Cyclobutyl Pyrimidine Dimers Research Articles

Beneficial effect of ζ-carotene-like compounds on acute UVB irradiation by alleviating inflammation and regulating intestinal flora.

ζ-Carotene is a key intermediate in the carotenoid pathway, but owing to its low content and difficulties in isolation, its application is restricted. In this study, three genes (pnCrtE, pnCrtB, and pnCrtP) in the carotenoid pathway of Antarctic moss were identified, recombined, and expressed in Escherichia coli (E. coli) BL21(DE3). The expression product was identified as one of the ζ-carotenes by UV absorbance spectrum, thin layer chromatography (TLC), and super-high-performance liquid chromatography-mass spectrum (UPLC-MS), and was called a ζ-carotene-like compound (CLC). Excessive exposure to ultraviolet B (UVB) irradiation is one of the main risk factors for skin photodamage. The purpose of this study was to investigate the preventive and therapeutic effects of CLC on UVB-induced skin photodamage in mice. In this paper, through histological examinations (hematoxylin-eosin, HE; Masson and TdT-mediated dUTP Nick-End Labeling, Tunel), biochemical index detection (reactive oxygen species, ROS; inflammatory factors; cyclobutyl pyrimidine dimers, CPDs and hyaluronic acid, HA), quantitative real time polymerase chain reaction (qRT-PCR), immunohistochemistry and intestinal content flora, etc., it is concluded that CLC has the potential to enhance skin antioxidant capacity by activating the nuclear transcription factor/antioxidant reaction element (Nrf2/ARE) pathway and also reduce skin inflammation and aging by inhibiting the mitogen-activated protein kinase (MAPK) pathway. Moreover, the regulation of intestinal flora may potentially mitigate skin damage induced by UVB radiation. This research not only developed a green and sustainable platform for the efficient synthesis of CLC but also laid a foundation for its application in functional food and medicine for skin resistance against UVB damage.

Topical Curcumin as Chemoprotector Against Photoproducts Production: The Role of Cyclobutyl Pyrimidine Dimers, 8-Hydroxy2'Deoxyguanosine Expression and Epidermal Hyperplasia in Acute and Chronic UVB-Induced Mice.

BackgroundUltraviolet B (UVB) exposure leads to formation of photoproducts leading to cellular damage. Prevention using sunscreen can sometimes be inadequate and can be an economic burden. Recent studies have suggested the photoprotective effect of curcumin.ObjectiveTo examine the acute and chronic photoprotective effect of topical curcumin, using cyclobutyl pyrimidine dimers (CPD) and 8-hydroxy2ʹdeoxyguanosine (8-OHdG) expression as markers of DNA-induced damage, and epidermal hyperplasia on UVB-induced mice.MethodsThree treatment groups were established. Group A (negative control) consisted of 5 mice, Group B and C were further divided into two categories to assess acute and chronic effects of topical curcumin and UVB radiation. Each consisted of six subgroups of five mice. Subgroup 1; UVB exposure only (positive control) subgroup 2; acetone and UVB exposure, subgroup 3–6; topical curcumin application of 100nM, 1µM, 10µM, and 100µM concentrations, respectively. In Group C, there were two categories that received 3x/week UVB exposure for three weeks which effects were being observed at 24 hours and 10 days after the last exposure. The topical curcumin dose was 2mg/mL/cm2 applied 30 minutes prior to 343mJ/cm2/day UVB irradiation. Skin biopsy was done one hour after the last UVB exposure for immunohistochemical and histopathology examinations.ResultsTopical curcumin showed a limited yet robust protective effect against CPD and 8-OHdG expression in Group B, while in Group C all concentrations showed significant CPD and 8-OHdG inhibition after 10 days of UVB exposure. The 10µM and 100µM concentrations showed the best epidermal hyperplasia inhibition effect (p<0.05). No significant differences were found in terms in efficacy either in single nor daily application.ConclusionTopical curcumin can prevent the formation of the photoproducts CPD and 8-OHdG and epidermal hyperplasia in both acute and chronic exposure in UVB-induced mice.

Effect of ultraviolet C emitted from KrCl excimer lamp with or without bandpass filter to mouse epidermis

It has been reported that 222-nm ultraviolet C (UVC) exerts a germicidal effect on bacteria and viruses as well as UV radiation emitted from a conventional germicidal lamp but is less toxic to the mammalian cells than that from a germicidal lamp. An excimer lamp filled with krypton chloride (KrCl) gas principally emits 222-nm UVC. However, the lamp also emits a wide band of wavelengths other than 222 nm, especially UVC at a longer wavelength than 222 nm and ultraviolet B, which cause DNA damage. There are some reports on the critical role of bandpass filters in reducing the harmful effect of UVC emitted from a KrCl excimer lamp in a human skin model and human subjects. However, the effectiveness of a bandpass filter has not been demonstrated in animal experiments. In the present study, mice were irradiated with UVC emitted from a KrCl excimer lamp with or without a bandpass filter. UVC emitted from an unfiltered KrCl lamp at doses of 50, 150 and 300 mJ/cm2 induced cyclobutyl pyrimidine dimer (CPD)-positive cells, whereas UVC emitted from a filtered lamp did not significantly increase CPD-positive cells in the epidermis. The present study suggested that the bandpass filter serves a critical role in reducing the harmful effect of emission outside of 222 nm to mouse keratinocytes.

Improved Spectral Purity of 222-nm Irradiation Eliminates Detectable Cyclobutylpyrimidine Dimers Formation in Skin Reconstructs even at High and Repetitive Disinfecting Doses.

UVC222 nm has germicidal effects with potential clinical applications. However, UVC irradiation is capable of inducing DNA damage like cyclobutylpyrimidine dimers (CPD). Although new devices have emission peaks in the short-wavelength region of UVC (~222 nm), the remaining "collateral" radiation at longer wavelengths could be harmful to human health. We investigated the DNA damage caused by far-UVC 222 nm KrCl exciplex radiation on human skin reconstructs after additional filtering using silica filters. The skin reconstructs were irradiated with 100 mJ cm-2 , 500 mJ cm-2 , and 3 × 500 mJ cm-2 unfiltered and filtered (230-270 nm suppressed) far-UVC or UVB (308 nm) radiation. UVB and non-filtered UVC irradiation induced a significant amount of CPDs, compared with the background. Filtered far-UVC lowered the CPD amount compared with unfiltered UVC and UVB treatments. Repetitive UVC irradiation did not result in the accumulation of CPDs compared with UVB treatment. Reduction in excess of 99.9% of E. coli, S. aureus and C. albicans was detected after applying far-UVC radiation. This identifies a therapeutic window in which microorganisms are killed but tissue is still alive and not damaged, which could give rise to new clinical applications.

Chronic irradiation with 222-nm UVC light induces neither DNA damage nor epidermal lesions in mouse skin, even at high doses.

Surgical site infections (SSIs) represent an important clinical problem associated with increased levels of surgical morbidity and mortality. UVC irradiation during surgery has been considered to represent a possible strategy to prevent the development of SSI. 254-nm UVC induces marked levels of DNA damage by generating cyclobutyl pyrimidine dimers (CPD) in microorganisms. However, this effect is elicited not only in microorganisms, but also in human cells, and chronic exposure to 254-nm UVC has been established to represent a human health hazard. In contrast, despite short wavelength-UVC light, especially 222-nm UVC, having been demonstrated to elicit a bactericidal effect, single irradiation with a high dose of 222-nm UVC energy has been reported to not induce mutagenic or cytotoxic DNA lesions in mammalian cells. However, the effect of chronic irradiation with a high dose of 222-nm UVC to mammalian cells has not been determined. In this study, it was demonstrated that large numbers of CPD-expressing cells were induced in the epidermis of mice following treatment with a small amount of single exposure 254-nm UVC, and then less than half of these cells reduced within 24 h. Chronic 254-nm UVC irradiation was revealed to induce sunburn and desquamation in mouse skin. Histological analysis demonstrated that small numbers of CPD-expressing cells were detected only in hyperkeratotic stratum corneum after chronic irradiation with a high dose of 254-nm UVC, and that significant hyperplasia and intercellular edema were also induced in the epidermis of mice. In contrast, chronic irradiation with 222-nm UVC light was revealed not to induce mutagenic or cytotoxic effects in the epidermis of mice. These results indicated that 222-nm UVC light emitted from the lamp apparatus (or device), which was designed to attenuate harmful light present in wavelengths of more than 230 nm, represents a promising tool for the reduction of SSI incidence in patients and hospital staff.

Extremophile DNA Photolyases: DNA Repair under Extreme Conditions

Adaptation to extreme environments is a seminal characteristic of life on Earth. Nowhere is this property more strongly evident than in single-celled organisms. Bacteria, archaea, and eukaryotes all have representatives that thrive in cold or hot environments. Many of these extremophile enzymes share significant amino acid sequence homology in spite of very different optimal growth temperatures (TG). Based on a simple amino acid sequence analysis it is unclear what adaptive changes are required to tailor homologous enzymes to function in extreme environments.A case in point is DNA photolyase (PL), a monomeric flavoprotein that binds UV-damaged DNA and repairs it by blue light activated picosecond electron transfer from a conserved flavin adenine dinucleotide cofactor (FADH¯) to the tightly bound cyclobutylpyrimidine dimer DNA lesion. These enzymes also include a second light-harvesting cofactor. Here we present a comparative analysis of three recombinant CPD photolyases, hyperthermophilic Sulfolobus solfataricus (TG=353K, rSsPL), mesophilic E. coli (TG=310K, rEcPL), and psychrophilic Colwellia psychrerythraea (TG=281K, rCpPL).All PLs utilize FADH¯ bound in a highly conserved site for repair. In addition, each PL demonstrates different properties for its 2nd cofactor. We used a variety of biochemical, physical, and molecular biological tools to compare these extremophile proteins with regard to repair yield, cofactor reduction potential and excited state properties. The difference in the stability of the redox state of the purified protein suggests different structural adaptations of each PL to their respective thermal environments. Denaturation studies reveal that rSsPL has a very stable structure, whereas rCpPL is extremely sensitive to its thermal and aqueous environments. A comparison of cofactor absorption and emission spectra reveal significant differences in how the flavin cofactor is bound in the binding pocket, in spite of significant amino acid sequence homology for this cavity across all proteins.

Modulation of the UVB-induced lethality by furocoumarins in Staphylococcus aureus

Furocumarins (FCs) are photoactive compounds capable of binding to DNA, and once excited by UVA light (∼365nm), they form photoadducts which can lead to mutagenicity and lethality. However, the biological effects of FCs combined with UVB light (312nm) is still little investigated. In the present study, the lethal effect of UVB light alone and combined with different concentrations of 8-methoxypsoralen (8-MOP), 4,5′,8-trimethylpsoralen (TMP) and 3-carbethoxypsoralen (3-CPs) was evaluated in a strain of Staphylococcus aureus. 8-MOP-UVB and TMP-UVB were more effective in inducing lethality compared to UVB alone, indicating that these FCs act as photosensitizing agents for UVB. The increase in concentration of 8-MOP resulted in a greater mortality. On the contrary, a decrease in mortality was found with an increase in TMP concentration. 3-CPs protected bacteria against damage induced by UVB, which can be attributed to the inhibition of cyclobutyl pyrimidine dimer formation. The different modulatory effects on lethality induced by UVB shown by the FCs tested could be related to differences in the specificity of each compound for particular nucleotide sequences, as well as other chemical characteristics of each molecule could influence the number and types of adducts formed, contributing to the photosensitizing or photoprotective effects observed.

Human replicative DNA polymerase δ can bypass T‐T (6‐4) ultraviolet photoproducts on template strands

DNA polymerase δ (Polδ) carries out DNA replication with extremely high accuracy. This great fidelity primarily depends on the efficient exclusion of incorrect base pairs from the active site of the polymerase domain. In addition, the 3'-5' exonuclease activity of Polδ further enhances its accuracy by eliminating misincorporated nucleotides. It is believed that these enzymatic properties also inhibit Polδ from inserting nucleotides opposite damaged templates. To test this widely accepted idea, we examined in vitro DNA synthesis by human Polδ enzymes proficient and deficient in the exonuclease activity. We chose the UV-induced lesions cyclobutyl pyrimidine dimer (CPD) and 6-4 pyrimidone photoproduct (6-4 PP) as damaged templates. 6-4 PP represents the most formidable challenge to DNA replication, and no single eukaryotic DNA polymerase has been shown to bypass 6-4 PP in vitro. Unexpectedly, we found that Polδ can perform DNA synthesis across both 6-4 PP and CPD even with a physiological concentration of deoxyribonucleotide triphosphates (dNTPs). DNA synthesis across 6-4 PP was often accompanied by a nucleotide deletion and was highly mutagenic. This unexpected enzymatic property of Polδ in the bypass of UV photoproducts challenges the received notion that the accuracy of Polδ prevents bypassing damaged templates.

The Extent of DNA Deformation in DNA Photolyase– Substrate Complexes: A Solution State Fluorescence Study

Cyclobutylpyrimidine dimers (CPDs) are the major UV photoproduct formed in DNA containing adjacent pyrimidines. These lesions can be repaired by DNA photolyase, a flavoprotein that utilizes blue light in a direct reversal of the cyclobutane ring. Previous studies have shown that the CPD is base flipped into the protein, with concomitant disruption of the substrate around the CPD. In this study, we use a fluorescent cytidine analog, pyrrolo-dC (PC), to probe how far base flipping propagates along the duplex. From these measurements, the degree of base destacking in the two bases flanking the adenines opposing the CPD appears to be minimal, which was consistent with the protein:substrate crystal structure. Fluorescence-detected melting temperatures for duplexes with and without a CPD were obtained, suggesting that a 5'-pyrimidine-PC-purine-3' motif is more stable than the 5'-purine-PC-pyrimidine-3' motif. This stability trend was reflected in the fluorescence intensities of ss-PC oligos but not for duplexes. The melting point depression due to the PC probe was found to be comparable to other popular fluorescent base analogs.

6MAP, a Fluorescent Adenine Analogue, Is a Probe of Base Flipping by DNA Photolyase

Cyclobutylpyrimidine dimers (CPDs) are formed between adjacent pyrimidines in DNA when it absorbs ultraviolet light. CPDs can be directly repaired by DNA photolyase (PL) in the presence of visible light. How PL recognizes and binds its substrate is still not well understood. Fluorescent nucleic acid base analogues are powerful probes of DNA structure. We have used the fluorescent adenine analogue 6MAP, a pteridone, to probe the local double helical structure of the CPD substrate when bound by photolyase. Duplex melting temperatures were obtained by both UV-vis absorption and fluorescence spectroscopies to ascertain the effect of the probe and the CPD on DNA stability. Steady-state fluorescence measurements of 6MAP-containing single-stranded and doubled-stranded oligos with and without protein show that the local region around the CPD is significantly disrupted. 6MAP shows a different quenching pattern compared to 2-aminopurine, another important adenine analogue, although both probes show that the structure of the complementary strand opposing the 5'-side of the CPD lesion is more destacked than that opposing the 3'-side in substrate/protein complexes. We also show that 6MAP/CPD duplexes are substrates for PL. Vertical excitation energies and transition dipole moment directions for 6MAP were calculated using time-dependent density functional theory. Using these results, the Förster resonance energy transfer efficiency between the individual adenine analogues and the oxidized flavin cofactor was calculated to account for the observed intensity pattern. These calculations suggest that energy transfer is highly efficient for the 6MAP probe and less so for the 2Ap probe. However, no experimental evidence for this process was observed in the steady-state emission spectra.

Differential Distortion of Substrate Occurs When It Binds to DNA Photolyase: A 2-Aminopurine Study

Cyclobutylpyrimidine dimers (CPDs) are formed between adjacent pyrimidines in DNA when it is exposed to ultraviolet light. CPDs can be directly repaired by DNA photolyase (PL) upon absorption of blue-green light. We have used the fluorescent adenine analogue 2-aminopurine (2Ap) to probe the local double-helical structure of the DNA substrate when it binds to the protein. Duplex melting temperatures and van't Hoff enthalpies were obtained by both UV-vis absorption and fluorescence spectroscopies to ascertain the effect of the probe and CPD on DNA stability. Steady-state fluorescence measurements of the single- and double-stranded oligos showed that the local region around the 5'-side of the CPD lesion was more disrupted and destacked than the 3'-side in substrate-protein complexes. These results were compared with those of a protein-substrate crystal structure, demonstrating that the crystal structure and solution-state studies are in agreement with regard to the differential distortions of the target DNA at the active site of the protein.

Repair of mitochondrial DNA in aging and carcinogenesis

Mitochondria are responsible for the generation of energy in the form of adenosine triphosphate. These organelles contain their own genetic material, mitochondrial (mt) DNA. This mtDNA has been hypothesized to play a role in the processes of aging and carcinogenesis. Initial reports have shown that there is no repair of cyclobutylpyrimidine dimers (CPD). More recent reports indicate however, that the mitochondrion contains several defence mechanisms against endogenous or exogenous damaging agents such as ultraviolet radiation or oxidative damage. The role of these defence mechanisms in the removal of mitochondrial DNA damage and the link to aging and carcinogenesis-associated processes are discussed in this review.

Protective Effect of Furocoumarins Against 254-nm Ultraviolet in Staphylococcus aureus

For Staphylococcus aureus, pretreatment with furocoumarins (FCs) protect cells against killing by far ultraviolet light (FUV; approximately 254 nm). This protective effect was evident in the repair-proficient, parental strain as well as in the repair-deficient variants in the following order of efficacy: 4,5'',8-trimethylpsoralen << 8-methoxypsoralen congruent with angelicin < 3-carbethoxypsoralen. The extent of protection was greater in the parental strain, indicating that despite the protective effect, a certain number of lethal lesions are nevertheless produced, which would be repaired with greater efficiency in such a strain than in the repair-deficient ones. This protective effect could be attribute to the inhibition of the formation of cyclobutyl pyrimidine dimers. Although the energy-transfer concept could explain the inhibition of pyrimidine dimer formation, and thus the protective effect of FC against FUV, we cannot rule out the possibility that the differences in degree of protection afforded by the FC employed here are related to a subtle and complex combination of effects.

CPD Photolyase Gene from Spinacia oleracea : Repair of UV-Damaged DNA and Expression in Plant Organs

The UV-B radiation contained in solar radiation has deleterious effects on plant growth, development and physiology. Specific damage to DNA caused by UV radiation involves the cyclobutyl pyrimidine dimers (CPD) and the pyrimidine (6-4) pyrimidone photoproducts. CPDs are repaired by CPD photolyase via a UV-A/blue light-dependent mechanism. The gene for the class II CPD photolyase has been cloned from higher plants such as Arabidopsis, cucumbers and rice. We isolated and characterized the cDNA and a genomic clone encoding the spinach class II CPD photolyase. The gene consisted of 3777 bases and 9 exons. The sequence of amino acids predicted from the nucleotide sequence of the cDNA of the gene was highly homologous to that of the higher plants listed above. When a photolyase-deficient Escherichia coli strain was transformed with the cDNA, photoreactivation activity was partially restored, by the illumination with photoreactivating light, resulting in an increased survival and decreased content of CPDs in the Escherichia coli genome. In both the male and female plants, the gene was highly expressed in leaves and flowers under the condition of 14-h light and 10-h dark cycle. The expression in the roots was quite low compared with the other organs.

UVB and UVA radiation-mediated damage to isolated and cellular DNA

Abstract The effects of solar light on cellular DNA are mostly explained by both direct excitation of nucleobases and photosensitized reactions that are mediated by UVB and UVA radiation, respectively. A large body of information is now available on the main photodynamic reactions to DNA, which involve guanine as the preferential target of both one-electron oxidation and singlet oxygen oxidation, as the result of type I and type II mechanisms. Most of the final products of the photosensitized reactions of guanine base in isolated DNA have been characterized, and comprehensive mechanisms for their formation have been proposed. Further insights into the mechanisms of solar radiation-induced modifications within cellular DNA have been gained from accurate measurement of the main photoproducts using recently designed sensitive chromatographic and biochemical methods. Thus, the distribution pattern of the 12 possible bipyrimidine photoproducts has been shown to be similar in the DNA of UVB-irradiated rodent and human cells. Cyclobutyl pyrimidine dimers are also generated at di-thymine and thymine-cytosine sites within nuclear DNA upon exposure to UVA radiation as the likely result of triplet energy transfer. In addition, oxidative reactions that involved mostly singlet oxygen, and to a lesser extent, .OH radicals are also implicated, although less efficiently.

Cis-syn thymidine dimer repair by DNA photolyase in real time.

DNA photolyase (PL) is a monomeric flavoprotein that repairs cyclobutylpyrimidine dimers (CPDs) via photoinduced electron transfer from a reduced flavin adenine dinucleotide cofactor (FADH(-)) to the bound CPD. We have used subpicosecond UV transient absorption spectroscopy to measure the electron-transfer and repair kinetics of Anacystis nidulans DNA photolyase with dimeric and pentameric oligothymidine substrates. Here we show that the electron-transfer lifetime is 32 +/- 20 ps for the pentameric substrate. Repair of the carbon-carbon double bonds (C=C) in the CPD is initiated in approximately 60 ps, and bond scission appears to be completed by 1500 ps. This suggests that the repair of the two C=C bonds proceeds sequentially and that the first bond scission has a much lower activation barrier than the second. Our experiments also suggest that the semiquinone FADH(*) cofactor is not reduced to its catalytically active FADH(-) state by substrate after repair but remains in the semiquinone state. In contrast to the longer substrate, the dinucleotide substrate produced a mixture of kinetics representing bound and unbound substrate.

DNA damage and photosynthesis in Antarctic and Arctic Sanionia uncinata (Hedw.) Loeske under ambient and enhanced levels of UV‐B radiation

AbstractThe response of the bipolar moss Sanionia uncinata (Hedw.) Loeske to ambient and enhanced UV‐B radiation was investigated at an Antarctic (Léonie Island, 67°35′ S, 68°20′ W) and an Arctic (Ny‐Alesund, 78°55′ N, 11°56′ E) site, which differed in ambient UV‐B radiation (UV‐BR: 280–320 nm) levels. The UV‐BR effects on DNA damage and photosynthesis were investigated in two types of outdoor experiments. First of all, sections of turf of S. uncinata were collected in an Arctic and Antarctic field site and exposed outdoors to ambient and enhanced UV‐BR for 2 d using UV‐B Mini‐lamps. During these experiments, chlorophyll a fluorescence, chlorophyll concentration and cyclobutyl pyrimidine dimer (CPD) formation were measured. Secondly, at the Antarctic site, a long‐term filter experiment was conducted to study the effect of ambient UV‐BR on growth and biomass production. Additionally, sections of moss turf collected at both the Antarctic and the Arctic site were exposed to UV‐BR in a growth chamber to study induction and repair of CPDs under controlled conditions. At the Antarctic site, a summer midday maximum of 2·1 W m−2 of UV‐BR did not significantly affect effective quantum yield (ΔF/Fm′) and the ratio of variable to maximal fluorescence (Fv/Fm). The same was found for samples of S. uncinata exposed at the Arctic site, where summer midday maxima of UV‐BR were about 50% lower than at the Antarctic site. Exposure to natural UV‐BR in summer did not increase CPD values significantly at both sites. Although the photosynthetic activity remained largely unaffected by UV‐B enhancement, DNA damage clearly increased as a result of UV‐B enhancement at both sites. However, DNA damage induced during the day by UV‐B enhancement was repaired overnight at both sites. Results from the long‐term filter experiment at the Antarctic site indicated that branching of S. uncinata was reduced by reduction of ambient summer levels of UV‐BR, whereas biomass production was not affected. Exposure of specimens collected from both sites to UV‐BR in a growth chamber indicated that Antarctic and Arctic S. uncinata did not differ in UV‐BR‐induced DNA damage. It was concluded that S. uncinata from both the Antarctic and the Arctic site is well adapted to ambient levels of UV‐BR.

Cyclobutylpyrimidine Dimer Base Flipping by DNA Photolyase

DNA Photolyase is a flavoprotein that uses light to repair cyclobutylpyrimidine dimers in DNA. From considerations of the crystal structure of the protein, it has been hypothesized that the dimer lesion is flipped out of the DNA double helix into the substrate binding pocket. We have used a fluorescent adenine analog, 2-aminopurine (2-Ap), as a probe of local double helical structure upon binding of the substrate to the protein. Our results show that the local structure around the thymidine lesion changes dramatically upon binding to Photolyase. This is consistent with base flipping of the lesion into the protein binding cavity with concomitant destacking of the opposing complementary 2-Ap nucleotide.

Bistranded oxidized purine damage clusters: induced in DNA by long-wavelength ultraviolet (290-400 nm) radiation?

Bistranded clustered DNA damages involving oxidized bases, abasic sites, and strand breaks are produced by ionizing radiation and radiomimetic drugs, but it was not known whether they can be formed by other agents, e.g., nonionizing radiation. UV radiation produces clusters of cyclobutyl pyrimidine dimers, photoproducts that occur individually in high yield. Since long-wavelength UV (290-400 nm) radiation induces oxidized bases, abasic sites, and strand breaks at low yields, we tested whether it also produces clusters containing these lesions. We exposed supercoiled pUC18 DNA to UV radiation with wavelengths of >290 nm (UVB plus UVA radiation), and assessed the induction of bistranded clustered oxidized purine and abasic clusters, as recognized by Escherichia coli Fpg protein and E. coli Nfo protein (endonuclease IV), respectively, as well as double-strand breaks. These three classes of bistranded clusters were detected, albeit at very low yields (37 Fpg-OxyPurine clusters Gbp(-1) kJ(-1) m(2), 8.1 double-strand breaks Gbp(-1) kJ(-1) m(2), and 3.4 Nfo-abasic clusters Gbp(-1) kJ(-1) m(2)). Thus, these bistranded OxyPurine clusters, abasic clusters, and double-strand breaks are not uniquely induced by ionizing radiation and radiomimetic drugs, but their level of production by UVB and UVA radiation is negligible compared to the levels of frequent photoproducts such as pyrimidine dimers.

Repair of cyclobutyl pyrimidine dimers in human skin: variability among normal humans in nucleotide excision and in photorepair.

Photoreactivation (PR) of cyclobutyl pyrimidine dimers (CPD) in human skin remains controversial. Recently Whitmore et al. (1) reported negative results of experiments using two photorepair light (PRL) sources on UV-irradiated skin of volunteers. However, their PRL sources induced substantial levels of dimers in skin, suggesting that the additional dimers formed could have obscured PR. We met a similar problem of dimer induction by a PRL source. We designed and validated a PRL source of sufficient intensity to catalyse PR, but that did not induce CPD, and used it to measure photorepair in human skin. Using a solar simulator filtered with three types of UV-filters, we found significant dimer formation in skin, quantified by number average length analysis using electrophoretic gels of isolated skin DNA. To prevent scattered UV from reaching the skin, we interposed shields between the filters and skin, and showed that the UV-filtered/shielded solar simulator system did not induce damage in isolated DNA or in human skin. We exposed skin of seven healthy human volunteers to 302 nm radiation, then to the improved PRL source (control skin areas were kept in the dark for measurement of excision repair). Using a high intensity PRL source that did not induce dimers in skin, we found that three of seven subjects carried out rapid photorepair of dimers; two carried out moderate or slow dimer photorepair, and three did not show detectable photorepair. Excision repair was similarly variable in these volunteers. Subjects with slower excision repair showed rapid photorepair, whereas those with rapid excision generally showed little or no photoreactivation.