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Enhanced repair of cyclobutane pyrimidine dimers and improved UV resistance in photolyase transgenic mice

2002; Springer Nature; Volume: 21; Issue: 17 Linguagem: Inglês

10.1093/emboj/cdf456

1460-2075

Wouter Schul,

DNA Repair Mechanisms

Article2 September 2002free access Enhanced repair of cyclobutane pyrimidine dimers and improved UV resistance in photolyase transgenic mice Wouter Schul Wouter Schul MGC, Department of Cell Biology and Genetics, Center for Biomedical Genetics, Erasmus University Rotterdam, PO Box 1738, 3000 DR, Rotterdam, The Netherlands Search for more papers by this author Judith Jans Judith Jans MGC, Department of Cell Biology and Genetics, Center for Biomedical Genetics, Erasmus University Rotterdam, PO Box 1738, 3000 DR, Rotterdam, The Netherlands Search for more papers by this author Yvonne M.A. Rijksen Yvonne M.A. Rijksen MGC, Department of Cell Biology and Genetics, Center for Biomedical Genetics, Erasmus University Rotterdam, PO Box 1738, 3000 DR, Rotterdam, The Netherlands Search for more papers by this author Kyra H.M. Klemann Kyra H.M. Klemann MGC, Department of Cell Biology and Genetics, Center for Biomedical Genetics, Erasmus University Rotterdam, PO Box 1738, 3000 DR, Rotterdam, The Netherlands Search for more papers by this author Andre P.M. Eker Andre P.M. Eker MGC, Department of Cell Biology and Genetics, Center for Biomedical Genetics, Erasmus University Rotterdam, PO Box 1738, 3000 DR, Rotterdam, The Netherlands Search for more papers by this author Jan de Wit Jan de Wit MGC, Department of Cell Biology and Genetics, Center for Biomedical Genetics, Erasmus University Rotterdam, PO Box 1738, 3000 DR, Rotterdam, The Netherlands Search for more papers by this author Osamu Nikaido Osamu Nikaido Division of Radiation Biology, Faculty of Pharmaceutical Sciences, Kanazawa University, Kanazawa, 920-0934 Japan Search for more papers by this author Satoshi Nakajima Satoshi Nakajima Department of Molecular Genetics, Institute of Development, Aging and Cancer, Tohoku University, Sendai, 980-8575 Japan Search for more papers by this author Akira Yasui Akira Yasui Department of Molecular Genetics, Institute of Development, Aging and Cancer, Tohoku University, Sendai, 980-8575 Japan Search for more papers by this author Jan H.J. Hoeijmakers Jan H.J. Hoeijmakers MGC, Department of Cell Biology and Genetics, Center for Biomedical Genetics, Erasmus University Rotterdam, PO Box 1738, 3000 DR, Rotterdam, The Netherlands Search for more papers by this author Gijsbertus T.J. van der Horst Corresponding Author Gijsbertus T.J. van der Horst MGC, Department of Cell Biology and Genetics, Center for Biomedical Genetics, Erasmus University Rotterdam, PO Box 1738, 3000 DR, Rotterdam, The Netherlands Search for more papers by this author Wouter Schul Wouter Schul MGC, Department of Cell Biology and Genetics, Center for Biomedical Genetics, Erasmus University Rotterdam, PO Box 1738, 3000 DR, Rotterdam, The Netherlands Search for more papers by this author Judith Jans Judith Jans MGC, Department of Cell Biology and Genetics, Center for Biomedical Genetics, Erasmus University Rotterdam, PO Box 1738, 3000 DR, Rotterdam, The Netherlands Search for more papers by this author Yvonne M.A. Rijksen Yvonne M.A. Rijksen MGC, Department of Cell Biology and Genetics, Center for Biomedical Genetics, Erasmus University Rotterdam, PO Box 1738, 3000 DR, Rotterdam, The Netherlands Search for more papers by this author Kyra H.M. Klemann Kyra H.M. Klemann MGC, Department of Cell Biology and Genetics, Center for Biomedical Genetics, Erasmus University Rotterdam, PO Box 1738, 3000 DR, Rotterdam, The Netherlands Search for more papers by this author Andre P.M. Eker Andre P.M. Eker MGC, Department of Cell Biology and Genetics, Center for Biomedical Genetics, Erasmus University Rotterdam, PO Box 1738, 3000 DR, Rotterdam, The Netherlands Search for more papers by this author Jan de Wit Jan de Wit MGC, Department of Cell Biology and Genetics, Center for Biomedical Genetics, Erasmus University Rotterdam, PO Box 1738, 3000 DR, Rotterdam, The Netherlands Search for more papers by this author Osamu Nikaido Osamu Nikaido Division of Radiation Biology, Faculty of Pharmaceutical Sciences, Kanazawa University, Kanazawa, 920-0934 Japan Search for more papers by this author Satoshi Nakajima Satoshi Nakajima Department of Molecular Genetics, Institute of Development, Aging and Cancer, Tohoku University, Sendai, 980-8575 Japan Search for more papers by this author Akira Yasui Akira Yasui Department of Molecular Genetics, Institute of Development, Aging and Cancer, Tohoku University, Sendai, 980-8575 Japan Search for more papers by this author Jan H.J. Hoeijmakers Jan H.J. Hoeijmakers MGC, Department of Cell Biology and Genetics, Center for Biomedical Genetics, Erasmus University Rotterdam, PO Box 1738, 3000 DR, Rotterdam, The Netherlands Search for more papers by this author Gijsbertus T.J. van der Horst Corresponding Author Gijsbertus T.J. van der Horst MGC, Department of Cell Biology and Genetics, Center for Biomedical Genetics, Erasmus University Rotterdam, PO Box 1738, 3000 DR, Rotterdam, The Netherlands Search for more papers by this author Author Information Wouter Schul1, Judith Jans1, Yvonne M.A. Rijksen1, Kyra H.M. Klemann1, Andre P.M. Eker1, Jan de Wit1, Osamu Nikaido2, Satoshi Nakajima3, Akira Yasui3, Jan H.J. Hoeijmakers1 and Gijsbertus T.J. van der Horst 1 1MGC, Department of Cell Biology and Genetics, Center for Biomedical Genetics, Erasmus University Rotterdam, PO Box 1738, 3000 DR, Rotterdam, The Netherlands 2Division of Radiation Biology, Faculty of Pharmaceutical Sciences, Kanazawa University, Kanazawa, 920-0934 Japan 3Department of Molecular Genetics, Institute of Development, Aging and Cancer, Tohoku University, Sendai, 980-8575 Japan ‡W.Schul and J.Jans contributed equally to this work *Corresponding author. E-mail: [email protected] The EMBO Journal (2002)21:4719-4729https://doi.org/10.1093/emboj/cdf456 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info During evolution, placental mammals appear to have lost cyclobutane pyrimidine dimer (CPD) photolyase, an enzyme that efficiently removes UV-induced CPDs from DNA in a light-dependent manner. As a consequence, they have to rely solely on the more complex, and for this lesion less efficient, nucleotide excision repair pathway. To assess the contribution of poor repair of CPDs to various biological effects of UV, we generated mice expressing a marsupial CPD photolyase transgene. Expression from the ubiquitous β-actin promoter allowed rapid repair of CPDs in epidermis and dermis. UV-exposed cultured dermal fibroblasts from these mice displayed superior survival when treated with photoreactivating light. Moreover, photoreactivation of CPDs in intact skin dramatically reduced acute UV effects like erythema (sunburn), hyperplasia and apoptosis. Mice expressing the photolyase from keratin 14 promoter photo reactivate CPDs in basal and early differentiating keratinocytes only. Strikingly, in these animals, the anti-apoptotic effect appears to extend to other skin compartments, suggesting the presence of intercellular apoptotic signals. Thus, providing mice with CPD photolyase significantly improves repair and uncovers the biological effects of CPD lesions. Introduction Absorption of UV light energy by DNA induces various types of lesions. Although single- and double-strand breaks, as well as DNA–protein cross-links, can occur, >99% of the UV-induced damage consists of chemical base modifications, with cyclobutane pyrimidine dimers (CPDs) and pyrimidine (6–4) pyrimidone photoproducts (6–4PPs) as the most frequent types of photolesions (Mitchell, 1988). CPDs and 6–4PPs occur at two adjacent pyrimidines in the DNA and affect proper base pairing, which results in interference with key cellular processes like transcription and replication. Lesions in the DNA can lead to reduction of RNA synthesis, arrest of cell cycle progression, and induction of apoptosis. Moreover, persisting DNA damage can give rise to gene mutations that may allow cells to escape from controlled growth, which may ultimately lead to cancer (Friedberg et al., 1995). To counteract the deleterious effects of DNA damage produced by endogenous and environmental genotoxic agents (including UV-induced lesions), all organisms have developed a complex network of repair systems with complementary substrate specificity that keeps the DNA under continuous surveillance (Friedberg et al., 1995; Hoeijmakers, 2001). Removal of photolesions from the DNA is performed by the versatile and evolutionary highly conserved nucleotide excision repair (NER) pathway. NER is a complex multi-step process, and involves the concerted action of 30 or so proteins to sequentially execute damage recognition, chromatin remodeling, excision of a small oligonucleotide containing the damage, and gap-filling DNA synthesis followed by strand ligation (Friedberg et al., 1995; Wood, 1996; de Laat et al., 1999; Hoeijmakers 2001). The NER system is comprised of two subpathways. Global genome NER (GG-NER) operates genome-wide, but has the disadvantage that certain types of damage (like UV-induced CPDs) are less well recognized and accordingly less efficiently repaired. To prevent such lesions hampering transcription too long by stalling RNA polymerase II, a distinct NER subpathway has evolved, called transcription-coupled repair (TC- NER). This process directs the repair machinery, preferentially to the blocked polymerase on the template strand of actively transcribed DNA, and operates as a selective backup system for lesions that are slowly, or not at all, repaired by GG-NER. Inherited defects in NER genes cause photosensitive disorders such as xeroderma pigmentosum (XP, subdivided over seven complementation groups, XP-A through to -G) and Cockayne syndrome (CS, complementation groups A and B; for a review, see Bootsma et al., 2001). All XP patients are deficient in GG-NER and, with the exception of XP-C and -E, also in TC-NER. A specific defect in TC-NER is encountered in CS (Venema et al., 1990; van Hoffen et al., 1993). XP and CS patients display an increased sensitivity of the skin to UV light. In addition, XP patients have a highly elevated risk of developing UV-induced skin cancer. Obviously, the repair of UV-induced DNA damage strongly reduces the many undesirable consequences of UV exposure. It has proven difficult to disentangle the individual contribution of different types of UV lesions (i.e. CPDs and 6–4PPs) to the range of deleterious effects exerted by UV irradiation. CPDs are known to be repaired much slower by NER than 6–4PPs (Mitchell, 1988). In NER-proficient human fibroblasts, repair of most of the CPD lesions introduced by a modest dose of UV light takes >24 h, while 6–4PPs are removed within a few hours (Bohr et al., 1985). The GG-NER system is responsible for CPD removal anywhere in the genome, whereas CPDs that stall elongating RNA polymerase are selectively eliminated by TC-NER (Bohr et al., 1985; Mellon et al., 1987; van Hoffen et al., 1993). Recognition and repair of CPDs by GG-NER requires p53-dependent upregulation of the p48 subunit of the XP-E p48-p125 UV-DDB heterodimer (Hwang et al., 1999). Importantly, the p48 gene is not induced in rodent cells (Hwang et al., 1998); therefore, repair of CPDs is limited to the transcribed strand of active genes by TC-NER (Bohr et al., 1985; van der Horst et al., 1997). In contrast to CPD repair, both human and rodent cells efficiently remove 6–4PPs from their genome (Mitchell, 1988; van Hoffen et al., 1995), mainly performed by GG-NER. In the epidermis of humans and mice, keratinocytes remove CPDs and 6–4PPs in the same fashion as cultured fibroblasts (Hanawalt et al., 1981; Mitchell et al., 1990; Ruven et al., 1994; Qin et al., 1995). Some studies have pointed to the CPDs as the most cytotoxic and carcinogenic lesions (Broughton et al., 1990; Nishigaki et al., 1998; Chiganças et al., 2000), but, to date, the actual magnitude of the biological effects provoked by the different types of UV-induced DNA lesions has remained unclear. Many organisms of all kingdoms mount an additional repair system to remove UV lesions called photoreactivation. In contrast to the complex NER pathway, photoreactivation is performed by photolyases that rapidly convert UV lesions back to the original undamaged bases in a simple enzymatic reaction using visible light as an energy source. To perform this reaction, photolyases are equipped with two different chromophoric co-factors. Depending on the photolyase, either 5,10-methyl tetrahydrofolate (MTHF) or 8-hydroxy-5-deazaflavin (8-HDF) serve as light-harvesting antennas that pass on energy to reduced FAD, the chromophore that acts as the reaction center in dimer splitting (for a review, see Yasui and Eker, 1997). Notably, photolyases show substrate specificity for either CPDs (CPD photolyase) or 6–4PPs (6–4PP photolyase; for a review, see Yasui and Eker, 1997). Photolyases occur in bacteria, lower eukaryotes, plants and many animals including marsupials. Remarkably, despite the strong conservation of photoreactivation, this repair mechanism is absent in placental mammals (e.g. humans and mice), implying that photolyase genes have been lost during evolution of this subclass. The inability to photoreactivate CPDs and 6–4PPs leaves placental mammals with only the NER system for removal of these photolesions. Here, we report the generation and characterization of mice that express the CPD photolyase transgene from the marsupial Potorous tridactylis either in all tissues or specifically in the basal keratinocytes of the epidermis. Results Generation of β-actin promoter-driven CPD photolyase transgenic mouse and cell lines To generate mice ubiquitously expressing a CPD photolyase transgene, we assembled a construct containing the P.tridactylis CPD photolyase cDNA (Yasui et al., 1994), preceded by a cytomegalovirus (CMV) enhancer containing the chicken β-actin promoter and hooked up to human genomic β-globin sequences, including exons 2 and 3, intron 2, the 3′ untranslated region and the polyadenylation signal (Figure 1A). After oocyte injections, we obtained two independent β-actin promoter-driven photolyase mouse lines, designated β-act-CPD-1 and β-act-CPD-2 (carrying two and three copies of the transgene, respectively; data not shown). FISH analysis of metaphase chromosome spreads of mouse dermal fibroblasts (MDFs) isolated from β-act-CPD-1 and β-act-CPD-2 animals revealed integration of the photolyase transgene on chromosome 3C and 15A2, respectively (data not shown). Figure 1.Expression of the β-actin CPD photolyase gene in transgenic mice. (A) Expression construct for the generation of β-act-CPD photolyase transgenic mice, containing the CMV enhancer and chicken β-actin promoter, the P.tridactylis CPD photolyase cDNA, and human genomic β-globin sequences, including exons 2 and 3, intron 2, the 3′ untranslated region and the polyadenylation signal. Arrows indicate the position of the primers used for the RT–PCR experiment. (B) RT–PCR on skin extracts of β-act-CPD photolyase transgenic mice results in a 300 bp band. (C) Immunoblot analysis of protein extracts (30 μg per lane) from cultured fibroblasts obtained from β-act-CPD photolyase transgenic mice. The arrow indicates the position of the 62 kDa photolyase protein. (D) Immunocytochemical detection of CPD photolyase in cultured mouse dermal fibroblasts, using an affinity-purified anti-CPD photolyase. Nuclei are visualized by DAPI staining. Download figure Download PowerPoint RT–PCR analysis of total skin RNA showed expression and correct splicing of the CPD photolyase mRNA (Figure 1B). In addition, immunoblot analysis of protein extracts from wild-type, β-act-CPD-1 and β-act-CPD-2 MDFs, using a polyclonal antibody raised against the Potorous CPD photolyase, showed a band of the expected size (61 kDa), indicating expression of the full-length protein (Figure 1C). Immunocytochemical analysis of these cultured MDFs revealed a fluorescent signal in the nuclei of transgenic cell lines only, indicating correct nuclear localization of the CPD photolyase protein, probably due to a putative nuclear localization signal sequence (24-ARKKRK-29; Yasui et al., 1994) in the N-terminus of the protein (Figure 1D). Taken together, these data demonstrate that the two independent CPD photolyase mouse lines properly express the transgene. To facilitate genotyping, to avoid the potential risk of adverse side-effects originating from inactivation of loci at the transgene integration site, and to obtain transgenic and wild-type animals at equal amounts, mouse strains were kept in a heterozygous state. Importantly, β-act-CPD photolyase transgenic mice are born at a Mendelian ratio are fertile and, up to the age of 1 year, do not show any overt phenotype. This indicates that insertion of the CPD photolyase transgene does not interfere with development, viability and fertility. Moreover, β-act-CPD-1 and β-act- CPD-2 cells and animals are indistinguishable for all the parameters tested. Photoreactivation of CPDs in cultured CPD photolyase fibroblasts The transgenically expressed CPD photolyase is expected to allow lesion-specific repair of CPDs upon exposure to photoreactivating light. To investigate light-dependent removal of CPDs in MDFs, we have applied an immunocytochemical assay using antibodies specific for either CPDs or 6–4PPs (Mori et al., 1991). Cells were irradiated with 20 J/m2 of UV-C light and, as expected for both antibodies, a bright immunofluorescent signal could be detected in the nucleus of UV-treated cells, while non-irradiated cells remained unlabeled. This confirms the presence of both CPDs and 6–4PPs immediately after UV exposure (Figure 2). When cells were subsequently kept in the dark for 1 h (thus withholding the photolyase from the energy source required for enzymatic activity), CPD and 6–4PP staining remained present. In line with the known repair rates for both photolesions in rodents (Bohr et al, 1985; Mitchell, 1988), this indicates that, in the first hour after UV exposure, TC-NER can only remove a small fraction of the induced photolesions. In marked contrast, however, after exposure of UV-irradiated cells to 1 h of photoreactivating light, CPD labeling was hardly detectable, while the signal for 6–4PPs was comparable with that observed in cells kept in the dark (Figure 2). The lesion-specific and light-dependent removal of CPDs clearly demonstrates that the marsupial CPD photolyase is enzymatically active in mouse cells. Figure 2.Photoreactivation of CPDs in cultured CPD photolyase transgenic fibroblasts. Induction of CPD and 6–4PP lesions in cultured MDFs from β-act-CPD-1 photolyase mice by 20 J/m2 of UV-C light and the effect of subsequent exposure of cells photoreactivating light for 1 h. Photolesions were detected by immunofluorescent labeling, using CPD- or 6–4PP-specific antibodies and FITC-conjugated goat anti-mouse antibodies. Nuclei were visualized by DAPI staining. Download figure Download PowerPoint In repair-proficient mammalian cells, UV-induced DNA lesions are exclusively repaired by NER. From the previous experiment, it is evident that CPDs in UV-C exposed transgenic MDFs are rapidly removed by the CPD photolyase. Accordingly, it is expected that cells show reduced UV-induced NER activity after photoreactivation of CPD lesions. NER activity can be measured in cultured cells by the unscheduled DNA synthesis (UDS) assay, which quantifies the incorporation of radiolabeled thymidine in newly synthesized DNA in repair patches. As shown in Figure 3A, the relative UDS level in β-act- CPD-1 MDFs that were exposed to UV light and subsequently kept in the dark for 1 h drops to 75% of the UDS activity measured immediately after exposure to UV light. This difference reflects the NER activity during the first hour following UV exposure. As predicted, relative UDS levels decreased even further when UV-exposed cells were given 1 h of photoreactivating light. The reduction in NER activity confirms the effectiveness of the CPD photolyase and reveals the fraction of UDS derived from CPD repair. Figure 3.Effect of photoreactivation of CPDs in MDFs on NER activity and cellular survival. (A) NER activity in cultured MDFs from β-act-CPD-1 photolyase mice exposed to 16 J/m2 of UV-C light and the effect of subsequent exposure of cells to 1 h of photoreactivating light. UDS was measured by the incorporation of [3H]thymidine, followed by counting of grains above nuclei of non-dividing cells. UDS levels are expressed as the percentage of activity observed immediately after UV exposure. (B) UV survival of primary β-act-CPD-1 MDFs after exposure to increasing doses of UV-C light, with or without 1 h exposure to photoreactivating light. Error bars indicate the standard error of the mean. Similar results were obtained with β-act-CPD-2 MDFs. Download figure Download PowerPoint Next, we studied whether the fast removal of CPDs renders MDFs supplemented with a CPD photoreactiva ting system less sensitive to UV-C light. As shown in Figure 3B, photoreactivation of the photolyase MDFs gave rise to a higher percentage of surviving cells over the whole range of UV doses tested when compared with non-photoreactivated transgenic and wild-type cells. The dose-reducing effect of the CPD photolyase is ∼2-fold, meaning that photoreactivation increases the UV survival to the level normally observed at half the UV dose. Thus, NER-proficient mouse cells significantly benefit from CPD photoreactivation. Photoreactivation of CPDs in the mouse skin To investigate whether the skin, the prime target of UV light in the intact animal, also profits from the addition of the expressed CPD photolyase transgene, we analyzed the repair of CPDs in the dermis and epidermis of β-actin promoter-driven CPD photolyase transgenic mice. To this end, the immunofluorescent assay used to visualize photolesions in cultured MDFs was adapted for use on skin sections (see Materials and methods). One-third of a depilated area on the back of photolyase mice was covered, while the remaining part was exposed to 1 minimal erythemal dose (MED; the UV dose at which erythema starts to appear) of UV-B light. Next, half of the UV-exposed area, as well as the non-UV-exposed area, was covered, while the remaining part of the skin was exposed to photoreactivating light for 3 h. As expected, the non-UV-exposed part of the skin did not show any CPD labeling (data not shown), whereas the UV-irradiated/non-photoreactivated part of the skin contained clear nuclear labeling of CPDs in the epidermis and the upper part of the dermis (Figure 4, top panels). Importantly, exposure of the UV-irradiated skin to photoreactivating light resulted in a strong decrease in CPD labeling in epidermis and dermis. In contrast, labeling of 6–4PPs was not reduced detectably upon photoreactivation (data not shown). These data clearly demonstrate that the ubiquitously expressed CPD photolyase is active in all epidermal and dermal cells and that the enzyme can specifically photoreactivate the majority of CPDs in these cells within 3 h following UV exposure, leaving the 6–4PPs unaffected. In addition, these data show that both UV-B light and photoreactiva ting light do penetrate the epidermis and reach into the dermis. Figure 4.Photoreactivation of CPDs in the skin of β-act-CPD photolyase transgenic mice. Induction of CPD lesions in the depilated dorsal skin of β-act-CPD-1 photolyase mice by 1 MED of UV-B light and the effect of subsequent exposure to photoreactivating light for 3 h. Photolesions were detected by immunofluorescent labeling, using CPD-specific antibodies and FITC-conjugated goat anti-mouse antibodies. Nuclei are visualized by DAPI staining. Download figure Download PowerPoint Reduction of acute skin effects by photoreactivation of CPDs Exposure of the skin to (repeated) UV light induces apoptosis in the epidermis, accompanied by redness (erythema, commonly known as sunburn), swelling (edema), and followed several days later by thickening of the epidermis (hyperplasia). To investigate the effect of CPD photoreactivation on induction of apoptosis, the depilated dorsal skin of CPD photolyase mice was exposed to a single dose of 1 MED of UV-B light and partly exposed to photoreactivating light for 3 h, as described in the previous section. Next, animals were kept in the dark and, after 40 h, skin samples were processed for analysis of apoptosis by a TUNEL assay. In the absence of photoreactivating light, we observed a strong induction of apoptosis in the epidermis of UV-exposed skin with few apoptotic dermal cells (Figure 5, middle panels), a staining pattern very similar to that observed in UV-exposed wild-type animals (data not shown). The non-UV-exposed area of the skin remained unstained (Figure 5, top panels), indicating that apoptosis was specifically induced by exposure to UV light. In marked contrast, the photoreactivated UV-exposed skin showed little or no apoptotic signal (Figure 5, bottom panels). Similar results were obtained when animals were exposed to 1.5 MED of UV-B, or when the TUNEL assay was performed 24 h after UV irradiation (data not shown). Thus, photoreactivation of CPDs in CPD photolyase transgenic mice clearly reduces the apoptotic response. Figure 5.Effect of CPD photoreactivation on UV-induced apoptosis in the skin of β-act-CPD photolyase transgenic mice. Induction of apoptosis in the depilated dorsal skin of β-act-CPD-1 mice exposed to 1 MED of UV-B light, without (middle panels) or with (bottom panels) subsequent treatment of animals with photoreactivating light for 3 h. Non-UV-exposed animals were used as a control (upper panels). Except for the photoreactivation step, animals were kept in the dark immediately after UV treatment. Apoptosis was measured 40 h after UV exposure by a TUNEL assay and nuclei were visualized by DAPI staining. Download figure Download PowerPoint To study the induction of erythema and hyperplasia, we exposed the depilated dorsal skin of CPD photolyase mice to 1.5 MED of UV-B light per day for four consecutive days. Animals were continuously kept in the dark, except for animals that received 3 h of photoreactivating light immediately after each UV exposure. Three days after the last UV exposure, a clear redness and swelling was observed on the back of UV-treated animals that had not been exposed to photoreactivating light (Figure 6A). In marked contrast, photoreactivated animals did not display detectable swelling and only showed slight discoloration of the skin when compared with the unirradiated transgenic mice used as a control (Figure 6A). Histological examination of skin sections from these mice revealed that photoreactivation strongly reduced formation of epidermal hyperplasia, as clearly visible in samples from the non-photoreactivated UV-exposed skin (Figure 6B). Figure 6.Effect of CPD photoreactivation on UV-induced erythema and hyperplasia in the skin of β-act-CPD photolyase transgenic mice. The depilated back of β-act-CPD-1 transgenic mice was exposed to UV-B light for four consecutive days (1.5 MED per day) and were either given 3 h of photoreactivating light or kept in the dark. As a control, non-UV-exposed animals were used. Animals were killed 3 days after the last exposure and, except for the photoreactivation step, had been kept in the dark throughout the experiment. (A) Appearance of the dorsal skin of non-UV-exposed (left), UV-exposed (middle) and UV-exposed/photoreactivated animals (right), showing clear erythema when photoreactivation of UV-exposed animals is omitted. (B) Representative examples of hematoxilin/eosin stained sections of the dorsal skin of the mice shown in (A). Note the thick epidermal layer in UV-exposed skin that had not received photoreactivating light, indicative of the induction of hyperplasia. Download figure Download PowerPoint Taken together, these data demonstrate that fast removal of CPDs by photoreactivation has a clear protective effect on the skin of the CPD photolyase transgenic mice. Photoreactivation of CPDs in basal keratinocytes The ubiquitous expression of the CPD photolyase from the β-actin promoter results in the photoreactivating light-dependent removal of CPDs from epidermis and upper dermis (Figure 4). Since basal keratinocytes are the prime target for UV-induced carcinogenesis, we generated a second expression construct in which the β-actin promoter and CMV enhancer were replaced by the human K14 promoter (Figure 7A), which, in the skin, has been reported to be only active in basal keratinocytes of the epidermis (Vassar et al., 1989). Using this construct, we obtained a transgenic mouse line (K14-CPD-1) containing ∼25 copies of the photolyase transgene, as determined by Southern blot analysis of mouse genomic DNA (data not shown). RT–PCR analysis of total skin RNA, using the same primer set previously applied for detection of the β-actin promoter-derived CPD photolyase transcript, revealed that the transgene is transcribed and properly spliced (Figure 7B). A similar result was obtained when cultured basal keratinocytes from this transgenic mouse line were used (Figure 7B). This shows that the CPD photolyase transgene is expressed in basal keratinocytes. Heterozygous transgenic animals were born at the expected Mendelian frequency, were fertile and did not show an aberrant phenotype up to the age of 1 year. Figure 7.Expression of the K14-CPD photolyase gene in transgenic mice. (A) Expression construct for the generation of K14-CPD photolyase transgenic mice, containing the human K14 promoter, the P.tridactylis CPD ph