The treatment with the probiotic Shewanella putrefaciens Pdp11 of specimens of Solea senegalensis exposed to high stocking densities to enhance their resistance to disease.

Aquaculture industry exposes fish to acute stress events, such as high stocking density, and a link between stress and higher susceptibility to diseases has been concluded. Several studies have demonstrated increased stress tolerance of fish treated with probiotics, but the mechanisms involved have not been elucidated. Shewanella putrefaciens Pdp11 is a strain isolated from healthy gilthead seabream (Sparus aurata L.) and it is considered as probiotics. The aim of this study was to evaluate the effect of the dietary administration of this probiotics on the stress tolerance of Solea senegalensis specimens farmed under high stocking density (PHD) compared to a group fed a commercial diet and farmed under the same conditions (CHD). In addition, during the experiment, a natural infectious outbreak due to Vibrio species affected fish farmed under crowding conditions. Changes in the microbiota and histology of intestine and in the transcription of immune response genes were evaluated at 19 and 30 days of the experiment. Mortality was observed after 9 days of the beginning of the experiment in CHD and PHD groups, it being higher in the CHD group. Fish farmed under crowding stress showed reduced expression of genes at 19 day probiotic feeding. On the contrary, a significant increase in immune related gene expression was detected in CHD fish at 30 day, whereas the gene expression in fish from PHD group was very similar to that showed in specimens fed and farmed with the conventional conditions. In addition, the dietary administration of S. putrefaciens Pdp11 produced an important modulation of the intestinal microbiota, which was significantly correlated with the high number of goblet cells detected in fish fed the probiotic diet.

Expression analysis of the nrdHIEF operon from Escherichia coli. Conditions that trigger the transcript level in vivo.

Escherichia coli has two aerobic ribonucleotide reductases encoded by the nrdAB and nrdHIEF operons. While NrdAB is active during aerobiosis, NrdEF is considered a cryptic enzyme with no obvious function. Here, we present evidence that nrdHIEF expression might be important under certain circumstances. Basal transcript levels were dramatically enhanced (25-75-fold), depending on the growth-phase and the growth-medium composition. Likewise, a large increase of >100-fold in nrdHIEF mRNA was observed in bacteria lacking Trx1 and Grx1, the two main NrdAB reductants. Moreover, nrdHIEF expression was triggered in response to oxidative stress, particularly in mutants missing hydroperoxidase I and alkyl-hydroperoxide reductase activities (69.7-fold) and in cells treated with oxidants (up to 23.4-fold over the enhanced transcript level possessed by cells grown on minimal medium). The mechanism(s) that triggers nrdHIEF expression remains unknown, but our findings exclude putative global regulators like RpoS, Fis, cAMP, OxyR, SoxR/S, or RecA. What we have learned about nrdHIEF expression indicates strong differences between its regulation and that of the nrdAB operon and of genes coding for components of both thioredoxin/glutaredoxin pathways. We propose that E. coli might optimize the responses to different stimuli by co-evolving the expression levels for its multiple reductases and electron donors.

Transcriptional regulation of glutaredoxin and thioredoxin pathways and related enzymes in response to oxidative stress.

We examined the in vivo expression of up to 16 genes encoding for components of both glutaredoxin and thioredoxin systems and for members of the OxyR and SoxRS regulons. We demonstrated that grxA (Grx1) transcription is triggered in bacteria lacking Trx1 (trxA) and GSH (gshA) in an OxyR-dependent manner. We also indicated that, unlike OxyR, SoxR is not constitutively activated in the oxidizing environment of trxA gshA mutants. We discovered that the lack of Trx1 plus GSH increases the steady-state levels of Trx reductase (trxB) and Trx2 (trxC) transcripts. This increase and the trxB and trxC up-regulation caused by the constitutive oxyR2 allele indicate that OxyR also plays a role in the regulation of the thioredoxin pathway. On the contrary, no change in the expression of genes for Trx1, Grx2, and Grx3 was observed. Transcription of nrdAB (RRase) was not induced by oxidative stress yet was induced by hydroxyurea (RRase inhibitor). Induction level was as the enhanced nrdAB basal expression of trxA grxA mutants, indicating that RRase operation without Trx1 and Grx1 must lead to disturbances sensed as those caused by hydroxyurea. We also demonstrated an inverse relation between nrdAB expression and that of genes coding for components of both glutaredoxin (grxA, gorA) and thioredoxin (trxB, trxC) systems.

Oxidative mutagenesis in Escherichia coli strains lacking ROS-scavenging enzymes and/or 8-oxoguanine defenses.

Escherichia coli strains with different combinations of null mutations in the katG, katE, sodA, sodB, fpg, and mutY genes were constructed to compare their spontaneous mutation frequencies and sensitivities to various oxidants with those of bacteria solely deficient in catalase (katG katE) or cytosolic superoxide dismutase (sodA sodB) and the parental strain possessing a full complement of these enzymes. The MutY DNA glycosylase represented the major protection against the mutagenic consequences of processes associated with normal aerobic metabolism. Spontaneous mutagenesis in MutY-lacking bacteria was not influenced by the absence of (A)BC excinuclease or the presence of MucAB proteins, a result consistent with 8-oxoguanine being a principal premutational lesion. In contrast, catalase and SOD represented the major protection against the genotoxic consequences of bursts of oxidative stress caused by reactive-oxygen-generating compounds. Therefore, only bacteria simultaneously defective in both katG and katE or sodA and sodB genes were hypersensitive with respect to mutability by peroxide and superoxide, respectively. These data suggest that oxidative lesions other than 8-oxoguanine contribute to mutagenesis by hydrogen peroxide and redox-cycling chemicals.

Overexpression of the human HAP1 protein sensitizes cells to the lethal effect of bioreductive drugs.

Abasic sites (AP sites) are generated in DNA either directly by DNA-damaging agents or by DNA glycosylases acting during base excision repair. These sites are repaired in human cells by the HAP1 protein, which, besides its AP-endonuclease activity, also possesses a redox function. To investigate the ability of HAP1 protein to modulate cell resistance to DNA-damaging agents, CHO cells were transfected with HAP1 cDNA, resulting in stable expression of the protein in the cell nuclei. The sensitivity of the transfected cells to the toxic effect of various agents, e.g. methylmethane sulfonate, bleomycin and H2O2, was not modified. However, the transfected cells became more sensitive to killing by mitomycin C, porfiromycin, daunorubicin and aziridinyl benzoquinone, drugs that are activated by reduction. To test whether the redox function of HAP1 protein was involved in this increased cytotoxicity, we have constructed a mutated HAP1 protein endowed with normal AP-endonuclease activity but deleted for redox function. When this mutated protein was expressed in the cells, elevated AP-endonuclease activity was measured, but sensitization to the lethal effects of compounds requiring bioreduction was no longer observed. These results suggest that HAP1 protein, besides its involvement in DNA repair, is able to activate bioreduction of alkylating drugs used in cancer chemotherapy.

Role of DNA repair by (A)BC excinuclease and Ogt alkyltransferase in the final distribution of LacI-d mutations induced by N-butyl-N-nitrosourea in Escherichia coli.

In the absence of nucleotide excision repair, the additional deficiency of the DNA alkyltransferase (ATase) encoded by the constitutive ogt gene of Escherichia coli caused a marked increase in mutation induction by N-butyl-N-nitrosourea (BNU). Irrespective of the presence or absence of the Ogt ATase, little mutagenic response was detected in Uvr+ bacteria in the concentration range 0-8 mM BNU, indicating that most premutagenic DNA lesions induced at these concentrations are efficiently recognized and repaired by the nucleotide excision repair system. Increased susceptibility to mutagenesis by BNU was detected in Uvr- Ogt+ bacteria, but the Uvr- Ogt- double mutant exhibited much higher sensitivity. These data suggest that the Ogt ATase can replace to a great extent the repair capacity of the (A)BC excinuclease. Forward mutations induced by 6 mM BNU within the initial part of the lacI gene of E.coli were recovered from Uvr+ Ogt-, Uvr- Ogt+ and Uvr- Ogt- bacteria. A total of 454 independent mutations were characterized by DNA sequence analysis. The BNU-induced spectra were dominated by G:C-->A:T transitions, consistent with the major role of the O6-alkylguanine miscoding lesion in mutagenesis by alkylating agents. Specific sites for G:C-->A:T transitions were recovered more or less frequently in one genetic background versus the others, giving statistically significant differences among the spectra (P < 10(-6)). We examined the influence of DNA repair by (A)BC excinuclease and Ogt ATase on the 5'-flanking base associated with the BNU-induced G:C-->A:T transitions; preferences different from those previously reported for other alkylnitrosoureas were detected. We discuss how these differences might be caused by BNU producing branched chain derivatives, in addition to the expected linear chain adducts, and by possible preferences with respect to both the initial distribution of O6-butylguanine lesions and their repairability.

Rat 7,8-dihydro-8-oxoguanine DNA glycosylase: substrate specificity, kinetics and cleavagemechanism at an apurinic site.

Reactive oxygen species produce different lesions in DNA. Among them, 7,8-dihydro-8-oxoguanine (8-oxoG) is one of the major oxidative products implicated in mutagenesis. This lesion is removed from damaged DNA by base excision repair, and genes coding for 8-oxoG-DNA glycosylases have been isolated from bacteria, yeast and human cells. We have isolated and characterized the cDNA encoding the rat 8-oxoG-DNA glycosylase (rOGG1). Expression of the cDNA in the fgp mutY Escherichia coli double mutant allowed the purification of the untagged rOGG1 protein. It excises 8-oxoG from DNA with a strong preference for duplex DNA containing 8-oxoG:C base pairs. rOGG1 also acts on formamidopyrimidine (FaPy) residues, and the K m values on 8-oxoG and FaPy residues are 18.8 and 9.7 nM, respectively. When acting on an oligonucleotide containing an 8-oxoG residue, rOGG1 shows a beta-lyase activity that nicks DNA 3' to the lesion. However, rOGG1 acts on a substrate containing an apurinic site by a beta-delta elimination reaction and proceeds through a Schiff base intermediate. Expression of rOGG1 in E.coli fpg mutY suppresses its spontaneous mutator phenotype.

Deficient DNA-ligase activity in the metabolic disease tyrosinemia type I.

Hereditary tyrosinemia type I (HT1) is an autosomal recessive inborn error of metabolism caused by the deficiency of fumarylacetoacetate hydrolase, the last enzyme in the tyrosine catabolism pathway. This defect results in accumulation of succinylacetone (SA) that reacts with amino acids and proteins to form stable adducts via Schiff base formation, lysine being the most reactive amino acid. HT1 patients surviving beyond infancy are at considerable risk for the development of hepatocellular carcinoma, and a high level of chromosomal breakage is observed in HT1 cells, suggesting a defect in the processing of DNA. In this paper we show that the overall DNA-ligase activity is low in HT1 cells (about 20% of the normal value) and that Okazaki fragments are rejoined at a reduced rate compared with normal fibroblasts. No mutation was found by sequencing the ligase I cDNA from HT1 cells, and the level of expression of the ligase I mRNA was similar in normal and HT1 fibroblasts, suggesting the presence of a ligase inhibitor. SA was shown to inhibit in vitro the overall DNA-ligase activity present in normal cell extracts. The activity of purified T4 DNA-ligase, whose active site is also a lysine residue, was inhibited by SA in a dose-dependent manner. These results suggest that accumulation of SA reduces the overall ligase activity in HT1 cells and indicate that metabolism errors may play a role in regulating enzymatic activities involved in DNA replication and repair.

Influence of DNA repair by (A)BC excinuclease and Ogt alkyltransferase on the distribution of mutations induced by n-propyl-N-nitrosourea in Escherichia coli.

In the absence of nucleotide excision repair, the additional deficiency of the DNA alkyltransferase (ATase) encoded by the constitutive ogt gene of Escherichia coli caused a marked increment in mutation induction by N-propyl-N-nitrosourea (PNU). Irrespective of the presence or the absence of the Ogt ATase, little mutagenic response was detected in Uvr+ bacteria in the concentration range 0-8 mM PNU, indicating that most premutagenic DNA lesions induced at these concentrations are efficiently recognized and repaired by the nucleotide excision repair system. Some increased susceptibility to mutagenesis by PNU was detected in Uvr- Ogt+ bacteria, but the Uvr- Ogt- double mutant exhibited much higher sensitivity. These data suggest that the Ogt ATase can replace to a great extent the repair capacity of the (A)BC excinuclease. Forward mutations induced by 6 mM PNU within the initial part of the lacl gene were recovered from Uvr+ Ogt-, Uvr- Ogt+, and Uvr- Ogt- bacteria. A total of 439 independent mutations were characterized by DNA sequence analysis. The PNU-induced spectra were dominated by G:C-->A:T transitions, consistent with the major role of the O6-alkylguanine miscoding lesion in mutagenesis by alkylating agents. Specific sites for G:C-->A:T transitions were recovered more or less frequently in one genetic background versus the others, giving statistically significant differences among the spectra (P < 10(-6)). We examined the influence of DNA repair by (A)BC excinuclease and Ogt ATase on the 5'-flanking base and DNA-strand associated with the PNU-induced G:C-->A:T transitions. Preferences different from those previously reported for the ethylating (ENU) and methylating (MNU) analogs were detected. We indicate that these differences might be caused by the PNU possibility of giving iso-propyl adducts, in addition to the expected n-propyl adducts, and by possible preferences in the initial distribution of these lesions as well as in their repair by the (A)BC excinuclease and the Ogt ATase of E. coli.

Bacterial and mammalian DNA alkyltransferases sensitize Escherichia coli to the lethal and mutagenic effects of dibromoalkanes.

Here we confirm and extend our previous studies demonstrating that the mutagenic potency of 1,2-dibromoethane (DBE) and dibromomethane (DBM) is markedly enhanced (not prevented) in bacteria expressing the O6-alkylguanine-DNA alkyltransferase (ATase) encoded by the Escherichia coli ogt gene. We demonstrate that, in close parallel with mutagenesis, the Ogt ATase sensitizes the bacteria to the lethal effects of these carcinogens, suggesting that one or more of the potentially mutagenic lesions induced by DBE and DBM in the presence of Ogt has additional lethal capacity. We further demonstrate that the sensitization to both lethality and mutagenesis by DBE and DBM is a property shared by other DNA alkyltransferases. This objective was accomplished by quantifying the induction of mutations and lethal events in ogt- ada- E. coli expressing an exogenous bacterial or mammalian ATase from a multicopy plasmid. Mammalian recombinant ATases enhanced the lethal and mutagenic actions of DBE and suppressed the lack of sensitivity of the vector-transformed bacteria to DBM. In most cases the order of effectiveness of the ATases ranked: murine > human > Ogt > rat. Further comparisons included the full-length Ada ATase from E. coli and a truncated Ada version (T-ada) that retains the O6-methylguanine binding domain of the protein. The full-length Ada ATase was effective in enhancing the lethality but not the mutagenicity induced by DBE and DBM. The T-ada ATase provided less sensitization than Ada to lethality by DBE, but of the three bacterial ATases T-ada yielded the highest sensitization to mutagenesis by this compound. T-ada and Ada ATases were in general less effective than the mammalian versions, with the exception of the rat recombinant ATase. The effectiveness of the different mammalian and bacterial ATases in promoting the deleterious actions of dibromoalkanes was compared with the effectiveness of these proteins in suppressing the lethal and mutagenic effects induced by N-nitroso-N-methylurea. The ability to sensitize E. coli to the lethal and mutagenic effects of DBE and DBM seems restricted to DNA alkyltransferase, since overexpression of thioredoxin (Trx) or glutaredoxin (Grx1) in ogt- ada- cells showed no effect, in spite of the reported potential of bioactive dihaloethane-derived species to alkylate Trx.

Mutational specificity of aflatoxin B1. Comparison of in vivo host-mediated assay with in vitro S9 metabolic activation.

An intrasanguineous host-mediated assay was used to determine the pattern of mutagenesis induced by the carcinogen aflatoxin B1 in the lacI gene of Escherichia coli recovered from rat liver. To investigate the influence of different types of metabolic activation, the mutation spectrum induced by AFB1 activated in vitro by a commercially prepared S9 microsomal fraction from Aroclor 1254-treated rats was also obtained. A total of 281 forward mutations affecting the N-terminal region of the lacI gene were characterized by DNA sequencing analysis. AFB1 induced similar type of mutations with similar site specificity when activated by the standard S9 fraction or by employing a rat host-mediated assay. These results indicate the ability of the in vitro S9 fraction to mimic the in vivo metabolism, suggesting that the same active metabolite, presumably AFB1 8,9-epoxide, is responsible for generating a similar pattern of DNA damage, as reflected in the similarity of mutational spectra. For both activation systems, most mutations (>90%) were base substitutions that occurred primarily at G:C pairs. Somewhat over one-half of G:C targeted substitutions were GC>TA transversions, other mutations being evenly divided between G:C>AT transitions and GC>CG transversions. The mutational specificity exhibited by activated AFB1 can be explained by incorporation of different bases opposite a single type of non-instructive lesion during error-prone DNA synthesis. To what extent the mutations are due to the main adduct (AFB1-N7-Gua), its imidazole-ring-opened derivative or an apurinic site remains unknown.

Contribution of ogt-encoded alkyltransferase to resistance to chloroethylnitrosoureas in nucleotide excision repair-deficient Escherichia coli.

We investigated the relative contribution of the two Escherichia coli DNA alkyltransferases (ATases) to the increased sensitivity of ATase-deficient bacteria to the mutagenic and lethal effects of chloroethylnitrosoureas (CNU). The ogtencoded protein was the principal determinant in resistance to the mutagenic effects of CNU in E.coli. Thus, only when the ogt gene was inactivated was sensitivity to mutagenesis greatly increased; the contribution of inactivation of the ada gene was relatively minor. Furthermore, induction of the adaptive response provided essentially no protection against CNU mutagenesis in either an ogt+ or ogt- background. Finally, overexpression of the ogt gene into ogt- ada- double mutants provided the greatest protection against CNU; introduction of the full-length or truncated ada gene was protective, but to a much lesser extent. Mammalian ATases were not as protective against mutation induction by CNU as Ogt, even though they were apparently expressed at higher level. In order of effectiveness the ATases ranked Ogt > human > truncated Ada = Ada > rat. This order was not observed in the protection against killing by 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea, where truncated Ada = human > Ogt > rat = Ada. Higher mutation frequency and toxicity were observed in uvr- mutants, suggesting that one or more of the potentially mutagenic and/or toxic lesions are also substrates for the excision repair proteins.

ogt alkyltransferase enhances dibromoalkane mutagenicity in excision repair-deficient Escherichia coli K-12.

We examined the role of the O6-alkylguanine-DNA alkyltransferase encoded by ogt gene in the sensitivity of Escherichia coli to the mutagenic effects of the dibromoalkanes, dibromoethane and dibromomethane, by comparing responses in ogt- bacteria to those in their isogenic ogt+ parental counterparts. The effects of the uvrABC excision-repair system, the adaptive response, mucAB and umuDC mutagenic processing, and glutathione bioactivation on the differential responses of ogt- and ogt+ bacteria were also studied. Mutation induction was monitored by measuring the frequency of forward mutations to L-arabinose resistance. Induced mutations occurred only in excision repair-defective strains and were totally (with dibromomethane) or substantially (with dibromoethane) dependent on the alkyltransferase (ATase) encoded by the ogt gene. An increased mutagenic response to both dibromoalkanes was also seen in ogt- bacteria that overexpressed the ogt protein from a multicopy plasmid, indicating that the differences in mutability between ogt+ and ogt- bacteria were not dependent on the ogt- null allele carried by the defective strain. The ATase encoded by the constitutive ogt gene was more effective in promoting dibromoalkane mutagenicity than the ada ATase induced by exposure to low doses of a methylating agent. The mutagenicity promoted by the ogt ATase was dependent on both glutathione bioactivation and SOS mutagenic processing. To our knowledge, this paper presents for the first time evidence that DNA ATases, in particular the ATase encoded by the ogt gene, can increase the mutagenic effects of a DNA-damaging agent. The mechanism of this effect has yet to be established.

Fpg protein protects Escherichia coli K-12 from mutation induction by the carcinogen 4-nitroquinoline 1-oxide.

This paper investigates the role of the fpg gene product in protecting Escherichia coli cells against the lethal and mutagenic effects of 4-nitroquinoline 1-oxide (4NQO). To this end, the araD81 mutation which make the cells sensitive to L-arabinose was combined with an fpg-1::Knr allele, in either an uvrA+ or uvrA-, umuC+ or umuC-, genetic background. Mutation induction was monitored by selecting forward mutations to L-arabinose resistance (Arar). The formamidopyrimidine-DNA glycosylase (Fpg protein) protected bacteria from 4NQO-induced mutagenesis since Fpg- defective cells showed greater Arar mutation induction than fpg+ bacteria did. This was confirmed since the increased sensitivity of the fpg- cells to mutagenesis by 4NQO was suppressed when the Fpg protein was overproduced by placing the fpg gene in a multicopy plasmid vector. The fpg- mutation had no detectable influence on 4NQO mutagenesis in a uvrA- genetic background, but its effect was magnified in umuC- cells. No influence on cell survival was observed after 4NQO treatment. Our data suggest that 8-hydroxyguanine, a non-lethal, non-bulky and directly miscoding lesion, might be responsible for the detected influence of Fpg protein expression on mutation induction by 4NQO. This is in agreement with the reported in vivo formation of 8-hydroxyguanine in cellular DNA after 4NQO exposure. The increased 4NQO-induction of GC to TA transversions on fpg- bacteria further support such a possibility. This work reinforces the role of Fpg protein in the bacterial defense against the mutagenicity by genotoxic agents.

Mutagenesis in Escherichia coli K-12 mutants defective in superoxide dismutase or catalase.

Escherichia coli K-12 strains with diminished levels of superoxide dismutase (SOD) due to inactivation of the sodA, sodB or sodA sodB genes were constructed in order to quantify the role of O2. in mutagenesis. Mutagenesis was monitored by selecting forward mutations to L-arabinose resistance (AraR). No sodA sodB mutant inability to grow in aerobic minimal medium was found, in contrast to that previously reported for a different E. coli wild-type genetic background. The role of SOD for coping with the damaging effects of superoxide became evident after the increase in intracellular O2-. flux by growing cells under hyperoxygenation, but particularly by using redox cycling compounds such as plumbagin, paraquat and menadione. Bacteria completely devoid of SOD activity showed very high levels of AraR-induced mutants at doses that were non-mutagenic for the SOD-proficient parental or the sodA or sodB single mutants. The mutagenicity of nifurtimox and quercetin were studied to further compare the responses of the SOD-deficient bacteria to those of their SOD-proficient counterparts. The relative importance of SOD and catalase for coping with the damaging effects of O2-. and H2O2 was quantified by comparing SOD-deficient bacteria with isogenic catalase-deficient cells (a katG katE double mutant). The mutagenicities of plumbagin and menadione were much higher in SOD-deficient than in catalase-deficient bacteria, in agreement with the role of the O2-. radical in the so-called metal-catalyzed Haber-Weiss reaction. The relevance of catalase in protecting against the damaging effects of H2O2 was evident from the hypersensitivity of the katG katE double mutant to the mutagenic and lethal effects of this oxidizing agent. It is concluded that the Ara mutagenicity assay combined with depletion in specific antioxidative enzymes could be a tool in establishing the extent to which DNA damage by oxygen radicals is relevant to mutagenesis.

Mutagenesis and DNA repair for alkylation damages in Escherichia coli K-12.

In this work we report on the isolation of an Escherichia coli K-12 mutation, which confers a high sensitivity to bacteria cells to mutagenesis by simple monofunctional alkylating agents. The mutation emerged spontaneously from a bacterial strain that already proved useful in various mutagenicity studies. By monitoring the influence of such a mutation on the frequency of induced mutation by ethylating (EMS, DES, ENU, ENNG) vs. methylating (MMS, DMS, MNU, MNNG) compounds, and on the in vivo repair capacity for different alkyl-DNA lesions (O6-alkG, N7-alkG, N3-meA), we conclude that the mutation should affect the gene (ogt) that encodes constitutive DNA repair alkyltransferase (ATase). Thus in the presence of ada, differences in mutagenicity were observed only with ethylating agents; the sensitization of cells to both the ethylating and methylating partners requiring, by contrast, the absence of the ada protein. These results support the reported in vitro substrate specificities for both ogt and ada ATases. The parental cells exhibited biphasic dose-response curves in accordance with the idea of low basal level saturation attributed to the uninducible ogt ATase. Deficient bacterial derivatives showed, by contrast, linear mutation induction responses. The in vivo removal of alkylated bases from DNA was measured in bacterial strains deficient in the excision repair pathway (delta uvrB) and unable to induce the adaptive response (ada::Tn10). The very low initial levels for O6-meG and O6-etG (1.1 and 0.2 molecules per cell, respectively) were readily repaired by the parental cells but remained unchanged in the hypermutable derivatives. This result suggests that in the absence of nucleotide excision repair and of the adaptive response, no alternative pathway, other than ogt, is available for the repair of the major mutagenic lesion, O6-alkG, at least during the first 4 hours after alkylation. Comparatively, no differences were found in the capacity to repair the major lethal adduct, N3-meA, in agreement with the fact that no effect on cell survival was detected. In conclusion, we propose that the biological significance of the ogt protein relies mainly on its ability to prevent mutagenesis by low levels of bulkier ethylation products (especially in the absence of uvr excision repair.(ABSTRACT TRUNCATED AT 400 WORDS)