Dimerization of pyrimidine bases following absorption of UV photons leads a wide variety of photoproducts. A first class damage are cyclobutane pyrimidine dimers (CPD), produced through a [2+2] cycloaddition between the C5-C6 double bonds of two adjacent pyrimidine bases. Four isomers are possible depending on the orientation of the bases with respect each other (trans/anti isomery) and depending on their position on opposite or on the same side of the cyclobutane ring (cis,syn isomery). All these isomers can be isolated with monomeric models but only the cis,syn diastereoisomer is produced in DNA. The second main class of UV-induced DNA photoproduct is the pyrimidine (6-4) pyrimidone photoproduct (64PP). This compound results from a [2+2] cycloaddition of the C5-C6 double bond of the 5’-end base to the C4 carbonyl of imino group of the 3’-end pyrimidine. Absorption of a second UVB or UVA photon by the pyrimidone moiety converts (6-4) photoproducts into their Dewar valance isomers. All these photoproducts can be produced at the 4 bipyrimidine dinucleotide in DNA (TT, TC, CT and CC).
Numerous assays are available for the quantification of UV-induced photoproducts. Although they all provide valuable information, none of them are both quantitative and allowed the simultaneous and individual quantification of all the possible DNA photoproducts. To this end, the LAN has developed an approach involving enzymatic hydrolysis of DNA followed by HPLC-MS/MS analysis. This technique is much milder than earlier chromatographic assays based on acidic hydrolysis of radiolabelled DNA. The HPLC-MS/MS approach was applied first to the study of the UVC and UVB photochemistry of isolated and cellular DNA. It was possible to provide an extensive distribution of the photoproducts. In human DNA, TT CPDs were found to be the major photoproducts. TC sites were also quite photoreactive with formation of both CPD and 64PP. The latter photoproduct was not significantly produced at either CT or CC sites which were both less photoreactive than TT and TC. Interestingly, this distribution was found to be strongly dependent on the GC content of the studied cells, as shown in bacteria. At high GC content, TT CPD becomes less frequent than the corresponding CC photoproduct. Experiments in isolated DNA also showed that the distribution was strongly dependent on the stability of the DNA duplex.
Comparison of the yield of formation of 8oxodGuo and TT CPD in primary cultures of human cutaneous cells exposed to UVA
We also investigated the still poorly understood genotoxic properties of more abundant UVA radiation (320-400 nm) that are poorly absorbed by DNA. Surprisingly, CPDs were found to be produced in significant yield in whole human skin exposed to UVA. The absence of 64PPs showed that the underlying mechanism was different from that triggered by UVB although observation of the formation of CPDs in isolated DNA exposed to UVA showed that a direct rather than photosensitized photoreaction was involved. A collaborative work with spectroscopists (LFP, IRAMIS, CEA-Saclay) supported a major role played by charge transfer excited states. Interestingly, CPDs were produced in larger amount than 8-oxo-7,8-dihydro-2'-deoxyguanosine, the most common oxidatively generated lesion, in human skin. This was unexpected because oxidative stress is often put forward as the major genotoxic pathway in the UVA range. It should be stressed that the contribution of oxidative stress was more significant in cultured melanocytes was larger than in other cell types. This observation is in agreement with the growing evidence of the free radical production upon UV-irradiation of melanin. Accordingly, the LAN participated in a study suggesting that oxidative lesions were responsible for induction of melanoma in UVA-exposed pigmented mice while albino ones did not get tumors.
The HPLC-MS/MS assay for UV-induced pyrimidine dimers was also applied to the investigation of DNA repair. First, the technique made possible the characterization of the repair kinetics of different classes of UVB-induced photoproducts in primary cultures of human skin cells and in human skin explants. As previously shown, 64PPs were the best repaired lesions. Dewar were found to be also rapidly removed. We could add that no difference in rate could be observed for the TT and TC derivatives. In sharp contrast, the four possible cyclobutane dimers exhibit very different repair efficiency in all sample types. The CT derivative is removed almost as fast as the 64PPs, while the TT CPD persists several days after irradiation. CC is also quite fast repaired, while TC CPD exhibits an intermediate behavior. Interestingly, these observations explain some of the mutagenesis features of UVB light. Indeed, mutagenic events at CT are almost absent while much more frequent at TC and to a lesser extent at CC. TT CPD is poorly mutagenic. We also investigated to repair of CPDs following UVA irradiation and observed that the rate of removal of cyclobutane pyrimidine dimers from skin DNA was lower than after exposure to UVB. This trend was confirmed in cultured keratinocytes. Preliminary exposure to UVA deduced the rate of repair of CPDs and 64PPs produced by a subsequent UVB irradiation. Further work is needed to understand the underlying processes.
Douki (2013) The variety of UV-induced pyrimidine dimeric photoproducts in DNA as shown by chromatographic quantification methods, Photochem Photobiol Sci 12, 1286-1302.
Mouret, Leccia, Bourrain, Douki and Beani (2011) Individual Photosensitivity of Human Skin and UVA-Induced Pyrimidine Dimers in DNA, J Invest Dermatol 131, 1539-1546.
Mouret, Forestier and Douki (2012) The specificity of UVA-induced DNA damage in human melanocytes, Photochem Photobiol Sci 11, 155-162.
Banyasz, Vaya, Changenet-Barret, Gustavsson, Douki and Markovitsi (2011) Base Pairing Enhances Fluorescence and Favors Cyclobutane Dimer Formation Induced upon Absorption of UVA Radiation by DNA, J Am Chem Soc 133, 5163-5165.
Mouret, Philippe, Gracia-Chantegrel, Banyasz, Karpati, Markovitsi and Douki (2010) UVA-induced cyclobutane pyrimidine dimers in DNA: a direct photochemical mechanism?, Org Biomolec Chem 8, 1706-1711.
Mouret, S., C. Philippe, J. Gracia-Chantegrel, A. Banyasz, S. Karpati, D. Markovitsi and T. Douki (2010) UVA-induced cyclobutane pyrimidine dimers in DNA: a direct photochemical mechanism?, Org. Biomolec. Chem. 8, 1706-1711.
Mouret, Charveron, Favier, Cadet and Douki (2008) Differential repair of UVB-induced cyclobutane pyrimidine dimers in cultured human skin cells and whole human skin, DNA Repair 7, 704-712.
Matallana-Surget, S., J. A. Meador, F. Joux and T. Douki (2008) Effect of the GC content of DNA on the distribution of UVB-induced bipyrimidine photoproducts, Photochem. Photobiol. Sci. 7, 794-801.
Douki (2006) Effect of denaturation on the photochemistry of pyrimidine bases in isolated DNA, J. Photochem. Photobiol. B: Biol. 82, 45-52.
Mouret, Baudouin, Charveron, Favier, Cadet and Douki (2006) Cyclobutane pyrimidine dimers are predominant DNA lesions in whole human skin exposed to UVA radiation, Proc. Natl. Acad. Sci. USA 103, 13765-13770.
Courdavault, Baudouin, Charveron, Canghilem, Favier, Cadet and Douki (2005) Repair of the three main types of bipyrimidine DNA photoproducts in human keratinocytes exposed to UVB and UVA radiations, DNA Repair 4, 836-844.
Courdavault, Baudouin, Charveron, Favier, Cadet and Douki (2004) Larger yield of cyclobutane dimers than 8 oxo-7,8-dihydroguanine in the DNA of UVA-irradiated human skin cells, Mutat. Res. 556, 135-142.
Douki, Reynaud-Angelin, Cadet and Sage (2003) Bipyrimidine photoproducts rather than oxidative lesions are the main type of DNA damage involved in the genotoxic effect of solar UVA radiation, Biochemistry 42, 9221-9226.
Douki and Cadet (2001) Individual determination of the yield of the main-UV induced dimeric pyrimidine photoproducts in DNA suggests a high mutagenicity of CC photolesions, Biochemistry 40, 2495-2501.
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