Isolation and characterization of Escherichia coli K-12 mutants unable to induce the adaptive response to simple alkylating agents.

When Esherichia coli cells are exposed to a low level of simple alkylating agents, they induce the adaptive response which renders them more resistant to the killing and the mutagenic effects of the same or other alkylating agents. This paper describes the isolation of one strain that was deficient in mutagenic adaptation and five that were deficient in both mutagenic and killing adaptation, confirming previous suggestions that killing and mutagenic adaptation are, at least to some extent, separable. These six strains have been called Ada mutants. They were more sensitive to the killing and mutagenic effects of N-methy-N'-nitro-N-nitrosoguanidine (MNNG) than the unadapted Ada+ parent. Thus, the adaptation pathway is responsible for circumventing some alkylation-induced damage even in cells that are preinduced. The increase in mutation frequency seen in Ada cells treated with MNNG was the same whether the cells were lexA+ or lexA, showing that the extra mutations found in Ada- strains do not depend upon the SOS pathway. Ada strains accumulated more O6-methyl guanine lesions than the Ada+ parent on prolonged exposure to MNNG, and this supports the idea that O6-methyl guanine is the most important lesion for MNNG-induced mutagenesis. The ada mutations have been shown to map in the 47 to 53-min region of the E. coli chromosome.     

Interaction of an antimutator gene with DNA repair pathways in Escherichia coli K-12

Summary A mutation in the purB gene of E. coli K-12, isolated and partially characterized by Geiger and Speyer (1977), confers a temperature sensitive requirement for adenine and an antimutator phenotype at 30°C. Several hypotheses about the mechanism of action of this mutation, named mud for mutation defective, were tested in the present work. The mud mutation has no effect upon the induction of the SOS response, so the antimutator phenotype is unlikely to be due to repression of mutagenic repair. Mud cells are resistant to the cytotoxic and mutagenic effects of alkylating agents such as MNNG, but this resistance is not due simply to derepression of the adaptive response. DNA isolated from mud cells is not undermethylated relative to DNA from purB ⁺ cells, so the antimutator phenotype of mud cannot be due to reduced hotspot base-substitution mutation at methylated cytosine residues. Nor is there a longer lag in post-replicative DNA methylation, which indicates that there is no enhancement of mismatch repair resulting from an extended time window for strand discrimination. Measurement of nucleotide pool levels demonstrated an elevation of dCTP in mud cells and a reduction of all other nucleoside triphosphates.This work was supported in part by Public Health Service grants numbers GM15697 and CA32182     

Prevalence of Pseudomonas aeruginosa FP plasmids which enhance spontaneous and uv-induced mutagenesis.

From work reported here and from previous studies 16 out of 53 (30%) FP plasmids (i.e. those plasmids that promote host chromosome transfer) of Pseudomonas aeruginosa are found to protect host cells against UV irradiation. 13 of these UV-protecting FP plasmids were tested to determine their mode of DNA repair and were found to contribute to error-prone repair because of their enhancement of UV-induced mutagenesis and in most instances spontaneous mutagenesis as well. Some of these plasmids were tested for their behaviour in a DNA polymerase I deficient (Pol^?) mutant of P. aeruginosa; the remainder could not be tested due to plasmid instability in the Pol^? mutant. 11 of these FP plasmids provided wild-type level of UV protection to the mutant. 4 of the plasmids tested (FP18, FP103, FP109 and FP111) were able to enhance the mutant's ability to host cell reactivate UV irradiated phage, though not to the level of the Pol⁺ parent. The presence of FP18 or FP111 in the Pol^? mutant did not increase polymerase I-like enzymatic activity. It is concluded that the plasmids do not confer a polymerase activity functionally equivalent to host DNA polymerase I. It is possible however, that the plasmids code for another polymerase or for a cofactor which interacts with a host polymerase, as seen by the partial restoration by FP plasmids of host-cell reactivation of UV-irradiated phage in the polymerase I deficient mutant.The mutagenic properties of those FP plasmids tested appears to be nonspecific because of their ability to mutate two host chromosomal genes, trpB1 and leu38 and an R plasmid gene, bla.The implications of the prevalence of FP plasmids in P. aeruginosa which enhance mutagenesis are discussed.     

Cross-resistance studies with V79 Chinese hamster cells adapted to the mutagenic or clastogenic effect of N-methyl-N′-nitro-N-nitrosoguanidine

When V79 cells are exposed to a single low dose of MNNG or MNU they acquire resistance to the mutagenic or to the clastogenic effect of the agents. Here the effect of MNNG pretreatment on mutagenesis (6-thioguanine resistance) and aberration formation in cells challenged with various mutagens/clastogens is reported. MNNG-adapted cells were resistant to the mutagenic effects of MNU and, to a lower extent, of EMS. No mutagenic adaptation was observed when MNNG-pretreated cells were challenged with MMS, ENU, MMC or UV.

Cells pretreated with a dose of MNNG which makes them resistant to the clastogenic effect of this compound were also resistant to the clastogenic activity of other methylating agents (MNU, MMS), but not so with respect to ethylating agents (EMS, ENU). Cycloheximide abolished the aberration-reducing effect of pretreatment. However, when given before the challenge dose of MNNG, MNU or MMS, it drastically enhanced the aberration frequency in both pretreated and non-pretreated cells. No significant enhancement of aberration frequency by cycloheximide was found for ethylating agents.

The results indicate that clastogenic adaptation is due to inducible cellular functions. It is concluded that mutagenic and clastogenic adaptation are probably caused by different adaptive repair pathways.     

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.     

The role of DNA polymerase I and the rec system in survival of bacteria and bacteriophages damaged by the photodynamic action of acridine orange

Summary The effect of acridine orange (AO)-sensitized photodynamic treatment (PD) was studied in various repair-deficient mutants of Salmonella typhimurium and Escherichia coli. Bacteria of either species carrying mutations in the polA gene and hence deficient in the enzyme DNA polymerase I were significantly more sensitive to PD-killing than polA ⁺ parent bacteria or phenotypically POL⁺ revertants of the polA strains (selected on the basis of resistance to methyl methanesulphonate). It therefore appears that DNA polymerase I plays an important role in cellular recovery from PD treatment. E. coli carrying a mutation in the recA gene was also more sensitive to PD-treatment than its parent strain, as was S. typhimurium carrying a mutation of the recA type. In S. typhimurium the rec mutant was somewhat less sensitive to PD-killing than the pol mutant even although it is much more sensitive to ultraviolet killing. E. coli strains with mutations in the recB and recC genes were intermediate in PD sensitivity between the recA and the parent strain. S. typhimurium and E. coli bacteria with mutations in the polA and recA genes showed reduced ability to host-cell reactivate PD-damaged bacteriophages ES 18 and c1, indicating that the polA ⁺ and recA ⁺ gene products also contribute to repair of bacteriophages damaged by PD treatment. It is suggested that the recombinational repair process is less important for recovery from PD than for recovery from UV, and that the primary contribution of the rec genes to recovery from PD may be in repair of single-strand gaps by repair resynthesis.     

The bacterial alkyltransferase-like (eATL) protein protects mammalian cells against methylating agent-induced toxicity

In both pro- and eukaryotes, the mutagenic and toxic DNA adduct O⁶-methylguanine (O⁶MeG) is subject to repair by alkyltransferase proteins via methyl group transfer. In addition, in prokaryotes, there are proteins with sequence homology to alkyltransferases, collectively designated as alkyltransferase-like (ATL) proteins, which bind to O⁶-alkylguanine adducts and mediate resistance to alkylating agents. Whether such proteins might enable similar protection in higher eukaryotes is unknown. Here we expressed the ATL protein of Escherichia coli (eATL) in mammalian cells and addressed the question whether it is able to protect them against the cytotoxic effects of alkylating agents. The Chinese hamster cell line CHO-9, the nucleotide excision repair (NER) deficient derivative 43-3B and the DNA mismatch repair (MMR) impaired derivative Tk22-C1 were transfected with eATL cloned in an expression plasmid and the sensitivity to N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) was determined in reproductive survival, DNA double-strand break (DSB) and apoptosis assays. The results indicate that eATL expression is tolerated in mammalian cells and conferes protection against killing by MNNG in both wild-type and 43-3B cells, but not in the MMR-impaired cell line. The protection effect was dependent on the expression level of eATL and was completely ablated in cells co-expressing the human O⁶-methylguanine-DNA methyltransferase (MGMT). eATL did not protect against cytotoxicity induced by the chloroethylating agent lomustine, suggesting that O⁶-chloroethylguanine adducts are not target of eATL. To investigate the mechanism of protection, we determined O⁶MeG levels in DNA after MNNG treatment and found that eATL did not cause removal of the adduct. However, eATL expression resulted in a significantly lower level of DSBs in MNNG-treated cells, and this was concomitant with attenuation of G2 blockage and a lower level of apoptosis. The results suggest that eATL confers protection against methylating agents by masking O⁶MeG/thymine mispaired adducts, preventing them from becoming a substrate for mismatch repair-mediated DSB formation and cell death.     

Two classes of Bacillus subtilis mutants deficient in the adaptive response to simple alkylating agents

Summary Six mutant strains of Bacillus subtilis hypersensitive to N-methyl-N-nitro-N-nitrosoguanidine (MNNG) were shown to be deficient in the adaptive response to MNNG and termed ada mutants (Morohoshi and Munakata 1985). All the mutations mapped between the attSPO2 and lin loci on the chromosome. The mutant and wild-type (ada ⁺) cells contained similar constitutive levels of O⁶-methylguanine-DNA methyltransferase activity. Pretreatment with low concentrations of MNNG increased the activity about nine-fold in the ada ⁺ cells, while it uniformly decreased the activity in the ada cells. The pretreatment of three mutants (ada-3, ada-4, and ada-6) as well as ada ⁺, augumented the activity of methylpurine-DNA glycosylase and rendered the cells resistant to the lethal and mutagenic effects of N-propyl- or N-butyl-N-nitro-N-nitrosoguanidine. With the rest of the mutant strains (ada-1, ada-2, and ada-5), neither of such responses was elicited by the pretreatment. Thus, the former ada strains seem to have a defect in the gene specifically involved in the induction of the methyltransferase, while the latter ada strains have a defect in the gene controlling the adaptive response as a whole.Abbreviations MNNG N-methyl-N-nitro-N-nitrosoguanidine - ENNG N-ethyl-N-nitro-N-nitrosoguanidine - PNNG N-propyl-N-nitro-N-nitrosoguanidine - MNU N-methyl-N-nitrosourea - MMS methyl methanesulphonate     

Enhanced Resistance to Nitrosoguanidine Killing and Mutagenesis in a DNA Gyrase Mutant of Escherichia coli

The role of DNA gyrase in handling DNA damages induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) was examined with two Escherichia coli strains, KL161 and KL166. The two strains are isogenic except that KL166 harbors a mutation at the nalA (gyrA) locus which specifies one of the two subunits of DNA gyrase. We treated the two strains with several different types of mutagenic agents and found the nalA strain to be highly resistant to MNNG-induced killing and mutagenic effects as compared with the parental strain. The MNNG resistance was specific, since the two strains were about equally sensitive to methyl methane sulfonate, ethyl methane sulfonate, and UV and gamma radiations. We pulse-labeled the two strains with [(3)H]uridine and (14)C-amino acids after MNNG treatment to analyze RNA and protein synthetic rates. The pulse-labeled proteins were also separated on polyacrylamide gels. The results show that pulse-labeled RNA and proteins persisted in the nalA strain but declined rapidly in the parental strain after MNNG treatment. We compared membrane-free nucleoid preparations from the two strains by sucrose density gradient centrifugation and found a difference in nucleoid organization between the two strains. The nucleoid of the nalA strain, unlike that of the parental strain, may have a highly ordered structure, as indicated by its resistance to ethidium bromide-induced relaxation. The ability of the two strains to express an adaptive response to MNNG was determined. We found that the resistance to MNNG killing and mutagenesis by the nalA strain cannot be further increased by adaptive treatment. These results suggest that an alteration in DNA gyrase may have profound effects on E. coli chromosome organization and base methylation by MNNG.     

DNA repair in Proteus mirabilis. VI. Plasmid (R46-) mediated recovery and UV mutagenesis.

Summary The expression of plasmid R46-mediated recovery and mutagenic function (s) was studied in P. mirabilis, which is normally either weakly or nonmutable after UV exposure. The plasmid was found to confer on P. mirabilis enhanced UV resistance as well as UV-induced mutability for various types of forward mutations and reversion of the thr273 mutation. The plasmid enhanced survival of UV-irradiated phages in P. mirabilis both in unirradiated host cells and with increased efficiency after UV-exposure of host cells, as is characteristic of UV-inducible phage reactivation. Spontaneous mutability of P. mirabilis harboring R46 was about 2 to 7 times higher than that of cells without plasmid, depending on the marker, repair type, and plating density of the cells used. All of these R46-mediated rescue and mutagenic functions require the rec672⁺ gene function.It is assumed that the plasmid R46 adds functions to P. mirabilis comparable to those deficient in umuC and uvm mutants of E. coli (Kato and Shinoura, 1977; Steinborn, 1978) and that P. mirabilis possesses functions homologous to those controlled in E. coli by the recA ⁺ and lexA ⁺ genes.The significance of plasmid-mediated rescue and mutagenic functions for bacteria which lack the misrepair branch of mutagenesis, is discussed.     

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.     

Protective effects of sodium selenite on killing and mutation by N-methyl-N''-nitro-N-nitrosoguanidine in E. coli.

Sodium selenite was found to protect Escherichia coli cells against killing and mutagenic effects of N-methyl-N′-nitro-N-nitrosoguanidine (MNNG). Such protective effects were not observed when cells were treated with N-methyl-N-nitrosourea (MNU). The protection by sodium selenite was not controlled by the ada gene, which is responsible for the repair of alkylated damage in DNA. A reduction of the amount of glutathione was found when cells were treated with sodium selenite, and glutathione is known to be involved in the methylation of DNA by MNNG, not by MNU. Reduced methylation by MNNG due to the reduction of the amount of glutathione caused by abundant sodium selenite was suggested to be the mechanism of protection.     

Characteristics of DNA repair of a UV-sensitive mutant (radC) of Dictyostelium discoideum

Summary A radiation-sensitive mutant, TW8(radC), of Dictyostelium discoideum is more sensitive to ultraviolet light (UV) killing than the parental wild strain NC4(RAD ⁺), but is resistant to 4-nitroquinoline 1-oxide (4NQO) at almost the same level as NC4. In TW8 amoebae, single-strand breaks of DNA molecules were hardly detectable immediately after UV irradiation, and the removal of pyrimidine dimers was depressed during the postirradiation incubation when compared with that of NC4 amoebae. After treatment with 4NQO, however, single-strand breaks were detected in TW8 amoebae. The almost complete rejoining of these breaks was also detected after the removal of 4HAQO-adducts. The TW8 amoebae have an efficient repair capacity against DNA damage caused by 4NQO, MMS, MMC and MNNG but not UV.Abbreviations 4NQO 4-nitroquinoline 1-oxide - MMS methyl methanesulphonate - MMC mitomycin C - MNNG N-methyl-N-nitro-N-nitrosoguanidine     

Mutagenic DNA repair in Escherichia coli. VII. Constitutive and inducible manifestations.

Incubation of E. coli WP2 in the presence of chloramphenicol (CAP) for 90 min before and 60 min after γ-irradiation had no effect on the induction of Trp⁺ mutations. Bacteria that had been treated with CAP for 90 min prior to UV irradiation showed normal or near normal yields of induced mutations to streptomycin or colicin E2 resistance. Most of these mutations lost their photoreversibility (indicating “fixation”) during continued incubation with CAP for a further 60 min after irradiation, during which time neither protein nor DNA synthesis was detectable. It is suggested that CAP-sensitive protein synthesis is not required for mutagenic (error-prone) repair of lesions in pre-existing DNA, arguing against an inducible component in this repair.In contrast the frequency of UV-induced mutations to Trp⁺ (largely at suppressor loci) was drastically reduced by CAP pretreatment, confirming the need for an active replication fork for UV-mutagenesis at these loci. It is known from the work of others that CAP given after UV abolishes mutagenesis at these loci. We conclude that CAP-sensitive protein synthesis (consistent with a requirement for an inducible function) is necessary for mutagenic repair only in newly-replicated DNA (presumably at daughter strand gaps) and not in pre-existing DNA. The data are consistent with but do not prove the hypothesis that CAP-sensitive and insensitive modes of mutagenesis reflect minor differences in the operation of a single basic mutagenic repair system.     

Evidence that N-methyl-N′-nitro-N-nitrosoguanidine induces adaptive response in Bacillus thuringiensis

Bacillus thuringiensis is shown to have an inducible error-free repair system for alkylation damage as found in Escherichia coli and Bacillus subtilis. Growth of cells in the presence of low concentrations of N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) induces an adaptive response which is characterized by an increase in resistance to killing and mutagenesis by challenge with higher concentrations of MNNG. In addition, we have noted with interest that adaptive low doses seem to produce lesions at a rate sufficient to induce an increase of mutation frequency, and inhibition of cell division. The possibility of an interaction between SOS and adaptive responses with these low doses of MNNG is discussed.     

Toxicity and mutagenicity of X-rays and [I]dUrd or [H]TdR incorporated in the DNA of human lymphoblast cells

We measured the toxicity and mutagenicity induced in human diploid lymphoblasts by various radiation doses of X-rays and two internal emitters. [¹²⁵I]iododeoxyuridine ([¹²⁵I]dUrd) and [³H]thymidine ([³H]TdR), incorporated into cellular DNA. [¹²⁵I]dUrd was more effective than [³H]TdR at killing cells and producing mutations to 6-thioguanine resistance (6TG^R). No ouabain-resistant mutants were induced by any of these agents. Expressing dose as total disintegrations per cell (dpc), the D₀ for cell killing for [¹²⁵I]dUrd was 28 dpc and for [³H]TdR was 385 dpc. The D₀ for X-rays was 48 rad at 37°C. The slopes of the mutation curves were approximately 75 × 10⁻⁸ 6TG^R mutants per cell per disintegration for [¹²⁵I]dUrd and 2 × 10⁻⁸ for [³H]TdR. X-Rays induced 8 × 10⁻⁸ 6TG^R mutants per cell per rad. Normalizing for survival, [¹²⁵I]dUrd remained much more mutagenic at low doses (high survival levels) than the other two agents. Treatment of the cells at either 37°C or while frozen at −70°C yielded no difference in cytotoxicity or mutation for [¹²⁵I]dUrd or [³H]TdR, whereas X-rays were 6 times less effective in killing cells at −70°C.Assuming that incorporation was random throughout the genome, the mutagenic efficiencies of the radionuclides could be calculated by dividing the mutation rate by the level of incorporation. If the effective target size of the 6TG^R locus is 1000–3000 base pairs, then the mutagenic efficiency of [¹²⁵I]dUrd is 1.0–3.0 and of [³H]TdR is 0.02–0.06 total genomic mutations per cell per disintegration. ¹²⁵I disintegrations are known to produce localized DNA double-strand breaks. If these breaks are potentially lethal lesions, they must be repaired, since the mean lethal dose (D₀) was 28 dpc. The observations that a single dpc has a high probability of producing a mutation (mutagenic efficiency 1.0–3.0) would suggest, however, that this repair is extremely error-prone. If the breaks need not be repaired to permit survival, then lethal lesions are a subset of or are completely different from mutagenic lesions.     

Mutagenic specificity of N′-methyl-N′-nitro-N-nitrosoguanidine in the gpt gene on a chromosome of Chinese hamster ovary cells and of Escherichia coli cells

Summary DNA base sequence changes induced by N-methyl-N-nitro-N-nitrosoguanidine (MNNG) mutagenesis have been determined for the Escherichia coli gpt gene stably incorporated in a chromosome of Chinese hamster ovary cells and in the chromosome of both growing and starving E. coli cells, instead of on a plasmid as in most previous studies. In the three cases, nearly all mutations were G: C to A: T transitions, with a 2-to 4-fold higher mutation rate, compared to other sites, at guanines flanked on the 5 side by another guanine. Mutagenic hot spots in these experiments were less prominent than in published results for MNNG mutagenesis of gpt and of other genes. A suggested explanation involves repair of O⁶meG. At low levels of mutagenic products, most are repaired and even small differences in the repair rates leads to large differences in the relative amounts of residual O⁶meG at various sites; in contrast, at high levels of mutagenic products there is little effect of repair on the distribution.Abbreviations MNNG N-methyl-N-nitro-N-nitrosoguanidine - MNU N-methyl-N-nitrosourea - O⁶meG O⁶-methylguanine - N7meG N7-methylguanine - CHO Chinese hamster ovary