Blanc SIMI 8 - Sciences de l'information, de la matière et de l'ingénierie : Chimie du solide, colloïdes, physicochimie

Photo-induced energy transfer in methylated DNA helices and its relevance to UV damage : an interactive theoretical-experimental study – DNAexciton

UV-induced DNA damage: the very first steps

It is well established today that absorption of UV radiation by DNA triggers chemical reactions that may provoke mutations of the genetic code, ultimately leading to skin cancer. Therefore, understanding this complex cascade of events is a challenge related to public health. The DNAexciton project focused on the very first events that take place between absorption of UV radiation and chemical reactions.

What happens to the energy of UV radiation from the moment of its absorption by DNA till the occurrence of chemical reactions?

The objective of DNAexciton was to study how the UV energy is redistributed among the DNA bases (adenine, thymine, guanine, cytosine), altering their electronic structure, and determine configurations that are favorable to the specific reactions. In particular, we examined two classes of reactions, resulting in the fusion of two bases (thymine and/or cytosine) giving rise to new entities: cyclobutanes dimers (CPDs) or 64 photo-products (64PP).

Experimentally, we followed the fate of the absorbed UV energy by spectroscopic methods; thanks to laser techniques we could detect fluorescence emission and/or absorption at times ranging from 100 fs to 1 ms after the absorption of UV photons. We also quantified the dimeric photoproducts produced following UV irradiation by analytical methods: liquid chromatography coupled to mass spectrometry. Finally, we rationalized our observations using various theoretical approaches: quantum chemistry, molecular dynamics and exciton theory. This is the first time that the UV-induced reactivity of DNA is investigated together with energy aspects in the frame of an interactive modeling–experiment study.

DNAexciton has clearly evidenced the collective behavior of DNA bases versus UV radiation, not only for model systems but also for natural DNA isolated in solution. Such a behavior arises from the proximity of the DNA building blocks within the double helix. It depends strongly on its internal motions which may be affected by external factor, as for example, the presence of salt in the aqueous environment. Due to this cooperativity, an important fraction of the energy deposited by a UV photon may be kept at the level of the bases 1000 times longer, compared to what would have happened in the absence of interaction among them. Moreover, the energy may migrate to specific sites reinforcing their propensity to react. This happens in the case of methylated cytosines, their methylation being a natural process involved in important biological functions.

The conclusions of DNAexciton are currently used in the frame of another ANR project, OPHID. Its objective is to characterize one-photon ionization of DNA and the resulting lesions of the genetic code.

DNAexciton produced 14 articles in international journals; the majority of them are cosigned by at least two partners. Some of them were published in the highly rated Journal of the American Chemical Society, while others have targeted the community of photobiologists. The scientific results were also presented in 12 international conferences (11 invited lectures).

key articles:

“Electronic Excitation Energy Transfer between Nucleobases of Natural DNA« Vayá, I., Gustavsson, T., Douki, T., Berlin, Y., Markovitsi, D. (2012) J. Am. Chem. Soc. 134, 11366-11368

“Electronic Excited States Responsible for Dimer Formation upon UV Absorption Directly by Thymine Strands: Joint Experimental and Theoretical Study” Banyasz, A., Douki, T., Improta, R., Gustavsson, T., Onidas, D., Vayá, I., Perron, M., Markovitsi, D. (2012), J. Am. Chem. Soc. 134, 14834–14845

«Cation Effect on the Electronic Excited States of Guanine Nanostructures Studied by Time-Resolved Fluorescence Spectroscopy« Hua, Y., Changenet-Barret, P., Improta, R., Vayá, I., Gustavsson, T., Kotlyar, A. B., Zikich, D., Šket, P., Plavec, J., Markovitsi, D. (2012) J Phys Chem C 116, 14682–14689

“Multi-Pathway Excited State Relaxation of Adenine Oligomers in Aqueous Solution: A Joint Theoretical and Experimental Study” Banyasz, A., Gustavsson, T., Onidas, D., Changenet-Barret, P., Markovitsi, D., Importa, R. (2013), Chem. Europ. J. 19, 3762-3774

«Effect of C5-methylation of cytosine on the photoreactivity of DNA: a joint experimental and computational study of TCG trinucleotides” Esposito, L,. Banyasz, A., Douki, T., Perron, M., Markovitsi, D., Improta, R. (2014) J. Am. Chem. Soc. 136, 10838-10841

“Stabilization of mixed Frenkel-charge transfer excitons extended across both strands of guanine-cytosine DNA duplexes” Huix-Rotllant, M. Brazard, J., Improta, R., Burghardt, I., Markovitsi, D. J. Phys. Chem. Lett. (2015) DOI:0.1021/acs.jpclett.5b00813

It is well established that absorption of UV radiation by DNA triggers photochemical reactions which may ultimately lead to skin cancer. Although
the final products of such reactions are well characterized, the fundamental processes preceding their formation are far from understood. In particular, little is known about the way that DNA methylation enhances the probability of the appearance of UV-induced carcinogenic mutations. DNA methylation, occurring at the 5 position of cytosine, is a natural process that plays an important biological role, for example, in gene expression. Despite the fact that 5-methyl cytosine (5-mC) represents only 2% of the bases in the human genome, it is involved in 40% of skin cancer occurrences. Understanding the mechanism of mutagenesis at a molecular level is one of today's grand challenges.

Very recent studies carried out by team 2 have provided some clues to the questions addressed above. It has been shown that the absorption spectrum of isolated 5m-C is red-shifted compared to the major DNA bases (adenine, thymine, guanine, cytosine). Moreover, femtosecond experiments revealed that ultrafast energy transfer occurs among bases in helices composed exclusively of adenine-thymine or guanine-cytosine base pairs. The energy transfer pathways are expected to be modified in the methylated species. 5-mC could thus act as a trap, leading to a localisation of excitation energy in certain parts of the double helix and thus enhancing reactivity (i.e., dimerisation).

The present proposal aims to obtain a precise picture of excitation transfer and trapping in methylated DNA, by combining state-of-the-art theoretical and experimental techniques. We propose to establish an interactive theory-experiment collaboration based upon (i) the theoretical description of the electronically excited, delocalized (excitonic) states of DNA and their dynamics, combining electronic structure information and quantum/classical molecular dynamics (team 1), (ii) optical spectroscopy (absorption, fluorescence), both steady-state and time-resolved, over a large time domain ranging from 100 fs to 100 ns (team 2) (iii) liquid chromatography coupled to mass spectrometry, in view of tracking the
photoproducts resulting from DNA damage (team 3).

The project brings together three groups with leading-edge know-how in complementary domains: (1) I. Burghardt is expert in the quantum-dynamical description of energy and charge transfer in complex systems. She will collaborate with several colleagues of international standing who are active in the modelling of DNA, i.e., Hans Lischka (University of Vienna) and Eric R. Bittner (University of Houston) on the electronic structure side, and Richard Lavery (University of Lyon) on the biomolecular dynamics simulation side. (2) The group of D. Markovitsi is the first experimental group to have provided evidence for the collective nature of the excited states of DNA helices. Furthermore, her group has demonstrated the role of conformational disorder (in collaboration with R. Lavery) and has made important steps in connecting spectroscopic properties to DNA photodamage (in collaboration with T. Douki). (3) Finally, the group of T. Douki is internationally known for its expertise in analysing nucleic acid lesions; the group has already carried out some preliminary studies of 5-mC photochemistry.

Since the first study of ultrafast excitation energy transfer (EET) in DNA, carried out by team 2 in 2005, the EET phenomenon has not been explicitly connected to the occurrence of UV-induced photodamage. The detailed understanding of these processes, at a molecular level, necessitates a synergy between experiment and theory that does not currently exist at an international level, and that we propose to create in the present project.

Project coordination


The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.



Help of the ANR 475,000 euros
Beginning and duration of the scientific project: - 36 Months

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