Liquid-holding and recovery from UV-induced damage in Eudorina elegans

Summary Eudorina elegans does not respond to liquid-holding or to postirradiation medium effects by changes in recovery.A decrease in survival ability is observed if a culture is starved prior to irradiation, or is incubated at 22°C rather than 32°C following UV irradiation. Eudorina loses the ability to photoreactivate UV damage within 10 to 48 h following irradiation, depending upon the pre-and post-UV culture conditions.The results are interpreted as indicating a failure of Eudorina elegans to carry out specific dark repair of UV damage. Some reactivation may occur during cellular DNA synthesis.Abbreviations used PR photoreactivation - LHR liquid holding recovery - LHP liquid holding protection - ERR excision-resynthesis-repair - BC complete medium - BM minimal medium - cfa colony forming ability - cfu colony forming units Supported by grants from the National Research Council of Canada # A4431.     

A study of the effect of caffeine upon excision repair of damaged DNA.

Cultured mammalian cells incur damage to their DNA when exposed to ultraviolet light or adduct-producing mutagens such as 4-nitroquinoline-1-oxide (4NQO). At least two processes are important in repair of such damage: post-replication repair and excision repair. Many researchers have reported that caffeine inhibits the former process, which occurs in connection with semiconservative DNA replication, especially in rodent cell lines such as mouse lymphoma or Chinese hamster. Excision repair is not generally considered caffeine-sensitive, although the data are somewhat conflicting because some studies had used rodent cells, which show little or no excision repair, or human cells in which alternate repair processes may have been operating.Human peripherhal blood lymphocytes from healthy donors were treated with UV light or 4NQO in order to produce pyrimidine dimers or adducts. Caffeine at concentrations of 0.75–3.0 mM was included in some cultures. The cells treated with caffeine were incubated for 90 min prior to mutagen treatment and for the entire period thereafter until cell harvests. [³H]Thymidine was added and the uptake quantitated as a measure of DNA repair. DNA replication was inhibited by hydroxyurea, so that only excision repair was measured by this method. Separate plates of cells not exposed to mutagens exhibited negligible or low thymidine uptakes.Following harvest, the cells were lysed and the DNA extracted. The DNA released was measured spectrophotometrically and then placed into liquid-scintillation counter (LSC) vials for measurement of incorporated radioactivity. Resulting cpm/μ DNA were compared for cells with and without caffeine. Lymphocytes from patients with systemic lupus erythematosus (SLE), who previously had demonstrated reduced levels of excision repair under these conditions, were also tested with caffeine. Caffeine did not inhibit repair by normal lymphocytes and the reduced repair seen in the SLE patients was not further reduced in its presence.In a series of pulse-chase experiments, some cells were treated with 4NQO and allowed to incubate with [³H]thymidine for 3 h and were harvested at the end of this period, while others were given a 13-h chase i n cold thymidine before harvest. The cpm/μg DNA for both groups were virtually identical, both in the presence and absence of 2.0 mM caffeine.     

Evaluation of UV damage at DNA level in <Emphasis Type="Italic">Nicotiana plumbaginifolia</Emphasis> protoplasts using single cell gel electrophoresis

The aim of the present study was to observe the induction and repair of single strand breaks (Ssbs) and double strand breaks (Dsbs) in mesophyll protoplasts of Nicotiana plumbaginifolia, irradiated with UV-C and cultured under light or dark conditions. DNA damage and repair was determined by the neutral and alkaline comet assay to reveal Dsbs and Ssbs respectively. Subculturing protoplasts for 4 h at low temperature was essential to reduce the amount of Dsbs to the detection limit of the assay procedure. Light-cultured protoplasts showed a significant increase of Ssbs and Dsbs compared to dark cultured protoplasts, in which the number of Ssbs and Dsbs remained very constant throughout the experiments. UV treatment significantly enhanced the levels of Ssbs and Dsbs in light and dark cultured protoplasts. On average, equal levels of DNA damage were observed under light or dark conditions. Formulations introduced to evaluate the contribution of UV-C or light treatment in repair kinetics of DNA damage, showed that the number of Ssbs, but not of Dsbs, evolved differently for light and dark cultured protoplasts. DNA repair was more rapidly observed under light conditions and occurred in different repair phases. Observations are discussed in relation to the involvement of chromatin remodelling, photosynthetic active radiation and DNA repair mechanisms.     

Stimulation of DNA repair and increased light output in response to UV irradiation in Escherichia coli expressing lux genes.

It has previously been suggested that the evolutionary drive of bacterial bioluminescence is a mechanism of DNA repair. By assessing the UV sensitivity of Escherichia coli, it is shown that the survival of UV-irradiated E. coli constitutively expressing luxABCDE in the dark is significantly better than either a strain with no lux gene expression or the same strain expressing only luciferase (luxAB) genes. This shows that UV resistance is dependent on light output, and not merely on luciferase production. Also, bacterial survival was found to be dependent on the conditions following UV irradiation, as bioluminescence-mediated repair was not as efficient as repair in visible light. Moreover, photon emission revealed a dose-dependent increase in light output per cell after UV exposure, suggesting that increased lux gene expression correlates with UV-induced DNA damage. This phenomenon has been previously documented in organisms where the lux genes are under their natural luxR regulation but has not previously been demonstrated under the regulation of a constitutive promoter.     

Genetic effects of acridine compounds.

Acridines and a very large number of acridine derivatives are used in enormous quantities both in medicine and industry. The mutagenic action of these compounds has been demonstrated in a wide variety of organisms and is known to occur both in the dark as well as in the presence of light (photodynamic action). At the molecular level, acridines have been shown to cause frameshift mutations of both the addition and deletion types, a characteristic which has been of tremendous help in elucidating the nature of the genetic code. These and various other biological effects of acridines, such as inhibition of DNA repair, curing of plasmids and cell-growth inhibition, are examined in this review.     

Combination of the mutation process with the sensitization and repair processes leading to increased frequencies of mutations in algal populations

The possibility of using a combination of the mutation process with the induction of the repair processes has been studied to increase the mutation frequencies in algal populations after UV-treatment. From this study it follows that the repair process induced by visible light is much more effective than the dark repair processes in the chlorococcal algae used. In these algae, visible light perhaps does not induce only those repair processes which affect their DNA, but probably also some recovery ones which affect their damaged structures and physiological functions. A suitable combination of the sensitization of algal cells by a DNA-base analogue before UV-treatment and the induction of the light repair and recovery processes resulted in a rather high increase of viable mutations in chlorococcal algae. These findings may be useful in the breeding of chlorococcal algae, which have no possibility of hybridization (except somatic).     

Induction and repair of DNA strand breaks in Bacteroides fragilis

Alkaline sucrose gradient sedimentation was used to establish whether strand breakage and repair take place in the DNA of UV-irradiated Bacteroides fragilis during the removal of pyrimidine dimers. A B. fragilis wild-type strain and two of its repair mutants, a mitomycin C sensitive mutant (MTC25) having wild-type levels of UV survival, and a UV-sensitive, mitomycin C sensitive mutant (UVS9), were investigated. Under anaerobic conditions, far-UV irradiation induced metabolically regulated strand breakage and resynthesis in the wild-type strain, but this was markedly reduced in both the MTC25 and UVS9 mutants. Approximately half of the strand breaks generated by the various strains were rejoined during further holding in buffer. Under replicating conditions, complete repair of strand breaks in the wild type was observed. Caffeine treatment under anaerobic conditions caused direct DNA strand breakage in B. fragilis cells but did not inhibit UV-induced breakage or repair.     

Repair of nonreplicating UV-irradiated DNA: cooperative dark repair by Escherichia coli uvr and phr functions.

The system previously used to study recombination of nonreplicating UV-irradiated phage lambda DNA was adapted to study UV repair. Irradiated phages infected undamaged homoimmune lysogens. Pyrimidine dimer content (by treatment with Micrococcus luteus UV endonuclease and alkaline sucrose sedimentation) and a biological activity endpoint (infectivity in transfection of uvrB recA recB spheroplasts) were followed. Unless room light was excluded during DNA extraction procedures, photoreactivation (Phr function) was significant. In uvr delta phr bacteria, repair, by both assays, was very low but not zero. Even when light was totally excluded, Phr function appeared to play a role in Uvr-mediated excision repair: both dimer removal and restoration of infectivity were two to five times as efficient in uvr+ phr+ bacteria as in uvr+ delta phr bacteria. Similarly, UV-irradiated phages plated with higher efficiencies on phr+ than delta phr bacteria even under totally dark conditions. In uvr phr+ repressed infections, removal of dimers from nonreplicating DNA did not increase infectivity as much as in uvr+ infections, suggesting a requirement for repair of nondimer photoproducts by the uvrABC system.     

Reparation of UV-damaged bacteriophage ed]

A repair of UV-damaged phage DNA in the "phage-host" system in accordance with the excision reparative mechanism is demonstrated by means of centrifugation in alkaline sucrose gradient of virulent 3H-thymidine labelled phage sd. The increase of the transfectants quantity of UV-irradiated DNA on uvr+ bacteria compatibly to uvr- bacteria evidences that the bacterial host participates in phage reparation. Caffeine inhibition of UV-irradiated phage sd survival confirms the participation of cell-host in reparation of UV-damaged phage.     

Marine Bacterial Isolates Display Diverse Responses to UV-B Radiation

The molecular and biological consequences of UV-B radiation were investigated by studying five species of marine bacteria and one enteric bacterium. Laboratory cultures were exposed to an artificial UV-B source and subjected to various post-UV irradiation treatments. Significant differences in survival subsequent to UV-B radiation were observed among the isolates, as measured by culturable counts. UV-B-induced DNA photodamage was investigated by using a highly specific radioimmunoassay to measure cyclobutane pyrimidine dimers (CPDs). The CPDs determined following UV-B exposure were comparable for all of the organisms except Sphingomonas sp. strain RB2256, a facultatively oligotrophic ultramicrobacterium. This organism exhibited little DNA damage and a high level of UV-B resistance. Physiological conditioning by growth phase and starvation did not change the UV-B sensitivity of marine bacteria. The rates of photoreactivation following exposure to UV-B were investigated by using different light sources (UV-A and cool white light). The rates of photoreactivation were greatest during UV-A exposure, although diverse responses were observed. The differences in sensitivity to UV-B radiation between strains were reduced after photoreactivation. The survival and CPD data obtained for Vibrio natriegens when we used two UV-B exposure periods interrupted by a repair period (photoreactivation plus dark repair) suggested that photoadaptation could occur. Our results revealed that there are wide variations in marine bacteria in their responses to UV radiation and subsequent repair strategies, suggesting that UV-B radiation may affect the microbial community structure in surface water.     

Saturation of Dark Repair Synthesis: Accumulation of Strand Breaks

Reversal of ultraviolet light damage to DNA by the dark repair system is limited. Experiments utilizing density and radioactive labels demonstrated that repair synthesis is not proportional to dose at doses above 200 ergs/mm². In addition, the number of residual excision induced gaps in Escherichia coli B/r hcr⁺ DNA increases with higher UV doses. The extent of repair is apparently limited by saturation of the repair synthesis step.     

Determination of pyrimidine dimers in Escherichia coli and Cryptosporidium parvum during UV light inactivation, photoreactivation, and dark repair

UV inactivation, photoreactivation, and dark repair of Escherichia coli and Cryptosporidium parvum were investigated with the endonuclease sensitive site (ESS) assay, which can determine UV-induced pyrimidine dimers in the genomic DNA of microorganisms. In a 99.9% inactivation of E. coli, high correlation was observed between the dose of UV irradiation and the number of pyrimidine dimers induced in the DNA of E. coli. The colony-forming ability of E. coli also correlated highly with the number of pyrimidine dimers in the DNA, indicating that the ESS assay is comparable to the method conventionally used to measure colony-forming ability. When E. coli were exposed to fluorescent light after a 99.9% inactivation by UV irradiation, UV-induced pyrimidine dimers in the DNA were continuously repaired and the colony-forming ability recovered gradually. When kept in darkness after the UV inactivation, however, E. coli showed neither repair of pyrimidine dimers nor recovery of colony-forming ability. When C. parvum were exposed to fluorescent light after UV inactivation, UV-induced pyrimidine dimers in the DNA were continuously repaired, while no recovery of animal infectivity was observed. When kept in darkness after UV inactivation, C. parvum also showed no recovery of infectivity in spite of the repair of pyrimidine dimers. It was suggested, therefore, that the infectivity of C. parvum would not recover either by photoreactivation or by dark repair even after the repair of pyrimidine dimers in the genomic DNA.     

UV survival of human mycoplasmas: evidence of dark reactivation in Mycoplasma buccale.

The inactivation by ultraviolet (UV) light irradiation of mycoplasma cells of five human strains was monitored by investigating the colony-forming ability. The survival curves of five strains tested indicated that the cells of Mycoplasma buccale only are single and homogenously susceptible to UV light. The effect of the repair inhibitor, caffeine, on the colony-forming ability of UV-irradiated cells was investigated with M. buccale because of its homogenous susceptibility to UV light. The colony formation of irradiated cells was markedly depressed by post-irradiation treatment with caffeine at concentrations that had little or no effect on the colony formation of unirradiated cells. The colony-forming units (CFU) of UV-irradiated cells which were kept in broth without caffeine in the dark increased without a lag as the time in the dark increased. The colony-forming ability of the irradiated cells completely recovered after 3 hr in the dark. However, when irradiated cells were kept in the presence of caffeine, no increase in their CFU was observed. The mode of action of caffeine on UV-irradiated cells closely resembles that described for other organisms which possess dark reactivation systems for UV-induced damage in deoxyribonucleic acid (DNA). Thus, the results obtained provide evidence for the existence of a dark repair function in M. buccale.     

Mutagenic and epigenetic influence of caffeine on the frequencies of UV-induced ouabain-resistant Chinese hamster cells

Caffeine, given as a post-treatment to UV-irradiated Chinese hamster cells in vitro, modified the frequency of induced mutations at the ouabain resistance locus. Mutation frequencies were increased when caffeine was added only for the DNA repair and mutation fixation period. When caffeine was added after the DNA repair and mutation fixation period, or immediately after DNA damage and for the entire repair and selection period, mutation frequencies were reduced. A hypothesis, given to explain both results, is that caffeine, by blocking a constitutive “error-free” postreplication repair process, allows an “error-prone” DNA repair process to produce many mutations. Moreover, caffeine, possibly by modifying C-AMP metabolism, causes a repression of induced mutations which, in effect, explains its anti-mutagenic and anti-carcinogenic properties.     

Kinetics of unscheduled DNA synthesis in UV-irradiated chicken embryo fibroblasts

Unscheduled DNA synthesis induced by 254-nm UV radiation in chicken embryo fibroblasts was examined for 24 h following irradiation, while cells were kept in the dark. The effect on this repair process of a 2-4 h exposure to photoreactivating light immediately after UV was studied. Initial [3H]thymidine incorporation in the light-treated cells was only slightly different from that in cells not exposed to light, but a distinct difference in rate and cumulative amount of unscheduled DNA synthesis was seen several hours after irradiation. By varying the UV dose and the time allowed for photoreactivation, the amount of dimers (determined as sites sensitive to a M. luteus UV-endonuclease) and non-dimers could be changed. The results of these experiments suggest that excision repair of dimers, rather than non-dimer products, is responsible for the unscheduled DNA synthesis seen after UV irradiation.     

Chlorella virus PBCV-1 encodes a homolog of the bacteriophage T4 UV damage repair gene denV.

The bacteriophage T4 denV gene encodes a well-characterized DNA repair enzyme involved in pyrimidine photodimer excision. We have discovered the first homologs of the denV gene in chlorella viruses, which are common in fresh water. This gene functions in vivo and also when cloned in Escherichia coli. Photodamaged virus DNA can also be photoreactivated by the host chlorella. Since the chlorella viruses are continually exposed to solar radiation in their native environments, two separate DNA repair systems, one that functions in the dark and one that functions in the light, significantly enhance their survival.     

DNA repair and survival in UV-irradiated chicken embryo fibroblasts

The role of the pyrimidine dimer in cell killing, DNA synthesis and repair has been studied by utilizing the light-requiring DNA-repair mechanism of photo- reactivation in UV-irradiated chicken-embryo fibroblasts. Survival, as measured by colony-forming ability at 41°C, is increased in cells left in the light. The initial inhibition of DNA synthesis by UV is much less in light-treated cells, and levels reach that of unirradiated controls much faster than when the cells are left in the dark. The number of endonuclease-sensitive sites (dimers)_measured by an assay with a crude extract from M. luteus, rapidly decreases as the cells are allowed to photoreactive. However, in the dark, significant amounts of repair also occur, but at a much lower rate and with a lag phase of several hours. Unscheduled DNA synthesis occurs to a similarly low extent in both dark- and light-treated cells, confirming the finding that some amount of excision repair occurs that is light-independent. When survival is examined as a function of the number of dimers present, the dimers, not the non-dimer products, appear to be responsible for cell killing. In this study, the removal of dimers in vivo by photoreactivation has made it possible to demonstrate directly that dimers are primarily responsible for the deleterious effects of UV on DNA synthesis and survival.     

Ultraviolet Irradiation of the Vegetative Cells of Dictyostelium discoideum

Experiments on the effect of ultraviolet (UV) light on the survival of vegetative Dictyostelium discoideum cells indicate that this is a relatively UV-resistant organism. Several factors suggest the presence of some type of repair process. Experiments to test for liquid-holding recovery and simple photoreactivation yielded negative results. Acriflavine and caffeine were utilized to possibly interfere with dark repair. Acriflavine produced no UV sensitization, but caffeine did cause a concentration-dependent decrease in survival of irradiated cells. When UV-irradiated cells were illuminated with photoreactivating light while suspended in caffeine, the survival increased above that for cells treated with caffeine alone, suggesting an overlap between lesions repaired by photorepair and dark repair. Growth experiments showed that UV light induced a dose-dependent division delay, followed by a period of retarded growth characterized by the presence of a constant fraction of nonviable cells in the irradiated population. The delayed exposure of cells to caffeine after irradiation showed that the magnitude of the caffeine sensitization diminished throughout the division-delay period. An action spectrum indicated probable nucleoprotein involvement in the induction of division delay. UV light retarded ribonucleic acid and protein synthesis and temporarily blocked deoxyribonucleic acid synthesis. However, synthesis of all three accelerated prior to the end of the division-delay period and then closely paralleled the increase in cell number.     

Conflicting effects of caffeine on apoptosis and clonogenic survival of human K1 thyroid carcinoma cell lines with different p53 status after exposure to cisplatin or UVc irradiation

Caffeine has been widely described as a chemo/radiosensitizing agent, presumably by inhibiting DNA repair, and affecting preferentially cells with an altered p53 status. We evaluated the effects of caffeine using isogenic and isophenotypic K1 cells derived from a papillary thyroid carcinoma and displaying either a wild type or a mutated p53 status. Apoptosis and clonogenic survival were examined after exposure of the cells to cisplatin or UVc irradiation. We find that at the most currently used concentration, 2mM, caffeine hinders cisplatin or UVc induced apoptosis in K1 cells. In addition, at this already barely achievable concentration in vivo, caffeine does not decrease their clonogenic survival. Hence in our cellular model, caffeine does not behave as a chemo- or a radiosensitizer. Although surprising, these results (1) are in agreement with the delayed G2/M block caused by caffeine that we previously observed in normal human fibroblasts and K1 cells and (2) allow us to elucidate some discrepancies concerning this molecule throughout the literature such as increase or decrease of apoptosis and clonogenic survival, activation or deactivation of molecules involved in DNA damage repair and proliferation inhibition but accelerated G2/M traverse.     

Caffeine-induced radiosensitization is independent of nonhomologous end joining of DNA double-strand breaks

After exposure to ionizing radiation, proliferating cells actively slow down progression through the cell cycle through the activation of checkpoints to provide time for repair. Two major complementary DNA double-strand break (DSB) repair pathways exist in mammalian cells, homologous recombination repair (HRR) and nonhomologous end joining (NHEJ). The relationship between checkpoint activation and these two types of DNA DSB repair pathways is not clear. Caffeine, as a nonspecific inhibitor of ATM and ATR, abolishes multi-checkpoint responses and sensitizes cells to radiation-induced killing. However, it remains unknown which DNA repair process, NHEJ or HRR, or both, is affected by caffeine-abolished checkpoint responses. We report here that caffeine abolishes the radiation-induced G(2)-phase checkpoint and efficiently sensitizes both NHEJ-proficient and NHEJ-deficient mammalian cells to radiation-induced killing without affecting NHEJ. Our results indicate that caffeine-induced radiosensitization occurs by affecting an NHEJ-independent process, possibly HRR.