Modification of the radiosensitivity of E. coli cells by factors affecting the yield of photoreactivated damage

A study was made of the influence of irradiation conditions on the yield of the photoreactivable damages in radiosensitive mutants of E. coli cells (E. coli WP2). Pyrimidine dimers were shown to occur in exrA- and recA- mutants irradiated under anoxic conditions, the survival of these mutants being modified depending on cell genotype. The processes of direct excitation of the molecules were involved in the formation of the damages observed. It can be assumed that the lesser oxygen effect observed in exrA- and partially in recA- mutants of E. coli WP2 cells is associated with a contribution of the photoreactivable damages to a lethal effect of ionizing radiation.     

Repair of Pyrimidine Dimer Damage Induced in Yeast by Ultraviolet Light

Crude extracts from ultraviolet (UV)-irradiated yeast cells compete with UV-irradiated transforming deoxyribonucleic acid (DNA) for photoreactivating enzyme. The amount of competition is taken as a measure of the level of cyclobutyl pyrimidine dimers in the yeast DNA. A calibration of the competition using UV-irradiated calf thymus DNA indicates that an incident UV dose (1,500 ergs/mm(2)) yielding 1% survivors of wild-type cells produces between 2.5 x 10(4) to 5 x 10(4) dimers per cell. Wild-type cells irradiated in the exponential phase of growth remove or alter more than 90% of the dimers within 220 min after irradiation. Pyrimidine dimers induced in stationary-phase wild-type cells appear to remain in the DNA; however, with incubation, they become less photoreactivable in vivo, although remaining photoreactivable in vitro. In contrast, exponentially growing or stationary-phase UV-sensitive cells (rad2-17) show almost no detectable alteration of dimers. We conclude that the UV-sensitive cells lack an early step in the repair of UV-induced pyrimidine dimers.     

Postreplication repair of DNA in chick cells: studies using photoreactivation.

During replication of DNA after ultraviolet irradiation, gaps are left in the newly-synthesized DNA strands in both bacterial and animal cells and these gaps are subsequently sealed by a process known as postreplication repair. In order to test whether it is the ultraviolet-induced pyrimidine dimers which are responsible for the production of these daughter-strand gaps in animal cells, we have used chick embryo fibroblasts. In these cells the pyrimidine dimers are photoreactivable, i.e. they can be split by an enzymatic process dependent on visible or near ultraviolet light. Our results indicate that chick cells possess a postreplication repair system similar to that in mammalian cells; gaps are produced in the newly-synthesized strands and then filled in. If the ultraviolet-irradiated cells are first photoreactivated to remove most of the dimers, the number of daughter-strand gaps produced is much less than without photoreactivation. This suggests that the dimers are indeed responsible for the formation of many of the gaps in the newly-synthesized DNA. Ultraviolet light also inhibits the overall rate of DNA synthesis. This inhibition is, however, only partly overcome by photoreactivation.     

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.     

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.     

Endonuclease-sensitive DNA modifications induced by acetone and acetophenone as photosensitizers.

Repair endonucleases, viz. endonuclease III, formamidopyrimidine-DNA glycosylase (FPG protein), endonuclease IV, exonuclease III and UV endonuclease, were used to analyse the modifications induced in bacteriophage PM2 DNA by 333 nm laser irradiation in the presence of acetone or acetophenone. In addition to pyrimidine dimers sensitive to UV endonuclease, 5,6-dihydropyrimidines (sensitive to endonuclease III) and base modifications sensitive to FPG protein were generated. The level of the last in the case of acetone was 50% and in the case of acetophenone 9% of the level of pyrimidine dimers. HPLC analysis of the bases excised by FPG protein revealed that least some of them were 8-hydroxyguanine (7,8-dihydro-8-oxoguanine). In the damage induced by direct excitation of DNA at 254 nm, which was analysed for comparison, the number of FPG protein-sensitive base modifications was only 0.6% of that of the pyrimidine dimers. Mechanistic studies demonstrated that the formation of FPG protein-sensitive modifications did not involve singlet oxygen, as the damage was not increased in D2O as solvent. Hydroxyl radicals, superoxide and H2O2 were also not involved, since the relative number of single strand breaks and of sites of base loss (AP sites) was much lower than in the case of DNA damage induced by hydroxyl radicals and since the presence of SOD or catalase had no effect on the extent of the damage. However, the mechanism did involve an intermediate that was much more efficiently quenched by azide ions than the triplet excited carbonyl compounds and which was possibly a purine radical. Together, the data indicate that excited triplet carbonyl compounds react with DNA not only by triplet-triplet energy transfer yielding pyrimidine dimers, but also by electron transfer yielding preferentially base modifications sensitive to FPG protein, which include 8-hydroxyguanine.     

Pyrimidine Dimers in the DNA of Paramecium aurelia

The production and fate of thymine-containing pyrimidine dimers in Paramecium aurelia DNA was investigated in three experimental series: production of dimers by UV irradiation, fate of dimers in the dark, and “loss of photoreactivability of dimers.” It is shown that cyclobutyl dimers are made by UV irradiation of Paramecium DNA in vivo, that because of cytoplasmic absorption the number of dimers made in DNA irradiated in vivo is much lower than in DNA irradiated in vitro, that dimers are lost from animals incubated in the dark after irradiation, and that all the dimers that remain in the animals can be destroyed by photoreactivating illumination. Since mutation induction is photoreactivable, these and previous photoreactivation data suggest that pyrimidine dimers are important in mutation induction in P. aurelia.     

The Role of Pyrimidine Dimers as Premutagenic Lesions: A Study of Targeted VS. Untargeted Mutagenesis in the lacI Gene of ESCHERICHIA COLI

We have employed conjugal transfer of an F' lac episome to examine targeted and untargeted mutagenesis in the lacI gene of Escherichia coli and to determine the relative importance of pyrimidine dimers as premutational UV lesions compared to (6-4) photoproducts that also may have a mutational role. This conjugal system allowed us to assess the premutagenic role of UV lesions independently from any role as inducers of SOS functions. F' DNA was transferred to an SOS-induced recipient strain from: unirradiated donor cells, UV-treated donor cells or donor cells that were irradiated and then exposed to photoreactivating light. The results indicate that SOS-related, untargeted events may account for as much as one-third of the nonsense mutations (i.e., base substitutions) recovered after undamaged F' DNA is transferred to UV-irradiated recipients. When the donor strain also is irradiated, in excess of 90% of the mutations detected following conjugation appear to be targeted. Photoreactivation of the UV-treated donors cells, prior to F' transfer to the SOS-induced recipient strain, demonstrated that in this experimental system virtually all recovered UV-induced mutations are targeted by photoreactivable lesions. We presume that these lesions are pyrimidine dimers because (6-4) photoproducts are not photoreactivable.     

Visible light (>395 nm) causes micronuclei formation in mammalian cells without generation of cyclobutane pyrimidine dimers

Solar radiation gives rise to DNA damage in mammalian cells not only directly by excitation of DNA, which generates predominantly pyrimidine dimers, but also indirectly by the excitation of endogenous photosensitizers, which causes oxidative DNA modifications. The latter mechanism has a low quantum yield, but it is the only one proceeding in the visible range of the spectrum. To investigate its relevance for the genotoxicity of sunlight, we have analysed the generation of micronuclei associated with the induction of oxidative DNA damage by visible light in melanoma cells and primary human skin fibroblasts. Similar yields of light-induced oxidative DNA base modifications sensitive to the repair glycosylase Fpg (7,8-dihydro-8-oxoguanine and other oxidative purine modifications) were observed in the normal fibroblasts and the malignant melanoma cells of the same donor. When irradiations were carried out at intervals to compensate for a photodecomposition of the endogenous chromophore, a significant generation of micronuclei was observed in both cell types. Cyclobutane pyrimidine dimers could be excluded to be responsible for the micronuclei induction at wavelengths >395 nm. Experiments with a cut-off filter indicate that the ratio of pyrimidine dimers and Fpg-sensitive oxidative modifications in irradiated cells not only reflects the relative contributions of direct and indirect mechanisms, but is also similar to the ratio by which the two mechanisms contribute to the generation of the micronuclei. The results suggest that indirectly generated oxidative DNA modifications can contribute significantly to the adverse effects of sunlight.     

UV light-induced DNA damage detection in the unicellular green alga <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis>

Exposure of cells to ultraviolet radiation (UVR) is one of the best studied and most used model system for the examination of the biological effects of DNA damage, its repair and tolerance. The major product after UVR treatment is cyclobutane pyrimidine dimer (TT, TC, CC). Pyrimidine dimers are repaired by a direct reversal called photoreactivation or by excision of damage in a process of nucleotide excision repair. Several methods have been developed for the detection and quantification of pyrimidine dimers in DNA. The technique of Small and Greimann, in which DNA is incubated with the pyrimidine dimer-specific endonuclease, was used for the analysis of mutant strains with impaired excision repair system of the unicellular green alga Chlamydomonas reinhardtii. Another method is based on the binding of specific monoclonal antibodies to pyrimidine dimers. The aim of our work was to compare these two techniques with the use of mutant strains of C. reinhardtii — uvsX1 and uvsX2 which are assumed to be deficient in DNA damage recognition. One of their traits was sensitivity to UVR which could be caused by breakdown of the excision repair pathway. The results suggest that the immuno-approach is suitable for the detection of DNA damage induced by UVR. Presented at the International Symposium Biology and Taxonomy of Green Algae V, Smolenice, June 26–29, 2007, Slovakia.     

UV-type damage associated with ionizing radiation: a review

The induction of UV-type damage by ionizing radiation in repair deficient strains of E. coli is reviewed. Both photoreactivable and non-photoreactivable types of damage can be observed. The induction of UV-type damage is largely independent of the presence of free-radical reactive agents (e.g. oxygen and thiols), but is dependent upon the energy of the photon--or electron--beam used, the radiation geometry and the optical absorbance of the extracellular medium. On the basis of calculations and experimental evidence, it is clear that one mechanism whereby such damage arises is through the generation of Cerenkov emission. However, small yields of UV-type damage can be produced using X-rays whose energy is below the threshold for production of Cerenkov emission. In this instance, the damage induction mechanism is thought to involve a direct excitation process.     

Comparison of DNA damage responses following equimutagenic doses of UVA and UVB: a less effective cell cycle arrest with UVA may render UVA-induced pyrimidine dimers more mutagenic than UVB-induced ones

Mechanisms of UVA-mutagenesis remain a matter of debate. Earlier described higher rates of mutation formation per pyrimidine dimer with UVA than with UVB and other evidence suggested that a non-pyrimidine dimer-type of DNA damage contributes more to UVA- than to UVB-mutagenesis. However, more recently published data on the spectra of UVA-induced mutations in primary human skin cells and in mice suggest that pyrimidine dimers are the most common type of DNA damage-inducing mutations not only with UVB, but also with UVA. As this rebuts a prominent role of non-dimer type of DNA damage in UVA-mutagenesis, we hypothesized that the higher mutation rate at UVA-induced pyrimidine dimers, as compared to UVB-induced ones, is caused by differences in the way UVA- and UVB-exposed cells process DNA damage. Therefore, we here compared cell cycle regulation, DNA repair, and apoptosis in primary human fibroblasts following UVB- and UVA-irradiation, using the same physiologic and roughly equimutagenic doses (100-300 J m(-2) UVB, 100-300 kJ m(-2) UVA) we have used previously for mutagenesis experiments with the same type of cells. ELISAs for the detection of pyrimidine dimers confirmed that much fewer dimers were formed with these doses of UVA, as compared to UVB. We found that cell cycle arrests (intra-S, G1/S, G2/M), mediated at least in part by activation of p53 and p95, are much more prominent and long-lasting with UVB than with UVA. In contrast, no prominent differences were found between UVA and UVB for other anti-mutagenic cellular responses (DNA repair, apoptosis). Our data suggest that less effective anti-mutagenic cellular responses, in particular different and shorter-lived cell cycle arrests, render pyrimidine dimers induced by UVA more mutagenic than pyrimidine dimers induced by UVB.     

UV-targeted dinucleotides are not depleted in light-exposed prokaryotic genomes

We have investigated the hypothesis that pyrimidine dinucleotides are avoided in light-exposed genomes as the result of selective pressure due to high ultraviolet (UV) exposure. The main damage to DNA produced by UV radiation is known to be the formation of pyrimidine photoproducts: it is estimated that about 10 dimers per minute are formed in an Escherichia coli chromosome exposed to the UV light in direct overhead sunlight at sea level. It is also known that on an E. coli chromosome exposed to UVb wavelengths (290-320 nm), pyrimidine photoproducts are formed in the following proportions: 59% TpT, 7% CpC, and 34% CpT plus TpC. We have analyzed all available complete prokaryotic genomes and the model organism Prochlorococcus marinus and have found that pyrimidine dinucleotides are not systematically avoided. This suggests that prokaryotes must have sufficiently effective protection and repair systems for UV exposure to not affect their dinucleotide composition.     

Demonstration of pyrimidine dimer-DNA glycosylase activity in vivo: bacteriophage T4-infected Escherichia coli as a model system.

An approach to the detection of pyrimidine dimer-DNA glycosylase activity in living cells is presented. Mutants of Escherichia coli defective in uvr functions required for incision of UV-irradiated DNA were infected with phage T4 denV+ or denV- (defective in the T4 pyrimidine dimer-DNA glycosylase activity). In the former case the denV gene product catalyzed the incision of UV-irradiated host DNA, facilitating the subsequent excision of thymine-containing pyrimidine dimers. Isolation of these dimers from the acid-soluble fraction of infected cells was achieved by a multistep thin-layer chromatographic system. Exposure of the dimers to irradiation that monomerizes pyrimidine dimers (direct photoreversal) resulted in the stoichiometric formation of free thymine. Thus, in vivo incision of UV-irradiated DNA dependent on a pyrimidine dimer-DNA glycosylase can be demonstrated.     

Repair of DNA-containing pyrimidine dimers

Ultraviolet light-induced pyrimidine dimers in DNA are recognized and repaired by a number of unique cellular surveillance systems. The most direct biochemical mechanism responding to this kind of genotoxicity involves direct photoreversal by flavin enzymes that specifically monomerize pyrimidine:pyrimidine dimers monophotonically in the presence of visible light. Incision reactions are catalyzed by a combined pyrimidine dimer DNA-glycosylase:apyrimidinic endonuclease found in some highly UV-resistant organisms. At a higher level of complexity, Escherichia coli has a uvr DNA repair system comprising the UvrA, UvrB, and UvrC proteins responsible for incision. There are several preincision steps governed by this pathway, which includes an ATP-dependent UvrA dimerization reaction required for UvrAB nucleoprotein formation. This complex formation driven by ATP binding is associated with localized topological unwinding of DNA. This same protein complex can catalyze an ATPase-dependent 5'----3'-directed strand displacement of D-loop DNA or short single strands annealed to a single-stranded circular or linear DNA. This putative translocational process is arrested when damaged sites are encountered. The complex is now primed for dual incision catalyzed by UvrC. The remainder of the repair process involves UvrD (helicase II) and DNA polymerase I for a coordinately controlled excision-resynthesis step accompanied by UvrABC turnover. Furthermore, it is proposed that levels of repair proteins can be regulated by proteolysis. UvrB is converted to truncated UvrB* by a stress-induced protease that also acts at similar sites on the E. coli Ada protein. Although UvrB* can bind with UvrA to DNA, it cannot participate in helicase or incision reactions. It is also a DNA-dependent ATPase.     

DNA damage spectra induced by photosensitization

DNA damage induced by photosensitization is not only responsible for the genotoxic effects of various types of drugs in the presence of light, but is also relevant for some of the adverse effects of sunlight, in particular in the UVA and visible range of the spectrum. The types of DNA modifications induced are very diverse and include pyrimidine dimers, covalent adducts, various base modifications generated by oxidation, single-strand breaks and (regular and oxidized) sites of base loss. The ratios in which the various modifications are formed (damage spectra) can be regarded as a fingerprint of the damaging mechanism. Here, we describe the damage spectra of various classes of photosensitizers in relation to the underlying damaging mechanisms. In mammalian cells irradiated with solar radiation, damage at wavelengths <400 nm is characteristic for a (not yet identified) endogenous type-I or type-II photosensitizer. In the UVA range, however, both direct DNA excitation and photosensitized damage appear to be relevant, and there are indications that other chromophore(s) are involved than in the visible range.     

Restriction enzyme cleavage of ultraviolet-damaged DNA

SV40 and pBR322 DNAs damaged by ultraviolet light were cleaved abnormally by several restriction enzymes because of damage to pyrimidines in the recognition sequences. The use of a tandemly duplicated plasmid provided a particularly sensitive target molecule for detecting pyrimidine dimers and other possible photoproducts. The relative efficiency with which cleavage was blocked (HindIII greater than TaqI greater than EcoRI greater than BamI greater than SalI much greater than Hha I, Hae III) corresponds approximately to the relative frequency of pyrimidine dimer formation in the recognition sequences, but at a slightly higher frequency in potential sites for the non-cyclobutane T-C product. The pyrimidine dimers appear to have a range of influence that extends 1 to 3 basepairs along the DNA molecule. These effects provide clues to the way DNA damage from mutagens and carcinogens can interfere with specific enzyme-DNA interactions.     

Ethidium bromide–DNA complex: Wavelength dependence of pyrimidine dimer inhibition and sensitized fluorescence as probes of excited states

Ethidium bromide inhibits the formation of ultraviolet-induced pyrimidine dimers in DNA. The efficiency of dimer inhibition increases with increasing energy of the exciting photons. The efficiency of energy transfer from the DNA singlet to the dye singlet, as monitored by sensitized fluorescence, is independent of wavelength. The efficiency of singlet–singlet transfer agrees with that for dimer inhibition at photon energies corresponding to excitation of the lowest singlet state of DNA. Our results support a model in which dimers are formed both directly from the singlet state and also from the triplet state, with triplets arising from higher vibrational levels of the singlet.     

Artificial and solar UV radiation induces strand breaks and cyclobutane pyrimidine dimers in Bacillus subtilis spore DNA

The loss of stratospheric ozone and the accompanying increase in solar UV flux have led to concerns regarding decreases in global microbial productivity. Central to understanding this process is determining the types and amounts of DNA damage in microbes caused by solar UV irradiation. While UV irradiation of dormant Bacillus subtilis endospores results mainly in formation of the "spore photoproduct" 5-thyminyl-5,6-dihydrothymine, genetic evidence indicates that an additional DNA photoproduct(s) may be formed in spores exposed to solar UV-B and UV-A radiation (Y. Xue and W. L. Nicholson, Appl. Environ. Microbiol. 62:2221-2227, 1996). We examined the occurrence of double-strand breaks, single-strand breaks, cyclobutane pyrimidine dimers, and apurinic-apyrimidinic sites in spore DNA under several UV irradiation conditions by using enzymatic probes and neutral or alkaline agarose gel electrophoresis. DNA from spores irradiated with artificial 254-nm UV-C radiation accumulated single-strand breaks, double-strand breaks, and cyclobutane pyrimidine dimers, while DNA from spores exposed to artificial UV-B radiation (wavelengths, 290 to 310 nm) accumulated only cyclobutane pyrimidine dimers. DNA from spores exposed to full-spectrum sunlight (UV-B and UV-A radiation) accumulated single-strand breaks, double-strand breaks, and cyclobutane pyrimidine dimers, whereas DNA from spores exposed to sunlight from which the UV-B component had been removed with a filter ("UV-A sunlight") accumulated only single-strand breaks and double-strand breaks. Apurinic-apyrimidinic sites were not detected in spore DNA under any of the irradiation conditions used. Our data indicate that there is a complex spectrum of UV photoproducts in DNA of bacterial spores exposed to solar UV irradiation in the environment.     

UV-induced damage and repair in centromere DNA of yeast

Summary The centromere is the region within a chromosome that is required for proper segregation during mitosis and meiosis. Lesions in this sequence represent a unique type of damage, as loss of function could result in catastrophic loss of the genetic material of an entire chromosome. We have measured the induction by ultraviolet (UV) light of pyrimidine dimers in a 2550-bp restriction fragment that includes the centromere region of chromosome III in Saccharomyces cerevisiae. Yeast cells were exposed to ultraviolet light, cellular DNA was gently extracted, and subsequently treated with a UV-specific endonuclease to cleave all pyrimidine dimers. The sites of UV-specific nuclease scission within the centromere were determined by separating the DNA according to molecular weight, transferring the fragments to nitrocellulose, and hybridizing to a radiolabeled 624-bp fragment homologous to the centromere DNA from chromosome III. Several hotspots were identified in chromatin DNA from cells, as well as in irradiated deproteinized DNA. Double strand damage due to closely opposed pyrimidine dimers was also observed. At biological doses (35% survival) there are approximately 0.1 to 0.2 pyrimidine dimers per centromere. These dimers are efficiently repaired in the centromere and surrounding region.