than were apn1 strains. Elimination of ETH1 in apn1 strains also increased spontaneous mutation rates 9- or 31-fold compared to the wild type as determined by reversion to adenine or lysine prototrophy, respectively. Transformation of apn1 eth1 cells with an expression vector containing ETH1 reversed the hypersensitivity to MMS and limited the rate of spontaneous mutagenesis. Expression of ETH1 in a dut-1 xthA3 Escherichia coli strain demonstrated that the gene product functionally complements the missing AP endonuclease activity. Thus, in apn1 cells where the major AP endonuclease activity is missing, ETH1 offers an alternate capacity for repair of spontaneous or induced damage to DNA that is normally repaired by Apn1 protein.     

Lack of the DNA glycosylases MYH and OGG1 in the cancer prone double mutant mouse does not increase mitochondrial DNA mutagenesis

Reactive oxygen species (ROS) are formed as natural byproducts during aerobic metabolism and readily induce premutagenic base lesions in the DNA. The 8-oxoguanine DNA glycosylase (OGG1) and MutY homolog 1 (MYH) synergistically prevent mutagenesis and cancer formation in mice. Their localization in the mitochondria as well as in the nucleus suggests that mutations in mitochondrial DNA (mtDNA) contribute to the carcinogenesis in the myh^?/?/ogg1^?/? double knockout mouse.In order to test this hypothesis, we analyzed mtDNA mutagenesis and mitochondrial function in young (1 month) and adult (6 months) wt and myh^?/?/ogg1^?/? mice. To our surprise, the absence of OGG1 and MYH had no impact on mtDNA mutation rates in these mice, even at the onset of cancer. This indicates that mtDNA mutagenesis is not responsible for the carcinogenesis of myh^?/?/ogg1^?/? mice. In line with these results, mitochondrial function was unaffected in the cancerous tissues liver and lung, whereas a significant reduction in respiration capacity was observed in brain mitochondria from the adult myh^?/?/ogg1^?/? mouse. The reduced respiration capacity correlated with a specific reduction (?25%) in complex I biochemical activity in brain mitochondria.Our results demonstrate that mtDNA mutations are not associated with cancer development in myh^?/?/ogg1^?/? mice, and that impairment of mitochondrial function in brain could be linked to nuclear DNA mutations in this strain. OGG1 and MYH appear to be dispensable for antimutator function in mitochondria.     

Complementation of DNA repair-deficient Escherichia coli by the yeast Apn1 apurinic/apyrimidinic endonuclease gene

The Saccharomyces cerevisiae APN1 gene encoding an AP endonuclease/3'-diesterase was engineered in vitro for expression in Escherichia coli. The expression vector directs the synthesis in E. coli of a Mr 40,500 protein that reacts with anti-Apn1 antibodies and has the DNA-repair activities characteristic of Apn1 isolated from yeast. A band corresponding to Apn1 was observed in DNA repair activity gels only with extracts of E. coli harbouring the APN1 expression plasmid. Expression of Apn1 conferred resistance to oxidants and alkylating agents in E. coli lacking exonuclease III and endonuclease IV. For H2O2 damage, this rescue effect was correlated with the repair of oxidative lesions in the bacterial chromosome by the Apn1 protein. Thus, Apn1 can function in bacteria in a manner similar to its proposed multiple functions in yeast.     

Repair of oxidative damage in mitochondrial DNA of Saccharomyces cerevisiae: involvement of the MSH1-dependent pathway

Mitochondrial DNA (mtDNA) is located close to the respiratory chain, a major source of reactive oxygen species (ROS). This proximity makes mtDNA more vulnerable than nuclear DNA to damage by ROS. Therefore, the efficient repair of oxidative lesions in mtDNA is essential for maintaining the stability of the mitochondrial genome. A series of genetic and biochemical studies has indicated that eukaryotic cells, including the model organism Saccharomyces cerevisiae, use several alternative strategies to prevent mutagenesis induced by endogenous oxidative damage to nuclear DNA. However, apart from base excision repair (BER), no other pathways involved in the repair of oxidative damage in mtDNA have been identified. In this study, we have examined mitochondrial mutagenesis in S. cerevisiae cells which lack the activity of the Ogg1 glycosylase, an enzyme playing a crucial role in the removal of 8-oxoG, the most abundant oxidative lesion of DNA. We show that the overall frequency of the mitochondrial oligomycin-resistant (Olir) mutants is increased in the ogg1 strain by about one order of magnitude compared to that of the wild-type strain. Noteworthy, in the mitochondrial oli1 gene, G:C to T:A transversions are generated approximately 50-fold more frequently in the ogg1 mutant relative to the wild-type strain. We also demonstrate that the increased frequency of Olir mutants in the ogg1 strain is markedly reduced by the presence of plasmids encoding Msh1p, a homologue of the bacterial mismatch protein MutS, which specifically functions in mitochondria. This suppression of the mitochondrial mutator phenotype of the ogg1 strain seems to be specific, since overexpression of the mutant allele msh1-R813W failed to exert this effect. Finally, we also show that the increased frequency of Olir mutants arising in an msh1/MSH1 heterozygote grown in glucose-containing medium is further enhanced if the cells are cultivated in glycerol-containing medium, i.e. under conditions when the respiratory chain is fully active. Taken together, these results strongly suggest that MSH1-dependent repair represents a significant back-up to mtBER in the repair of oxidative damage in mtDNA.     

Schizosaccharomyces pombe encodes a mutated AP endonuclease 1

Mutagenic and cytotoxic apurinic/apyrimidinic (AP) sites are among the most frequent lesions in DNA. Repair of AP sites is initiated by AP endonucleases and most organisms possess two or more of these enzymes. Saccharomyces cerevisiae has AP endonuclease 1 (Apn1) as the major enzymatic activity with AP endonuclease 2 (Apn2) being an important backup. Schizosaccharomyces pombe also encodes two potential AP endonucleases, and Apn2 has been found to be the main repair activity, while Apn1 has no, or only a limited role in AP site repair. Here we have identified a new 5' exon (exon 1) in the apn1 gene and show that the inactivity of S. pombe Apn1 is due to a nonsense mutation in the fifth codon of this new exon. Reversion of this mutation restored the AP endonuclease activity of S. pombe Apn1. Interestingly, the apn1 nonsense mutation was only found in laboratory strains derived from L972 h(-) and not in unrelated isolates of S. pombe. Since all S. pombe laboratory strains originate from L972 h(-), it appears that all experiments involving S. pombe have been conducted in an apn1(-) mutant strain with a corresponding DNA repair deficiency. These observations have implications both for future research in S. pombe and for the interpretation of previously conducted epistatis analysis.     

The role of yeast DNA 3'-phosphatase Tpp1 and rad1/Rad10 endonuclease in processing spontaneous and induced base lesions

Tpp1 is a DNA 3'-phosphatase in Saccharomyces cerevisiae that is believed to act during strand break repair. It is homologous to one domain of mammalian polynucleotide kinase/3'-phosphatase. Unlike in yeast, we found that Tpp1 could confer resistance to methylmethane sulfonate when expressed in bacteria that lack abasic endonuclease/3'-phosphodiesterase function. This species difference was due to the absence of delta-lyase activity in S. cerevisiae, since expression of bacterial Fpg conferred Tpp1-dependent resistance to methylmethane sulfonate in yeast lacking the abasic endonucleases Apn1 and Apn2. In contrast, beta-only lyases increased methylmethane sulfonate sensitivity independently of Tpp1, which was explained by the inability of Tpp1 to cleave 3' alpha,beta-unsaturated aldehydes. In parallel experiments, mutations of TPP1 and RAD1, encoding part of the Rad1/Rad10 3'-flap endonuclease, caused synthetic growth defects in yeast strains lacking Apn1. In contrast, Fpg expression led to a partial rescue of apn1 apn2 rad1 synthetic lethality by converting lesions into Tpp1-cleavable 3'-phosphates. The collected experiments reveal a profound toxicity of strand breaks with irreparable 3' blocking lesions, and extend the function of the Rad1/Rad10 salvage pathway to 3'-phosphates. They further demonstrate a role for Tpp1 in repairing endogenously created 3'-phosphates. The source of these phosphates remains enigmatic, however, because apn1 tpp1 rad1 slow growth could be correlated with neither the presence of a yeast delta-lyase, the activity of the 3'-phosphate-generating enzyme Tdp1, nor levels of endogenous oxidation.     

On the excess of G --> T transversions in the p53 gene in lung cancer cell lines. Reply to Pfeifer and Hainaut

Our recent retrospective analysis of the lung cancer-associated p53 mutation data [Mutat. Res. 508 (2002) 1] showed the possibility of (i) inhibiting action of tobacco smoke on repair of G --> T primary lesions in the non-transcribed strand of the p53 gene and (ii) the origin of new p53 mutations, predominantly G --> T transversions, in lung cancer cell lines apparently unexposed to tobacco smoke. In summary, our arguments suggest that (i) in addition to polycyclic aromatic hydrocarbons (PAH)-DNA adducts there exist other lung cancer-specific, rather than smoke-specific sources of G --> T transversions and (ii) a direct mutagenic action is not the only smoke-associated cause of the prevalence of this class of p53 mutations in lung cancer. In the subsequent critical commentary [Mutat. Res. 526 (2003) 39], Pfeifer and Hainaut suggested our arguments to be "incompatible with available evidence". We would like to address their critique, and appreciate the editors of Mutation Research giving us an opportunity to do so.