Studies on the reactivation of bacteria photodamaged by psoralen

Summary In photoreaction with the pyrimidine bases (thymine, cytosine, uracil) as well as with nucleic acids (DNA, RNA) a C₄-cycloaddition of furocoumarins to the 5.6-double bond of pyrimidine bases takes place. The simple photoadduct furocoumarin-pyrimidine base can be split by reirradiation at wavelengths shorter than 334 nm. Reactivation of bacterial cells photodamaged by psoralen (365 nm) was tried experimentally. However, reirradiation at shorter wavelengths and with visible light of the psoralen-inactivated bacterial cells was without any effect. The inability of the shorter wavelengths to repair this photodamage was probably due to a filter effect of DNA for such wavelengths, as shown by experiments on a DNA-psoralen combination. On the other hand the observed ability of psoralen to form inter-strand cross-linkages in the photoreaction with DNA may be significant for explaining the absence of photoreactivation when the inactivated bacterial cells are irradiated with visible light.     

A Light-Dependent Pathway for the Elimination of UV-Induced Pyrimidine (6-4) Pyrimidinone Photoproducts in Arabidopsis

Light-dependent repair of UV-induced cyclobutane pyrimidine dimers (CPDs) and pyrimidine (6-4) pyrimidinone dimers (6-4 products) was investigated in an excision repair-deficient Arabidopsis mutant. As previously described, exposure to broad-spectrum lighting was found to greatly enhance the rate of repair of CPDs. We demonstrate that 6-4 products are also efficiently eliminated in a light-dependent manner and that this photoreactivation of 6-4 products occurs independently of the previously described 6-4 product dark repair pathway. The light-dependent repair of both 6-4 products and CPDs occurs in the presence of blue light (435 nm) but not upon exposure to light of longer wavelengths. We also found that high-level expression of the CPD-specific photoreactivating activity in the Arabidopsis seedling requires induction by exposure to light prior to as well as during the period of repair while the 6-4 photoreactivating activity is constitutively expressed. This differential regulation of the photoreactivating activities suggests that the Arabidopsis seedling produces at least two distinct photolyases: one specific for CPDs and the other specific for 6-4 products.     

Cyanobacteria toxins in the Salton Sea

Background

Sequenced archaeal genomes contain a variety of bacterial and eukaryotic DNA repair gene homologs, but relatively little is known about how these microorganisms actually perform DNA repair. At least some archaea, including the extreme halophile Halobacterium sp. NRC-1, are able to repair ultraviolet light (UV) induced DNA damage in the absence of light-dependent photoreactivation but this 'dark' repair capacity remains largely uncharacterized. Halobacterium sp. NRC-1 possesses homologs of the bacterial uvrA, uvrB, and uvrC nucleotide excision repair genes as well as several eukaryotic repair genes and it has been thought that multiple DNA repair pathways may account for the high UV resistance and dark repair capacity of this model halophilic archaeon. We have carried out a functional analysis, measuring repair capability in uvrA, uvrB and uvrC deletion mutants.

Results

Deletion mutants lacking functional uvrA, uvrB or uvrC genes, including a uvrA uvrC double mutant, are hypersensitive to UV and are unable to remove cyclobutane pyrimidine dimers or 6–4 photoproducts from their DNA after irradiation with 150 J/m² of 254 nm UV-C. The UV sensitivity of the uvr mutants is greatly attenuated following incubation under visible light, emphasizing that photoreactivation is highly efficient in this organism. Phylogenetic analysis of the Halobacterium uvr genes indicates a complex ancestry.

Conclusion

Our results demonstrate that homologs of the bacterial nucleotide excision repair genes uvrA, uvrB, and uvrC are required for the removal of UV damage in the absence of photoreactivating light in Halobacterium sp. NRC-1. Deletion of these genes renders cells hypersensitive to UV and abolishes their ability to remove cyclobutane pyrimidine dimers and 6–4 photoproducts in the absence of photoreactivating light. In spite of this inability to repair UV damaged DNA, uvrA, uvrB and uvrC deletion mutants are substantially less UV sensitive than excision repair mutants of E. coli or yeast. This may be due to efficient damage tolerance mechanisms such as recombinational lesion bypass, bypass DNA polymerase(s) and the existence of multiple genomes in Halobacterium. Phylogenetic analysis provides no clear evidence for lateral transfer of these genes from bacteria to archaea.     

Induction of sister-chromatid exchanges in ICR 2A frog cells exposed to 265-313 nm monochromatic ultraviolet wavelengths and photoreactivating light

Exposure of ICR 2A frog cells to 265 nm, 289 nm, 302 nm or 313 nm monochromatic ultraviolet (UV) wavelengths induced the formation of sister-chromatid exchanges (SCEs). However, treatment of cells with photoreactivating light (PRL) following the UV irradiations resulted in a lower level of SCEs compared with cells incubated in the dark. Hence, it can be concluded that pyrimidine dimers are the principal photoproducts responsible for the induction of SCEs in cells exposed to 265-313 nm UV due to the specificity of DNA photolyase for the light-dependent monomerization of dimers in DNA. It was also found that the maximum yield of induced SCEs in 313 nm-irradiated cells was only about 7 SCEs per cell whereas the plateau values for the shorter wavelengths were approximately 15-20 SCEs per cell. In addition, treatment of cells with 313 nm plus 265 nm light resulted in a lower level of SCEs than in cells exposed to 265 nm UV alone. These results can be interpreted in the context of a replication model for SCE, in which the high level of non-dimer damages produced in the DNA of 313 nm-irradiated cells inhibits the induction of SCEs by the pyrimidine dimers that are also produced by this wavelength.     

Photoreactivation of UV induced cell killing, chromosome aberrations, sister chromatid exchanges, mutations and pyrimidine dimers in Xenopus laevis fibroblasts

Summary Fibroblasts from Xenopus laevis, which possess photoreactivating enzyme were used to study the influence of photoreactivating light on the frequency of pyrimidine dimers in DNA, chromosomal aberrations, sister chromatid exchanges, cell killing and the induction of gene mutations (ouabain-resistance) induced by 254 nm ultraviolet irradiation. The frequency of all biological endpoints studied were reduced following exposure to photoreactivating light parallel to the reduction in the frequencies of pyrimidine dimers (determined as endonuclease sensitive sites). However there was not always an absolute quantitative relationship between the reduction in the frequency of pyrimidine dimers and the reduction in the biological effects. This probably reflects a fast fixation process for the biological effects prior to removal of the dimers by photoreactivation.Abbreviations UV ultraviolet - PR photoreactivating - ESS endonuclease sensitive site - SCE sister chromatid exchanges - BrdUrd 5-brothodeoxyuridine     

Genetic exchanges caused by ultraviolet photoproducts in phage λ DNA molecules: The role of DNA replication

Summary Genetic recombination induced by structural damage in DNA molecules was investigated in E. coli K12 () lysogens infected with genetically marked phage . Photoproducts were induced in the phage DNA before infection by exposing them either to 313 nm light in the presence of acetophenone or to 254 nm light. To test the role of the replication of the damaged phage DNA on the frequency of the induced recombination, both heteroimmune and homoimmune crosses were performed.First, samples of a heteroimmune phage imm434 P80 exposed to these treatments were allowed to infect cells lysogenic for prophage cI857 P3. Phage DNA replication and maturation took place, and the resulting progeny phages were assayed for the frequency of P ⁺ recombinants. Recombination was less frequent in infected cells exposed to visible light and in wild type cells able to perform excision repair than in excision-defective lysogens. Therefore, much of the induced recombination can be atributed to the pyrimidine dimers in the phage DNA, the only photoproducts known to be dissociated by photoreactivating enzyme.Second, in homoimmune crosses, samples of similarly treated homoimmune P3 phages were allowed to infect lysogens carrying cI857 P80. Replication of the phage DNA containing ultraviolet photoproducts was repressed by immunity, and was futher blocked by the lack of the P gene product needed for replication. The lysogens were purified and scored for both colony forming ability and for P ⁺ recombinant prophages. The 254 nm photoproducts increased the frequency of recombination in these homimmune crosses, even though phage DNA replication was blocked. Irradiation with 313 nm light and acetophenone M, which produces dimers and unknown photoproducts, was not as effective per dimer as the 254 nm light.It is concluded from these results that certain unidentified 254 nm photoproducts can cause recombination even in the absence of DNA replication. They are not pyrimidine dimers, as they are not susceptible to excision repair or photoreactivation. In contrast, pyrimidine dimers appear to cause recombination only when the DNA containing them undergoes replication.     

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.     

Photoreactivating enzyme from Escherichia coli: appearance of new absorption on binding to ultraviolet irradiated DNA.

The photoreactivating enzyme, PRE, monomerizes pyrimidine dimers in DNA in a light requiring reaction (lambda greater than 300 nm). However, the purified PRE from E. coli has no well-defined absorption band for lambda greater than 300 nm. Using absorption difference spectroscopy, we show that when PRE is mixed with ultraviolet-irradiated DNA, new absorption appears in the spectral region required for catalysis. There is a concomitant decrease in the absorption of the mixture for wavelength less than 300 nm. The hyperchromicity for lambda greater than 300 nm is true absorption, not an artifact due to light scattering. Both the hyperchromicity (lambda greater than 300 nm) and hypochromicity (lambda less than 300 nm) can be reversed by irradiation of 365 nm with identical first-order kinetics. We estimate the molar extinction coefficient of the new absorption to be 6900 +/- 1400 at 350 nm. We conclude that the PRE from E. coli does not possess a distinct "chromophore" which by itself is entirely responsible for the absorption of photoreactivating light. Instead, new absorption results when PRE binds its substrate, dimer-containing DNA.     

Analysis of the excision repair of nondimer DNA damage induced by solar ultraviolet radiation in ICR 2A frog cells

The excision repair of solar uv-induced nondimer DNA damage was examined in ICR 2A frog cells through the use of the bromodeoxyuridine (BrdUrd) photolysis assay. A relatively pure population of nondimer DNA photoproducts was induced by irradiation of ICR 2A cells with the Mylar-filtered solar ultraviolet (uv) wavelengths produced by a fluorescent sunlamp followed by exposure to photoreactivating light (PRL) which removes most of the small yield of pyrimidine dimers induced by this treatment. Cultures of cells were also exposed to 254 nm uv, which induces primarily dimers, and 60Co gamma rays. Through use of a modification of the BrdUrd photolysis assay possessing enhanced sensitivity, it was found that the solar uv-induced nondimer DNA damage was repaired by a short patch repair mechanism in which less than approximately 20 nucleotides are inserted into a repaired region. Similar results were also obtained for gamma-irradiated cells. In contrast, excision repair of 254-nm-induced dimers was accomplished by a long-patch process in which an average of about 180 nucleotides are inserted into the repaired sites.     

Ultraviolet radiation-induced lethality and repair of pyrimidine dimers in fish embryos

Pimephales promelas (fathead minnow) embryos were used to show a correlation between induction of pyrimidine dimers in DNA and embryo death. Embryo killing was measured by a lack of heart-beat and blood circulation at 48 h post-ultraviolet radiation (UVR). When the embryos were exposed to various doses of UVR from a FS-40 sunlamp followed by exposure to photoreactivating light (PRL) (320-400 nm), the number of pyrimidine dimers decreased significantly. The photorepair of dimers was accompanied by a substantial increase in embryo survival. When embryo killing was examined as a function of the number of dimers present, dimers were identified as a major lesion involved in UVR-induced killing in these fish embryos. This in vivo study on photoreactivation treatment of fish embryos shows a direct association between UVR-induced pyrimidine dimers and embryo killing. In addition, when embryos were held in the dark for 9 h after UVR, 50% of the dimers were removed by excision repair.     

Photorepair of ultraviolet radiation-induced pyrimidine dimers in corneal DNA

The induction and photorepair of pyrimidine dimers in DNA have been measured in the ultraviolet-irradiated, corneal epithelium of the marsupial, Monodelphis domestica, using damage-specific nucleases from Micrococcus luteus in conjunction with agarose gel electrophoresis. We observed that FS-40 sunlamps (280-400 nm) induced 7.2 +/- 1.0 X 10(-5) pyrimidine dimers per kilobase (kb) of DNA per J/m2. Following 100 J/m2, 50% and greater than 90% of the dimers were photorepaired during a 10- and 30-min exposure to photoreactivating light (320-400 nm), respectively. In addition, approximately 70% and approximately 60% of the dimers induced by 300 and 500 J/m2, respectively, were repaired by a 60-min exposure to photoreactivating light. The capacity of the corneal epithelium of M. domestica to photorepair pyrimidine dimers identifies this animal as a potentially useful model with which to determine whether pyrimidine dimers are involved in pathological changes of the irradiated eye.     

Photoreactivation of pyrimidine dimers in the DNA of normal and xeroderma pigmentosum cells.

Photoproducts formed in the DNA of human cells irradiated with ultraviolet light (uv) were identified as cyclobuytl pyrimidine dimers by their chromatographic mobility, reversibility to monomers upon short wavelength uv irradiation, and comparison of the kinetics of this monomerization with that of authentic cis-syn thymine-thymine dimers prepared by irradiation of thymine in ice. The level of cellular photoreactivation of these dimers reflects the level of photoreactivating enzyme measured in cell extracts. Action spectra for cellular dimer photoreactivation in the xeroderma pigmentosum line XP12BE agree in range (300 nm to at least 577 nm) and maximum (near 400 nm) with that for photoreactivation by purified human photoreactivating enzyme. Normal human cells can also photoreactivate dimers in their DNA. The action spectrum for the cellular monomerization of dimers is similar to that for photoreactivation by the photoreactivating enzyme in extracts of normal human fibroblasts.     

Photoreactivation of UV damage in Escherichia coli uvrA6: lethality is more effectively reversed than either premutagenic lesions or SOS induction

The effect of cyclobutyl pyrimidine dimers on cytotoxicity, induction of synthesis of the RecA and UmuC proteins, and mutagenesis was studied in Escherichia coli uvrA6 cells possessing excess amounts of photoreactivating enzyme. Exposure of 254 nm ultraviolet-irradiated (10 J/m2) cells to radiation from daylight fluorescent lamps reduced the amounts of thymine-containing dimers in a photoreactivating fluence-dependent manner, up to about 90% reduction at 5 min exposure. Of the lethal ultraviolet damage, 85% was photoreactivable (i.e. cyclobutyl pyrimidine dimers) and 15% was non-photoreactivable. An incident fluence of 1 J/m2 resulted in approximately a 5-fold increase in the synthesis of the RecA and UmuC proteins, as compared to the spontaneous level. If the UV-irradiated cell suspensions were illuminated with a fluorescent lamp at a dose which resulted in the full photoreactivation of viability, the yields of both proteins were reduced to 60% of the non-photoreactivated control cells. Furthermore, photoreactivation was shown to be more effective in the repair of lethal damage than in the repair of premutational damage. These experiments suggest that, among lethal damages, non-photoreactivable damage plays a more important role in both induction of the SOS functions and mutagenesis in uvrA6 cells than do cyclobutyl pyrimidine dimers.     

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.     

The excision of pyrimidine dimers from DNA of ultraviolet irradiated yeast

Summary It is shown that pyrimidine dimers formed by ultraviolet light in the DNA of haploid Saccharomyces cerevisae are removed under the influence of photoreactivating light and also in the dark under growth conditions. The integrity of the rad 1 locus is necessary for the dark-removal of dimers.     

Studies on the influence of photoreactivation on the frequencies of UV-induced chromosomal aberrations, sister-chromatid exchanges and pyrimidine dimers in chicken embryonic fibroblasts

Chicken embryonic fibroblasts, which possess photoreactivating enzyme were used to study the influence of photoreactivating light on the induction of pyrimidine dimers, sister-chromatid exchanges (SCEs) and chromosomal aberrations by 254 nm UV. While photoreactivation (PR) efficiently removed most of the induced dimers (75-95%), the frequencies of SCEs and chromosomal aberrations were reduced only by about 30-65%, in parallel experiments. Since pyrimidine dimers are the only photoreactivable photolesions known, the reduction in the frequencies of SCEs and chromosomal aberrations on PR has been interpreted as due to disappearance of pyrimidine dimers, implying that these lesions are the primary events responsible for the induction of the biological end points studied. The possible reasons for the lack of quantitative relationship between the frequencies of dimers and the frequencies of SCEs and chromosomal aberrations are discussed.     

<Emphasis Type="Italic">In vivo</Emphasis> Role of the UV-Endonuclease from <Emphasis Type="Italic">Micrococcus luteus</Emphasis> in the Repair of DNA

THE UV-endonuclease enzyme of Micrococcus luteus causes single strand breaks in vitro adjacent to or one nucleotide removed from pyrimidine dimers produced in DNA by ultraviolet irradiation^1–4. Excision of the dimers is catalysed by a second enzyme, the UV-exonuclease^1,5. We now report the isolation of strains of M. luteus which possess altered levels of UV-endonuclease. Mutants lacking this enzyme are incapable of excising thymine-containing dimers while strains with decreased UV-endonuclease activity repair more slowly than wild type cells.     

Damage and repair in mammalian cells after exposure to non-ionizing radiations : I. Ultraviolet and visible light irradiation of cells of the rat kangaroo (Potorous tridactylus) and determination of photorepairable damage in vitro

Cornea cells of the rat kangaroo or “potoroo” (Potorous tridactylus) were exposed to far-UV (254 or 302 nm) radiation, with or without subsequent illumination by near-UV or visible light. The DNA of these cells was extracted and tested for the presence of photoproducts binding yeast photoreactivating enzyme (PRE). The criterion for the latter was competitive inhibition of an in vitro photorepair system, consisting of UV-irradiated transforming DNA of Haemophilus influenzae and an extract containing yeast PRE. The effects on repair kinetics of the transforming DNA indicate that in UV-irradiated potoroo cornea cells up to approximately 90% of photorepairable DNA damage can be photorepaired within 15 min. However, the extent of cellular photorepair, assessed by the reduction in competitive inhibition of the in vitro repair system depends appreciably on experimental parameters during photoreactivating treatment. Control experiments with non-UV-irradiated cells indicated that, depending on specific conditions, the photoreactivating treatment itself produces a varying amount of DNA damage, which reacts with the PRE in vitro. To avoid most of this kind of damage, cells are nitrogen-gassed and kept at 5°C during illumination, and the photoreactivating light must not contain wavelengths shorter than 380–400 nm. Our results show that wavelengths >470 nm are still very effective, whereas wavelengths >555 nm are ineffective in photorepairing potoroo DNA. For unknown reasons, one particular strain of potoroo cornea cells lost its potential for photorepair. Treatment of unirradiated potoroo cells, or their extracted DNA, with hydrogen peroxide also results in competitive inhibition of photorepair in vitro, resembling that observed after near-UV illumination. Because of the occurrence of synergistic effects it is not clear whether the damage only interacts with PRE or can actually be photorepaired under appropriate conditions.

The results presented in this paper suggest that the expression of photorepair in mammalian cells, unlike that in prokaryotes, greatly depends on a number of experimental parameters, including the spectral composition of photoreactivating light. Apparently superposition of damage by the photoreactivating treatment itself is the critical factor. This may explain experimental discrepancies existing in different laboratories studying photorepair in UV-irradiated cells of placental mammals.     

Photoreactivation, Excision, and Strand-Rejoining Repair in R Factor-Containing Minicells of Escherchia coli K-12

Chromosomeless “minicells” are formed by misplaced cell fissions near the polar extremities of an Escherichia coli K-12 mutant strain. Resistance (R)-factor deoxyribonucleic acid (DNA) can be introduced into minicells by segregation from an R⁺ (R64-11) derivative of the original mutant. We have assessed the ability of R⁺ minicells to correct defects produced in their plasmid DNA by ultraviolet (UV) and gamma radiations. Minicells harboring plasmid DNA, in comparison with their repair-proficient minicell-producing parents, possess (i) an equal competence to rejoin single-strand breaks induced in DNA by gamma rays, (ii) a reduced capacity for the photoenzymatic repair of UV-induced pyrimidine dimers, and (iii) a total inability to excise dimers, apparently owing to a deficiency in UV-specific endonuclease activity responsible for mediating the initial incision step in excision repair. Assuming that the DNA repair properties of R⁺ minicells reflect the concentration of repair enzymes located in the plasmid-containing polar caps of entire cells, these findings suggest that: (i) the enzymes responsible for rejoining single-strand breaks are distributed throughout the cell; (ii) photoreactivating enzyme molecules tend to be concentrated near bacterial DNA and to a lesser extent near plasmid DNA; and (iii) UV-specific endonuclease molecules are primarily confined to the central region of the E. coli cell and, thus, seldom segregate with R-factor DNA into minicells.     

Water- and temperature-dependence of DNA damage and repair in the fruticose lichen Cladonia arbuscula ssp. mitis exposed to UV-B radiation

The induction of cyclobutane pyrimidine dimers (CPDs) by ultraviolet‐B radiation (UV‐B, 280–315 nm) and repair mechanisms were studied in the lichen Cladonia arbuscula ssp. mitis exposed to different temperatures and water status conditions. In addition, the development and repair of CPDs were studied in relation to the different developmental stages of the lichen thallus podetial branches. Air‐dried lichen thalli exposed to UV‐B radiation combined with relatively high visible light (HL, 800 μmol m^?2 s^?1; 400–700 nm) for 7 days showed a progressive increase of CPDs with no substantial repair, although HL was present during and after irradiation with UV‐B. Fully hydrated lichen thalli, that had not been previously exposed to UV‐B radiation for 7 days, were given short‐term UV‐B radiation treatment at 25°C, and accumulated DNA lesions in the form of CPDs, with repair occurring when they were exposed to photoreactivating conditions (2 h of 300 μmol m^?2 s^?1, 400–700 nm). A different pattern was observed when fully hydrated thalli were exposed to short‐term UV‐B radiation at 2°C, in comparison with exposure at 25°C. High levels of CPDs were induced at 2°C under UV‐B irradiation, without significant repair under subsequent photoreactivating light. Likewise, when PAR (300 μmol m^?2 s^?1) and UV‐B radiation were given simultaneously, the CPD levels were not lowered. Throughout all experiments the youngest, less differentiated parts of the lichen thallus – namely ‘tips’, according to our arbitrary subdivision – were the parts showing the highest levels of CPD accumulation and the lowest levels of repair in comparison with the older thallus tissue (‘stems’). Thus the experiments showed that Cladonia arbuscula ssp. mitis is sensitive to UV‐B irradiation in the air‐dried state and is not able to completely repair the damage caused by the radiation. Furthermore, temperature plays a role in the DNA damage repairing capacity of this lichen, since even when fully hydrated, C. arbuscula ssp. mitis did not repair DNA damage at the low temperatures.