Replication and repair of UV-induced mutational lesions in chemostat cultures of Escherichia coli WP2 Hcr

Mutation to resistance to bacteriophage T5 was studied in chemostat cultures of Escherichia coli strain WP2 Hcr exposed to ultraviolet radiation (UV). The results are in generally good agreement with those obtained earlier by Bridges and Munson for UV-induced reversion to tryptophan independence in exponentially growing cultures of the same strain: expressed mutant yields followed a dose-squared response, mutations were not expressed before approximately one generation after exposure to UV, there was a slow disappearance of dimers especially noticeable in slowly growing and stationary cultures, and the first replication gave rise to duplex mutants in both strands. Several new results were also obtained. In addition to expressed mutant yields, induction of mutational capacity was also observed to follow a dose-squared response, indcating that the response is not an artifact of selection or repair. Induction also increased with growth rate, apparently as the square of the number of genes for T5-sensitivity per cell. It is suggested that mutagenesis is proportional to the number of genes per cell, that recombination is also proportional to the number of genes per cell, and that the number of mutational lesions is proportional to the product of the two. These results also provide evidence that DNA replication occurs near the end of the cell cycle in slowly growing cultures. Under all growth conditions, latent mutant concentrations mutational capacity) decreased by a factor of two with each successive division. Latent mutants were, however, photoreversible for only the first two generations. If mutagenesis occurs as a recombinant event between two mutational lesions, then the results also indicate that these lesions are separated, on the average, by no more than a single cistron.     

Near-UV mutagenesis: photoreactivation of 365-nm-induced mutational lesions in Escherichia coli WP2s.

Reversion to tryptophan independence induced by 365-nm and 254-nm radiation was studied in Escherichia coli WP2s (B/r trp uvrA). Under aerobic conditions, the mutant frequency responses was of the fluence-square or "two-hit" type at both 365 and 254 nm when revertants were assayed on minimal agar supplemented with 2% nutrient broth (SEM plates). In contrast, when mutants were assayed on minimal agar supplemented with tryptophan only, the revertant yield was reduced to very low values at 365 nm, whereas values substantially greater than with SEM plates were obtained at 254 nm. Premutational lesions induced by both 365-nm and 254-nm radiation were photoreactivated more than 10-fold when assayed on SEM plates, implicating pyrimidine dimers as premutational lesions at both wavelengths. The strong photoreactivation of 365-nm-induced mutagenesis contrasted strikingly with the complete absence of photoreactivation of 365-nm-induced lethality in this strain.     

The bacterial tryptophan reverse mutation assay with Escherichia coli WP2

The Escherichia coli WP2 tryptophan reverse mutation assay detects trp(-) to trp(+) reversion at a site blocking a step in the biosynthesis of tryptophan prior to the formation of anthranilic acid. The different WP2 strains all carry the same AT base pair at the critical mutation site within the trpE gene. The assay is currently used by many laboratories in conjunction with the Ames Salmonella assay for screening chemicals for mutagenic activity. In general the WP2 strains are used as a substitute for, or as an addition to Salmonella strain TA102 which also carries an AT base pair at the mutation site. The assay is also recommended together with the Ames assay for data submission to regulatory agencies. National and international guidelines have been established for performing these mutagenicity assays.The E. coli WP2 assay procedures are the same as those described elsewhere in this volume for the Ames Salmonella assay (Mortelmans and Zeiger, 2000) with the exception that limited tryptophan instead of limited histidine is used. This chapter is an addendum to the previous chapter and the reader should refer to the previous chapter for details regarding experimental procedures and assay design.     

The Escherichia coli WP2/WP100 rec assay for detection of potential chemical carcinogens

46 chemicals of various classes and structures, including 30 known animal carcinogens, were evaluated for genotoxic effects using the Escherichia coli rec assay with strains WP2 (wild-type) and WP100 (uvrA⁻¹recA⁻) in qualitative and quantitative spot tests and in quantitative suspension tests. The rec assay detected 17 of 30 known carcinogens as genotoxic agents, including mitomycin C and diethylnitrosamine, both negative in the Salmonella/Ames test as utilized in these studies. The rec assay in conjunction with the Salmonella/Ames test 30 known carcinogens as genotoxic agents. Azo/aminoazo carcinogens showed little genotoxicity, and the aromatic amine 2-acetylaminofluorene was non-genotoxic in the rec assay. The rec assay was more effective than pol tests with E. coli strains W3110/p3478 and strains WP2/WP67. Effectiveness of the rec assay was related to the DNA repair-defective nature of the uvrA⁻ recA⁻ genotype of strain WP100.     

Elimination of lethal and pre-mutational DNA lesions during the photoreactivation of UV-irradiatedEscherichia coli

The comparison of the frequency oftrp ⁺ revertants ofEscherichia coli B/r Hcr⁺ thy trp after UV-irradiation on the one hand and after UV-irradiation plus photoreactivation on the other showed that both photoreversible pyrimidine dimers of the cyclobutane type and the non-photoreversible DNA lesions cause, at equal lethal effects, alsotrp ⁺ reversions with the same efficiency. If lethal, the pyrimidine dimers may thus be conceived as primary pre-mutational lesions.     

Evidence that dimers remaining in preinduced Escherichia coli B/r Hcr+ become insensitive after DNA replication to the extract from Micrococcus luteus.

In Escherichia coli B/r Her+ irradiated with two separate fluences, dimer excision is prematurely interrupted. The present study was designed to follow tha fate of dimers remaining unexcised. The results imply that these dimers (or distortions containing dimers) are transformed on replication from the state of sensitivity to the state of insensitivity to endonuclease from Micrococcus luteus. This conclusion is based on the following findings: (a) dimers were radiochromatographically detectable in DNA replicated after UV, which indicated that they were tolerated on replication. (b) Similar amounts of dimers were detected radiochromatographically both in DNA remaining unreplicated and DNA twice replicated after UV, This along with the low transfer of parental label into daughter DNA, indicated that dimers remained in situ in parental chains. (c) Immediately after UV, all parental DNA contained numerous sites sensitive to the extract from M. luteus. 2 h after UV, a portion of parental DNA still contained a number of endonuclease-sensitive (Es) sites, while another portion of parental DNA and all daughter DNA were free of Es sites. (d) The occurrence of parental DNA free of Es sites was not temporally correlated with dimer excision, but with the first round of DNA replication. (e) The amount of DNA free of Es sites corresponded to the amount of replicated DNA. (f) Separation of replicated and unreplicated DNA, and detection of Es sites in both portions separately showed that the replicated DNA was almost free of Es sites, whereas unreplicated DNA contained a number of such sites.     

Pyrimidine Pools and Macromolecular Composition of Pyrimidine-Limited Escherichia coli

The growth rate of a pyrimidine-requiring strain was controlled by limiting the concentration of exogenous orotic acid. As the steady state, pyrimidine-limited growth rate was decreased, the intracellular pyrimidine pools and the total nucleic acid per unit mass of culture also decreased. The ratio of deoxyribonucleic acid to protein remained constant, whereas the ratio of ribonucleic acid to protein decreased 30% over a threefold variation in growth rate (50- to 150-min doubling times). The intracellular uridine triphosphate and cytosine triphosphate pools also decreased (although not coordinately), and the pyrimidine biosynthetic enzymes were derepressed. Cell size was unaffected by pyrimidine-mediated variation of the growth rate.     

Excision of pyrimidine dimers in toluene-treated Escherichia coli.

Toluene-treated cells were used for examining excision of pyrimidine dimers in Escherichia coli strains W3110, DM845 (uvrA-), P3478 (polA-), and KS5064 (polAex1). Excision occurring in toluene-treated cells is rapid, adenosine 5'-triphosphate dependent, and requires the uvrA gene function. In strains lacking either the polymerizing or 5' leads to 3' exonucleolytic activity of deoxyribonucleic acid polymerase I, excision does occur. However, both in vivo and in vitro, the excision in such strains is initially slower than wild type.     

The pyrogallol related compounds reduce UV-induced mutations in Escherichia coli B/r WP2

Plant components with bio-antimutagenic activity were screened on UVC (254 nm)-induced mutagenesis using E. coli B/r WP2. The components with a pyrogallol moiety including gallic acid, (-)-epicatechin gallate (ECG), (-)-epigallocatechin (EGC) and (-)-epigallocatechin gallate (EGCG) reduced the mutation induction, but other components such as caffeic acid, chlorogenic acid and quercetin did not. The above compounds with a pyrogallol moiety were also effective on UVAB (295-400 nm)-induced mutagenesis, while they showed little effect on N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-induced mutagenesis. As this bio-antimutagenic effect was not seen in the DNA excision-repair-deficient strains WP2s and ZA159, the activity by the above plant components might be based on the promotion of the excision-repair system in E. coli B/r WP2.     

Pyrimidine dimers are not the principal pre-mutagenic lesions induced in lambda phage DNA by ultraviolet light

Experiments were performed to examine the role of cyclobutyl pyrimidine dimers in the process of mutagenesis by ultraviolet (u.v.) light. Lambda phage DNA was irradiated with u.v. and then incubated with an Escherichia coli photoreactivating enzyme, which monomerizes cyclobutyl pyrimidine dimers upon exposure to visible light. The photoreactivated DNA was packaged into lambda phage particles, which were used to infect E. coli uvr- host cells that had been induced for SOS functions by ultraviolet irradiation. Photoreactivation removed most toxic lesions from irradiated phage, but did not change the frequency of induction of mutations to the clear-plaque phenotype. This implies that cyclobutyl pyrimidine dimers can be lethal, but usually do not serve as sites of mutations in the phage. The DNA sequences of mutants derived from photoreactivated DNA showed that almost two-thirds (16/28) were transitions, the same fraction found for u.v. mutagenesis without photoreactivation. These results show that in this system, the lesion inducing transitions (the major type of u.v.-induced mutation) is not the cyclobutyl pyrimidine dimer; a strong candidate for a mutagenic lesion is the Pyr(6-4)Pyo photoproduct. On the other hand, photoreactivation of SOS-induced host cells before infection with u.v.-irradiated phage reduced mutagenesis substantially. In this case, photoreversal of cyclobutyl dimers serves to reduce expression of the SOS functions that are required in the process of targeted u.v. mutagenesis.     

Determination of the initial rate of antimutagenic repair in UV-irradiated WP2 Escherichia coli cells]

The initial rates of antimutagenic dark repair were measured in Escherichia coli WP2 trpE65 cells irradiated by UV-light (11 J/m2) and then incubated in liquid media of various compositions. Samples were taken from suspension of incubated bacteria every 5 min following irradiation, mixed with acriflavine to block further repair and plated onto the selective medium containing acriflavine (1 micrograms/ml) to score the Trp+ mutations. The initial rate of antimutagenic repair was estimated from the kinetics of disappearance of mutations in several successive probes. It appeared to depend on the composition of a medium, to establish just after placing irradiated bacteria onto the medium and to decrease significantly in irradiated cells incubated under conditions favourable for growth. The decrease was not due to inhibition of postreplicative repair and was not caused by casaminoacids as such, but by combination of growth factors that provided the intensive protein synthesis. The decrease could be responsible for a strong mutational response of bacteria to irradiation because it secures the survival of premutagenic lesions in DNA till mutation fixation. It is suggested that metabolic regulation of the antimutagenic repair activity exists, based on an active switch of the energy flows required for several parallel metabolic pathways that proceed in irradiated cells.     

Mutation induction in Escherichia coli WP2 uvrA by Cerenkov emission associated with 137Cs gamma irradiation

Evidence is presented for the mutation of the tryptophan-requiring bacterial strain Escherichia coli WP2 uvrA from auxotrophy to prototrophy, and from streptomycin sensitivity to resistance, by Cerenkov emission associated with 137Cs gamma irradiation. Furthermore, the data strongly suggest a more than additive interaction between the gamma-induced damage and that induced by Cerenkov emission for both mutations scored. An additional observation is that mutant yields (expressed as mutants/10(7) survivors) show a dependence on the number of viable cells plated for both uv (254 nm) and Cerenkov-induced mutations, but not for those induced by gamma irradiation. This demonstrates another similarity between uv- and Cerenkov-induced damage.