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.     

Mutagenic interaction between near-(365 nm) and far-(254 nm)ultraviolet radiation in repair-proficient and excision-deficient strains of Escherichia coli.

The mutational interaction between radiation at 365 and 254 nm was studied in various strains of E. coli by a mutant assay based on reversion to amino-acid independence in full nutrient conditions. In the two repair-proficient strains (K12 AB 1157 and B/r), pre-treatment with radiation at 365 nm strongly suppressed the induction of mutations by far-UV, a phenomenon accompanied by a strong lethal interaction. The frequency of mutations induced by far-UV progressively declined with increasing dose of near-UV. Far-UV-induced mutagenesis to T5 resistance was almost unaltered by pre-treatment with near-UV. In AB 1886 uvrA there was no lethal interaction between the two wavelengths but the mutagenic interaction was synergistic. This synergism was maximal at a 365-nm dose of 8 X 10(5) J m-2. It is proposed that in the wild-type strain, cells containing potentially mutagenic lesions are selectively eliminated from the population because of abortive excision of an error-prone repair-inducing signal. In excisionless strains, 365-nm radiation may be less damaging to the error-prone than to the error-free post-replication repair system. Alternatively, mutation may be enhanced because of the occurrence of error-prone repair of 365-nm lesions by a system that is not induced in the absence of 254-nm radiation.     

Mutation induction by monochromatic 254-nm and 365-nm radiation in strains of Escherichia coli that differ in repair capability

Mutation to tryptophan independence after exposure to radiation at the monocrhomatic wavelengths of 254 and 365 nm was studied and compared in 7 strains of Escherichia coli B/r that differ in repair capability. Efficient mutation induction was obtained with both 254-nm and 365-nm radiation with strains WP2 (wild-type), WP2s (uvrA), WP6s (polA uvrA). Mutants were not induced at either wavelength in the lexA strain WP5 or the recA strains WP10 and WP100. These results support the induction of mutants with 365-nm radiation through the error-prone (SOS) pathway of postreplication repair. Log-log plots of tryptophan revertant data at 254 nm showed the expected slopes of approximately 2.0 over the entire influence range tested. In contrast, similar plots of revertant data at 365 nm were complex in all cases tested: at low fluence values (survival greater than 0.5) in all cases where reversion occurred the slopes were approximately 1.0, while at higher fluences (survival less than 0.5) the slopes of the log-log plots were approximately 3.0 with strains WP2s and WP6s, approximately 4.0 with strain WP6 and approximately 6.0 with strain WP2. Differential sensitivity of components of excision and postreplication repair systems to 365-nm radiation may account for the 2-part mutation curves obtained with uvr⁺ rec⁺ lex⁺ strains. It is proposed that efficient error-free repair of mutational lesions occurs at 365-nm fluences below 2–4×10⁵ J m²⁻; at greater 365-nm fluences, error-free excision repair may be selectively inhibited, forcing a greater fraction of mutational lesions to be processed by the error-prone component of the postreplication repair system. The similarity of the mutational responses of WP2s and WP6 at 365 nm supports the selective inhibition of error-free excision repair.     

Comparative mutagenesis and interaction between near-ultraviolet (313- to 405-nm) and far-ultraviolet (254-nm) radiation in Escherichia coli strains with differing repair capabilities.

Comparative mutagenesis and possible synergistic interaction between broad-spectrum (313- to 405-nm) near-ultraviolet (black light bulb [BLB]) radiation and 254-nm radiation were studied in Escherichia coli strains WP2 (wild type), WP2s (uvrA), WP10 (recA), WP6 (polA), WP6s (polA uvrA), WP100 (uvrA recA), and WP5 (lexA). With BLB radiation, strains WP2s and WP6s demonstrated a high level of mutagenesis, whereas strains WP2, WP5, WP6, WP10, and WP100 did not demonstrate significant mutagenesis. In contrast, 254-nm radiation was mutagenic in strains WP2, WP2s, WP6, and WP6s, but strains WP5, WP10, and WP100 were not significantly mutated. The absence of mutagenesis by BLB radiation in lexA and recA strains WP10, WP5, and WP100 suggests that lex+ rec+ repair may play a major role in mutagenesis by both BLB and 254-nm radiation. The hypothesis that BLB radiation selectively inhibits rec+ lex+ repair was tested by sequential BLB-254-nm radiation. With strain WP2, a fluence of 30 J/m2 at 254 nm induced trp+ revertants at a frequency of 15 X 10(-6). However, when 10(5) J/m2 or more of BLB radiation preceded the 254-nm exposure, no trp+ revertants could be detected. A similar inhibition of 254-nm mutagenesis was observed with strain WP6 (polA). However, strains WP2s (uvrA) and wP6s (polA uvrA) showed enhanced 254-nm mutagenesis when a prior exposure to BLB radiation was given.     

Loss of photoreversibility for UV mutation in E. coli using 405 nm or near-UV challenge

E. coli mutagenized with germicidal ultraviolet light (UV) were incubated to allow for development of mutation-fixation processes. Fixation was estimated from the effects on mutation frequency of photoreactivation challenge during the first 60 min post-UV. Two different light sources were used for photoreactivation, one providing effective light primarily at 405 nm and another providing a broad range of near-UV around 365 nm. Kinetics for the loss of photoreversibility (LOP) were determined. The times for completion of LOP in wild-type cells indicated one fixation process for back mutation and another for de novo or converted suppressor mutation regardless of the light source. Using 405-nm light for photoreactivation, the LOP kinetics for back mutation and de novo suppressor mutation in uvrA cells were similar. Hence, classical observations were confirmed here. Immediately post-UV all mutation frequencies were more sensitive to near-UV than 405-nm light. Experiments with rel cells supported the idea that growth delay and inhibition of induced lexA-coordinated responses may be responsible for this early, pronounced sensitivity to photoreactivation by near UV. For back mutation and de novo suppressor mutation, the sensitivity to 405-nm light was initially small and actually increased for 10-15 min. Possibly genome conformation changes are induced by UV and this affects the efficiency of photoenzymatic monomerization of 405-nm light during the first 10-15 min after irradiation.     

Evidence for photoenzymatically repairable, lethal "non-dimer" photoproducts, formed in E. coli cells by 365-nm radiation or by sunlight greater than 360 nm.

Three pairs of E. coli strains with different dark-repair potentials, viz. H/r30 and H/r30-R, H_s30 H_s30-R, CSR 603 and AB 2480, have been investigated for their survival after exposure to 254-nm and 365-nm radiation. Each pair consists of a non-photorepairable (phr^?) and a photorepairable (phr⁺) strain of otherwise identical or similar genotype. At 254 nm, the mean inactivation fluence (F_0.37) is for the dark-repair proficient phr^? strain (H/r30) 300–750 times greater than for the completely dark-repair deficient phr^tt- strain (CSR 603), but at 365 nm the F_0.37's differ by a factor of only 5–10. Comparison of survival curves of phr^? and phr⁺ strains indicates that, at all three levels of dark-repair potential, lethal damage resulting from 365-nm exposure is extensively photorepaired by the same wavelength. Qualitatively similar effects were observed with sunlight from which all wavelengths < 360 nm were filtered out. Furthermore, we have shown that fluences of 365-nm radiation used in our experiments do not damage the enzymatic dark-repair systems themselves. These results seem compatible with one another only if one assumes that photoenzymatic repair is capable of abolishing lethal DNA damage other than the common types of cyclobutane dipyrimidines occurring after 254-nm irradiation.     

Similar ultraviolet action spectra for the protection by glycerol of transforming DNA against single-strand breaks and inactivation of biological activity

An action spectrum for the protection of purified DNA by glycerol against the induction of single-strand breaks in the DNA by ultraviolet (uv) light is described. Protection was not observed below 300 nm, was maximal between 334 and 365 nm, and decreased at 405 nm. This spectrum closely matched the spectrum for the protection by glycerol against the inactivation of biological transforming activity by near uv, described previously. Also, deviations from the reciprocity rule are similar for inactivation of transforming activity and for induction of DNA breaks by 365-nm radiation. That is, the deviations for the two end points are quantitatively the same, such that high fluence rates are less effective than low fluence rates.     

Lack of induction of non-targeted mutations in intact bacteriophage by UVB (313 nm), UVA (334 nm, 365 nm) and visible (405 nm) irradiation of host cells

Mutation to virulence has been measured in intact bacteriophage lambda 15 infected into host cells pre-treated with UVC (254 nm), UVB (313 nm), UVA (334 nm, 365 nm) or visible (405 nm) radiations. We have confirmed that UVC radiation leads to a large enhancement (maximum enhancement factor of 140 in wild-type) of the background spontaneous mutation frequency (non-targeted mutagenesis) and have further shown that this is at least partially dependent on excision repair (maximum enhancement factor of 14 in uvrA strain). In contrast, UVB (313 nm) radiation enhances the mutation frequency by less than a factor of 2. Longer wavelength UVA radiation (334 nm, 365 nm) actually reduces the mutation frequency to 25% of the background levels presumably by reducing the levels of viral replication occurring in the host cells. A visible wavelength (405 nm) has no effect on mutation frequency over the fluence range employed.     

Mutagenic action of monochromatic UV radiation in the solar range on human cells

Mutations to ouabain resistance (selecting for base modifications at the co-dominant Na+K+-dependent ATP-ase locus) and thioguanine resistance (selecting for a wide range of genetic changes at the recessive hypoxanthine-guanine phosphoribosyl transferase locus) were measured in a repair-proficient human lymphoblastoid line with defined monochromatic radiations in the UVC (254 nm), UVB (302 nm, 313 nm), UVA (334 nm, 365 nm) and visible (405 nm) ranges. No mutations were detected at wavelengths in the range 334-405 nm. At 254 nm and 313 nm, both mutations to thioguanine resistance and survival were consistent with those expected from the relative levels of cyclobutane-type pyrimidine dimers induced. However, at 313 nm, the ratio of ouabain-resistant to thioguanine-resistant mutants is 10 times higher than at 254 nm, indicating that a unique type of pre-mutagenic base damage is induced at the longer wavelength. Radiation in the UVA (334 nm) range reduced the induction of mutations by a UVC (254 nm) wavelength at both mutation markers. These results suggest, first, that distinct types of biologically expressed genetic damage may be induced in the UVB region of sunlight and, second, that strong interactions may occur between the different wavelength regions of sunlight that can modify the expression of this genetic damage in human cells.     

Mutagenesis and cytotoxicity in human epithelial cells by far- and near-ultraviolet radiations: action spectra

Action spectra were determined for cell killing and mutation by monochromatic ultraviolet and visible radiations (254-434 nm) in cultured human epithelial P3 cells. Cell killing was more efficient following radiation at the shorter wavelengths (254-434 nm) than at longer wavelengths (365-434 nm). At 254 nm, for example, a fluence of 11 Jm-2 gave 37% cell survival, while at 365 nm, 17 X 10(5) Jm-2 gave equivalent survival. At 434 nm little killing was observed with fluences up to 3 X 10(6) Jm-2. Mutant induction, determined at the hypoxanthine-guanine phosphoribosyltransferase locus, was caused by radiation at 254, 313, and 365 nm. There was no mutant induction at 334 nm although this wavelength was highly cytotoxic. Mutagenesis was not induced by 434 nm radiation, either. There was a weak response at 405 nm; the mutant frequencies were only slightly increased above background levels. For the mutagenic wavelengths, log-log plots of the mutation frequency against fluence showed linear regressions with positive slopes of 2.5, consistent with data from a previous study using Escherichia coli. The data points of the action spectra for lethality and mutagenesis were similar to the spectrum for DNA damage at wavelengths shorter than 313 nm, whereas at longer wavelengths the lethality spectrum had a shoulder, and the mutagenesis spectrum had a secondary peak at 365 nm. No correlation was observed for the P3 cells between the spectra for cell killing and mutagenesis caused by wavelengths longer than 313 nm and the induction of DNA breakage or the formation of DNA-to-protein covalent bonds in these cells.     

Non-specific incision of DNA due to the presence of 8-methoxypsoralen photoinduced interstrand cross-links in Saccharomyces cerevisiae

The repair of DNA interstrand cross-links (CL) induced by 8-methoxypsoralen (8-MOP) plus UVA irradiation was analyzed by the alkaline step elution technique. A double-exposure protocol was used with 8-MOP, starting with exposure to monochromatic 405-nm radiation inducing only DNA monoadducts (MA), followed, after washing out of unbound 8-MOP molecules, by a second exposure to 365-nm radiation inducing varying relative amounts of CL at a constant level of total photoadducts. In the range of doses used for the second exposure, repair of CL took place; however, in the presence of increased relative amounts of CL induced non-specific incision of DNA occurred. This endonucleolytic cleavage appears to be related to the increased mutagenic and recombinogenic effects observed at increased levels of CL.     

Action spectra for killing and mutation of Chinese hamster cells exposed to mid- and near-ultraviolet monochromatic light

We have examined the response of Chinese hamster V79 cells to monochromatic light of selected wavelengths in the mid- to near-UV region, using cell survival and induction of mutants resistant to 6-thioguanine (6-TG) or ouabain (OUA) as end points. As the wavelength increased from 313 to 405 nm, the induction of mutants resistant to 6-TG and to OUA decreased to a greater degree than did cell survival. Cells resistant to OUA were induced with considerably lesser efficiency at wavelengths of 313 and 334 nm than cells resistant to 6-TG. No mutants resistant to either 6-TG or OUA were induced by 405-nm light, and no mutants resistant to OUA were induced by 365-nm light. Thus, cell killing and mutation induction have different action spectra, and furthermore, action spectra for mutation induction at the HGPRT and Na+/K+-ATPase loci are different from each other. These observations imply important differences in the cellular mechanisms, and/or lesions, for cell inactivation, induction of 6-TG and OUA resistance for V79 cells exposed to near-UV monochromatic light.     

Gamma-ray- and UV-sensitive strains of a radioresistant cell line: isolation and cross-sensitivity to other agents

Two gamma-ray-sensitive and two ultraviolet (UV)-sensitive variants were isolated from the gamma-ray- and UV-resistant TN-368 lepidopteran insect cell line. The isolation was performed by inducing mutations in the TN-368 cells using ethyl methanesulfonate, growing them for an expression period, irradiating with 137Cs gamma rays or 254-nm UV radiation, allowing cells to incorporate 5-bromodeoxyuridine (BrdU) in the presence of hydroxyurea (DNA repair synthesis), and finally irradiating with 365-nm UV radiation to cause DNA strand breakage at sites of BrdU incorporation with the intent of killing those cells that have undergone DNA repair synthesis and sparing those cells which, for a variety of reasons, did not. The survival of the Cs2 and Cs7 variants exposed to X rays is significantly different from the parent TN-368 line at the P less than 0.0001 level. The survival of the UV10 and UV19 variants exposed to UV radiation is different from the parent at the P less than 0.0001 and P less than 0.003 levels, respectively. In cross-sensitivity testing of the gamma-ray-sensitive variants, only Cs2 is more sensitive to 254-nm UV and only Cs7 is more sensitive to 44 degrees C heating; both are sensitive to PUVA. The UV-sensitive mutants are both sensitive to X irradiation, PUVA, and mitomycin C. However, UV10 is not sensitive to 44 degrees C heating while UV19 is, making UV19 the only variant strain sensitive to all agents examined. Despite the isolation procedure which was intended to select for DNA repair-deficient cells, the results suggest that a more general mechanism is responsible for the sensitivity of the variant cells to the agents tested.     

Near-ultraviolet radiation blocks SOS responses to DNA damage in Escherichia coli

Summary Escherichia coli cells in which the recA promoter is fused to a lac structural gene, (Mu) Mud(Ap,lac)::rec, were irradiated with two far-ultraviolet light wavelengths (254 and 290 nm), selected monochromatic near-ultraviolet (NUV) wavelengths 313 nm, 334 nm, 365 nm, or broad band solar-UV (290–420 nm) from a solar simulator. Irradiation with the two far-ultraviolet wavelengths was followed by high yields of -galactosidase, lambda prophage induction, and Weigle reactivation. These end points were not observed after irradiation with the selected NUV wavelengths or the broad spectrum solar-UV. Thus, neither broad spectrum solar-UV nor monochromatic NUV wavelengths resulted in the derepression of the recA promoter. Further, prior exposure of the cells either to the selected monochromatic NUV wavelengths or to solar-UV inhibited (a) the induction of -galactosidase by subsequent 254-nm radiation, (b) subsequent 254-nm induction of lambda prophage, (c) Weigle reactivation, and (d) mutation frequency. These observations are consistent with the hypothesis that NUV blocks subsequent recA protease action. Offprint requests to: Ms. Susan Barr, Editor, Division of Biological and Medical Research, Argonne National Laboratory, Argonne, IL 60439, USA     

Ultraviolet and violet light: attractive orientation cues for the Indian meal moth, Plodia interpunctella

The Indian meal moth (IMM), Plodia interpunctella (Hübner) (Lepidoptera: Pyralidae), engages in long-distance or foraging flights in the twilight hours of the scotophase when blue light dominates the irradiance spectrum of the sky. We tested the hypothesis that IMM uses wavelengths of visible blue/violet light as orientation cues that trigger phototactic responses. In four-choice laboratory experiments, blue light (400–475 nm) was significantly more effective than green (475–600 nm), orange (575–700 nm), or red (590–800 nm) light in attracting males and mated females. In subsequent experiments that tested light emitting diodes (LEDs) emitting peak wavelengths in the blue/violet-light range, the 405-nm 'violet' LED was significantly more effective than the 435-, 450-, or 470-nm 'blue' LED in attracting males as well as virgin and mated females. In electroretinogram recordings, the 405-nm wavelength elicited significantly stronger receptor potentials from female and male eyes than the 350-nm (UV) wavelength, and in a behavioral experiment it significantly enhanced the known attractiveness of UV light. Equal attraction of IMMs to 405-nm LEDs at 600–700 µW/cm² with or without UV light, and significantly stronger attraction to a 405-nm LED than to a 350-nm LED at maximum light intensities, suggest that the deployment of violet instead of UV light could become one of several management tactics for control of IMMs.     

Induction of DNA-protein crosslinks in melanotic cloudman S91 mouse melanoma cells and EMT6 mouse mammary carcinoma cells by monochromatic 254 and 405 nm light

The rates of induction of DNA-protein crosslinks (DPC) by monochromatic radiation at 254 and 405 nm were compared in melanotic S91 mouse melanoma cells and EMT6 mouse mammary carcinoma cells. At 254 nm, the rates of induction of DPC are the same in the two cell lines, whereas, at 405 nm, the rate of induction of DPC in the melanotic cells is considerably less than that in the nonmelanotic cells. Since the major difference in the two cell lines with respect to absorption is melanin, the latter finding implies that intracellular melanin can protect against this DNA damage caused by a component of environmental carcinogenic solar radiation.     

Uridine 5'-phosphate photohydrate formation in irradiated Escherichia coli 30S ribosomes: photochemistry and relation to ribonucleic acid-protein cross-linking

Irradiation of aqueous buffered solutions of Escherichia coli 30S ribosomes with doses of 254-nm radiation greater than 10(19) quanta causes formation of uridine 5'-phosphate (UMP) photohydrates in ribosomal 16S RNA (rRNA). The number of molecules of UMP photohydrate formed at doses less than 2 x 10(20) quanta is linearly dependent on dose of absorbed 254-nm radiation. Maximum UMP photohydrate formation is dependent on initial ribosome concentration. When solutions containing 1 A260 unit of 30S ribosomes/mL were irradiated with greater than 2 x 10(20) quanta of 254-nm radiation, maximum photohydrate formation was equal to 47 residues/ribosome. Irradiation of solutions containing 2 A260 units/mL with greater than 7 x 10(20) quanta caused formation of 102 UMP photohydrates/ribosome. These values correspond to conversion of either 15 or 33%, respectively, of the total UMP content of 30S ribosome 16S rRNA to photohydrates. Target theory analysis of UMP photohydration in 30S ribosomes showed that UMP photohydrates are formed by single-hit kinetics from two photochemically distinct precursors. Of the total 16S rRNA UMP residues, 10% was included in the most rapidly (low dose) reacting fraction. The respective photohydration cross sections are 0.014 (low dose) and 0.0095 cm2/muEinstein (high dose) for ribosome solutions containing 2 A260 units/mL. UMP photohydrate content of irradiated 30S ribosomes was compared with that of previous data for the extent of RNA-protein cross-linking at equivalent doses of absorbed 254-nm radiation. This comparison showed that at least two UMP photohydrates form per RNA-protein cross-linking event in 30S ribosomes irradiated with a dose of 254-nm radiation (1.5 x 10(19) quanta), which causes cross-linking of only three ribosomal proteins to 16S rRNA.     

Lethal effects of pyrimidine dimers induced at 365 nm in strains of E. coli differing in repair capability

Photoreactivation (PR) after 365-nm inactivation was measured in four strains of Escherichia coli differing in repair capability. Photoreactivation was observed in the recA strains K12 and AB2480 and K12 AB2463 indicating a significant role of pyrimide dimers in the lethal action of 365-nm radiation in these strains. Significant PR was not observed in the uvrA strain, K12 AB1886, or in the repair proficient strain, K12 AB1157, after 265-nm inactivation. Biological evidence indicated that stationary phase cells had not lost the capacity for photo-enzymatic repair after fluences of 365-nm radiation of 2 × 10⁶ J/m⁻² or less. It is proposed that pyrimidine dimers, although induced, are not significant 365-nm lethal lesions in uvrA and wild-type strains because of their efficient dark repair.     

Repair and recombination of nonreplicating UV-irradiated phage DNA in E. coli II. Stimulation of RecF-dependent recombination by excision repair of cyclobutane pyrimidine dimers and of other photoproducts

Summary Three aspects of recombination of UV-irradiated nonreplicating lambda phage DNA were addressed: the photoproduct(s) responsible, the role of UvrABC-mediated excision repair, and the dependence on RecF function.Cyclobutane pyrimidine dimers appeared responsible for some recombination because photoreactivation reduced the frequency of 254-nm-stimulated recombination and because photosensitized 313-nm irradiation stimulated recombination. Other photoproducts seemed recombinogenic as well, because high fluences of 254-nm irradiation stimulated recombination considerably more, per cyclobutane dimer induced, than photosensitized 313-nm irradiation, and because photoreactivation did not eliminate 254-nm stimulated recombination. For both treatments, much, but not all, of the recombination was UvrABC-dependent. Recombination was mostly RecF-dependent, but was not affected by recB recC or recE mutationsThe first paper in this series is Hays et al., (1985)     

UV-Induced destruction of light-harvesting complexes from purple bacterium Chromatium minutissimum

We studied UV-induced photodestruction of the native forms of bacteriochlorophyll a (Bchl a) from chromatophores and light harvesting complexes (LHC) of the sulphur photosynthetic bacterium Chromatium minutissimum. Irradiation of chromato- phores with 365-nm light (Soret band) or 280-nm light (absorption region of aromatic amino acids) led to the destruction of all long-wavelength forms of Bchl a. The quantum yields of photodestruction produced by the 280-nm light was higher than that produced by the 365-nm light. For the spectral forms of Bchl a absorbing at 850 nm and 890 nm, the difference was about one order of magnitude, and for the form absorbing at 800 nm the difference was almost two orders of magnitude. Similar UV sensitivity was observed for the Bchl a forms from isolated LHC. As a rule, the quantum yields of photodestruction induced by UV irradiation at 280 nm were about 100-1000 times higher (approximately 10(-3)-10(-4)) than that upon red light irradiation (approximately 10(-6)-10(-7)). We found that irradiation of chromatophores at 280 nm resulted in a crosslink between the core and peripheral LHC.