Depto. de Microbiologia, Instituto de Ciências Biomédicas, Universidade de São Paulo, São Paulo, Brazil. cfmmenck@usp.br
Photolyases are enzymes involved in the repair of ultraviolet light−induced DNA damage when activated by light absorption. The generation of photolyase transgenic mice now suggests that an improved understanding of the mechanisms of skin damage and carcinogenesis induced by sunlight is in the offing.
Photorepair is one of the most efficient systems for removing the major ultraviolet- (UV) induced DNA damages, cyclobutane pyrimidine dimer (CPD) and pyrimidine (6−4) pyrimidone photoproducts (6−4PPs). Specific photolyases revert each of these lesions to the original undamaged bases, using visible light as an energy source. These enzymes appeared early in evolution, and are present in the three domains of life, Archaea, Bacteria and Eukarya, which attests to their fundamental role in protecting organisms from UV light.
In vertebrates, photolyases are found in fish, amphibians, birds and mammals. Curiously, however, our ancestors seem to have lost this powerful protection mechanism after the split of marsupials and placental mammals (approximately 170 million years ago1), as CPD-specific photolyases have been found in marsupials, but no photorepair has been observed in placental mammals, including mouse and man (Fig. 1). The recently published work by Wouter Schul and colleagues in the EMBO Journal2 has finally changed this evolutionary destiny. Specifically, the authors generated transgenic mice expressing a CPD-photolyase from the rat kangaroo (Potorous tridactylus). Of course, this was not done to build a better mouse, but to improve our understanding of the biological effects of CPDs and other UV lesions, especially in the skin.
Of mice and marsupials The fact that the marsupial photolyase functions to remove CPDs efficiently in mice, as reported by Schul et al.2, is an interesting observation. These results, together with previous in vitro experiments with cultured cell lines3,
4, indicate that cells from placental organisms still provide the necessary conditions for CPD-photolyase action. Transgenic mice provide the means of investigating the protective action of CPD-photolyase directly in UV-B−irradiated skin.
Schul et al.2 irradiated the depilated backs of transgenic mice with UV-B light, and then tested for photorepair. The results are clear: photorepair reduces the effects that are commonly observed in the skin of humans who are inadvertently exposed to long periods of sunlight, including erythema (sunburn), edema and hyperplasia. The authors observed an efficient light-dependent reduction of CPDs (detected by specific antibodies) in skin cells. Damage removal correlated with a reduction of apoptotic cells in UV-B irradiated epidermis and dermis, as well as skin redness and swelling. Histological analysis also revealed that photoreactivation strongly reduces thickening of the epidermis (hyperplasia). The data imply that lesions eliminated by photolyase, CPDs, are the main cause of the deleterious consequences of UV-B light in skin.
As is the case for most new findings, the work presented by Schul et al.2 is both exciting and unexpected. As basal keratinocytes may be directly implicated in skin carcinogenesis, the authors also generated mice that express CPD-photolyase under the control of the human K14 promoter, which is normally active only in a specific window of keratinocyte differentiation. CPD photorepair was seen mainly in keratinocytes located in the basal region of epidermis, as lesions were still detected in fibroblasts of the dermis and in the differentiated cells of the epidermis, thus confirming that photolyase was expressed and active only in the basal and early differentiating keratinocytes. The surprise came when, under the same experimental conditions, no apoptotic cells were observed in the skin, even at the basal dermis and the differentiated epidermis where the CPDs were still present. The authors interpret this to be the result of intercellular anti-apoptotic signaling from the keratinocytes to other cell types. Although this is an interesting explanation, further study is needed to clarify this fascinating question. Low expression of the CPD-photolyase gene in dermis and differentiated epidermis could reduce the number of CPD lesions below that which is required to induce apoptosis, although the lesions would still be detected by immunological methods. Alternatively, the CPDs could be removed preferentially from specific chromatin sites, such as transcriptionally active genes, which may constitute essential signals for apoptosis5.
Photolyases and DNA repair Photolyase transgenic mice open new avenues to improve our understanding of the effects of light on skin, potentially answering questions not completely solved in vitro (Fig. 2). This includes the longstanding question of the relative roles of the two major UV-induced DNA lesions, CPDs and 6−4PPs. The specificity of photolyases points to CPDs as directly involved in the acute effects of UV in skin. However, transgenic mice expressing a functional 6−4PP-photolyase (from Arabidopsis thaliana) have been generated by the same group and, together with the double transgenic mice (CPD- and 6−4PP−photolyases), may contribute to discriminating the effects of each of these two lesions (and eventually other minor UV-induced lesions) in UV−light irradiated skin.
Figure 2. Schematic representation of the approaches used to distinguish the consequences of UV-induced lesions with photolyases.
The two types of lesions represented in a may be removed by specific photolyases and visible light. Initial experiments were done with cultured cells (b) and, more recently, with transgenic mice2 (c). Some consequences of UV light (such as apoptosis) have already been investigated, but the use of transgenic mice will direct experiments of light-induced skin cancer and immunosuppression, with considerable impact on human health. The colored boxes highlight UV light effects that may be further elucidated by the use of transgenic mice.
Photolyase transgenic mice promise many other insights (Fig. 2). Generation of such mice in a DNA repair−deficient background is just a matter of time. This will be especially useful in the case of nucleotide excision repair (NER) knockout mice6, including those that mimic human photosensitive disorders such as xeroderma pigmentosum (XP, with seven complementation groups, XPA through XPG) and Cockayne syndrome (CS, complementation groups CSA and CSB). These individuals are NER-deficient (NER is the main dark DNA repair for UV lesions), and people with XP are at a high risk for developing skin cancer. The genes involved in these disorders are known to affect two NER subpathways: one that repairs lesions in the whole genome (global genome repair, GGR) and the other that removes lesions from the transcribed strand of active genes (transcription coupled repair, TCR). The different roles of GGR and TCR in the biological effects of DNA damage are also open questions. The availability of photolyase mice that are lacking only GGR (XPC) or TCR (CSB) or both (XPA) will certainly produce a clearer picture of how NER protects mouse skin DNA from light. This is especially exciting, as mice have inefficient GGR for CPD when compared to humans, but very efficient GGR for 6−4PPs7. It will be possible to distinguish the consequences of these lesions employing the specific photolyases.
Light-induced skin cancer Although photorepair clearly reduces acute skin effects in mice, other aspects still require investigation. Skin carcinogenesis is an obvious endpoint to be dissected with photolyase mice, especially those unable to carry out DNA repair. UV-B irradiation of the skin is linked to carcinogenesis and has been known to suppress immunological responses, through mechanisms not yet fully understood8. The combination of photolyase-filled liposomes and light was recently shown to protect human skin from UV-B radiation−induced immunosupression9, implicating CPDs as causal agents. Similarly, topical application of liposomes containing a bacterial DNA repair enzyme, T4 endonuclease V, has been shown to reduce pretumoral keratosis in individuals with XP10. Experiments with the different combinations of photolyase mice and XP or CS deficiencies will be anxiously awaited, as they will certainly have an impact on our current knowledge of light-induced skin cancer.
