Quantitative analysis of UV photolesions suggests that cyclobutane pyrimidine dimers produced in mouse skin by UVB are more mutagenic than those produced by UVC

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UV-Induced Structural Changes of Model DNA Helices Probed by Optical Spectroscopy

International audienceChemical alterations of DNA, if not repaired, may lead to carcinogenic mutations. Structural modifications of the helix around the lesion enable its recognition by repair enzymes. We have used absorption spectroscopy and mass spectrometry to detect structural changes provoked by cyclobutane thymine dimers, the major lesion induced by UV radiation. We found that formation of a cyclobutane dimer in the model duplex (dA)20 · (dT)20 destroys base stacking on the adenine strand. The physical background of this novel approach is the existence of charge-transfer states among neighboring bases, whose contribution to the hypochromism of the helix disappears following destacking

Fluorescence of Natural DNA: From the Femtosecond to the Nanosecond Time Scales

International audienceThe fluorescence of calf thymus DNA is studied by steady-state and time-resolved spectroscopy combining fluorescence upconversion and time-correlated single photon counting. The fluorescence spectrum is very similar to that of a stoichiometric mixture of monomeric chromophores, arising from bright ππ* states, and contrasts with the existing picture of exciplex emission in natural DNA. Yet, the DNA fluorescence decays span over five decades of time, with 98% of the photons being emitted at times longer than 10 ps. These findings, in association with recent studies on model duplexes, are explained by the involvement of dark states, possibly related to charge separation, serving as a reservoir for the repopulation of the bright ππ* states

太陽光中のUVA/UVBの比率の季節差がDNA損傷（6-４）型光産物のDewar型光産物への光異性化の効率に影響を及ぼす

The UVA and UVB components of sunlight can produce three classes of bipyrimidine DNA photolesions [cyclobutane pyrimidine dimers (CPDs), pyrimidine (6-4) pyrimidone photoproducts (6-4PPs) and related Dewar valence isomers (DewarPPs)]. The UVA/UVB ratio of sunlight is high in winter and low in summer in the Northern Hemisphere. Since UVB radiation produces 6-4PPs and UVA radiation converts them into DewarPPs through photoisomerization, it is expected that there may be differences in the photoisomerization of 6-4PPs between summer and winter, although that has never been documented. To determine that, isolated DNA was exposed to natural sunlight for 8 h in late summer and in winter, and absolute levels of the three classes of photolesions were quantified using calibrated ELISAs. It was found that sunlight produces CPDs and 6-4PPs in DNA at a ratio of about 9:1 and converts approximately 80% of 6-4PPs into DewarPPs within 3 h. Moreover, photoisomerization is more efficient in winter than in late summer after sunlight irradiation for the same duration, at similar solar UV doses and with the same induction level of CPDs. These results demonstrate that seasonal differences in the UVA/UVB ratio influence the efficiency of the photoisomerization of 6-4PPs into DewarPPs.博士（医学）・甲第775号・令和3年3月15日© 2020 The Authors. Photochemistry and Photobiology published by Wiley Periodicals LLC on behalf of American Society for Photobiology. This is an open access article under the terms of the Creative Commons Attribution License(https://creativecommons.org/licenses/by/4.0/), which permits use, distribution and reproduction in any medium, provided the original work is properly cited

Effect of C5-Methylation of Cytosine on the UV-Induced Reactivity of Duplex DNA: Conformational and Electronic Factors

International audienceC5-methylation of cytosines is strongly correlated with UV-induced mutations detected in skin cancers. Mutational hot-spots appearing at TCG sites are due to the formation of pyrimidine cyclobutane dimers (CPDs). The present study, performedfor the model DNA duplex (TCGTA)3·(TACGA)3 and the constitutive single strands, examines the factors underlying the effect of C5-methylation on pyrimidine dimerization at TCG sites. This effect is quantified for the first time by quantum yields ϕ.They were determined following irradiation at 255, 267, and 282 nm and subsequent photoproduct analysis using HPLC coupled to mass spectrometry. C5-methylation leads to an increase of the CPD quantum yield up to 80% with concomitant decrease of that of pyrimidine(6−4) pyrimidone adducts (64PPs) by at least a factor of 3. The obtained ϕ values cannot be explained only by the change of the cytosine absorption spectrum upon C5-methylation. The conformational and electronic factors that may affect the dimerization reaction are discussed in light of results obtained by fluorescence spectroscopy,molecular dynamics simulations, and quantum mechanical calculations. Thus, it appears that the presence of an extra methyl on cytosine affects the sugar puckering, thereby enhancing conformations of the TC step that are prone to CPD formation but less favorable to 64PPs. In addition, C5-methylation diminishes the amplitude of conformational motions in duplexes; in the resulting stiffer structure, ππ* excitations may be transferred from initially populated exciton states to reactive pyrimidines giving rise to CPDs

Base Pairing Enhances Fluorescence and Favors Cyclobutane Dimer Formation Induced upon Absorption of UVA Radiation by DNA

[EN] The photochemical properties of the DNA duplex (dA)(20) center dot (dT)(20) are compared with those of the parent single strands. It is shown that base pairing increases the probability of absorbing UVA photons, probably due to the formation of charge-transfer states. UVA excitation induces fluorescence peaking at similar to 420 nm and decaying on the nanosecond time scale. The fluorescence quantum yield, the fluorescence lifetime, and the quantum yield for cyclobutane dimer formation increase upon base pairing. Such behavior contrasts with that of the UVC-induced processes.We thank Mrs. Si. Karpati and M. Perron for their help, Dr. R. lmprota for helpful discussions, and the French Agency for Research (ANR PCV07_ 194999) for financial support. I.V. acknowledges the Conselleria de Educacion-Generalitat Valenciana (VALi+D program, No. 20100331).Banyasz, A.; Vayá Pérez, I.; Changenet-Barret, P.; Gustavsson, T.; Douki, T.; Markovitsi, D. (2011). Base Pairing Enhances Fluorescence and Favors Cyclobutane Dimer Formation Induced upon Absorption of UVA Radiation by DNA. Journal of the American Chemical Society. 133:5163-5165. doi:10.1021/ja110879m5163516513

UV-induced formation of the thymine-thymine pyrimidine (6-4) pyrimidone photoproduct - a DFT study of the oxetane intermediate ring opening

International audienceThe mechanism by which the hypothetical oxetane/azetidine intermediate formed during the photochemical process leading to pyrimidine (6-4) pyrimidone photoproducts when DNA is submitted to UV radiation opens is investigated computationally by DFT using a 5′-TT-3′ dinucleoside monophosphate as a structural model. First, the feasibility of an intramolecular mechanism involving one proton transfer inducing opening of the oxetane ring is examined. It results in a very high Gibbs energy of activation (+166 kJ mol−1) and quite a low Gibbs energy of reaction (−35 kJ mol−1). The protonation state of the phosphate group is shown to have little effect while the bulk effect of an aqueous environment modeled by the Polarizable Continuum Model method lowers slightly the activation barrier (by about 10-20 kJ mol−1), not enough to explain the fact that the oxetane intermediate is not observed experimentally. Then the catalytic effect of water molecules on the reaction pathway is studied by including either 1 or 2 assisting water molecules in the chemical system. The resulting activation barrier is considerably lowered and in the most favorable situation - a phosphate group deprotonated and 2 assisting water molecules - the Gibbs energy activation is as low as +44 kJ mol−1 and the Gibbs energy of reaction is quite favorable: −79 kJ mol−1, suggesting that in biological systems the oxetane ring opening process proceeds with explicit intervention of water molecules from the environment

UVA-induced cyclobutane pyrimidine dimers in DNA: a direct photochemical mechanism?

International audienceThe carcinogenic action of UVA radiation is commonly attributed to DNA oxidation mediated by endogenous photosensitisers. Yet, it was recently shown that cyclobutane pyrimidine dimers (CPD), well known for their involvement in UVB genotoxicity, are produced in larger yield than oxidative lesions in UVA-irradiated cells and skin. In the present work, we gathered mechanistic information on this photoreaction by comparing formation of all possible bipyrimidine photoproducts upon UVA irradiation of cells, purified genomic DNA and dA20:dT20 oligonucleotide duplex. We observed that the distribution of photoproducts, characterized by the sole formation of CPD and the absence of (6-4) photoproducts was similar in the three types of samples. The CPD involving two thymines represented 90% of the amount of photoproducts. Moreover, the yields of formation of the DNA lesions were similar in cells and isolated DNA. In addition, the effect of the wavelength of the incident photons was found to be the same in isolated DNA and cells. This set of data shows that UVA-induced cyclobutane pyrimidine dimers are formed via a direct photochemical mechanism, without mediation of a cellular photosensitiser. This is possible because the double-stranded structure increases the capacity of DNA bases to absorb UVA photons, as evidenced in the case of the oligomer dA20:dT20. These results emphasize the need to consider UVA in the carcinogenic effects of sunlight. An efficient photoprotection is needed that can only be complete by completely blocking incident photons, rather than by systemic approaches such as antioxidant supplementation

UV-radiation-induced formation of DNA bipyrimidine photoproducts in Bacillus subtilis endospores and their repair during germination

The spore photoproduct (SP) is the main DNA lesion after UV-C irradiation, and its repair is crucial for the resistance of spores to UV. The aims of the present study were to assess the formation and repair of bipyrimidine photoproducts in spore DNA of various Bacillus subtilis strains using a sensitive HPLC tandem mass spectrometry assay. Strains deficient in nucleotide excision repair, spore photoproduct lyase, homologous recombination (recA), and with wild-type repair capability were investigated. Additionally, one strain deficient in the formation of major small, acid-soluble spore proteins (SASPs) was tested. In all SASP wild-type strains, UV-C irradiation generated almost exclusively SP (>95 %) but also a few by-photoproducts. In the major SASP-deficient strain, SP and by-photoproducts were generated in equal quantities. The status of the UV-induced bipyrimidine photoproducts was determined at different stages of spore germination. After a germination time of 60 min, >75% of the SP was repaired in wild-type strains and in the SASP-deficient strain, while half of the photoinduced SP was removed in the recA-deficient strain. SP-lyase-deficient spores repaired < 20% of the SP produced. Thus, SP lyase, with respect to nucleotide excision repair, has a remarkable impact on the removal of SP upon spore germination. [Int Microbiol 2007; 10(1):39-46

Non-additive genotoxic effets of mixtures of polycyclic aromatic hydrocarbons applied to human cells

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