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Distribution and Repair of Bipyrimidine Photoproducts in Solar UV-irradiated Mammalian Cells

POSSIBLE ROLE OF DEWAR PHOTOPRODUCTS IN SOLAR MUTAGENESIS*
Open AccessPublished:September 01, 2000DOI:https://doi.org/10.1016/S0021-9258(19)61437-7
      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.
      CPD
      cyclobutane pyrimidine dimers
      (6–4)PP
      pyrimidine (6-4) pyrimidone photoproducts
      DewarPP
      Dewar photoproducts
      8-oxodGuo
      8-oxo-7,8-dihydro-2′-deoxyguanosine
      SSL
      simulated solar light
      NER
      nucleotide excision repair
      TCR
      transcription-coupled repair
      GGR
      global genomic repair
      IDB
      immuno-dot-blot
      W
      watt(s)
      CHO
      Chinese hamster ovary
      kbp
      kilobase pair(s)
      PBS
      phosphate-buffered saline
      TPBS
      phosphate-buffered saline plus Tween 20
      NFM
      nonfat milk
      Overwhelming evidence associates the steadily increasing incidence of skin cancer with an increased exposure to the UV components of sunlight (
      • IARC
      ). The UV induction of DNA damage is unambiguously an essential step in photocarcinogenesis (
      • Brash D.E.
      ). 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 (
      • Brash D.E.
      • Rudolph J.A.
      • Simon J.A.
      • Lin A.
      • McKenna G.J.
      • Baden H.P.
      • Halperin A.J.
      • Ponten J.
      ,
      • Daya-Grosjean L.
      • Dumaz N.
      • Sarasin A.
      ). 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 (
      • Mitchell D.L.
      • Pfeifer G.P.
      • Taylor J.-S.
      • Zdzienicka M.Z.
      • Nikaido O.
      ). The Dewar photoproducts (DewarPP), which are valence isomers of (6-4)PP formed via photoisomerization at wavelengths around 320 nm (
      • Taylor J.-S.
      • Lu H.-F.
      • Kotyk J.J.
      ,
      • Mitchell D.L.
      • Rosenstein B.S.
      ), 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.
      • Freeman S.E.
      • Hacham H.
      • Gange R.W.
      • Maytum D.J.
      • Sutherland J.C.
      • Sutherland B.M.
      ) 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 (
      • Tyrrell R.M.
      • Keyse S.M.
      ,
      • Kochevar I.E.
      • Dunn D.A.
      ). 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.
      • Sage E.
      for review; see also Refs.
      • Peak M.J.
      • Peak J.G.
      • Carnes B.A.
      ,
      • Alapetite C.
      • Wachter T.
      • Sage E.
      • Moustacchi E.
      ,
      • Kielbassa C.
      • Roza L.
      • Epe B.
      ,
      • Douki T.
      • Perdiz D.
      • Gróf P.
      • Kuluncsics Z.
      • Moustacchi E.
      • Cadet J.
      • Sage E.
      ). However, the direct effect of UVA has only recently been described (
      • Kielbassa C.
      • Roza L.
      • Epe B.
      ,
      • Douki T.
      • Perdiz D.
      • Gróf P.
      • Kuluncsics Z.
      • Moustacchi E.
      • Cadet J.
      • Sage E.
      ,
      • Kuluncsics Z.
      • Perdiz D.
      • Brulay E.
      • Muel B.
      • Sage E.
      ).
      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 (
      • Friedberg E.C.
      • Walker G.C.
      • Siede W.
      ). 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 (
      • Friedberg E.C.
      • Walker G.C.
      • Siede W.
      ).
      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 (
      • Drobetsky E.A.
      • Moustacchi E.
      • Glickman B.W.
      • Sage E.
      ,
      • Drobetsky E.A.
      • Turcotte J.
      • Chateauneuf A.
      ,
      • Sage E.
      • Lamolet B.
      • Brulay E.
      • Moustacchi E.
      • Chateauneuf A.
      • Drobetsky E.A.
      ). 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 (
      • Kuluncsics Z.
      • Perdiz D.
      • Brulay E.
      • Muel B.
      • Sage E.
      ).
      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 (
      • Douki T.
      • Perdiz D.
      • Gróf P.
      • Kuluncsics Z.
      • Moustacchi E.
      • Cadet J.
      • Sage E.
      ). 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 (
      • Clingen P.H.
      • Arlett C.F.
      • Roza L.
      • Mori T.
      • Nikaido O.
      • Green M.H.L.
      ,
      • Clingen 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.
      ,
      • Chadwick C.A.
      • Potten C.S.
      • Nikaido O.
      • Matsunaga T.
      • Proby C.
      • Young A.R.
      ). Clingen and colleagues (
      • Clingen 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.
      ) 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.

      RESULTS

      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 (
      • Mori T.
      • Nakane M.
      • Hattori T.
      • Matsunaga T.
      • Ihara M.
      • Nikaido O.
      ,
      • Matsunaga T.
      • Hatakeyama Y.
      • Ohta M.
      • Mori T.
      • Nikaido O.
      ), as described under “Experimental Procedures.” To establish the relationship between the antibody binding site and the absolute amount of photoproduct formed, a calibration procedure was developed.

      Quantification of the Bipyrimidine Photoproducts

      Antibody luminescence intensity was calibrated with respect to the absolute amount of CPD and (6-4)PP on the irradiated plasmid DNA according to the following protocol. Various amounts of CPD and (6-4)PP were produced by irradiating supercoiled plasmid DNA with various doses of UV irradiation (UVC or UVB). The number of CPD or (6-4)PP per plasmid molecule was determined as a function of the dose, using purified, specific DNA repair enzymes and a plasmid relaxation assay. In parallel, photoproducts were detected by using specific TDM-2 and 64M-2 antibodies in the IDB assay, and the relative luminescence intensity was determined as a function of the dose. These two determinations allowed us to establish a linear relationship between the luminescence signal and the number of CPD or (6-4)PP per kbp of DNA. The calibration curves were then used to calculate the amount of CPD or (6-4)PP in the genomic DNA of irradiated cells.
      To calibrate the luminescence signal of the TDM-2 antibody, plasmid DNA was UVB-irradiated and the number of CPD was determined following digestion with T4 DenV protein in the plasmid relaxation assay (data not shown). The luminescence signal induced by IDB increased as a function of the UVB dose. A calibration curve of the luminescence intensity as a function of CPD per kbp is given in Fig.1 A.
      Figure thumbnail gr1
      Figure 1Calibration curves for the antibody luminescence signals. A–C, TDM-2 (A), 64M-2 (B), and DEM-1 (C) antibodies. Each value represents the mean ± S.E. of three independent experiments.
      Since a specific enzyme for (6-4)PP was not available, a two-step protocol was employed to quantify this photolesion. We used the UvrABC endonucleolytic complex from E. coli, which recognizes a number of different lesions, including the UV-induced photoproducts, CPD, (6-4)PP, and DewarPP (
      • Svoboda D.L.
      • Smith C.A.
      • Taylor J.-S.A.
      • Sancar A.
      ). Prior to incision by the UvrABC complex, CPD were photoreversed by E. coli photolyase plus light (365 nm). To avoid the presence of DewarPP in the DNA substrate, UVC radiation was used as the damaging agent and the enzymatic reactions were performed immediately after UVC exposure of the plasmid. As a matter of fact, the photoreactivating light by itself did not generate a detectable amount of (6-4)PP or DewarPP. Other UV photolesions, i.e. monomeric cytosine hydrates, which can be recognized by UvrABC proteins, are at least 100 times less frequent than CPD and, thus, were not expected to interfere with the quantification of the dimeric photoproducts at the doses used (see Ref.
      • Sage E.
      for review). The complete photoreversion of CPD was verified using the T4 DenV protein (data not shown). In addition, (6-4)PP were detected by IDB on the same irradiated plasmid DNA samples, allowing us to establish the calibration dose-response curve for the 64M-2 antibody luminescence signal (Fig. 1 B).
      The procedure for quantifying DewarPP is described in Fig.2. It is based on the photoisomerization of (6-4)PP into DewarPP by radiation around 320 nm (
      • Taylor J.-S.
      • Lu H.-F.
      • Kotyk J.J.
      ,
      • Mitchell D.L.
      • Rosenstein B.S.
      ). Since TT and TC (6-4)PP exhibit maximal absorption of radiation between 310 and 340 nm (
      • Taylor J.-S.
      • Lu H.-F.
      • Kotyk J.J.
      ,
      • Smith C.A.
      • Taylor J.-S.
      ), we used for photoconversion an unfiltered broad-band UVA source whose emitted radiation was composed of at least 15% of the 320–340 nm wavelengths. Genomic DNA from CHO cells was irradiated at various doses of UVB, and an aliquot of the irradiated DNA was subjected to a first IDB analysis with 64M-2 and DEM-1 antibodies. The remaining DNA was subsequently re-irradiated with a UVA source for photoconversion (see “Experimental Procedures”) and subjected to a second IDB analysis with the same antibodies. According to the calibration curve for the 64M-2 antibody shown in Fig. 1 B, the decrease in luminescence obtained with the 64M-2 antibody after photoisomerization is a measure of the number of photoisomerized (6-4)PP. Assuming a quantitative conversion of (6-4)PP into DewarPP by the photoisomerization process (
      • Taylor J.-S.
      • Lu H.-F.
      • Kotyk J.J.
      ,
      • Douki T.
      • Cadet J.
      ), the increase in luminescence obtained with the DEM-1 antibody can be expressed as the number of DewarPP produced or (6-4)PP lost by photoisomerization. Since several UVB doses were employed, the calibration curve for luminescence signal obtained with DEM-1 antibodies could be established (Fig.1 C). It must be noted that the UVA dose for photoisomerization was not sufficient to elicit the formation of either (6-4)PP or DewarPP (see below).
      Figure thumbnail gr2
      Figure 2Strategy for the quantification of Dewar photoproducts.

      CPD Induction

      The CPD induction obtained with the four types of radiation is displayed in Fig. 3. The saturation observed for UVB and to a lesser extent for UVC is likely to be due to photoreversion of this lesion. Results in TableI indicate that the rate of CPD formation by UVB radiation was 100 times lower than for UVC, and about 1000 times higher than for UVA. When only the UV portion (6.8% of the total emitted energy) is taken into account in the determination of the yield of CPD, SSL was 100 times less efficient than UVB in producing CPD. Such yields of CPD in the DNA of irradiated cells were very similar to those obtained on isolated plasmid DNA irradiated under the same conditions (
      • Kuluncsics Z.
      • Perdiz D.
      • Brulay E.
      • Muel B.
      • Sage E.
      ). In addition, the maximal SSL and UVA doses used in these experiments correspond to a sunlight exposure of 3.5–4 h in summer at zenith in Paris, in terms of production of CPD (
      • Kuluncsics Z.
      • Perdiz D.
      • Brulay E.
      • Muel B.
      • Sage E.
      ).
      Figure thumbnail gr3
      Figure 3Quantification of cyclobutane pyrimidine dimers in DNA of CHO cells exposed to UVC, UVB, SSL, and UVA radiation. Two independent series of cells were used for each type of radiation. Two IDB experiments were performed for each DNA sample, which was loaded in triplicate. Each point represents the mean ± S.E. of 12 values. Means were compared using a three-way analysis of variance (dose, irradiation experiments, IDB) with three repeated measurements for one factor (IDB). Dose effect on DNA damage formation was considered as statistically significant whenp < 0.05.
      Table IYields of formation of CPD, (6-4)PP, and DewarPP in the DNA of cells exposed to UV radiation
      Lesion/kbp/J · m−2Ratio
      CPD(6–4)PPDewarPP(6–4)/CPDDewar/CPDDewar/(6–4)
      UVC2.2 × 10−25.3 × 10−3ND1:4
      UVB2.0 × 10−42.4 × 10−52.9 × 10−61:81:701:8
      UVA3.0 × 10−7NDND
      SSL
      The yields of photoproducts were calculated taking into account only the UV region (6.8%) of the whole emitted radiation.
      2.1 × 10−63.7 × 10−71.3 × 10−71:61:161:3
      The yields were calculated from the slope of the regression lines given in Figs. Figure 3, Figure 4, Figure 5. For UVC- and UVB-induced CPD and (6-4)PP, only the linear parts of the curves were used. ND, not detectable at the physiological doses used.
      1-a The yields of photoproducts were calculated taking into account only the UV region (6.8%) of the whole emitted radiation.
      Since the formation of CPD by UVA radiation has been a matter of debate for over 20 years, we calculated the relative efficiency of different regions of the UV spectrum for the production of this damage using the action spectrum reported by Kielbassa et al. (
      • Kielbassa C.
      • Roza L.
      • Epe B.
      ) and the spectral distributions of our UV sources (
      • Kuluncsics Z.
      • Perdiz D.
      • Brulay E.
      • Muel B.
      • Sage E.
      ). TableII shows that the majority of CPD (86.7%) formed with our UVA irradiation device were produced at wavelengths above 345 nm and that the very weak UVB emission did not contribute significantly to their formation (5 × 104% of total emission). Furthermore, since a WG345 Schott filter was used to eliminate UVC and UVB wavelengths, the contribution of UVA2 wavelengths (320–345 nm) to the formation of this photolesion was low (1.8%). In contrast, among CPD produced by SSL, 89% were produced by the UVB component and 11% in the UVA range that comprised 8% by UVA2 wavelengths (320–345 nm). As expected, virtually all the CPD (99.6%) induced by the UVB source were formed by the absorption of photons in the 290–320-nm range of the spectrum.
      Table IIPhysical characteristics (% irradiance) and calculated biological effectiveness (% CPD yield) of the UV sources used
      RegionSource
      UVBUVASSL
      UVB (290–320 nm)Irradiance (%)38.80.00774.6
      CPD yield (%)99.611.589.1
      UVA (320–400 nm)Irradiance (%)61.2100 95.4
      CPD yield (%)0.488.510.9
      UVA2 (320–345 nm)Irradiance (%)43.90.724.2
      CPD yield (%)0.41.88.2
      UVA1 (345–400 nm)Irradiance (%)19.499.371.8
      CPD yield (%)086.73.5
      The details of the calculations have been extensively described by Douki et al. (
      • Douki T.
      • Perdiz D.
      • Gróf P.
      • Kuluncsics Z.
      • Moustacchi E.
      • Cadet J.
      • Sage E.
      ). Values of spectral irradiances for the various intervals are based on measurements of the spectral irradiances in 1-nm steps for the given UV source. CPD spectral effectiveness was calculated on the basis of the action spectrum for CPD formation from Ref.
      • Kielbassa C.
      • Roza L.
      • Epe B.
      .
      These data clearly demonstrate that the production of CPD not only depends on the energy distribution and the dose received by the sample, but also on the interaction between DNA and each type of photon. We next calculated an enhancement factor for the relative efficiency of the 290–320 nm wavelengths in producing CPD. TableIII gives the ratio of irradiance in the UVB region to irradiance in the UVA range for the different lamps and for comparison, the ratio of CPD production in the UVB and UVA regions. Interestingly, the very low contribution of UVB energy in the filtered emission spectrum of our UVA lamp (the ratio UVB/UVA equals 7.7.105) leads to a relatively high efficiency of CPD formation (a ratio UVB/UVA of 0.13). In other words, the rare UVB photons emitted by our UVA source are far more efficient than the abundant UVA photons in producing CPD. Indeed, an enhancement factor of 1695 was calculated. This enhancement factor is only 406 and 169 for the UVB source and the solar simulator, respectively. In addition, it appears that even though the filtered emission of our UVB lamp is composed of about 60% of UVA wavelengths, the biological effect studied here is purely a UVB-induced effect (Tables II and III). Altogether, these calculations substantiate the importance of the spectral distribution of a given source in the manifestation of a biological effect.
      Table IIIRelative spectral effectiveness for CPD formation
      Source
      UVB, UVA, and SSL correspond to the UV sources used in the experiments, and as specified under “Experimental Procedures.”
      UVBUVASSL
      Irradiance UVB/UVA
      Ratio of the sum of irradiances between 290 and 320 nm (UVB) to those between 320 and 400 nm (UVA) for the different UV sources.
      0.6357.68 × 10−50.048
      CPD UVB/UVA
      Ratio of CPD yields due to UVBversus UVA region. The CPD yields were calculated as the products of the measured spectral distribution of the given source and the CPD's spectral sensitivity for the interval considered.
      2570.138.2
      Enhancement factor
      The enhancement factor represents the ratios of CPD yields normalized by the irradiance ratios. It highlights the relative importance of the UVB component of a lamp's emission spectrum for CPD induction.
      4061695169
      3-a UVB, UVA, and SSL correspond to the UV sources used in the experiments, and as specified under “Experimental Procedures.”
      3-b Ratio of the sum of irradiances between 290 and 320 nm (UVB) to those between 320 and 400 nm (UVA) for the different UV sources.
      3-c Ratio of CPD yields due to UVBversus UVA region. The CPD yields were calculated as the products of the measured spectral distribution of the given source and the CPD's spectral sensitivity for the interval considered.
      3-d The enhancement factor represents the ratios of CPD yields normalized by the irradiance ratios. It highlights the relative importance of the UVB component of a lamp's emission spectrum for CPD induction.

      (6–4)PP and DewarPP Induction

      Under our conditions, (6-4)PP were readily produced by UVC, UVB, and SSL (Fig.4) but not by UVA radiation. Fig.5 shows the linear induction of DewarPP as a function of UVB and SSL doses. DewarPP was not detected after UVC or UVA exposure. Using the same UVA doses as those used for eliciting CPD, both (6-4)PP and DewarPP were detected in trace amounts when a more sensitive IDB method was employed (data not shown). For reliable quantification, very high, non-biologically relevant UVA doses would have been necessary. Table I indicates that UVB is about 2 × 102 times less effective than UVC and about 60 times more efficient than SSL in producing (6-4)PP, but only 20 times more efficient than SSL for DewarPP formation.
      Figure thumbnail gr4
      Figure 4Quantification of pyrimidine (6-4) pyrimidone in the DNA of CHO cells exposed to UVC, UVB, and SSL radiation. Irradiation experiments and data analysis were performed as described in the legend of Fig. .
      Figure thumbnail gr5
      Figure 5Quantification of Dewar photoproducts in DNA of CHO cells exposed to UVB and SSL radiation. Irradiation experiments and data analysis were performed as described in the legend of Fig. .

      Relative Distribution of Bipyrimidine Photoproducts at Different UV Wavelengths

      Table I demonstrates that the relative induction of the three bipyrimidine photolesions varies depending on the type of radiation. For example, the ratio of CPD to (6-4)PP is about 4–5 for UVC and about 8 and 6 for UVB and SSL respectively. This is in favor of a partial photoisomerization of (6-4)PP into DewarPP by long UV wavelengths. Indeed, the ratio of (6-4)PP to DewarPP is about 8 for UVB and 3 for SSL. These data demonstrate that DewarPP are relatively more frequent in cellular DNA upon SSL exposure than upon UVB irradiation. Consequently, this last photolesion should be taken into account when considering the genotoxicity of sunlight.

      Repair Rates of CPD, (6-4)PP, and DewarPP

      In order to compare the rates of removal of CPD, (6-4)PP, and DewarPP from the genomic DNA of NER-proficient CHO cells exposed to UVB, UVA radiation, or SSL, the photoproducts were measured at various times after exposure by the IDB assay. In general, a unique dose of radiation was chosen to ensure the detection of all three photolesions and their progressive removal in the same cellular population. Cells received 1 kJ·m2 UVB radiation, 1000 kJ·m2 UVA radiation, and 306 kJ·m2 SSL (corresponding to 3.5–4 h of sun exposure at midday in summer in term of CPD induction (Ref.
      • Kuluncsics Z.
      • Perdiz D.
      • Brulay E.
      • Muel B.
      • Sage E.
      )). These treatments led to survival rates of 15%, 30%, and 10%, respectively. In order to provide a sufficient initial amount of DewarPP and to follow the fate of these lesions, a dose of 5 kJ·m2 UVB radiation was also used.
      Fig. 6 A shows that CPD induced by UVA were slowly and inefficiently removed in CHO cells. At similar survival levels (10–30%), their repair rates were not significantly different from the rates of CPD produced by UVB or SSL (Fig.6 A). However, UVB radiation leads to a large excess of CPD (
      • Kielbassa C.
      • Roza L.
      • Epe B.
      ,
      • Douki T.
      • Perdiz D.
      • Gróf P.
      • Kuluncsics Z.
      • Moustacchi E.
      • Cadet J.
      • Sage E.
      ), whereas UVA radiation and SSL produce substantial amounts of 8-oxodGuo and/or single strand breaks (
      • Peak M.J.
      • Peak J.G.
      • Carnes B.A.
      ,
      • Kielbassa C.
      • Roza L.
      • Epe B.
      ,
      • Douki T.
      • Perdiz D.
      • Gróf P.
      • Kuluncsics Z.
      • Moustacchi E.
      • Cadet J.
      • Sage E.
      ). This indicates that the presence of oxidative DNA lesions (which are formed at levels similar to those of CPD after UVA (
      • Douki T.
      • Perdiz D.
      • Gróf P.
      • Kuluncsics Z.
      • Moustacchi E.
      • Cadet J.
      • Sage E.
      ) and are rapidly repaired) does not interfere with the repair of CPD. The CPD induction is 0.22 and 0.25 per kbp for UVB and UVA irradiation, respectively (Table IV), which corresponds to the level of CPD obtained with about 15 J·m2UVC. Between 51% and 76% of CPD were still present 24 h after cell exposure to the different UV sources. This high residual level of CPD is probably not due to saturation of NER pathways since (6-4)PP were removed quickly and efficiently under the same conditions (see below).
      Figure thumbnail gr6
      Figure 6Repair of CPD (A), (6-4)PP (B), and DewarPP (C) in CHO cells. Cells were analyzed at different times after receiving 1 kJ·m2 (A and B) or 5 kJ·m2 (C) UVB (diamond), 1000 kJ·m2 UVA (circle), and 306 kJ·m2 SSL (square). Each data point is the mean value calculated from four IDB assays from two independent irradiation experiments, in which samples were dotted in duplicate on membrane.Bars represent the standard error of the mean values.Insets shows representative IDB assays on genomic DNA from UVB (A and B) or SSL (C) irradiated cells.
      Table IVNumber of bipyrimidine photoproducts (per kbp) immediately after irradiation and at t = 24 h repair time
      PhotoproductsRadiationPhotoproducts per kbp% Remaining damage
      t = 0 ht = 24 h
      %
      CPDUVB (1 kJ · m−2)0.220.1568
      SSL (306 kJ · m−2)0.670.3451
      UVA (1000 kJ · m−2)0.250.1976
      (6–4)PPUVB (1 kJ · m−2)0.02400
      SSL (306 kJ · m−2)0.110.0044
      DewarPPUVB (5 kJ · m−2)0.0150.00747
      SSL (306 kJ · m−2)0.03060.02478
      Quantitatively, the (6-4)PP lesion represents the second major type of UV-induced photolesion (Tables I and IV). Fig. 6 B shows that 90% of (6-4)PP were removed 6 h after exposure and that removal was almost complete after 24 h. The repair kinetics of cells exposed to UVB and SSL are undistinguishable and fit a first-order exponential decay function. The half-life of (6-4)PP is between 2 and 2.5 h. These data confirm that (6-4)PP are rapidly and efficiently eliminated compared with the poor repair of CPD (
      • Friedberg E.C.
      • Walker G.C.
      • Siede W.
      ).
      The capacity of CHO cells to remove DewarPP was also examined after exposure to UVB and SSL. The two repair kinetics shown in Fig.6 C are not statistically different (p > 0.05), and resemble those of CPD. The repair rate of DewarPP follows a two-step process. The repair rates of CPD and DewarPP (Fig. 6,A and C) do not exhibit significant differences.

      Relationship between Induction of Bipyrimidine Photoproducts and Cytotoxicity

      Since the role of each of the three bipyrimidine photoproducts in UV-induced lethality may vary depending on the cell type (
      • Clingen 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.
      ), we examined the response of the repair-proficient CHO AT3–2 cells. Survival, assayed by determining clonogenic efficiencies, was plotted as a function of the amount of CPD, (6-4)PP, and DewarPP produced by UVB radiation and SSL, which produce significant amounts of all three lesions (Fig. 7, A,B, and C). Insets in Fig. 7 show that cell killing was well correlated with the formation of CPD (r = −0.71, p < 0.05) and also of (6-4)PP (r = −0.68, p < 0.05). Since we observed that 90% of (6-4)PP were removed within 6 h after UV exposure, the contribution of this lesion to the loss of the clonogenic efficiency of the irradiated cells is questionable. This discrepancy may be due to the parallel induction of CPD and (6-4)PP (
      • Rosenstein B.
      • Mitchell D.L.
      ). However, high correlation coefficients for CPD and (6-4)PP were observed while investigating the role of these lesions in the lethality of the repair-deficient UVL9 cell line upon UVB and SSL irradiation. Ther values were −0.85 and −0.77 (p < 0.05) for CPD and (6-4)PP, respectively. In the absence of repair, the lethality at a given dose of radiation depends on the intrinsic cytotoxicity of the lesion and its amount. Consequently, both lesions seem to be cytotoxic.
      Figure thumbnail gr7
      Figure 7Correlation between CPD (A), (6-4)PP (B), and DewarPP (C) induction and the survival of NER-proficient AT3–2 cells irradiated by UVB (diamond) and SSL (square). Bars represent the standard errors of the mean values. Ininsets, all the experimental points are considered to be a single population, and the corresponding regression line is obtained.r 2 is the square of the correlation coefficient from the linear regression. The correlation is statistically significant (p < 0.05) for CPD and (6-4)PP.
      There was no statistically significant correlation between the induction of DewarPP and the lethality of irradiated NER+cells (r = −0.25, p > 0.05), whereas a statistically significant but loose correlation was observed in irradiated NER cells (r = − 0.64,p = 0.05). This weak (or null for NER+) correlation may not be surprising considering the low production of DewarPP in comparison to CPD (Table I), at least in the case of UVB exposure (ratio 1:70). DewarPP formation depends on the formation of (6-4)PP, but the action spectra of these lesions are very different (see in the discussion). The LD10 values (amount of damage leading to 10% survival, or lethal dose) calculated for SSL-irradiated NER cells are 0.12 CPD, 0.015 (6-4)PP, and 0.006 DewarPP per kbp. This apparently suggests that the former photolesion is the least cytotoxic. However, the ratios between the LD10 values for both the NER+ and NER cell lines follow exactly the same ratios for the induction of these lesions upon SSL exposure. It seems that the lethality is governed by the extent of each lesion and not only by the intrinsic cytotoxicity of these lesions. These data clearly demonstrate that quantitative analysis of damage induction is important for our understanding of how each photoproduct contributes to biological repercussions of solar UV radiation.

      DISCUSSION

      We quantitatively analyzed the induction of the three bipyrimidine photoproducts, i.e. CPD, (6-4)PP, and DewarPP, in CHO cells exposed to the different UV components of solar radiation. The present study provides information about the absolute amounts of three photolesions produced by various regions of the UV spectrum and about how efficiently these types of DNA damage are produced by certain wavelengths. Furthermore, the repair rate of each of these photolesions was also examined at the genomic level.
      The yield of each of the bipyrimidine photoproducts was assessed using previously described monoclonal antibodies TDM-2, 64M-2, and DEM-1 (
      • Mori T.
      • Nakane M.
      • Hattori T.
      • Matsunaga T.
      • Ihara M.
      • Nikaido O.
      ,
      • Matsunaga T.
      • Hatakeyama Y.
      • Ohta M.
      • Mori T.
      • Nikaido O.
      ). These and similar monoclonal or polyclonal antibodies have already been largely used for the in vitro or in situdetection of these photolesions and to study their repair as well (
      • Mitchell D.L.
      • Rosenstein B.S.
      ,
      • Clingen P.H.
      • Arlett C.F.
      • Roza L.
      • Mori T.
      • Nikaido O.
      • Green M.H.L.
      ,
      • Clingen 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.
      ,
      • Chadwick C.A.
      • Potten C.S.
      • Nikaido O.
      • Matsunaga T.
      • Proby C.
      • Young A.R.
      ,
      • Eveno 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.
      ,
      • Roza L.
      • van der Wulp K.J.M.
      • MacFarlane S.J.
      • Lohman P.H.M.
      • Baan R.A.
      ,
      • Vink A.A.
      • Bergen Henegouwen J.B.A.
      • Nikaido O.
      • Baan R.A.
      • Roza L.
      ,
      • Fekete A.
      • Vink A.A.
      • Gaspar S.
      • Berces A.
      • Modos K.
      • Ronto G.
      • Roza L.
      ,
      • Mitchell D.L.
      • Greinert R.
      • de Gruijl F.R.
      • Guikers K.L.H.
      • Breitbart E.W.
      • Byrom M.
      • Gallmeier M.M.
      • Lowery M.G.
      • Volkmer B.
      ). The TDM-2 antibody, specific for CPD, binds preferentially to TT dimers, recognizes CT dimers and binds to a lesser extent to CC and TC dimers (
      • Mori T.
      • Nakane M.
      • Hattori T.
      • Matsunaga T.
      • Ihara M.
      • Nikaido O.
      ). TC (6-4)PP (
      • Douki T.
      • Zalizniak T.
      • Cadet J.
      ) and expectedly TC DewarPP predominate among these types of photoproducts and are ideal substrates for the 64M-2 and DEM-1 antibodies, respectively, but these antibodies can also recognize TT derivatives (
      • Matsunaga T.
      • Hatakeyama Y.
      • Ohta M.
      • Mori T.
      • Nikaido O.
      ). It thus appears that these antibodies are suitable for providing a good estimate of the yields of formation of these photolesions. In accord with earlier observations (
      • Clingen P.H.
      • Arlett C.F.
      • Roza L.
      • Mori T.
      • Nikaido O.
      • Green M.H.L.
      ,
      • Clingen 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.
      ,
      • Chadwick C.A.
      • Potten C.S.
      • Nikaido O.
      • Matsunaga T.
      • Proby C.
      • Young A.R.
      ,
      • Mori T.
      • Nakane M.
      • Hattori T.
      • Matsunaga T.
      • Ihara M.
      • Nikaido O.
      ,
      • Vink A.A.
      • Bergen Henegouwen J.B.A.
      • Nikaido O.
      • Baan R.A.
      • Roza L.
      ,
      • Fekete A.
      • Vink A.A.
      • Gaspar S.
      • Berces A.
      • Modos K.
      • Ronto G.
      • Roza L.
      ), we found that they were sensitive enough to detect the photoproducts induced at physiologically relevant doses of radiation, i.e. those that gave low toxicity in cultured cells or those of the normal human environment. However, in contrast to other studies (
      • Clingen 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.
      ,
      • Rosenstein B.S.
      • Mitchell D.L.
      ), the detection method reported here allowed us to examine for the induction of all three bipyrimidine photoproducts within the same dose range and for each of the UV sources. Importantly, the three photolesions could be quantified in the same irradiated DNA sample.

      CPD and UVA Radiation: Role in Solar Mutagenesis

      The yields of CPD formed by UVC and UVB radiation shown in Table I are in excellent agreement with those reported in the literature (
      • Roza L.
      • van der Wulp K.J.M.
      • MacFarlane S.J.
      • Lohman P.H.M.
      • Baan R.A.
      ,
      • Mitchell D.L.
      • Jen J.
      • Cleaver J.E.
      ,
      • Mullaart E.
      • Lohman P.H.M.
      • Vijg J.
      ,
      • Thomas D.C.
      • Okumoto D.S.
      • Sancar A.
      • Bohr V.A.
      ,
      • Zhang X.
      • Rosenstein B.S.
      • Wang Y.
      • Lebwohl M.
      • Mitchell D.L.
      • Wei H.
      ,
      • Cadet J.
      • Anselmino C.
      • Douki T.
      • Voituriez L.
      ). In addition, we observed induction of CPD by UVA radiation in mammalian cells (Fig. 3). This confirms our previous observation using plasmid DNA (
      • Kuluncsics Z.
      • Perdiz D.
      • Brulay E.
      • Muel B.
      • Sage E.
      ), and is in agreement with other studies (
      • Kielbassa C.
      • Roza L.
      • Epe B.
      ,
      • Zhang X.
      • Rosenstein B.S.
      • Wang Y.
      • Lebwohl M.
      • Mitchell D.L.
      • Wei H.
      ,
      • Tyrrell R.M.
      ). We further demonstrated that CPD are produced mostly by UVA1 wavelengths in the interval between 345–400 nm, which corresponds to the spectral irradiance of our UVA source (Table II). The yield of CPD produced by UVA follows exactly the DNA absorption at these wavelengths (
      • Sutherland J.C.
      • Griffin K.P.
      ) and Setlow's action spectrum (
      • Setlow R.B.
      ). Thus, compared with UVC, 105 higher doses of UVA are necessary to produce the same amount of CPD. Despite this difference, these UVA doses are still biologically relevant since they correspond to a few hours of sunlight exposure at zenith in summer (
      • Kuluncsics Z.
      • Perdiz D.
      • Brulay E.
      • Muel B.
      • Sage E.
      ). Similarly, (6-4)PP and DewarPP can also be produced by UVA radiation and detected at trace levels.
      D. Perdiz and E. Sage, unpublished data.
      Interestingly, we show in Table II that about 10% of CPD induced by SSL irradiation was formed by the UVA component. This is an additional argument indicating that UVA radiation may partly contribute to the DNA damage induced by sunlight, and consequently, UVA may participate to solar light mutagenesis more than previously expected. Indeed, a majority of the mutations induced by sunlight and found in skin tumors (
      • Brash D.E.
      • Rudolph J.A.
      • Simon J.A.
      • Lin A.
      • McKenna G.J.
      • Baden H.P.
      • Halperin A.J.
      • Ponten J.
      ,
      • Dumaz N.
      • Drougard C.
      • Sarasin A.
      • Daya-Grosjean L.
      ,
      • Bodak N.
      • Queille S.
      • Avril M.F.
      • Bouadjar B.
      • Drougard C.
      • Sarasin A.
      • Daya-Grosjean L.
      ) are GC to AT transitions and tandem double CC to TT mutations, all of which can be attributed to bipyrimidine photoproducts (mainly to CPD in repair-proficient non-XP individuals). Furthermore, the mutational specificity of UVA comprises a large amount of GC to AT transition located at bipyrimidine sites (
      • Drobetsky E.A.
      • Turcotte J.
      • Chateauneuf A.
      ,
      • Robert C.
      • Muel B.
      • Benoit A.
      • Dubertret L.
      • Sarasin A.
      • Stary A.
      ). This class of mutation is over-represented (65% of all events) in excision repair-deficient cells (
      • Sage E.
      • Lamolet B.
      • Brulay E.
      • Moustacchi E.
      • Chateauneuf A.
      • Drobetsky E.A.
      ). Such base changes are likely to be due to the CPD produced by UVA.

      DewarPP, a Major Lesion Induced by Sunlight

      The yield of (6-4)PP formation after UVC radiation (Table I) is in good agreement with that reported in Ref.
      • Thomas D.C.
      • Okumoto D.S.
      • Sancar A.
      • Bohr V.A.
      (no value available either for UVB or DewarPP). We observed that (6-4)PP were 200 times less efficiently formed by UVB than by UVC, which is in accordance with the published action spectrum for the induction of CPD and (6-4)PP (
      • Rosenstein B.
      • Mitchell D.L.
      ). DewarPP were not formed after UVC radiation at biologically relevant doses (Ref.
      • Clingen P.H.
      • Arlett C.F.
      • Roza L.
      • Mori T.
      • Nikaido O.
      • Green M.H.L.
      , and this report). However, they were produced by broad-band UVB (Ref.
      • Clingen 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.
      , and this report) and, moreover, were more extensively induced by natural (
      • Clingen P.H.
      • Arlett C.F.
      • Roza L.
      • Mori T.
      • Nikaido O.
      • Green M.H.L.
      ) or simulated sunlight (this report), reflecting the maximal absorption of (6-4)PP, which lies between 310 and 340 nm (
      • Taylor J.-S.
      • Lu H.-F.
      • Kotyk J.J.
      ,
      • Smith C.A.
      • Taylor J.-S.
      ).
      The comparative rates of the formation of CPD, (6-4)PP and DewarPP were 1:0.25 for UVC, 1:0.12:0.014 for UVB, and 1:0.18:0.06 for SSL. This illustrates that the distribution of the different classes of photolesion varies depending on the UV source, as already observed (
      • Clingen P.H.
      • Arlett C.F.
      • Roza L.
      • Mori T.
      • Nikaido O.
      • Green M.H.L.
      ,
      • Clingen 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.
      ,
      • Fekete A.
      • Vink A.A.
      • Gaspar S.
      • Berces A.
      • Modos K.
      • Ronto G.
      • Roza L.
      ), and clearly demonstrates that the biological effects of sunlight cannot be deduced solely on the basis of studies with UVC. Furthermore, DewarPP was formed with high yield, making it the third major photolesion induced by SSL (about 10 times more frequent than 8-oxodGuo (Ref.
      • Douki T.
      • Perdiz D.
      • Gróf P.
      • Kuluncsics Z.
      • Moustacchi E.
      • Cadet J.
      • Sage E.
      ) or cytosine hydrates (Ref.
      • Cadet J.
      • Anselmino C.
      • Douki T.
      • Voituriez L.
      )). This indicates that this lesion may contribute in a previously unsuspected manner to the effects produced by sunlight exposure.

      Differences in Kinetics and Efficiencies of CPD, DewarPP, and (6-4)PP Removal

      All three lesions are most likely eliminated by the NER process (
      • Svoboda D.L.
      • Smith C.A.
      • Taylor J.-S.A.
      • Sancar A.
      ), although the repair mechanism of DewarPP has not been elucidated in mammalian cells. We first observed that the rate of removal of all of these photolesions did not depend on the source of irradiation. This indicates that the presence of relatively large amounts of single strand breaks and 8-oxodGuo or other oxidized bases in UVA- or SSL- irradiated cells (
      • Peak M.J.
      • Peak J.G.
      • Carnes B.A.
      ,
      • Alapetite C.
      • Wachter T.
      • Sage E.
      • Moustacchi E.
      ,
      • Kielbassa C.
      • Roza L.
      • Epe B.
      ,
      • Douki T.
      • Perdiz D.
      • Gróf P.
      • Kuluncsics Z.
      • Moustacchi E.
      • Cadet J.
      • Sage E.
      ,
      • Rosenstein B.S.
      • Mitchell D.L.
      ) does not impair the processing of bipyrimidine photoproducts by the cellular machinery. On the other hand, SSB and oxidative damage are processed by repair pathways other than NER, i.e. base excision repair (
      • Wilson D.M.
      • Thompson L.H.
      ). In human cells, it has been shown that most strand breaks and a large proportion of oxidized bases are repaired within 1 and 4 h, respectively (
      • Rosenstein B.S.
      • Mitchell D.L.
      ,
      • Weiss R.B.
      • Gallagher P.E.
      ,
      • Collins A.R.
      • Ai-guo M.
      • Duthie S.J.
      ). The rate of removal of base damage is similar to that observed for (6-4)PP.
      The repair rate of CPD has been extensively studied in both rodent and human cells. We observed that, at most, 20% of CPD was eliminated after a repair time of 6 h and 25–50% after 24 h. This is within the range of published repair times for global genomic repair in cultured rodent cells or rodent skin under similar conditions of irradiation (
      • Vink A.A.
      • Bergen Henegouwen J.B.A.
      • Nikaido O.
      • Baan R.A.
      • Roza L.
      ,
      • Mullaart E.
      • Lohman P.H.M.
      • Vijg J.
      ,
      • Regan J.D.
      • Thompson L.H.
      • Carrier W.L.
      • Weber C.A.
      • Francis A.A.
      • Zdzienicka M.Z.
      ). In contrast, 70% of CPD are removed by TCR from expressed genes after 24 h (
      • Vreeswijk M.P.G.
      • van Hoffen A.
      • Westland B.E.
      • Vrieling H.
      • van Zeeland A.A.
      • Mullenders L.H.F.
      ). In accordance with the literature (
      • Mitchell D.L.
      ), we report fast and efficient removal of (6-4)PP in CHO cells after UVB and SSL, i.e. 90% removal within the 6-h period following irradiation (versus 3 h in human cells; Refs.
      • Eveno 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.
      ,
      • Rosenstein B.S.
      • Mitchell D.L.
      ,
      • Tung B.S.
      • McGregor W.G.
      • Wang Y.-C.
      • Maher V.M.
      • McCormick J.J.
      , and
      • Nakagawa A.
      • Kobayaschi N.
      • Muramatsu T.
      • Yamashina Y.
      • Shirai T.
      • Hashimoto M.W.
      • Ikenaga M.
      • Mori T.
      ). In contrast, only 50–60% of (6-4)PP were lost from active genes within the 6-h period following exposure of CHO cells to 30–40 J·m2 UVC (
      • Thomas D.C.
      • Okumoto D.S.
      • Sancar A.
      • Bohr V.A.
      ,
      • Vreeswijk M.P.G.
      • van Hoffen A.
      • Westland B.E.
      • Vrieling H.
      • van Zeeland A.A.
      • Mullenders L.H.F.
      ). Hairless mouse epidermis was even less efficient in removing (6-4)PP after treatment of the animals with a unique UVB dose (
      • Vink A.A.
      • Bergen Henegouwen J.B.A.
      • Nikaido O.
      • Baan R.A.
      • Roza L.
      ). Furthermore, a chronic low dose of UVB exposure significantly reduced the excision repair of both CPD and (6-4)PP in mice (
      • Mitchell D.L.
      • Greinert R.
      • de Gruijl F.R.
      • Guikers K.L.H.
      • Breitbart E.W.
      • Byrom M.
      • Gallmeier M.M.
      • Lowery M.G.
      • Volkmer B.
      ).
      DewarPP have received much less attention than other forms of DNA damage. Indeed there is only one report dealing with its repair rate (
      • Rosenstein B.S.
      • Mitchell D.L.
      ). We found that the repair rate of DewarPP was very similar to that of CPD, but much lower than that of (6-4)PP. After a repair time of 6 h, CHO cells did not remove more than 15–30% of DewarPP, and the repair efficiency was no more than 20–50% after 24 h. Using radioimmunoassay, Rosenstein and Mitchell (
      • Rosenstein B.S.
      • Mitchell D.L.
      ) observed a slightly faster repair rate of DewarPP in human skin fibroblasts than in the rodent cells described here. This is in agreement with reported differences in the repair kinetics between the two species (
      • Friedberg E.C.
      • Walker G.C.
      • Siede W.
      ).
      A conformational basis for the differences in the repair rates of the three bipyrimidine photoproducts may be tentatively considered. Most likely, the recognition of the photolesion by NER proteins, which bind to sites of DNA damage, is the rate-limiting step in the repair process. Most of the investigations report greater DNA unwinding by (6-4)PP and DewarPP than by cis-syn CPD, and a smaller distortion of DNA by DewarPP than by (6-4)PP (
      • Hwang G.-S.
      • Kim J.-K.
      • Choi B.-S.
      ,
      • Jing Y.
      • Kao J.F.L.
      • Taylor J.-S.
      ,
      • Taylor J.S.
      • Garrett D.S.
      • Cohrs M.P.
      ). This is in agreement with the 10-fold higher rate of excision of (6-4)PP over CPD by E. coli UvrABC excinuclease (
      • Svoboda D.L.
      • Smith C.A.
      • Taylor J.-S.A.
      • Sancar A.
      ) and human cell extracts (
      • Szymkowski D.E.
      • Lawrence C.W.
      • Wood R.D.
      ) and with the relative affinities of DNA damage binding proteins,E. coli UvrA and human UV-damaged DNA binding (UV-DDB) protein for (6-4)PP, DewarPP, and CPD (10, 4–2.8, and 1, respectively; Ref.
      • Reardon J.T.
      • Nichols A.F.
      • Keeney S.
      • Smith C.A.
      • Taylor J.-S.
      • Linn S.
      • Sancar A.
      ). However, DewarPP are excised at the same rate as (6-4)PP by UvrABC excinuclease (
      • Svoboda D.L.
      • Smith C.A.
      • Taylor J.-S.A.
      • Sancar A.
      ), and at the same rate and extent as CPD in CHO cells (Fig. 6, A and C). The fast removal of (6-4)PP by GGR may be due to the tight binding of XPC/hHR23B to the highly distorted region carrying (6-4)PP, whereas UV-DDB activity, which is absent from certain xeroderma pigmentosum E groups and hamster cells (
      • Hwang B.J.
      • Ford J.M.
      • Hanawalt P.C.
      • Chu G.
      ), may be required for targeting CPD and DewarPP for GGR. It is tempting to speculate that DewarPP, as well as CPD, are efficiently repaired by TCR but not by GGR, and that (6-4)PP are mainly repaired by GGR. Another possibility could be that, due to the distortion of the DNA helix at the DewarPP site, the DewarPP is still able to bind XPC/hHR23B and other associated proteins, but with lower affinity compared with the affinity of these proteins for (6-4)PP. Thus, according to this hypothesis, the DewarPP lesion would be repaired by GGR, but with slow kinetics.

      Biological Relevance of DewarPP

      The relative contribution of CPD and (6-4)PP to UVC- and solar UV- induced mutagenesis has been a matter of debate for almost 20 years (
      • Haseltine W.A.
      ). Studies on site specific mutagenesis in E. coli and yeast has helped to elucidate the intrinsic mutagenicity of the three bipyrimidine photoproducts (
      • Banerjee S.K.
      • Christensen R.B.
      • Lawrence C.W.
      • Leclerc J.E.
      ,
      • Leclerc J.E.
      • Borden A.
      • Lawrence C.W.
      ,
      • Smith C.A.
      • Wang M.
      • Jiang N.
      • Che L.
      • Zhao X.
      • Taylor J.S.
      ,
      • Horsfall M.J.
      • Lawrence C.W.
      ). In short, (6-4)PP and DewarPP are more mutagenic than the corresponding CPD. In eucaryotic cells, it is likely that these lesions produce mutations via error-prone bypass by DNA polymerase ζ lacking proofreading activity (see Ref.
      • Wood R.D.
      for review and Ref.
      • Nelson J.R.
      • Lawrence C.W.
      • Hinkle D.C.
      ).
      As our results show that DewarPP are no longer quantitatively negligible compared with CPD lesions, especially at TC sites, it is legitimate to question about biological contribution of this type of lesion to solar UV-mutagenesis. Considering the fast and efficient repair of (6-4)PP and the slow and inefficient removal of CPD and DewarPP, most of the mutations that we observed in the repair-proficient CHO cells after SSL (
      • Drobetsky E.A.
      • Moustacchi E.
      • Glickman B.W.
      • Sage E.
      ) would appear to be due to CPD and DewarPP. Indeed, it has been previously emphasized that (6-4)PP are mainly responsible for the mutations in repair-deficient, but not in repair-proficient cells (
      • Mitchell D.L.
      • Pfeifer G.P.
      • Taylor J.-S.
      • Zdzienicka M.Z.
      • Nikaido O.
      ). The SSL-induced mutation spectrum in CHO cells was particularly marked by an increase in tandem double CC to TT events; such mutations are very rare after UVC (
      • Drobetsky E.A.
      • Moustacchi E.
      • Glickman B.W.
      • Sage E.
      ,
      • Drobetsky E.A.
      • Turcotte J.
      • Chateauneuf A.
      ,
      • Sage E.
      • Lamolet B.
      • Brulay E.
      • Moustacchi E.
      • Chateauneuf A.
      • Drobetsky E.A.
      ). At such sites, CPD are readily formed after SSL, representing about 20% of the total CPD content (
      • Sage E.
      ). Since we do not know the intrinsic mutagenicity of CC CPD in mammalian cells, and based on the fact that TT (6-4)PP and TT DewarPP, but not TT CPD, produced some tandem double mutations (
      • Smith C.A.
      • Wang M.
      • Jiang N.
      • Che L.
      • Zhao X.
      • Taylor J.S.
      ), it is tempting to suggest that at least part of the CC to TT double events occurring after irradiation of CHO cells with SSL are due to DewarPP. Interestingly, such mutational events have also been recovered in the mutated p53 gene of skin tumors from normal and XP individuals exposed to sunlight (
      • Brash D.E.
      • Rudolph J.A.
      • Simon J.A.
      • Lin A.
      • McKenna G.J.
      • Baden H.P.
      • Halperin A.J.
      • Ponten J.
      ,
      • Daya-Grosjean L.
      • Dumaz N.
      • Sarasin A.
      ). The determination of the yield of formation of these photolesions by the different UV components of solar light should help to estimate the risks associated with exposure to environmental sunlight or artificial sources such as sunbeds.

      Acknowledgments

      We are grateful to Yvette Rolland for excellent technical assistance and to Dr. P. Hughes and Y. Cohen for critical reading of the manuscript.

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