Acclimation and repair of DNA damage in recombinant bioluminescent Escherichia coli

AIMS: The aim of this study is to understand different adaptive responses in bacteria caused by three different mutagens, namely, an intercalating agent, an alkylating agent and a hydroxylating agent, and the repair systems according to the type of DNA damage, that is, DNA cross-linking and delayed DNA synthesis, alkylation and hydroxylation of DNA. A recombinant bioluminescent Escherichia coli, DPD2794 with the recA promoter fused to luxCDABE originating from Vibrio fischeri, was used in this study. METHODS AND RESULTS: The recombinant bioluminescent E. coli strain DPD2794, containing a recA promoter fused to luxCDABE from V. fischeri, was used to detect adaptive and repair responses to DNA damage caused by mitomycin C (MMC), and these responses were compared with those when the cells were induced with N-methyl-N-nitro-N-nitrosoguanidine (MNNG) and hydrogen peroxide (H2O2). The response ratio between the induced samples and that of the controls decreased suddenly when the induced culture was used in further inductions, indicating a possible adaptive response to DNA damage. DNA damage, or the proteins produced, because of MMC addition does not appear to be completely resolved until the seventh sub-culture after the initial induction, whereas simple damage, such as the base modification caused by MNNG and H2O2, appears to be repaired rapidly as evidenced by the quick recovery of sensitivity. CONCLUSIONS: These results suggest that it takes more time to completely repair DNA damage caused by MMC, as compared with a simple repair such as that required for the damage caused by MNNG and H2O2. Therefore, repair of the damage caused by these three mutagens is controlled by different regulons, even though they all induced the recA promoter. SIGNIFICANCE AND IMPACT OF THE STUDY: Using a bioluminescent E. coli harbouring a recA promoter-lux fusion, it was found that different adaptive responses and repair systems for DNA damage caused by several mutagens exists in E. coli.     

Chemical and UV mutagenesis in Zymomonas mobilis

Mutagenesis of the facultative anaerobe Zymomonas mobilis was accomplished by three different mutagens. Ultra-violet (UV) irradiation, whose effectiveness relies on misrepair of damaged DNA via an error-prone pathway, was a poor mutagen for this organism. Ethyl methane sulphonate (EMS) gave results very similar to UV-irradiation. N-methyl-N-nitro-N-nitrosoguanidine (MNNG), which is believed to act by multiple mutagenic mechanisms, was the most powerful mutagen, always resulting in a large number of mutants of all types examined (i.e. auxotrophs, antibiotic resistant, heavy metal resistant and ultraviolet sensitive). Reversion frequencies of MNNG-induced mutants were very low. Evidence is provided that mutagenesis of Z. mobilis is affected by photoreactivation, adaptive response and error-prone repair mechanisms. Moreover, cells treated with alkylating agents and allowed to recover under anaerobic conditions clearly demonstrated that anaerobiosis plays a significant role in repair, but not in the induction of mutants.     

Growth-phase-dependent response to DNA damage in poly(ADP-ribose) polymerase deficient cell lines: basis for a new hypothesis describing the role of poly(ADP-ribose) polymerase in DNA replication and repair

We have studied the role of poly(ADP-ribose) polymerase in the repair of DNA damage induced by x-ray and N-methyl N-nitro-N-nitrosoguanidine (MNNG) by using V79 chinese hamster cells, and two derivative mutant cell lines, ADPRT54 and ADPRT351, that are deficient in poly(ADP-ribose) polymerase activity. Under exponentially growing conditions these mutant cell lines are hypersensitive to x-irradiation and MNNG compared to their parental V79 cells which could be interpreted to suggest that poly(ADP-ribose) polymerase is involved in the repair of DNA damage. However, the level of DNA strand breaks induced by x-irradiation and MNNG and their rates of repair are similar in all the cell lines, thus suggesting that it may not be the difference in strand break formation or in its rate of repair that is contributing to the enhanced cell killing in exponentially growing poly(ADP-ribose) polymerase deficient cell lines. In contrast, under growth-arrested conditions, all three cell lines become similarly sensitive to both x-irradiation and MNNG, thus suggesting that poly(ADP-ribose) polymerase may not be involved in the repair of DNA damage in growth-arrested cells. These paradoxical results could be interpreted to suggest that poly(ADP-ribose) polymerase is involved in DNA repair in a cell-cycle-dependent fashion, however, it is functionally active throughout the cell cycle. To resolve this dilemma and explain these results and those obtained by many others, we propose that the normal function of poly(ADP-ribose) polymerase is to prevent DNA recombination processes and facilitate DNA ligation.     

Induction of theEscherichia coli UVM response by oxidative stress

UVM (ultravioletmodulation of mutagenesis) is a recently describedrecA-independent, inducible mutagenic phenomenon in which prior UV irradiation ofEscherichia coli cells strongly enhances mutation fixation at a site-specific 3-N⁴-ethenocytosine (C) lesion borne on a transfected single-stranded M13 DNA vector. Subsequent studies demonstrated that UVM is also induced by alkylating agents, and is distinct from both the SOS response and the adaptive response to alkylation damage. Because of the increasing significance being attributed to oxidative DNA damage, it is interesting to ask whether this class of DNA damage can also induce UVM. By transfecting M13 vector DNA bearing a site-specificC lesion into cells pretreated with inducing agents, we show here that the oxidative agent H₂O₂ is a potent inducer of UVM, and that the induction of UVM by H₂O₂ does not requireoxyR-regulated gene expression. UVM induction by H₂O₂ appears to be mediated by DNA damage, as indicated by the observation of a concomitant reduction in cellular toxicity and UVM response in OxyR^c cells. Available evidence suggests that UVM represents a generalized cellular response to a broad range of chemical and physical genotoxicants, and that DNA damage constitutes the most likely signal for its induction.     

Mutagenic and error-free DNA repair in Streptomyces

Summary Two mutants of Streptomyces fradiae defective in DNA repair have been characterized for their responses to the mutagenic and lethal effects of several chemical mutagens and ultraviolet (UV) light. S. fradiae JS2 (mcr-2) was more sensitive than wild type to agents which produce bulky lesions resulting in large distortions of the double helix [i.e. UV-light, 4-nitroquinoline-1-oxide (NQO), and mitomycin C (MC)] but not to agents which produce small lesions [i.e. hydroxylamine (HA), methyl methanesulfonate (MMS), ethyl methanesulfonate (EMS) and N-methyl-N-nitro-N-nitrosoguanidine (MNNG)]. JS2 expressed a much higher frequency of mutagenesis induced by UV-light at low doses and thus appeared to be defective in an error-free excision repair pathway for bulky lesions analogous to the uvr ABC pathway of Escherichia coli. S. fradiae JS4 (mcr-4) was defective in repair of damage by most agents which produce small or bulky lesions (i.e., HA, NQO, MMS, MNNG, MC, and UV, but not EMS). JS4 was slightly hypermutable by EMS and MMS but showed reduced mutagenesis by NQO and HA. This unusual phenotype suggests that the mcr-4 ⁺ protein plays some role in error-prone repair in S. fradiae.     

Helicobacter pylori infection can modulate the susceptibility of gastric mucosa cells to MNNG

The pathogenesis of stomach cells can be associated with their susceptibility to exogenous dietary irritants, like nitrosamines such as dimethylnitrosamines (DMNA), and to the effects of non-dietary factors, including Helicobacter pylori infection. We used N-methyl-N’-nitro N-nitrosoguanidyne (MNNG) as a surrogate agent that induces a spectrum of DNA damage similar to DMNA. Using the alkaline comet assay, we showed that antioxidants — vitamins C and E, quercetin, and melatonin — reduced the genotoxic effect of MNNG in H. pylori-infected and non-infected human gastric mucosa cells (GMCs). To compare the sensitivity of the stomach and the blood, the experiment was also carried out in peripheral blood. We observed a higher level of DNA damage induced by MNNG in H. pylori-infected than in noninfected GMCs. We did not note any difference in the efficacy of the repair of the damage in either type of GMC. H. pylori infection may play an important role in the pathogenesis of GMCs, as it can modulate their susceptibility to dietary mutagens/carcinogens, thus contributing to gastric cancer.     

Mutagenic DNA repair in Escherichia coli. VIII. Involvement of DNA polymerase III in constitutive and inducible mutagenic repair after ultraviolet and gamma irradiation.

Summary Mutagenic repair in Escherichia coli after ultraviolet (UV) irradiation has previously been shown to require a function of DNA polymerase III. In contrast, no effect of incubating a polC temperature-sensitive strain at 42° has been found after gamma irradiation. Thus at present there is no direct evidence for the involvement of polymerase III in gamma ray mutagenesis. This could, however, merely reflect the stability of the premutational lesion during the period of polymerase III insufficiency such that mutagenic repair is resumed on the plate during subsequent incubation at permissive temperature.It was previously suggested that an inducible factor might interact with polymerase III to enable it to polymerise in an error-prone way in daughter strand gaps opposite non-coding lesions in the template strand. A temperature-resistant revertant (CM 792) of a temperature-sensitive polC strain (CM 731) has been isolated which has properties expected of a strain in which the polymerase III complex is no longer susceptible to the inducible co-factor. Its UV sensitivity, spontaneous mutation rate and mutagenic response to ethyl methanesulphonate are all normal or near normal, also the rates of mutation to prototrophy after gamma irradiation and to streptomycin resistance after UV. These latter mutations are believed to arise through constitutive mutagenic repair at sites in pre-existing DNA. In contrast, the rate of UV-induced mutation to prototrophy due to changes at ochre suppressor loci is greatly depressed and no Weigle-reactivation of bacteriophage T3 is observable; both these effects are believed to result from the action of inducible mutagenic repair in newly-replicated DNA. It is suggested that the 3 to 5 exnnuclease activity of the polymerase III complex in CM 792 may not be susceptible to inhibition by an inducible factor and so continues to remove mismatched bases inserted in newly-replicated DNA opposite damage template sites thus preventing the fixation of errors as mutations.     

Induction of transversion mutations in Escherichia coli by N-methyl-N''-nitro-N-nitrosoguanidine is SOS dependent.

Escherichia coli alkA mutants, which are deficient for an inducible DNA glycosylase, 3-methyladenine-DNA glycosylase II, are sensitive to mutagenesis by low doses of the alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). As many as 90% of the alkA-dependent mutations induced by MNNG are also umuC+ dependent and thus are due to DNA lesions that are substrates for the mutagenic functions of the SOS response. A great number of these mutations are base substitutions at A . T sites, particularly A . T transversions. We discuss which DNA lesions may be responsible for these mutations. Our results show that the induction of 3-methyladenine-DNA glycosylase II, which occurs as part of the adaptive response to alkylating agents such as MNNG, significantly reduces the mutagenicity as well as the lethality of alkylation damage.     

Defective excision repair in a mutant of Micrococcus radiodurans hypermutable by some monofunctional alkylating agents

Summary The lethal and mutagenic effects of methyl methanesulphonate (MMS), ethyl methanesulphonate (EMS), and N-methyl-N-nitro-N-nitrosoguanidine (MNNG) can be dissociated in a mitomycin C (MTC)-sensitive mutant, strain 302, of Micrococcus radiodurans.As regards lethality 302 is extremely sensitive, compared with the wild type, to MTC and decarbamoyl MTC (DCMTC), slightly sensitive to EMS, MNNG, nitrous acid, 7-bromomethylbenz {} anthracene (BrMBA), and N-acetoxy-N-2-acetylaminofluorene (AAAF), and resistant to MMS, hydroxylamine, and ICR 191G. As regards mutability it is, compared to the wild type, very sensitive to MMS, EMS, and MNNG, and slightly sensitive to hydroxylamine and nitrous acid but not to any other agent examined.Alkaline sucrose gradient studies indicate that 302 does not incise DNA containing BrMBA adducts, although it does incise DNA damaged by AAAF but probably not to the same extent as wild type.We put forward the hypothesis that the hypermutability of 302 is due to the non-removal of bases or nucleotides, modified in exocyclic positions, which have altered base-pairing capabilities, while lethality results from the non-removal of bases or nucleotides, also modified in exocyclic positions, which no longer form hydrogen-bonded base pairs.     

Random mutagenesis and use of 2-deoxy-D-glucose as an antimetabolite for selection of α-amylase-overproducing mutants of Aspergillus oryzae

By using 2-deoxy-D-glucose, selection of different mutants of Aspergillus oryzae PTCC 5164, which were produced by random mutagenesis by u.v. radiation, nitrous acid and N-methyl-N-nitro-N-nitrosoguanidine (MNNG), was studied. 2-Deoxy-D-glucose, a well-known antimetabolite, was used to isolate derepressed mutants. The mutational and lethal effects of these mutagens on conidia of A. oryzae were compared and the frequency distribution of isolated mutants, in the presence of 2-deoxy-D-glucose, was determined. Potent mutants, which produced higher dextrinizing and saccharogenic activities, were isolated. The best strain was a result of mutagenesis by nitrous acid, which produced 6.73 times more dextrinizing and 5.13 times more saccharogenic activity than the parent strain. In general, the mutants obtained by nitrous acid and u.v. were more potent than those obtained by MNNG.     

PI 3 kinase related kinases-independent proteolysis of BRCA1 regulates Rad51 recruitment during genotoxic stress in human cells

Background

The function of BRCA1 in response to ionizing radiation, which directly generates DNA double strand breaks, has been extensively characterized. However previous investigations have produced conflicting data on mutagens that initially induce other classes of DNA adducts. Because of the fundamental and clinical importance of understanding BRCA1 function, we sought to rigorously evaluate the role of this tumor suppressor in response to diverse forms of genotoxic stress.

Methodology/Principal Findings

We investigated BRCA1 stability and localization in various human cells treated with model mutagens that trigger different DNA damage signaling pathways. We established that, unlike ionizing radiation, either UVC or methylmethanesulfonate (MMS) (generating bulky DNA adducts or alkylated bases respectively) induces a transient downregulation of BRCA1 protein which is neither prevented nor enhanced by inhibition of PIKKs. Moreover, we found that the proteasome mediates early degradation of BRCA1, BARD1, BACH1, and Rad52 implying that critical components of the homologous recombinaion machinery need to be functionally abrogated as part of the early response to UV or MMS. Significantly, we found that inhibition of BRCA1/BARD1 downregulation is accompanied by the unscheduled recruitment of both proteins to chromatin along with Rad51. Consistently, treatment of cells with MMS engendered complete disassembly of Rad51 from pre-formed ionizing radiation-induced foci. Following the initial phase of BRCA1/BARD1 downregulation, we found that the recovery of these proteins in foci coincides with the formation of RPA and Rad51 foci. This indicates that homologous recombination is reactivated at later stage of the cellular response to MMS, most likely to repair DSBs generated by replication blocks.

Conclusion/Significance

Taken together our results demonstrate that (i) the stabilities of BRCA1/BARD1 complexes are regulated in a mutagen-specific manner, and (ii) indicate the existence of mechanisms that may be required to prevent the simultaneous recruitment of conflicting signaling pathways to sites of DNA damage.     

DNA repair in pollen: Range of mutagens inducing repair, effect of replication inhibitors and changes in thymidine nucleotide metabolism during repair

Summary Pollen of Petunia hybrida carry out DNA repair during the first two hours of germination when certain mutagens are included in the germination medium. This repair, detected readily as unscheduled DNA synthesis, since there is no replicative DNA synthesis in Petunia pollen, can be induced by the chemical mutagens N-methyl-N-nitro-N-nitrosoguanidine, 4-nitroquinoline-1-oxide, azaserine and methyl methanesulphonate. These compounds are all considered to be capable of direct covalent interaction with DNA. Mutagens requiring metabolic activation before interaction with DNA did not induce DNA repair synthesis in pollen. The practice of solubilizing water-insoluble chemical mutagens with dimethyl sulphoxide did not prove practical, due to the extremely harmful effects of dimethyl sulphoxide on pollen. Pretreatment of pollen before germination with pure ether, however, had no harmful effect on either repair or pollen germination. Therefore water-insoluble, ether-soluble mutagens were tested by pretreatment of the pollen with mutagens in ether solution. By this means it was shown that the direct-acting mutagen, diethyl sulphate, would also bring about unscheduled DNA synthesis in pollen, while 2-acetylaminofluorence and dimethyl-p-aminobenzene, both requiring metabolic activation, did not do so. Inhibitors of DNA replicative synthesis, hydroxyurea, azaserine, azauridine and fluorodeoxyuridine did not inhibit unscheduled DNA synthesis brought about by N-methyl-N-nitro-N-nitrosoguanidine. On the contrary, these compounds stimulated repair synthesis to varying degrees, hydroxyurea having the greatest effect. Pollen uptake of ³H-thymidine and the amount of radioactive label subsequently appearing in dTMP and dTDP+dTTP was increased by 4-nitroquinoline-1-oxide. Partial inhibition of these increases and of 4-nitroquinoline-1-oxide induced repair synthesis by 3,5-cyclic AMP suggested that thymidine:AMP phosphotransferase rather than thymidine kinase was responsible for thymidine phosphorylation in pollen. Enzyme assays on pollen extracts confirmed this.     

Herbivory damage does not indirectly influence the composition or excretion of aphid honeydew

Recent research has shown that extrafloral nectar secretion by plants, which also attracts ants, is a defense inducible by herbivory damage. Our research addresses the question of whether plants can manipulate the honeydew secretions of homopterans as an inducible defense in response to herbivory in the same manner as extrafloral nectaries. We investigated changes in honeydew composition and excretion rate by the facultatively ant-attended aphid Chaitophorus saliniger Shinji (Homoptera: Aphididae) depending on the presence or absence of herbivory by caterpillars (Clostera anastomosis L.; Lepidoptera: Notodontidae) on their host plant, the willow Salix gilgiana Seemen. Our results found no evidence to suggest that herbivory damage to the aphids host plant causes significant changes in aphid population growth, honeydew droplet volume, volume of honeydew excreted per aphid per hour, or composition of three abundant sugars in aphid honeydew as a result of herbivory damage to the aphids host plant, suggesting that, in this particular system, the plant is not manipulating the aphids honeydew output for its own benefit.     

Isolation and characterization of DNA repair deficient mutants from Penicillium chrysogenum

Summary Mutants sensitive to far ultraviolet light (UV) and 4-nitroquinoline-1-oxide (4NQO) have been isolated from Penicillium chrysogenum NRRL 1951. Two strains HP500 and HP508 are examined in detail. Their cross sensitivity to and altered mutation by UV and 4NQO suggests that damage caused by both agents is repaired through similar pathways in Penicillium chrysogenum. Strain HP500 is refractive to UV and 4NQO mutagenesis and is likely to be defective in an error-prone mechanism of repair. Mutation by N-methyl-N-nitro-N-nitrosoguanidine (MNNG) in HP500 is also reduced, indicating involvement of an error-prone UV repair process in MNNG mutagenesis in Penicillium chrysogenum. Strain HP508 shows an increase of forward mutation rate up to 4.5 times over that of the wild-type, when compared at similar surviving fractions and is also hypermutable by 4NQO. The repair defect present in strain HP508 has been demonstrated by its inability to remove DNA sites sensitive to single strand specific nuclease during post-irradiation incubation of protoplasts.     

Inducible DNA-repair systems in yeast: competition for lesions

DNA lesions may be recognized and repaired by more than one DNA-repair process. If two repair systems with different error frequencies have overlapping lesion specificity and one or both is inducible, the resulting variable competition for the lesions can change the biological consequences of these lesions. This concept was demonstrated by observing mutation in yeast cells (Saccharomyces cerevisiae) exposed to combinations of mutagens under conditions which influenced the induction of error-free recombinational repair or error-prone repair. Total mutation frequency was reduced in a manner proportional to the dose of 60Co-gamma- or 254 nm UV radiation delivered prior to or subsequent to an MNNG exposure. Suppression was greater per unit radiation dose in cells gamma-irradiated in O2 as compared to N2. A rad3 (excision-repair) mutant gave results similar to wild-type but mutation in a rad52 (rec-) mutant exposed to MNNG was not suppressed by radiation. Protein-synthesis inhibition with heat shock or cycloheximide indicated that it was the mutation due to MNNG and not that due to radiation which had changed. These results indicate that MNNG lesions are recognized by both the recombinational repair system and the inducible error-prone system, but that gamma-radiation induction of error-free recombinational repair resulted in increased competition for the lesions, thereby reducing mutation. Similarly, gamma-radiation exposure resulted in a radiation dose-dependent reduction in mutation due to MNU, EMS, ENU and 8-MOP + UVA, but no reduction in mutation due to MMS. These results suggest that the number of mutational MMS lesions recognizable by the recombinational repair system must be very small relative to those produced by the other agents. MNNG induction of the inducible error-prone systems however, did not alter mutation frequencies due to ENU or MMS exposure but, in contrast to radiation, increased the mutagenic effectiveness of EMS. These experiments demonstrate that in this lower eukaryote, mutagen exposure does not necessarily result in a fixed risk of mutation, but that the risk can be markedly influenced by a variety of external stimuli including heat shock or exposure to other mutagens.     

Induction of gene conversion in yeast cells continuously cultured at high radiation background

The induction of genetic damage was investigated by culturing diploid yeastSaccharomyces cerevisiae D7 cells continuously at radiation levels ranging from 0.383 µSv/h to 1.275 mSv/h by selecting appropriate concentrations of tritiated water in the growth medium. These radiation levels correspond to 3–10000 times the natural background. Parameters such as growth kinetics, gene conversion frequency at background radiation and after a challenging dose of acute gamma-radiation or alkylating agentN-methyl-N-nitro-N-nitrosoguanidine (MNNG) were assessed. The gene conversion frequency in most of the assays was in the range of 5–10 convertants per 10⁶ cells, as in the case of controls. However, a number of the cultures showed conversion frequencies above 20 per 106 viable cells. This stochastic phenomenon occurred more frequently in cells which were incubated at higher radiation levels and for longer durations. This suggests that radiation is responsible for the phenomenon. When subculturing continued beyond 900 h, gene conversion frequencies reverted back to normal values in all cultures in spite of elevated background radiation levels, thus suggesting an adaptive response. The generation time of the cells was 78 min in all cultures irrespective of the radiation level. The response of the cells cultured at elevated background radiation levels to subsequent challenging treatment with gamma-radiation or MNNG was identical to that of the control cultures. Our results suggest that in eukaryotic yeast, low-level radiation may induce an adaptive response to chronic radiation, whereas no such response could be detected when the cells were challenged with acute high-dose exposure or with MNNG.     

Retardation of cell cycle progression in yeast cells recovering from DNA damage: A study at the single cell level

Summary Pedigree analyses of individual yeast cells recovering from DNA damage were performed and time intervals between morphological landmark events during the cell cycle (bud emergence and cell separation), were recorded for three generations. The associated nuclear behavior was monitored with the aid of DAPI staining. The following observations were made: (1) All agents tested (X-rays, MMS, EMS, MNNG, nitrous acid) delayed the first bud emergence after treatment, which indicates inhibition of the initiation of DNA replication. (2) Cells that survived X-irradiation progressed further through the cell cycle in a similar way to control cells. (3) Progress of chemically treated cells became extremely asynchronous because surviving cells stayed undivided for periods of varying length. (4) Prolongation of the time between bud emergence and cell separation was most pronounced for cells treated with the alkylating agents MMS and EMS. This is interpreted as retardation of ongoing DNA synthesis by persisting DNA adducts. (5) Cell cycle prolongation in the second and third generation after treatment was observed only with MMS treated cells. (6) In all experiments, individual cells of uniformly treated populations exhibited highly variable responses.Abbreviations DAPI 4,6-diamidino-2-phenyl-indole - EMS ethyl methanesulfonate - MMS methyl methanesulfonate - MNNG N-methyl-N-nitro-N-nitrosoguanidine     

DNA lesions that signal the induction of radioresistance and DNA repair in yeast

DNA recombinational repair, and an increase in its capacity induced by DNA damage, is believed to be the major mechanism that confers resistance to killing by ionizing radiation in yeast. We have examined the nature of the DNA lesions generated by ionizing radiation that induce this mechanism, using two different end points: resistance to cell killing and ability of the error-free recombinational repair system to compete for other DNA lesions and thereby suppress chemical mutation. Under the various conditions examined in this study, the "maximum" inducible radiation resistance was increased approximately 1.5- to 3-fold and suppression of mutation about 10-fold. DNA lesions produced by low-LET gamma rays at doses greater than about 20 Gy given in oxygen were shown to be more efficient, per unit dose, at inducing radioresistance to killing than were lesions produced by neutrons (high-LET radiation). This suggests that DNA single-strand breaks are more important lesions in the induction of radioresistance than DNA double-strand breaks. Oxygen-modified lesions produced by gamma rays (low-LET radiation) were particularly efficient as induction signals. DNA damage due to hydroxyl radicals (OH.) derived from the radiolytic decomposition of H2O produced lesions that strongly induced this DNA repair mechanism. Similarly, OH. derived from aqueous electrons (e-aq) in the presence of N2O also efficiently induced the response. Cells induced to radioresistance to killing with high-LET radiation did not suppress N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-generated mutations as well as cells induced with low-LET radiation, supporting the conclusion that the type of DNA damage produced by low-LET radiation is a better inducer of recombinational repair. Surprisingly, however, cells induced with gamma radiation in the presence of N2O that became radioresistant to killing were unable to suppress MNNG mutations. This result indicates that OH. generated via e-aq (in N2O) may produce unusual DNA lesions which retard normal repair and render the system unavailable to compete for MNNG-generated lesions. We suggest that the repairability of these unique lesions is restricted by either their chemical nature or topological accessibility. Attempted repair of these lesions has lethal consequences and accounts for N2O radiosensitization of repair-competent but not incompetent cells. We conclude that induction of radioresistance in yeast by ionizing radiation responds variably to different DNA lesions, and these affect the availability of the induced recombinational repair system to deal with subsequent damage.     

Inducible error-prone repair in yeast. Suppression by heat shock

The production of reversion mutations in wild-type, diploid Saccharomyces cerevisiae by the alkylating agents N-methyl-N'-nitro- N-nitrosoguanidine (MNNG) and methylnitrosourea (MNU) was suppressed in cells previously treated with a heat shock, or the protein synthesis inhibitor, cycloheximide. The same cells previously treated with a heat shock, or the protein synthesis inhibitor, cycloheximide. The same treatment after mutagen exposure did not lower the induced mutation frequency. In split-dose experiments, a first MNNG exposure prevented subsequent heat (or cycloheximide) treatment from blocking mutation by a second, later mutagen exposure. These data suggest that, in yeast, MNNG or MNU induces an error-prone DNA-repair system, and that this induction is blocked by protein-synthesis inhibitors. The specificity of this system for different types of DNA damage was investigated using a variety of other mutagenic agents. A prior heat shock did not suppress mutation produced by exposure to ethyl methanesulfonate, ethylnitrosourea, 8-methoxypsoralen + UVA, or gamma-radiation. Partial suppression was observed in cells exposed to methyl methanesulfonate or to 254-nm ultraviolet light. These results indicate that, unlike the SOS system of E. coli, this inducible error-prone process of yeast is responsive to only certain mutagens. Heat shock suppression of mutation produced by MNNG exposure was also demonstrated in wild-type haploid cells, as well as haploid strains mutant in representative genes of the RAD52 epistasis group (rad52, rad53, rad54), the RAD3 epistasis group (rad1, rad2, rad3) and the RAD6 epistasis group (rad9, rad18). The rad6 mutant itself was immutable with MNNG and therefore untestable by these techniques. These data indicate that this error-prone repair system is not absolutely dependent on the integrity of the RAD52 (recombination) or the RAD3 (excision) systems, or on at least some parts of the RAD6 system.     

Action ofN-methyl-N′-nitro-N-nitrosoguanidine inRhizobium trifolii

Rhizobium trifolii was highly resistant to the lethal effect ofN-methyl-N-nitro-N-nitrosoguanidine (MNNG), but it was sensitive to the mutagenic action of this chemical. A concentration of 500g/ml yields a survival of between 1% and 10%, which allows us to obtain a higher number of mutants than lower concentrations that yield higher survival rates. Lethal damage produced by nitrosoguanidine was repaired, and repair is inhibited by acriflavine.