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• W3118381401 abstract "In order to better understand the relative contribution of the different UV components of sunlight to solar mutagenesis, the distribution of the bipyrimidine photolesions, cyclobutane pyrimidine dimers (CPD), (6-4) photoproducts ((6–4)PP), and their Dewar valence photoisomers (DewarPP) was examined in Chinese hamster ovary cells irradiated with UVC, UVB, or UVA radiation or simulated sunlight. The absolute amount of each type of photoproduct was measured by using a calibrated and sensitive immuno-dot-blot assay. As already established for UVC and UVB, we report the production of CPD by UVA radiation, at a yield in accordance with the DNA absorption spectrum. At biologically relevant doses, DewarPP were more efficiently produced by simulated solar light than by UVB (ratios of DewarPP to (6-4)PP of 1:3 and 1:8, respectively), but were detected neither after UVA nor after UVC radiation. The comparative rates of formation for CPD, (6-4)PP and DewarPP are 1:0.25 for UVC, 1:0.12:0.014 for UVB, and 1:0.18:0.06 for simulated sunlight. The repair rates of these photoproducts were also studied in nucleotide excision repair-proficient cells irradiated with UVB, UVA radiation, or simulated sunlight. Interestingly, DewarPP were eliminated slowly, inefficiently, and at the same rate as CPD. In contrast, removal of (6-4) photoproducts was rapid and completed 24 h after exposure. Altogether, our results indicate that, in addition to CPD and (6-4)PP, DewarPP may play a role in solar cytotoxicity and mutagenesis. In order to better understand the relative contribution of the different UV components of sunlight to solar mutagenesis, the distribution of the bipyrimidine photolesions, cyclobutane pyrimidine dimers (CPD), (6-4) photoproducts ((6–4)PP), and their Dewar valence photoisomers (DewarPP) was examined in Chinese hamster ovary cells irradiated with UVC, UVB, or UVA radiation or simulated sunlight. The absolute amount of each type of photoproduct was measured by using a calibrated and sensitive immuno-dot-blot assay. As already established for UVC and UVB, we report the production of CPD by UVA radiation, at a yield in accordance with the DNA absorption spectrum. At biologically relevant doses, DewarPP were more efficiently produced by simulated solar light than by UVB (ratios of DewarPP to (6-4)PP of 1:3 and 1:8, respectively), but were detected neither after UVA nor after UVC radiation. The comparative rates of formation for CPD, (6-4)PP and DewarPP are 1:0.25 for UVC, 1:0.12:0.014 for UVB, and 1:0.18:0.06 for simulated sunlight. The repair rates of these photoproducts were also studied in nucleotide excision repair-proficient cells irradiated with UVB, UVA radiation, or simulated sunlight. Interestingly, DewarPP were eliminated slowly, inefficiently, and at the same rate as CPD. In contrast, removal of (6-4) photoproducts was rapid and completed 24 h after exposure. Altogether, our results indicate that, in addition to CPD and (6-4)PP, DewarPP may play a role in solar cytotoxicity and mutagenesis. cyclobutane pyrimidine dimers pyrimidine (6-4) pyrimidone photoproducts Dewar photoproducts 8-oxo-7,8-dihydro-2′-deoxyguanosine simulated solar light nucleotide excision repair transcription-coupled repair global genomic repair immuno-dot-blot watt(s) Chinese hamster ovary kilobase pair(s) phosphate-buffered saline phosphate-buffered saline plus Tween 20 nonfat milk Overwhelming evidence associates the steadily increasing incidence of skin cancer with an increased exposure to the UV components of sunlight (1IARC Monographs on the Evaluation of Carcinogenic Risks to Human: Solar and UV Radiation. IARC 55, Lyon, France1992Google Scholar). The UV induction of DNA damage is unambiguously an essential step in photocarcinogenesis (2Brash D.E. Trends Genet. 1997; 13: 410-414Abstract Full Text PDF PubMed Scopus (268) Google Scholar). In the mutated p53 tumor suppressor gene of skin cancer, the majority of mutations harbor the “UV mutational signature,” i.e. C → T transitions and CC → TT tandem double mutations, which occur at sites where major DNA photoproducts are formed (3Brash D.E. Rudolph J.A. Simon J.A. Lin A. McKenna G.J. Baden H.P. Halperin A.J. Ponten J. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 10124-10128Crossref PubMed Scopus (1730) Google Scholar, 4Daya-Grosjean L. Dumaz N. Sarasin A. J. Photochem. Photobiol. B Biol. 1995; 28: 115-126Crossref PubMed Scopus (140) Google Scholar). The photolesions that are readily produced at these sites by UVC (254 nm) and UVB (280–320 nm), such as cyclobutane pyrimidine dimers (CPD)1 and pyrimidine (6-4) pyrimidone photoproducts ((6–4)PP), are thought to represent the predominant forms of premutagenic damage (5Mitchell D.L. Pfeifer G.P. Taylor J.-S. Zdzienicka M.Z. Nikaido O. Frontier of Photobiology. Elsevier Science Publishers, Amsterdam1993: 337-344Google Scholar). The Dewar photoproducts (DewarPP), which are valence isomers of (6-4)PP formed via photoisomerization at wavelengths around 320 nm (6Taylor J.-S. Lu H.-F. Kotyk J.J. Photochem. Photobiol. 1990; 51: 161-167Crossref PubMed Scopus (106) Google Scholar, 7Mitchell D.L. Rosenstein B.S. Photochem. Photobiol. 1987; 45: 781-786Crossref PubMed Scopus (61) Google Scholar), have received much less attention to date. Until recently, the investigation of DNA photolesions has been predominantly conducted using UVC at 254 nm or UVB. However, wavelengths lower than 290 nm do not reach the Earth's surface due to absorption by the stratosphere. Consequently, the human population is mostly exposed to longer wavelength UVB (λ > 295 nm; Ref. 8Freeman S.E. Hacham H. Gange R.W. Maytum D.J. Sutherland J.C. Sutherland B.M. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 5605-5609Crossref PubMed Scopus (256) Google Scholar) and UVA (320–400 nm) radiation, which constitute about 5% and 95%, respectively, of the solar spectrum. The relatively weak UVB component is believed to be responsible for most of the biological effects of sunlight, which are mediated by direct absorption of UVB by DNA. Since UVA is poorly absorbed by DNA, its genotoxic effect has been attributed to indirect photosensitizing reactions (9Tyrrell R.M. Keyse S.M. J. Photochem. Photobiol. B Biol. 1990; 4: 349-361Crossref PubMed Scopus (265) Google Scholar, 10Kochevar I.E. Dunn D.A. Bioorganic Photochemistry. John Wiley & Sons, New York1990: 273-315Google Scholar). At the DNA level, photosensitization induces oxidative damage either by charge transfer from excited endogenous chromophores, or by reactions with reactive oxygen species that are generated at these wavelengths. Indeed, photo-induced oxidative DNA modifications, such as single strand breaks, DNA-protein cross-links, alkali-labile sites, 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodGuo) have been observed (see Ref. 11Sage E. Photochem. Photobiol. 1993; 57: 163-174Crossref PubMed Scopus (214) Google Scholar for review; see also Refs. 12Peak M.J. Peak J.G. Carnes B.A. Photochem. Photobiol. 1987; 45: 381-387Crossref PubMed Scopus (161) Google Scholar, 13Alapetite C. Wachter T. Sage E. Moustacchi E. Int. J. Radiat. Biol. 1996; 69: 359-369Crossref PubMed Scopus (143) Google Scholar, 14Kielbassa C. Roza L. Epe B. Carcinogenesis. 1997; 18: 811-816Crossref PubMed Scopus (484) Google Scholar, 15Douki T. Perdiz D. Gróf P. Kuluncsics Z. Moustacchi E. Cadet J. Sage E. Photochem. Photobiol. 1999; 70: 184-190Crossref PubMed Scopus (107) Google Scholar). However, the direct effect of UVA has only recently been described (14Kielbassa C. Roza L. Epe B. Carcinogenesis. 1997; 18: 811-816Crossref PubMed Scopus (484) Google Scholar, 15Douki T. Perdiz D. Gróf P. Kuluncsics Z. Moustacchi E. Cadet J. Sage E. Photochem. Photobiol. 1999; 70: 184-190Crossref PubMed Scopus (107) Google Scholar, 16Kuluncsics Z. Perdiz D. Brulay E. Muel B. Sage E. J. Photochem. Photobiol. B Biol. 1999; 49: 71-80Crossref PubMed Scopus (190) Google Scholar). In mammalian cells, UV-induced bipyrimidine photoproducts are removed via nucleotide excision repair (NER), either by transcription-coupled repair (TCR) or global genomic repair (GGR), while oxidative photolesions and single strand breaks are most likely eliminated by base excision repair (17Friedberg E.C. Walker G.C. Siede W. DNA Repair and Mutagenesis. ASM Press, Washington, D. C.1995Google Scholar). The repair kinetics and efficiencies of UV-induced damage vary considerably from one type of photolesion to another, and between species, such as rodent and human. For example, (6-4)PP are rapidly and efficiently removed in both organisms, probably via GGR, whereas CPD are repaired rather slowly and incompletely (17Friedberg E.C. Walker G.C. Siede W. DNA Repair and Mutagenesis. ASM Press, Washington, D. C.1995Google Scholar). The mutational specificities of UVC, UVB, and UVA radiation and simulated solar light (SSL) were previously determined at theaprt locus in Chinese hamster ovary (CHO) cells that were proficient or deficient in DNA repair (18Drobetsky E.A. Moustacchi E. Glickman B.W. Sage E. Carcinogenesis. 1994; 15: 1577-1583Crossref PubMed Scopus (21) Google Scholar, 19Drobetsky E.A. Turcotte J. Chateauneuf A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 2350-2354Crossref PubMed Scopus (229) Google Scholar, 20Sage E. Lamolet B. Brulay E. Moustacchi E. Chateauneuf A. Drobetsky E.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 176-180Crossref PubMed Scopus (100) Google Scholar). Briefly, it was found that: 1) in the NER-proficient cell line, GC → AT transitions and CC →TT tandem double mutations were the major types of events following SSL exposure, whereas the former type of mutation largely predominated following UVB irradiation; 2) while GC → AT transitions still contributed up to 27% of the changes after UVA exposure, they were no longer preponderant; 3) in contrast, in the NER-deficient cell line, GC → AT transitions, all of which occurred at bipyrimidine sites, represented a large proportion of the mutational events induced by UVA. Altogether, these results suggested the formation of bipyrimidine photoproducts by UVA radiation and a role for such damage in UVA-induced mutagenesis. Indeed, we later observed a significant production of CPD in plasmid DNA irradiated with UVA (16Kuluncsics Z. Perdiz D. Brulay E. Muel B. Sage E. J. Photochem. Photobiol. B Biol. 1999; 49: 71-80Crossref PubMed Scopus (190) Google Scholar). In order to better understand the relative contribution of the different UV components to solar mutagenesis, we previously examined their respective DNA damage profiles. In particular, we investigated the relative spectral effectiveness in the induction of CPD and 8-oxodGuo. We showed that CPD are formed at least as frequently as 8-oxodGuo by UVA radiation (15Douki T. Perdiz D. Gróf P. Kuluncsics Z. Moustacchi E. Cadet J. Sage E. Photochem. Photobiol. 1999; 70: 184-190Crossref PubMed Scopus (107) Google Scholar). Here, using specific antibodies, we determine the yield of three bipyrimidine photoproducts, CPD, (6-4)PP, and DewarPP, in the genomic DNA of CHO cells exposed to physiologically relevant doses of UVC, UVB, UVA, and SSL. An estimation of the levels of these three types of photolesions was previously obtained by immunostaining cells irradiated with UVC, broad or narrow band UVB, or natural sunlight with monoclonal antibodies similar to those used here (21Clingen P.H. Arlett C.F. Roza L. Mori T. Nikaido O. Green M.H.L. Cancer Res. 1995; 55: 2245-2248PubMed Google Scholar, 22Clingen P.H. Arlett C.F. Cole J. Waugh A.P.W. Lowe J.E. Harcourt S.A. Hermanova N. Roza L. Mori T. Nikaido O. Green M.H.L. Photochem. Photobiol. 1995; 61: 163-170Crossref PubMed Scopus (59) Google Scholar, 23Chadwick C.A. Potten C.S. Nikaido O. Matsunaga T. Proby C. Young A.R. J. Photochem. Photobiol. B Biol. 1995; 28: 163-170Crossref PubMed Scopus (106) Google Scholar). Clingen and colleagues (22Clingen P.H. Arlett C.F. Cole J. Waugh A.P.W. Lowe J.E. Harcourt S.A. Hermanova N. Roza L. Mori T. Nikaido O. Green M.H.L. Photochem. Photobiol. 1995; 61: 163-170Crossref PubMed Scopus (59) Google Scholar) assigned the increase in arbitrary gray scale values to the induction of antibody binding sites and estimated the relative production of (6-4)PP and DewarPP by assuming an equal luminescence signal for all three antibodies. However, their data took into account neither the very different affinities of the three antibodies for their respective substrates nor the possibility of multimeric forms of the immunoglobulins. In addition, comparison of the photolesion induction by various UV lamps was made possible only by expressing the fluence rates as dimer-equivalent fluences. In contrast to estimating relative values, determination of the absolute amount of different damage due to UV radiation allows a comparison of the biological effectivenesses of two or more types of photobiological action, e.g. induction of CPD and other types of photolesion. This is not the case for the determination of relative lesion induction, even if it can provide a precise action spectrum for a given photobiological effect. In this report, we describe the results of a method that allows a determination of the absolute amounts of these photoproducts. This method relies on the calibration of the immunochemical signals for each antibody using specific DNA repair enzymes. We present the first absolute determination of all three bipyrimidine photoproducts using one detection method. Our data show that the distribution of the bipyrimidine photolesions varies greatly depending on the wavelength region considered, and we demonstrate that UVA radiation produces significant amounts of CPD at doses comparable to those of human exposure. In addition, the relatively high production of DewarPP by SSL observed in this study, suggests that DewarPP may be a biologically relevant photolesion. However, the cytotoxic and mutagenic properties of this damage are not well established, mainly due to the difficulties in detecting DewarPP. In this respect, we analyzed the capacity of CHO cells to remove DewarPP, as well as CPD and (6-4)PP, using the same immunological approach. Furthermore, in order to investigate the possible effects of other types of lesions, i.e. DNA strand breaks and oxidative DNA damage, on the repair of the bipyrimidine photoproducts, CHO cells were irradiated with UVB, UVA radiation, or SSL. We demonstrate that DewarPP are removed with similar kinetics and efficiency as CPD, although they are valence isomers of (6-4)PP. In contrast, the removal of (6-4)PP is almost complete within 6 h. The similarity between the responses of DewarPP and CPD suggests that DewarPP may contribute significantly to the mutagenesis that occurs after exposure to sunlight. UVC irradiation was performed using a germicidal lamp emitting primarily at 254 nm. Broad band UVB irradiation (290–320 nm) was carried out with a set of six 15-W fluorescent tubes (Vilber Lourmat, Torcy, France) having a spectral irradiance very similar to that of FS20 lamps. The incident light was filtered through a Schott WG 305 cut-off filter (thickness, 2 mm), which efficiently blocks contaminating wavelengths below 290 nm. Polychromatic UVA radiation was obtained from an Osram Ultramed 400-W gas discharge lamp, emitting through a 2-cm water layer held in Pyrex glass, and through a Schott WG345 filter (thickness, 3 mm) and an anticaloric KG1 filter. The delivered radiation comprised 60.8% UVA radiation (320–400 nm) including 0.05% UVA2 wavelengths (320–340 nm), 39.2% visible light, and less than 5 × 10−4% UVB (280–320 nm). The simulated solar light (SSL) was produced by a 2500-W xenon compact arc lamp (Conrad-Hanovia Inc., Newark, NJ) and passed through a Schott WG320 filter (thickness, 3 mm). The incident light was composed of 0.8% UVB, 6% UVA, 44.5% visible light, 48.7% infrared, and less than 10−5 % UVC. The proportions of UVB, UVA, visible light, and infrared in natural terrestrial sunlight are approximately 0.3%, 5.1%, 62.7%, and 31.9%, respectively. Emission spectra after filtration were previously presented (13Alapetite C. Wachter T. Sage E. Moustacchi E. Int. J. Radiat. Biol. 1996; 69: 359-369Crossref PubMed Scopus (143) Google Scholar, 16Kuluncsics Z. Perdiz D. Brulay E. Muel B. Sage E. J. Photochem. Photobiol. B Biol. 1999; 49: 71-80Crossref PubMed Scopus (190) Google Scholar, 18Drobetsky E.A. Moustacchi E. Glickman B.W. Sage E. Carcinogenesis. 1994; 15: 1577-1583Crossref PubMed Scopus (21) Google Scholar). Percentage spectral irradiance values, which were normalized for the UV region (290–400 nm) only, are given under “Results.” UVC fluence rate (0.16 J·m−2·s−1) was measured with a Latarjet dosimeter, and the irradiation time did not exceed 4 min. UVB (12.5 J·m−2·s−1) and UVA (135 J·m−2·s−1) fluence rates were measured with a radiometer VLX 3W equipped with interferential filters (Vilber Lourmat, Torcy, France); the exposure time did not exceed 15 and 120 min, respectively. According to a YSI Kettering 65A thermopile (Yellow Spring Instruments, OH), the fluence rate for SSL was 1250 J·m−2·s−1. Irradiation lasted no longer than 45 min. The UV wavelengths contributed to approximately 6.8% of the total energy measured with the thermopile. CHO cell lines AT3–2 and UVL9 (kindly provided by G. Adair, University of Texas, Smithville, USA), which are, respectively, proficient and deficient in nucleotide excision repair (mutated in ERCC1gene), were used. Cells were routinely grown in α-minimal essential medium containing 10% fetal calf serum (Life Technologies, Inc.) and 16 μg/ml gentallin. 2–4 h prior to irradiation, 107cells were seeded on 60-mm dishes (Costar). Cells were then washed and irradiated with UVB, UVA, and SSL in phosphate-buffered saline (PBS) on ice to prevent repair during exposure. Irradiation with UVC was performed at room temperature, since short exposures were required. For photolesion quantification, NER− cells were used to avoid repair during long irradiation periods. After irradiation, cells, maintained on ice, were immediately scrapped from the dishes into ice-cold PBS buffer and pellets were stored at −20 °C until use. For repair experiments, confluent NER+ cells received 1 kJ·m−2 UVB, 1000 kJ·m−2 UVA, or 4500 kJ·m−2 SSL (equivalent to 306 kJ·m−2 UV energy, a conversion that was used throughout the experiments described here). The duration of irradiation corresponded to 160 s, 120 min, and 45 min, respectively. To study the repair of the DewarPP after UVB exposure, a dose of 5 kJ·m−2 was given to ensure sufficient induction of this photoproduct. In order to get enough DNA for the immuno-dot-blot (IDB) assay, one dish per repair time was used. Immediately after irradiation, cells were scrapped into cold PBS buffer and one half was pelleted and stored at −20 °C for the determination of the photolesion at time t = 0, whereas the other half was allowed to undergo repair for a set time in fresh medium at 37 °C. At times t = 2, 4, 6, and 24 h after irradiation, cells were harvested, washed, pelleted, and stored at −20 °C. At the repair time of 24 h, cellular growth was not observed. For DNA extraction, cells were lysed for 1 h at 37 °C in lysis buffer (20 mm Tris-HCl, pH 8, 20 mm NaCl, 20 mm EDTA, and 0.5% SDS). Samples were then incubated 4 h at 37 °C with 100 μg/ml RNase A, 5 units/ml RNase T1, and overnight at 37 °C with 10 μl of proteinase K (25 mg/ml, Roche Molecular Biochemicals). DNA was purified by two extractions with phenol and chloroform/isoamyl alcohol (25:24:1 v/v/v) and further precipitated by the cold ethanol procedure. The amount of DNA was determined spectrophotometrically (Shimadzu UV-160A spectrophotometer) on the basis of its absorbance at 260 nm. Cyclobutane pyrimidine dimers, (6-4)PP, and DewarPP were detected by using TDM-2, 64M-2, and DEM-1 monoclonal antibodies, respectively (24Mori T. Nakane M. Hattori T. Matsunaga T. Ihara M. Nikaido O. Photochem. Photobiol. 1991; 54: 225-232Crossref PubMed Scopus (389) Google Scholar, 25Matsunaga T. Hatakeyama Y. Ohta M. Mori T. Nikaido O. Photochem. Photobiol. 1993; 57: 934-940Crossref PubMed Scopus (64) Google Scholar). For IDB analysis (slightly modified from Ref.26Eveno E. Bourre F. Quilliet X. Chevallier-Lagente O. Roza L. Eker A.P.M. Kleijer W.J. Nikaido O. Stefanini M. Hoeijmakers J.H.J. Bootsma D. Cleaver J.E. Sarasin A. Mezzina M. Cancer Res. 1995; 55: 4325-4332PubMed Google Scholar), a triplicate of 500 ng of heat-denatured DNA per dot was loaded (Hybri-dot Manifold, Life Technologies, Inc.) on nitrocellulose membrane (0.2 μm, BA83; Schleicher & Schuell). After blotting, the dots were rinsed twice with 100 μl of PBS. Membranes were saturated overnight at 4 °C in PBS containing 5% nonfat dry milk (NFM) and 0.1% Tween 20 (Sigma) and then incubated for 1 h at 37 °C with TDM-2, 64M-2, or DEM-1 antibody (dilution 1/1000, 1/250, and 1/1000, respectively, in 0.5% NFM, 0.1% Tween 20, PBS). After extensive washing with 0.5% NFM, 0.1% Tween 20, PBS (NFM-TPBS), membranes were incubated 1 h at room temperature with a 1/2000 dilution of a second anti-mouse horseradish peroxidase-conjugated antibody (Calbag, San Francisco, CA) in NFM-TPBS buffer. Blots were then washed extensively with NFM-TPBS buffer, and peroxidase activity was revealed with the enhanced chemiluminescence blotting detection system (RPN2106, ECL™, Amersham Pharmacia Biotech). Membranes were immediately exposed to x-ray films (Kodak XAR) for different times depending on the antibody (for a given antibody, the same exposure time was always used). Relative luminescence intensity was determined using a Biocom image analyzer and Macroautorag software (Biocom, Les Ulis, France). In repair experiments, a streptavidin/biotin system was used to increase the luminescence signal for detection of the (6-4)PP after UVB. In this case, after incubation with 64M-2 antibodies in TPBS, membranes were washed extensively with TPBS and incubated 1 h at room temperature with biotinylated second antibody (1/5000 in TPBS, Calbag). Next, membranes were washed three times for 15 min with TPBS, incubated 30 min with streptavidin-peroxidase solution (1/5000 in TPBS), and further processed as described above. For each repair time within a given experiment, the percentage of remaining lesions was deduced by comparing the decrease in the luminescence intensity with the luminescence at time t = 0 h using the same set of irradiated cells. The fraction of damage remaining at various times was expressed as a mean value of eight determinations as follows: two series of irradiation experiments and two IDB assays per experiment were performed, and each sample was dotted in duplicate. The pZ189 plasmid DNA (5500 base pairs) was UVB-irradiated on ice in 10 mm sodium phosphate buffer, pH 7.5, at doses ranging between 0.5 and 5 kJ·m−2. The number of CPD per plasmid was determined by measuring the conversion of irradiated supercoiled plasmid (form I) DNA to the open circular (form II) following digestion with the pyrimidine dimer-specific enzyme T4 endonuclease V (DenV protein, obtained from Applied Genetics and from Dr. J. Brouwer (Leiden University, The Netherlands)). Briefly, 150 ng of pZ189 DNA was incubated for 30 min at 37 °C in 10 μl of reaction buffer (50 mm KH2PO4, 100 mm NaCl, 1 mm EDTA, 1 mm dithiothreitol, 0.1 mg/ml bovine serum albumin) with or without 0.2 μg of DenV protein. Samples were then electrophoresed through a 0.8% agarose gel in the presence of ethidium bromide. Photographic negatives of the gels were scanned using a Biocom image analyzer. The number of enzyme-sensitive sites per plasmid as a function of the dose was calculated from the Poisson distribution and corrected for differential binding of ethidium bromide to supercoiled versus relaxed DNA. CPD were also detected by TDM-2 antibody in irradiated plasmids after linearization and denaturation (triplicates of 500 ng) using the IDB assay as described above. The linearization procedure ensured complete denaturation of the plasmid molecules. The luminescence intensity was determined as a function of the dose. Finally, a calibration curve, which represented the luminescence intensity as a function of CPD/kbp, was obtained and used to calculate the number of CPD in the genomic DNA of irradiated cells. 400 ng of pCAT plasmid DNA (4610 base pairs; Promega, Madison, WI) was subjected to 10–40 J·m−2 UVC radiation. CPD were removed under UVA illumination by incubation of (250 ng) irradiated DNA for 1 h in 50 mm Tris-HCl, pH 7.4, 50 mm NaCl, 1 mm EDTA, 10 mm dithiothreitol, in the presence of 30 ng of photolyase from Escherichia coli (a generous gift from Dr. A. Sancar, University of North Carolina, Chapel Hill, NC). The UVA source used was a HPW 125 Philips lamp, which emits mainly at 365 nm; a dose of 50 kJ·m−2 was necessary for complete photoreversion of CPD. An aliquot of 100 ng of the treated plasmid was then digested with DenV protein to check for the completion of the photoreversion. After photoreversion of CPD, remaining photolesions (essentially (6-4)PP) were detected by UvrABC endonuclease. Briefly, DNA (125 ng) was incubated 5 min at 37 °C with 0.75 pmol of UvrA protein and 1.8 pmol of UvrB protein in reaction buffer (50 mm Tris-HCl, pH 7.5, 10 mmMgCl2, 85 mm KCl, 1 mmdithiothreitol, and 2 mm ATP). Then, 0.75 pmol of UvrC protein was added for 30 min at 37 °C and the reaction was stopped by adding 0.05% SDS and heating for 5 min at 65 °C. The samples were then electrophoresed, stained, photographed, and analyzed as described above. The number of (6-4)PP per kbp could therefore be established as a function of the UVC doses, which were chosen to produce less than one (6-4)PP per plasmid molecule. At each step a control reaction was performed on irradiated or unirradiated plasmid DNA in the absence of the protein. In the case of UvrABC digestion, a mock reaction was also carried out in the presence of only two proteins. In parallel, (6-4)PP were also detected in UVC-irradiated plasmid by monoclonal 64M-2 antibody by using the IDB assay, as described above. Similarly, the luminescence intensity obtained with 64M-2 antibody was determined as a function of the UVC dose, and a calibration curve (luminescence intensity as a function of (6-4)PP/kbp) was established. Genomic DNA from cells exposed to UVB radiation at doses ranging from 2 to 10 kJ·m−2 was used as substrate. Triplicates of 500 ng of DNA/dose were then subjected to IDB using 64M-2 and DEM-1 antibodies. The exact number of initial (6-4)PP per kbp was calculated from the calibration curve obtained using the 64M-2 antibody. In order to convert (6-4)PP into their DewarPP isomers, the remaining DNA was re-irradiated with a single dose (450 kJ·m−2) of UVA radiation (Osram Ultramed 400-W gas discharge lamp without cut-off filters; thus, at least 0.3% of the emitted photons were in the range of 295–316 nm; see above). This DNA was again subjected to IDB with the two antibodies. The number of (6-4)PP that disappeared following isomerization was deduced from the calibration curve for the 64M-2 antibody luminescence signal. Since one DewarPP is assumed to result from the photoisomerization of one (6-4)PP at wavelengths around 320 nm (6Taylor J.-S. Lu H.-F. Kotyk J.J. Photochem. Photobiol. 1990; 51: 161-167Crossref PubMed Scopus (106) Google Scholar, 27Douki T. Cadet J. J. Photochem. Photobiol. B Biol. 1992; 15: 199-213Crossref PubMed Scopus (63) Google Scholar), the number of (6-4)PP that disappeared during the UVA re-irradiation step corresponded to the number of DewarPP produced. For each initial UVB dose, the increase in the luminescence intensity obtained with the DEM-1 antibody after the photoisomerization process was correlated with the number of DewarPP per kbp, thus establishing a calibration curve for the DEM-1 luminescence signal. For cell survival determination, the CHO repair-proficient and deficient cells were irradiated by UVB radiation (0–5 kJ·m−2) or SSL (0–306 kJ·m−2) in PBS in 60-mm Petri dishes as described above, washed with PBS and trypsinized. Aliquots of 102 to 106 cells were seeded in triplicate into 60- or 100-mm dishes. After 10 days of incubation in growth medium, colonies were stained with methylene blue in methanol and counted. Survival was calculated as the ratio of the cloning efficiencies of irradiated over unirradiated cells × 100. The surviving fraction was plotted versus the amount of each of the three photoproducts. To calculate the correlation coefficient, all the experimental points obtained with each source of irradiation were considered as a single population, and the corresponding linear regression was determined. The correlation between damage induction and cell survival was considered as statistically significant whenp < 0.05 (from correlation table, for a risk of first order equal to 0.05 and a degree of freedom = n − 2; Ref. 28Schwartz D. Methodes Statistiques à l'Usage des Médecins et des Biologistes. Flammarion, Paris1963Google Scholar). We first determined the yield of formation of the three bipyrimidine photoproducts, CPD, (6-4)PP, and DewarPP in cellular DNA irradiated with the different UV components of solar radiation. NER-deficient CHO cells were irradiated with UVC, UVB, UVA radiation, and SSL. Bipyrimidine photoproducts were revealed by using the IDB technique with TDM-2, 64M-2, and DEM-1 antibodies, which recognize CPD, (6-4)PP, and DewarPP, respectively (24Mori T. Nakane M. Hattori T. Matsunaga T. Ihara M. Nikaido O. Photochem. Photobiol. 1991; 54: 225-232Crossref PubMed Scopus (389) Google Scholar, 25Matsunaga T. Hatakeyama Y. Ohta M. Mori T. Nikaido O. Photochem. Photobiol. 1993; 57: 934-940Crossref PubMed Scopus (64) Google Scholar), as described under “Experimental Procedures.” To establish the relationship between the antibody binding site and the absolute amount of photoproduct formed, a c" @default.
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