Photoreactivation reverses ultraviolet radiation induced premutagenic lesions leading to frameshift mutations in Escherichia coli

Summary The effect of photoreactivation of the ultraviolet radiation induced reversion of a trpE9777 frameshift mutation was studied in a uvrA6 derivative of Escherichia coli K12. Two different photoreactivation treatments were used, one providing a single flash of photoreactivating light and another providing 10 min of light from fluorescent lamps. The reversion frequency of the trpE9777 frameshift mutation was strongly reduced when subsequently exposed to visible light. The dose modification factor (the ratio of equally effective doses), for cells challenged with single-flash photoreactivation, for survival and induction of reversion to Trp⁺ was 3.6 and 3.4, respectively. UV induction of RecA protein synthesis was not reversed by a single flash of photoreactivation. The dose modification factor for 10 min of fluorescent lamp photoreactivation for survival and for induction of reversion to Trp⁺ was 6.5 and 6.3, respectively. The dose modification factor for 10 min of photoreactivation for induction of RecA protein was 1.7–2.5. Photoreactivation decreased the reversion of trpE9777 and increased survival to the same extent. We concluded that cyclobutyl pyrimidine dimers are the premutagenic lesions of UV mutagenesis of the trpE9777 allele in a uvrA6 background.     

Effect of flash photoreactivation on Escherichia coli recA induction by ultraviolet light

Excision-deficient Escherichia coli, carrying the gene for the photolyase on a multicopy plasmid, were irradiated with ultraviolet (UV) light then photoreactivated by illumination delivered from a camera flash unit. Such instantaneous illumination monomerizes only cyclobutane pyrimidine dimers already bound by the photolyase. Whereas the lethal effect of UV light and the number of C-to-T transition-type mutations induced by UV irradiation were both significantly reduced by subsequent irradiation with a single flash of light, single-flash photoreactivation did not reverse the induction of the recA gene by UV light. The results indicate, therefore, that non-photoreactivable DNA lesions play a role in recA induction.     

Double mutations induced in Escherichia coli by ultraviolet light.

Double mutations to azide resistance and to bacteriophage T5 resistance of genes separated by more than 50 kilobases were induced in Escherichia coli WP2s in chemostat cultures by exposure to a single low dose of ultraviolet light. Frequencies of induced double mutations were three orders of magnitude greater than would be predicted by chance. Reversions from azide resistance and phage resistance occurred independently, showing that that the double mutation was not due to pleiotropic effects of a single gene mutation. These results support earlier findings which show that low doses of ultraviolet light induce multiple gene mutations in Bacillus subtilis over a similarly broad range.     

Conjugation is not required for adaptive reversion of an episomal frameshift mutation in Escherichia coli.

Adaptive reversion of a lac allele on an F' episome in a strain of Escherichia coli is dependent on the RecA-BCD pathway for recombination and is enhanced by conjugal functions. However, conjugation, i.e., transfer of the episome, whether between distinct populations of cells or between newly divided siblings, does not contribute to the mutational process.     

New mutations in cloned Escherichia coli umuDC genes: novel phenotypes of strains carrying a umuC125 plasmid

The umuDC locus of Escherichia coli is required for most mutagenesis by UV and many chemicals. Mutations in E. coli umuDC genes cloned on pBR322-derived plasmids wer e isolated by two methods. First, spontaneously-arising mutant umuDC plasmids that failed to confe cold-sensitive growth on a lexA51(Def) strain were isolated by selection. Second, mutant umuDC plasmids that affected apparent mutant yield after UV-irradiation in a strain carrying umuD⁺C⁺ in the chromosome were isolated by screening hydroxylamine-mutagenized umuD⁺C⁺ plasmids. pBR322-derived umuD⁺C⁺ plasmids inhibited the induction of the SOS response of lexA⁺ strains as measured by expression of din::Mu dl(lac) Ap) fusionsbut most mutant plasmids did not. Mutant plasmids defective in complementation of chromosomal umuD44, umuC36, or both were found among those selected for failure to confer cold-sensitivity, whereas those identified by the screening procedure yielded mostly mutant plasmids with more complex phenotypes. We studied in greater detail a plasmid pLM109, carrying the umuC125 mutation. This plasmid increased the sensitivity of lexA⁺ strainsto killing by UV-irradiation but was able to complement the deficiencies of umuC mutants in UV mutagenesis. pLM109 failed to confer cold-sensitive growth on lexA(Def) strains but inhibited SOS induction in lexA⁺ strains. The effect of pLM109 on the UV sensitivity of lexA(Def)strains was similar to that of the parental umuD⁺C⁺ plasmid. The mutation responsible for the phenotypes of pLM109 was localized to a 615-bp fragment. DNA sequencing revealed that the umuC125 mutation was a G:C → A:T transition that changed codon 39 of umuC from GCC → GTC thus changing Ala39 to Val39. The implications of the umuC125 mutation for umuDC-dependent effects on UV-mutagenesis and cell survival after UV damage are discussed.     

Construction of a umuDC operon substitution mutation in Escherichia coli.

Using a specialized transducing lambda phage, the umuDC operon of Escherichia coli was deleted and replaced with the chloramphenicol acetyltransferase gene. The delta (umuDC)595::cat mutation was subsequently transferred by generalized P1 transduction into a variety of genetic backgrounds. It is concluded that the UmuDC proteins, which are normally required for inducible mutagenesis, are not essential for cell survival.     

Induction of -2 frameshift mutations by 2-nitrofluorene, N-hydroxyacetylaminofluorene, and N-2-acetylaminofluorene in reversion assays in Escherichia coli strains differing in permeability and acetyltransferase activity

The mutagenicity of 2-nitrofluorene (NF), N-hydroxyacetylaminofluorene (N-OH-AAF), and N-2-acetylaminofluorene (AAF) was measured in strains of Escherichia coli that contain a lacZ allele that reverts by -2 frameshift mutations from CG(5) to CG(4). Mutagenesis was compared in a strain having wild-type permeability and metabolism, a strain with increased permeability caused by a lipopolysaccharide-defective (LPS(d)) mutation, a strain with N- and O-acetyltransferase (NAT/OAT) activity conferred by the Salmonella nat gene on plasmid pYG219, and a strain carrying both an LPS(d) mutation and pYG219. The LPS(d) mutation facilitated the measurement of mutagenicity but was not absolutely required, in that lower levels of mutagenicity were detected in LPS(+) strains. The NAT/OAT activity conferred by pYG219 strongly potentiated the mutagenicity of NF and N-OH-AAF. Surprisingly, AAF was mutagenic in the NAT/OAT LPS(d) strain without an exogenous P450 metabolic activation system. Its activity may be ascribable to the detection of a directly mutagenic impurity by the highly sensitive strain or to a low level of metabolic activation by the bacteria under the assay conditions. The findings add to the evidence that the lacZ allele derived from E. coli strain CC109 is an effective indicator of -2 frameshift mutagenesis and that strains expressing high levels of NAT/OAT activity are highly sensitive in monitoring the mutagenicity of nitroarenes and aromatic amides.     

Action spectra for photoreactivation of ultraviolet-irradiated Escherichia coli and Streptomyces griseus

Action spectra for photoreactivation (light-induced recovery from ultraviolet radiation injury) of Escherichia coli B/r and Streptomyces griseus ATCC 3326 were determined. The spectral region explored was 365 to 700 mµ. The action spectrum for S. griseus differed from that for E. coli, indicating that the chromophores absorbing reactivating energy in the two species were not the same. Reactivation of S. griseus occurred in the region 365 mµ (the shortest wave length studied) to about 500 mµ, with the most effective wave length lying near 436 mµ. This single sharp peak in the spectrum at 436 mµ suggested the Soret band typical of porphyrins. Reactivation of E. coli occurred in the region 365 to about 470 mµ, with the most active wave length lying near 375 mµ. The single, non-pronounced peak near 375 was probably not due to a Soret band, and the identification of the substance absorbing reactivating light in E. coli is uncertain. In neither species was the region 500 to 700 mµ active. The implications of these action spectra and their differences are discussed.     

Thermal resistance to photoreactivation of ultraviolet light induced mutations in the lacI gene of E. coli ung

Ultraviolet light (UV) induced mutations in the lacI gene of Escherichia coli are thought to be targeted by DNA photoproducts. A number of reports suggest that both cyclobutyl pyrimidine dimers and pyrimidine (6−4) pyrimidone photoproducts may be involved. To investigate the potential contribution of each of these DNA photoproducts to mutagenesis in the lacI gene, we held UV-irradiated cells at a temperature of 44°C for 75 min and then exposed them to photoreactivating light (PR). This protocol is expected to preferentially deaminate specifically those cytosines that are contained in cyclobutyl dimers and subsequently monomerize the dimers to yield uracils in the DNA. In a strain deficient for uracil-DNA glycosylase (Ung⁻), these uracils would not be removed and a G : C → A : T transition would result at the site of the dimer. This protocol resulted in the enhancement of amber nonsense mutations that result from transitions at potential cytosine-containing dimer sites. The enhanced mutation frequencies resulting from this procedure were used to estimate the probability of dimer formation at the individual sites. A comparison of the dimer distribution with the mutation frequencies following UV alone suggests that both cyclobutyl dimers and (6−4) photoproducts contribute to UV-mutagenesis in the lacI gene. In addition, we conclude that the frequency of mutation at any particular site not only reflects the occurrence of DNA damage, but also the action of metabolic processes that are responsible for DNA repair and mutagenesis.     

Isolation and characterization of mutants of Escherichia coli deficient in induction of mutations by ultraviolet light.

Summary Mutants of E. coli defective in susceptibility to UV-induction of mutations were isolated by direct screening for their UV nonmutable phenotype (Umu^–). Screening of about 30,000 mutagenized clones of a uvrB ^– derivative of AB1157 yielded six Umu^– strains. The mutants can be classified into three groups by the location of the mutations, umuA, umuB and umuC. Mutations umuA and umuB are, respectively, mapped close to lexA and recA genes and mutations at both loci partially reduce UV mutagenesis. The locus of umuC is between hemA and purB and the mutations at this new locus result in a moderate increase of UV sensitivity. The mutation diminishes UV mutagenesis and UV reactivation of phage without affecting the inducibility of phophage nor the inhibition of cell division following UV irradiation. Related properties of an isogenic strain of a recF ^– mutant are compared with those of umuC ^–.     

Intragenic reversion mutations that improve export of maltose-binding protein in Escherichia coli malE signal sequence mutants

Escherichia coli strains harboring malE signal sequence point mutations accumulate export-defective precursor maltose-binding protein (MBP) in the cytoplasm. Beginning with these mutants, a number of spontaneous intragenic revertants have been obtained in which export of the MBP to the periplasm is either partially or totally restored. With a single exception, each of the reversion mutations resulted in an increase in the overall hydrophobicity of the signal peptide hydrophobic core by one of five different mechanisms. In some revertants, MBP export was achieved at a rate comparable to the wild type MBP; in other cases, the rate of MBP export was significantly slower than wild type. The results indicate that the overall hydrophobicity of the signal peptide, rather than the absolute length of its uninterrupted hydrophobic core, is a major determinant of MBP export competency. An alteration at residue 19 of the mature MBP also has been identified that provides fairly efficient suppression of the export defect in the adjacent signal peptide, further suggesting that important export information may reside in this region of the precursor protein.     

The early detection of frameshift mutations induced by a food-borne carcinogen in rats: a new tool for molecular epidemiology

The accumulation of genetic changes is considered as the main factor that determines the development of cancer. Recent progresses in genetics and molecular biology led to the discovery of many new molecular markers and to the development of techniques able to monitor these markers. As a consequence, molecular epidemiology has emerged as a powerful approach to study the ternary relationship between the environment, the behaviour and the genetic predisposition of each individual. Susceptibility to cancer is determined at different levels such as the genetic polymorphism of enzymes involved in the activation and detoxification of carcinogens, the polymorphism of genes that maintains the genome stability, like those involved in DNA repair or recombination processes, and finally the polymorphism in oncogenes or tumour suppressor genes. Consequently, the full assessment of each individual's genetic predisposition is a long and difficult task. As the accumulation of mutations in somatic cells integrates all these parameters, its measurement would facilitate the evaluation of the individual predisposition status, provided that a marker common to a large spectrum of carcinogens could be found. Our current studies on the molecular mechanisms of carcinogen-induced mutagenesis has revealed that G-rich repetitive sequences are mutational hot spots for several major classes of environmental genotoxins such as aromatic and heterocyclic amines, polycyclic hydrocarbons and oxidative agents. We thus consider the possibility that these sequences form a new class of biomarkers for carcinogen exposure. In order to validate this hypothesis, we designed a sensitive PCR-based assay able to detect specific mutations induced by a common food-borne carcinogen in the colon epithelium of rats exposed for a short period to this carcinogen. This assay is sensitive enough to allow early detection of induced mutations and therefore allows to differentiate between unexposed animal and those exposed for a period as short as 1 week.