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 of Haemophilus influenzae transforming DNA in vitro with near-ultraviolet radiation: action spectrum

Mutations were produced in purified transforming DNA from Haemophilus influenzae by near-UV radiation and were assayed as mutants among cells transformed with irradiated DNA. The maximum efficiency of mutation induction was at around 334 nm, and the efficiency dropped off steeply at lower and higher wavelengths. The difference between the action spectrum for mutation and that for the oxygen-independent inactivation of transforming DNA, which had a shoulder at 365 nm, indicates that there are different lesions involved in the inactivating and mutagenic effects of near-UV. The presence of histidine during irradiation enhanced the mutagenic effect at 334 and 365 nm, although it protected against inactivation at 365 nm. The effective near-UV wavelengths for in vitro mutation are to some extent the same as the effective wavelengths for mutation in vivo reported previously. These findings indicate that mutations are produced in vivo by near-UV with DNA as the primary target molecule rather than by a secondary non-photochemical reaction between DNA and some other cell component.     

Relative effectiveness of the 300–320 nm spectral region of sunlight for the production of primary lethal damage in E. coli cells

Cell inactivation by sunlight exposure has been studied in E. coli CSR 603 (uvrA recA phr), a K12 derivative which is deficient in all known repair systems. Under suitable conditions, unfiltered sunlight inactivates these cells to 10⁻³ survival within 30 sec. The effects of unfiltered sunlight have been compared with those of sunlight filtered through 1-cm layers of aqueous caffeine solutions ranging in concentration from 1 to 20 mg/ml. In the wavelength region of solar emission below 320 nm, which is most critical for DNA damage, the transmission of these liquid filters changes from 10 to 90% within 8-nm intervals. Thus our results permit minimum estimates for the fraction of lethal lesions produced by the solar spectrum below certain wavelengths. In an experiment analyzed in this manner more than 80% of primary lethal lesions are caused by wavelengths <312 nm, and more than 50% by wavelengths <306 nm, while the contribution of wavelengths >380 nm to primary lethal damage is below 1%.     

The influence of the wavelength of ultraviolet radiation on survival,mutation induction and DNA repair in irradiated chinese hamster cells

Chinese hamster ovary cells were used to compare the cytotoxicity and mutagenicity of far-UV radiation emitted by a low-pressure mercury, germicidal lamp (wavelength predominantly 254 nm) with that of near-UV radiation emitted by a fluorescent lamp with a continuous spectrum (Westinghouse “Sun Lamp”), of which only the radiation with wavelengths greater than 290 nm or greater than 310 nm was transmitted to the cells. The radiation effects were compared on the basis of an equal number of pyrimidine dimers, the predominant lesion induced in DNA by far-UV, for the induction of which much more energy is needed with near-UV than with 254-nm radiation.The numbers of dimers induced were determined by a biochemical method detecting UV-endonuclease-susceptible sites. The equivalence of these sites with pyrimidine dimers was established, qualitatively and quantitatively, in studies with enzymic photoreactivation in vitro and chromatographic analysis of dimers.On the basis of induced dimers, more cells were killed by >310-nm UV than by >290-nm UV; both forms of radiation were more cytotoxic than 254-nm UV when equal numbers of dimers were induced. Moreover, 5–6 times as many mutants were induced per dimer by >310-nm UV than by >290-nm UV; the latter appeared approximately as mutagenic as 254-nm UV. The differences in lethality and mutagenicity were not caused by differences in repair of dimers: cells with an equal number of dimers induced by either 254-nm or near-UV showed the same removal of sites susceptible to a UV endonuclease specific for dimers, as well as an identical amount of repair replication.The results indicate that near-UV induces, besides pyrimidine dimers, other lesions that appear to be of high biological significance.     

Action spectrum for lethality of near-UV light on Haemophilus influenzae and lack of mutation.

Mutation and inactivation of H. influenzae have been measured following irradiation at various near-UV wavelengths. Inactivation takes place most readily at 334 nm (but is unaffected by absence of excision or postreplication repair), and decreases markedly at longer wavelengths. No induced mutations to resistance to novobiocin or streptomycin or to ability to utilize protoporphyrin instead of hemin were detected at any of the wavelengths used. There were also no detectable induced mutations in an excision-defective strain after 334-nm irradiation. These results are in contrast to the in vitro mutation of purified transforming DNA we previously observed.     

Lethal and potentially lethal lesions induced by radiation--a unified repair model

A model of radiation action is described which unifies several of the major existing concepts which have been applied to cell killing. Called the lethal and potentially lethal (LPL) model, it combines the ideas of lesion interaction, irreparable lesions caused by single tracks, linear lesion fixation, lesion repair via first-order kinetics, and binary misrepair. Two different kinds of lesions are hypothesized: irreparable (lethal) and repairable (potentially lethal) lesions. They are tentatively being identified with DNA double-strand breaks of different severity. Two processes compete for depletion of the potentially lethal lesions: correct repair following first-order kinetics, and misrepair following second-order kinetics. Fixation of these lesions can also occur. The model applies presently only to plateau (stationary)-phase cells. Radiobiological phenomena described include effects of low dose rate, high LET, and repair kinetics as measured with repair inhibitors such as hypertonic solution and beta-arabinofuranosyladenine (beta-araA). One consequence of the model is that repair of sublethal damage and the slow component of the repair of potentially lethal damage are two manifestations of the same repair process. Hypertonic treatment fixes a completely new class of lesions which normally repair correctly. Another consequence of the model is that the initial slope of the survival curve depends on the amount of time available for repair after irradiation. The "dose-rate factor" occurring in several linear-quadratic formulations is shown to emerge when appropriate low-dose and long-repair-time approximations are made.     

Hijacking of the mismatch repair system to cause CAG expansion and cell death in neurodegenerative disease

Mammalian cells have evolved sophisticated DNA repair systems to correct mispaired or damaged bases and extrahelical loops. Emerging evidence suggests that, in some cases, the normal DNA repair machinery is "hijacked" to become a causative factor in mutation and disease, rather than act as a safeguard of genomic integrity. In this review, we consider two cases in which active MMR leads to mutation or to cell death. There may be similar mechanisms by which uncoupling of normal MMR recognition from downstream repair allows triplet expansions underlying human neurodegenerative disease, or cell death in response to chemical lesion.     

Comparative study of the lethal effects of near-UV light (360 nm) and 8-methoxypsoralen plus near-UV on plasmid DNA

We have studied the lethality produced on pBR322 by near-UV radiation and by 8-Methoxypsoralen plus near-UV (PUV treatment). Samples of pBR322 DNA were irradiated with increasing fluences of 360 nm-light either in the absence or presence of 400 molecules of 8-Methoxypsoralen (8-MOP) per plasmid molecule. We have estimated to what extent the global lethality of PUVA treatment is due to the presence of psoralen adducts in DNA or to radiation itself. In order to analyse the involvement of DNA repair mechanisms in the removal of plasmid lesions, several strains of E. coli (differing in their repair capacities) were used as recipients of the treated plasmids. Results showed that excision and recombination participate in the repair of near-UV-induced plasmid lesions. Repair of PUV-induced lesions showed an even greater requirement of the excision pathway. Besides, a slight increase on plasmid mutation frequencies was observed after near-UV or PUV treatment in wild type and uvrA cells. Estimation of the contribution of 8-MOP to the global lethality of PUV treatment showed that only the excision pathway was involved in removing psoralen adducts from plasmid DNA, suggesting the involvement of the recombinational pathway in the repair of near-UV-derived lesions.     

Endonuclease III and endonuclease IV protect Escherichia coli from the lethal and mutagenic effects of near-UV irradiation.

In contrast to the DNA damage caused by far-UV (lambda < 290 nm), near-UV (290 < lambda < 400 nm) induced DNA damage is partially oxygen dependent, suggesting the involvement of reactive oxygen species. To test the hypothesis that enzymes that protect cells from oxidative DNA damage are also involved in preventing near-UV mediated DNA damage, isogenic strains deficient in one or more of exonuclease III (xthA), endonuclease IV (nfo), and endonuclease III (nth) were exposed to increasing levels of far-UV and near-UV. All strains, with the exception of the nth single mutant, were found to be hypersensitive to the lethal effects of near-UV relative to a wild-type strain. A triple mutant strain (nth nfo xthA) exhibited the greatest sensitivity to near-UV-mediated lethality. The triple mutant was more sensitive than the nfo xthA double mutant to the lethal effects of near-UV, but not far-UV. A forward mutation assay also revealed a significantly increased sensitivity for the triple mutant compared to the nfo xthA deficient strain in the presence of near-UV. However, the triple mutant was no more sensitive to the mutagenic effects of far-UV than a nfo xthA double mutant. These data suggest that exonuclease III, endonuclease IV, and endonuclease III are important in protection against near-UV-induced DNA damage.     

Interactions between uv radiation of different energies in the inactivation of bacteria.

A strong lethal interaction was observed between various monochromatic wavelengths (254, 334, 365, and 405 nm) in the repair-proficient E. coli K-12 strain AB 1157, except in the case of preexposure to 405-nm radiation which resulted in a protection against the inactivation resulting from subsequent exposure to 365-or 254-nm radiations. The results may be tentatively explained by assuming two classes of DNA lesions and two classes of damage to repair (reversible and inrreversible) whose proportions vary according to wavelength.     

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.     

Progress in the analysis of urinary oxidative DNA damage

Oxidative DNA damage has been implicated to be important in the pathogenesis of many diseases, including cancer and heart disease. The assessment of damage in various biological matrices, such as DNA, serum, and urine, is vital to understanding this role and subsequently devising intervention strategies. Despite the numerous techniques to measure oxidative DNA damage products in urine, it remains unclear what these measurements truly represent. Sources of urinary lesions may include the diet, cell death, and, of most interest, DNA repair. Were it possible to exclude the two former contributions, a noninvasive assay for DNA repair would be invaluable in the study of DNA damage and disease. This review highlights that, although progress has been made, significant work remains. Diet, cell death, and repair need continued examination to further elucidate the kinetics of lesion formation and clearance in vivo. Studies from our laboratory and others are making appreciable progress towards the interpretation of urinary lesion measurements along with the development of urinary assays to evaluate DNA repair. Upon establishment of these details, urinary oxidative DNA damage measurements may become more than a reflection of generalized oxidative stress.     

Split-dose recovery is due to the repair of DNA double-strand breaks

DNA double-strand breaks are the molecular lesions the repair of which leads to the reappearance of the shoulder observed in split-dose experiments. This conclusion is based on results obtained with the help of a diploid yeast mutant rad 54-3 which is temperature-conditional for the repair of DNA double-strand breaks. Two repair steps must be met to yield the reappearance of the shoulder on a split-dose survival curve: the repair of double-strand breaks during the interval between two doses and on the nutrient agar plate after the second dose. In yeast lethality may be attributable to either an unrepaired double-strand break (i.e. a double-strand break is a potentially lethal lesion) or to the interaction of two double-strand breaks (misrepair of double-strand breaks). Evidence is presented that the two cellular phenomena of liquid holding recovery (repair of potentially lethal damage) and of split-dose recovery (repair of sublethal damage) are based on the repair of the same molecular lesion, the DNA double-strand break.     

The induction and repair of DNA damage and its influence on cell death in primary human fibroblasts exposed to UV-A or UV-C irradiation

Irradiation with UV-A of normal human fibroblasts in phosphate-buffered saline induced cell death, measured as lack of colony-forming ability. A specially filtered sunlamp, emitting wavelengths greater than 330 nm, was used as UV-A source. After UV-A irradiation, single-strand breaks (alkali-labile bonds) could be detected in DNA; these lesions were rapidly repaired. The induction of these single-strand breaks was almost eliminated when irradiation was performed in the presence of catalase. However, catalase, when present during UV-A irradiation, did not reduce cell death of the fibroblasts. Excision repair, monitored as unscheduled DNA synthesis, was induced strongly by irradiation with UV-C (predominantly 254 nm), but could not be detected after UV-A irradiation. Moreover, very little accumulation of incision breaks during post-irradiation incubation with hydroxyurea and 1-beta-D-arabinofuranosylcytosine (araC) was detected after UV-A. This is consistent with the low amount of pyrimidine dimers (measured as UV-endonuclease susceptible sites) induced by UV-A. Xeroderma pigmentosum fibroblasts of complementation group A, which are extremely sensitive to UV-C irradiation, showed the same sensitivity to UV-A as normal fibroblasts. The results indicate that lethality by UV-A wavelengths greater than 330 nm is caused by lesions other than single-strand breaks (alkali-labile bonds) and pyrimidine dimers.     

Probing for DNA damage with β-hairpins: similarities in incision efficiencies of bulky DNA adducts by prokaryotic and human nucleotide excision repair systems in vitro

Nucleotide excision repair (NER) is an important prokaryotic and eukaryotic defense mechanism that removes a large variety of structurally distinct lesions in cellular DNA. While the proteins involved are completely different, the mode of action of these two repair systems is similar, involving a cut-and-patch mechanism in which an oligonucleotide sequence containing the lesion is excised. The prokaryotic and eukaryotic NER damage-recognition factors have common structural features of β-hairpin intrusion between the two DNA strands at the site of the lesion. In the present study, we explored the hypothesis that this common β-hairpin intrusion motif is mirrored in parallel NER incision efficiencies in the two systems. We have utilized human HeLa cell extracts and the prokaryotic UvrABC proteins to determine their relative NER incision efficiencies. We report here comparisons of relative NER efficiencies with a set of stereoisomeric DNA lesions derived from metabolites of benzo[a]pyrene and equine estrogens in different sequence contexts, utilizing 21 samples. We found a general qualitative trend toward similar relative NER incision efficiencies for ～65% of these substrates; the other cases deviate mostly by ～30% or less from a perfect correlation, although several more distant outliers are also evident. This resemblance is consistent with the hypothesis that lesion recognition through β-hairpin insertion, a common feature of the two systems, is facilitated by local thermodynamic destabilization induced by the lesions in both cases. In the case of the UvrABC system, varying the nature of the UvrC endonuclease, while maintaining the same UvrA/B proteins, can markedly affect the relative incision efficiencies. These observations suggest that, in addition to recognition involving the initial modified duplexes, downstream events involving UvrC can also play a role in distinguishing and processing different lesions in prokaryotic NER.     

DNA repair is responsible for the presence of oxidatively damaged DNA lesions in urine

The repair of oxidatively damaged DNA is integral to the maintenance of genomic stability, and hence prevention of a wide variety of pathological conditions, such as aging, cancer and cardiovascular disease. The ability to non-invasively assess DNA repair may provide information regarding repair pathways, variability in repair capacity, and susceptibility to disease. The development of assays to measure urinary DNA lesions offered this potential, although it rapidly became clear that possible contribution from diet and cell turnover may influence urinary lesion levels. Whilst early studies attempted to address these issues, up until now, much of the data appears conflicting. However, recent work from our laboratories, in which human volunteers were fed highly oxidatively modified 15N-labelled DNA demonstrates that diet does not appear to contribute to urinary levels of 8-hydroxyguanine and 7,8-dihydro-8-oxo-2'-deoxyguanosine. Furthermore, we propose that a number of literature reports form an argument against a contribution from cell death. Indeed we, and others, have presented evidence, which strongly suggests the involvement of cell death to be minimal. Taken together, these data would appear to rule out various confounding factors, leaving DNA repair pathways as the principal source of urinary purine, if not DNA, lesions enabling such measurements to be used as indicators of repair.     

The role of DNA double strand breaks in ionizing radiation-induced killing of eukaryotic cells.

A widely accepted assumption in radiobiology is that ionizing radiation kills cells by inducing forms of damage in DNA structures that lead to the formation of lethal chromosome aberrations. One goal of radiation biology research is the identification of these forms of DNA damage, the characterization of the mechanisms involved in their repair and the elucidation of the processes involved in their transformation to chromosome damage. In recent years, evidence has accumulated implicating DNA double stranded breaks as lesions relevant for cell killing. Here, the available information on this topic is reviewed together with the methods most commonly used to quantitate induction and repair of this type of lesion. The presentation concludes with an outline of present research directions and future goals.     

Radiation-induced potentially lethal damage: DNA lesions susceptible to fixation

The various postirradiation incubation conditions reported to uncover potentially lethal damage (PLD) induced by ionizing radiation are outlined and critically discussed. The process of damage fixation is the most characteristic determinant in distinguishing between PLD and other forms of damage (lethal or non-lethal). The results compiled indicate the induction of two forms of PLD (termed alpha- and beta-PLD). Evidence is presented that repair and fixation of alpha-PLD may underlie the variation in radiosensitivity observed through the cycle. Beta-PLD appears to be sensitive only to postirradiation treatment in anisotonic sale solutions. Results obtained at the DNA and chromosome level, under conditions allowing repair or causing fixation of PLD, are reviewed and combined together to devise a qualitative model that outlines a possible sequence of events from damage fixation at the DNA level, to damage fixation at the chromosome level and, ultimately, to cell death. It is suggested that damage uncovered at the cellular level as potentially lethal, comprises DNA dsb (single, pairs or groups) and that fixation is mediated by forces transmitted to the double helix through alteration (local or general) in chromatin conformation. Changes in chromatin conformation are caused either as a result of the cell's progression through the cycle or in response to a postirradiation treatment. The fixation process leads to the induction of chromosome aberrations. The validity of the concept of PLD in in vivo systems is shown, and the possible importance of PLD repair in radiation therapy is reviewed. The concept of PLD is compared to the concept of sublethal damage, and the possibility that similar molecular lesions underlie both types of damage is discussed.     

Roles of sequential ubiquitination of PCNA in DNA-damage tolerance

Living organisms not only repair DNA damage induced by environmental agents and endogenous cellular metabolites, but have also developed mechanisms to survive in the presence of otherwise lethal lesions. DNA-damage tolerance (DDT) is considered such a mechanism that resumes DNA synthesis in the presence of replication-blocking lesions. Recent studies revealed that DDT in budding yeast is achieved through sequential ubiquitination of DNA polymerase processivity factor, proliferating cell nuclear antigen (PCNA). It is generally believed that monoubiquitinated PCNA promotes translesion DNA synthesis, whereas polyubiquitinated PCNA mediates an error-free mode of lesion bypass. This review will discuss how ubiquitinated PCNA modulates different means of lesion bypass.