Repair of closely opposed cyclobutyl pyrimidine dimers in UV-sensitive human diploid fibroblasts

An enzyme-sensitive site assay has been used to examine the fate of closely opposed pyrimidine dimers (bifilar enzyme-sensitive sites) in fibroblasts from individuals afflicted with various genetic disorders that confer increased cellular sensitivity to UV radiation. The disappearance of bifilar enzyme-sensitive sites was found to be normal in cells from individuals with Fanconi's anemia, Cockayne's syndrome, dyskeratosis congenita and the variant form of xeroderma pigmentosum. The rate of bifilar enzyme-sensitive site removal in XP cells assigned to complementation group C was reduced by an amount similar to that observed for the repair of isolated dimers. Our results indicate that the initiation of repair at closely opposed dimers is slow in XP-C cells but normal in all other cells examined.     

Bifilar enzyme-sensitive sites in ultraviolet-irradiated DNA are indicative of closely opposed cyclobutyl pyrimidine dimers.

Incubation of UV-irradiated DNA with pyrimidine dimer-DNA glycosylase in cell-free lysates prepared from Micrococcus luteus results in the appearance of double-strand breaks. It has previously been assumed that such double-strand breaks result from cleavage at closely opposed dimers. We have used hybrid molecules of bacteriophage T7 DNA comprised of two unirradiated strands, two UV-irradiated strands, or one unirradiated and one UV-irradiated strand to test this hypothesis. Bifilar cleavage was observed only with molecules consisting of two irradiated strands and no bifilar cleavage was observed after the monomerization of pyrimidine dimers by enzymatic photoreactivation. Our results indicate that at least 80% of the double-strand breaks result from cleavage at closely opposed dimers and that the induction of dimers in one strand does not influence the induction of dimers at closely opposed positions in the complementary strand of a DNA double helix.     

A sensitive, enzymatic assay for the detection of closely opposed cyclobutyl pyrimidine dimers induced in human diploid fibroblasts

A sensitive, enzymatic assay has been developed for the detection of closely opposed cyclobutyl pyrimidine dimers induced in UV-irradiated human diploid fibroblasts. In this assay closely opposed dimers are detected as bifilar enzyme-sensitive sites. Single-strand incisions are made at the positions of individual pyrimidine dimers through the action of M. luteus pyrimidine dimer-DNA glycosylase. Incisions at closely opposed dimers, effectively expressed as double-strand breaks, are quantified from the resulting reduction in DNA double-strand molecular weight as determined by velocity sedimentation through neutral sucrose density gradients. The stability of the bacteriophage lambda cos site under our reaction conditions indicates that opposed incisions must be relatively close to be expressed as a double-strand break. The dose response for the induction of bifilar enzyme-sensitive sites in mammalian cells was found to be complex but can be approximated by a function that increases as the 1.2-1.4 power of UV dose. The frequency of bifilar enzyme-sensitive sites observed decreased during postirradiation incubation of excision-repair-proficient human diploid fibroblasts with less than 20% still detectable at 24 h after irradiation with 5 J/m2 (254 nm). By comparison, over 80% of the bifilar enzyme-sensitive sites induced in fibroblasts assigned to xeroderma pigmentosum complementation group A remained detectable 24 h after irradiation. The implications of these results for models addressing the induction and repair of closely opposed pyrimidine dimers are discussed.     

Induction and repair of closely opposed pyrimidine dimers in Saccharomyces cerevisiae

Pyrimidine dimer-DNA glycosylase activity prepared from Micrococcus luteus has been used to develop an enzyme-sensitive site assay for the detection and quantification of closely opposed pyrimidine dimers in the nuclear DNA of UV-irradiated yeast. With this assay, closely opposed dimers were found to be induced as a linear function of dose from 0 to 200 J/m2 (254 nm). Closely opposed dimer frequencies decreased during the incubation of UV-irradiated, excision repair-proficient cells under liquid-holding conditions in the dark and during post-irradiation exposure of excision-deficient cells to photoreactivating light. Incubation of excision-deficient cells in the dark had no effect on the frequency of closely opposed dimers for up to 16 h. These results indicate that closely opposed dimers in UV-irradiated yeast are subject to repair by enzymatic photoreactivation and/or by dark-repair processes dependent, at least in part, upon functions necessary for normal excision repair. The genetic and biochemical implications of these results are discussed.     

Quantitation of ultraviolet radiation-induced cyclobutyl pyrimidine dimers in DNA by video and photographic densitometry

We have compared video and photographic methods for calculating the number of ultraviolet radiation (uv)-induced pyrimidine dimers in DNA from the bacteriophage T7 exposed to uv (0 to 800 J/m2) from an FS40 sunlamp. DNA was incubated with a pyrimidine dimer-specific Micrococcus luteus uv endonuclease, subjected to alkaline agarose gel electrophoresis, neutralized, and stained with ethidium bromide, and the DNA fluorescence was recorded either with a video camera or on photographic film. The slopes of the dose-response curves for the number of uv-endonuclease-sensitive sites per 10(3) bases (pyrimidine dimers) was 1.2 (+/- 0.1) X 10(-4) uv-endonuclease-sensitive sites per J/m2 for the video analysis and 1.3 (+/- 0.04) X 10(-4) uv-endonuclease-sensitive sites per J/m2 for the photographic analysis. Results for pyrimidine dimer determination by either method were statistically comparable.     

Wavelength dependence of biological damage induced by UV radiation on bacteria

The biological effects of UV radiation of different wavelengths (UVA, UVB and UVC) were assessed in nine bacterial isolates displaying different UV sensitivities. Biological effects (survival and activity) and molecular markers of oxidative stress [DNA strand breakage (DSB), generation of reactive oxygen species (ROS), oxidative damage to proteins and lipids, and the activity of antioxidant enzymes catalase and superoxide dismutase] were quantified and statistically analyzed in order to identify the major determinants of cell inactivation under the different spectral regions. Survival and activity followed a clear wavelength dependence, being highest under UVA and lowest under UVC. The generation of ROS, as well as protein and lipid oxidation, followed the same pattern. DNA damage (DSB) showed the inverse trend. Multiple stepwise regression analysis revealed that survival under UVA, UVB and UVC wavelengths was best explained by DSB, oxidative damage to lipids, and intracellular ROS levels, respectively.     

Inhibition of semiconservative DNA synthesis in ICR 2A frog cells by pyrimidine dimers and nondimer photoproducts induced by ultraviolet radiation

DNA synthesis was examined in ultraviolet (uv)-irradiated ICR 2A frog cells in which either pyrimidine dimers or nondimer photoproducts represented the major class of DNA lesions. Dimers were induced by exposure of cells to 254 nm uv, while nondimer photoproducts were induced by irradiation of cells with uv produced by a fluorescent sunlamp (FSL) that was filtered through 48A Mylar (removes wavelengths less than 310 nm). The FSL-irradiated cultures were also treated with photoreactivating light (PRL) which removed most of the small number of dimers induced by the irradiation, leaving a relatively pure population of nondimer photoproducts. In addition, cells were exposed to 60Co gamma rays. The cultures were pulse-labeled and the size distribution of the DNA synthesized was estimated using both sucrose gradient sedimentation and alkaline step elution. Using either of these techniques, it was found that the presence of dimers resulted in a reduction principally in the synthesis of high molecular weight (MW) DNA. In contrast, nondimer photoproducts caused a strong inhibition in the synthesis of low MW DNA, as was also observed in gamma-irradiated cells. Hence the induction of pyrimidine dimers in DNA mainly affected the elongation of replicons, whereas nondimer lesions primarily caused an inhibition of replicon initiation.     

Enhanced DNA repair of cyclobutane pyrimidine dimers changes the biological response to UV-B radiation

The goal of DNA repair enzyme therapy is the same as that for gene therapy: to rescue a defective proteome/genome by introducing a substitute protein/DNA. The danger of inadequate DNA repair is highlighted in the genetic disease xeroderma pigmentosum. These patients are hypersensitive to sunlight and develop multiple cutaneous neoplasms very early in life. The bacterial DNA repair enzyme T4 endonuclease V was shown over 25 years ago to be capable of reversing the defective repair in xeroderma pigmentosum cells. This enzyme, packaged in an engineered delivery vehicle, has been shown to traverse the stratum corneum, reach the nuclei of living cells of the skin, and enhance the repair of UV-induced cyclobutane pyrimidine dimers (CPD). In such a system, changes in DNA repair, mutagenesis, and cell signaling can be studied without manipulation of the genome.     

Excision of pyrimidine dimers from the DNA of Neurospora

Summary Germinated conidia of Neurospora have been monitored for their ability to excise pyrimidine dimers. Dimer concentration was measured in DNA extracted immediately after UV treatment, and it was compared to that of DNA from cells which had a post-UV incubation before extraction. Two methods were used to assay dimer level in DNA: 1) measurement of the number of single-strand breaks (as revealed in alkaline sucrose gradients) produced by a dimer-specific endonuclease; 2) monitoring the ability to compete for binding to dimer-specific antibodies in a radioimmuno assay. Both methods showed efficient excision of dimers by wild-type and by uvs-2, even though an earlier study had reported that uvs-2 was unable to excise dimers.UV-induced mutation shows a dose-rate effect: acute UV yields several times as many mutations as does the same dose of chronic UV. There is a parallel effect on dimer accumulation. The concentration of dimers at the conclusion of the UV treatment shows a strong correlation with the resultant mutation frequency.     

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.     

Immunological detection of UV induced cyclobutane pyrimidine dimers and (6-4) photoproducts in DNA from reference bacteria and natural aquatic populations

UV light-caused DNA damage is a widespread field of study. As UV light has the biological effect of inactivating or killing bacteria, it is used for water disinfection. Due to this application, it is important to study the DNA damage efficiencies and regeneration capacities in bacteria after UV irradiation. Two monoclonal antibodies, anti-CPD and anti-(6-4) PP, were applied for an immunoassay of UV-induced DNA modifications. Cyclobutane pyrimidine dimer (CPD) and 6-4 photoproduct (6-4 PP) were detected in the reference bacteria Pseudomonas aeruginosa and Enterococcus faecium, and in natural water communities. The antibody-mediated detection signals increased with the UV doses from 100-400 J/m². Here, the CPD-specific signals were stronger than the (6-4) PP-specific signals. These immunological results were in accordance with parallel cultivation experiments. All UV-irradiated bacteria showed a reduction of their growth rate depending on UV application by several orders of magnitudes.The immunoassay was also applied to three types of natural aquatic habitats with different cell densities. Besides artificial UV irradiation, it was possible to visualize natural sunlight effects on these natural bacterial communities. Light-dependent and dark repair processes were distinguished using the established immunological assays. The antibody-mediated analyses presented are fast methods to detect UV-induced DNA lesions and repair capacities in selected bacterial species as well as in entire natural mixed populations.     

Quantification of ultraviolet-C irradiation induced cyclobutane pyrimidine dimers and their removal in Beauveria bassiana conidiospore DNA

Ultraviolet (UV) radiation-induced DNA damage leading to entomopathogenic fungal inactivation is commonly measured by viability counts. Here we report the first quantification of UV-induced cyclobutane pyrimidine dimers (CPD) in DNA of the entomopathogenic fungus, Beauveria bassiana. Changes in the mobility of UV-C irradiated DNA were resolved with CPD specific bacteriophage T4 endonuclease V and alkaline agarose gel electrophoresis. The maximum number of CPD formed in B. bassiana DNA in vitro by UV-C irradiation was 28 CPD/ 10 kb after 720 J/m2 dose. The maximum number of CPDs formed in B. bassiana conidiospore DNA irradiated in vivo was 15 CPD/10 kb after 480 J/m2 dose and was quantified from conidiospores that were incubated to allow photoreactivation and nucleotide excision repair. The conidiospores incubated for photoreactivation and nucleotide excision repair showed decreased number of CPD/10 kb DNA and a higher percent survival of conidiospore populations than conidiospores not allowed to repair.     

Dose response, wavelength dependence and rate of excision of ultraviolet radiation-induced pyrimidine dimers in mouse skin DNA

The yield of thymine-containing dimers produced in mouse skin DNA in vivo by 290 nm ultraviolet radiation was shown to increase with dose up to around 2000 J/m2 and subsequently at a much slower rate up to 8000 J/m2. The study of wavelength dependence of dimer formation in skin indicated that 290 nm was the most effective wavelength of those investigated, followed by 300, 280 and 260 nm, with 310 nm being by far the least effective. A reduction in the number of dimers present in skin DNA was shown to occur by 24 h post-irradiation in a dose-dependent manner. A significant percentage of the dimers was, however, found to persist in the skin until at least 72 h post-irradiation.     

Sequence specificity of cyclobutane pyrimidine dimers in DNA treated with solar (ultraviolet B) radiation.

Cyclobutane pyrimidine dimers were quantified at the sequence level after irradiation with solar ultraviolet (UVB) and nonsolar ultraviolet (UVC) light sources. The yield of photoproducts at specific sites was dependent on the nucleotide composition in and around the potential lesion as well as on the wavelength of ultraviolet light used to induce the damage. Induction was greater in the presence of 5' flanking pyrimidines than purines; 5' guanine inhibited induction more than adenine. UVB irradiation increased the induction of cyclobutane dimers containing cytosine relative to thymine homodimers. At the single UVC and UVB fluences used, the ratio of thymine homodimers (T mean value of T) to dimers containing cytosine (C mean value of T, T mean value of C, C mean value of C) was greater after UVC compared to UVB irradiation.