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

Excited State Pathways Leading to Formation of Adenine Dimers

International audienceThe reaction intermediate in the path leading to UV-induced formation ofadenine dimers A=A and AA* is identified for the first time quantum mechanically, usingPCM/TD-DFT calculations on (dA)2 (dA: 2′deoxyadenosine). In parallel, its fingerprint isdetected in the absorption spectra recorded on the millisecond time-scale for the singlestrand (dA)20 (dA: 2′deoxyadenosine)

Populations and Dynamics of Guanine Radicals in DNA strands—Direct versus Indirect Generation

International audienceGuanine radicals, known to be involved in the damage of the genetic code and aging, are studied by nanosecond transient absorption spectroscopy. They are generated in single, double and four-stranded structures (G-quadruplexes) by one and two-photon ionization at 266 nm, corresponding to a photon energy lower than the ionization potential of nucleobases. The quantum yield of the one-photon process determined for telomeric G-quadruplexes (TEL25/Na+) is (5.2 ± 0.3) × 10−3, significantly higher than that found for duplexes containing in their structure GGG and GG sequences, (2.1 ± 0.4) × 10−3. The radical population is quantified in respect of the ejected electrons. Deprotonation of radical cations gives rise to (G-H1)• and (G-H2)• radicals for duplexes and G-quadruplexes, respectively. The lifetimes of deprotonated radicals determined for a given secondary structure strongly depend on the base sequence. The multiscale non-exponential dynamics of these radicals are discussed in terms of inhomogeneity of the reaction space and continuous conformational motions. The deviation from classical kinetic models developed for homogeneous reaction conditions could also be one reason for discrepancies between the results obtained by photoionization and indirect oxidation, involving a bi-molecular reaction between an oxidant and the nucleic acid

Guanine Radicals Generated in Telomeric G-Quadruplexes by Direct Absorption of Low-Energy UV Photons: Effect of Potassium Ions

International audienceThe study deals with the primary species, ejected electrons, and guanine radicals, leading to oxidative damage, that is generated in four-stranded DNA structures (guanine quadruplexes) following photo-ionization by low-energy UV radiation. Performed by nanosecond transient absorption spectroscopy with 266 nm excitation, it focusses on quadruplexes formed by folding of GGG(TTAGGG)$_3$ single strands in the presence of K$^+$ ions, TEL21/K$^+$. The quantum yield for one-photon ionization (9.4 × 10$^{−3}$) was found to be twice as high as that reported previously for TEL21/Na$^+$. The overall population of guanine radicals decayed faster, their half times being, respectively, 1.4 and 6.7 ms. Deprotonation of radical cations extended over four orders of magnitude of time; the faster step, concerning 40% of their population, was completed within 500 ns. A reaction intermediate, issued from radicals, whose absorption spectrum peaked around 390 nm, was detected

Deprotonation dynamics of guanine radical cations

Special Issue Invited ReviewInternational audienceThis review is dedicated to guanine radical cations (G$^+$)$^•$ that are precursors to oxidatively generated damage to DNA. (G$^+$)$^•$ are unstable in neutral aqueous solution and tend to lose a proton. The deprotonation process has been studied by time-resolved absorption experiments in which (G$^+$)$^•$ radicals are produced either by an electron abstraction reaction, using an external oxidant, or by low-energy/low-intensity photoionization of DNA. Both the position of the released proton and the dynamics of the process depend on the secondary DNA structure. While deprotonation in duplex DNA leads to (G-H1)$^•$ radicals, in guanine quadruplexes the (G-H2)$^•$ analogues are observed. Deprotonation in monomeric guanosine proceeds with a time constant of ~60 ns, in genomic DNA is completed within 2 µs and spans from at least 30 ns to over 50 µs in guanine quadruplexes. Such a deprotonation dynamics in four-stranded structures, extended over more than three decades of times, is correlated with the anisotropic structure of DNA and the mobility of its hydration shell. In this case, commonly used second order reaction models are inappropriate for its description

Nanoparticle growth probed by time-resolved luminescence spectroscopy

International audienceOxide nanoparticles (NP) are currently drawing a lot of attention because of their diverse applications such as catalysis, drug delivery, sensing and as luminescent materials. Clearly, optimizing the synthesis of such oxide nanoparticles is of high importance. In particular, the manufacturing of crystalline oxide nanoparticles in aqueous solution at room-temperature is industrially very appealing. However, this requires a detailed understanding of all the different phases involved in their formation from the initial homogeneous solution to the final crystalline mineral phase. This complex process is loosely described by the classical nucleation theory, which relies upon a single step description of the formation of the primary centers ("seeds") by means of dynamic and stochastic association of the ions in solution. In this picture, the ions must overcome the free energy barrier before developing to a crystal of critical size which can grow to a mature crystal. However, there is evidence of the existence of non-classical intermediates like clusters, nanoscale amorphous precipitates, and other complex precursors in the liquid phase. The existence of a transient amorphous network with a two-level structuration was indeed confirmed by Fleury et al in the case of Eu:YVO$_4$ nanoparticles using In situ X-ray scattering and steady-state fluorescence techniques

Topology Controls the Electronic Absorption and Delocalization of Electron Holes in Guanine Quadruplexes

International audienceGuanine quadruplexes (G4) are four‐stranded DNA structures involved in biological processes and are promising candidates for potential nanotechnological applications. This study examines how the G4 topology affects the electronic absorption and the delocalization of electron holes, which play a key role in charge transport and oxidative damage. Combining transient absorption spectroscopy with PCM/TD‐DFT calculations both parallel (P) and antiparallel (A) G4 are investigated, which are formed, respectively, by association of four TGGGGT strands and folding of the human telomeric sequence GGG(TTAGGG)3. The experimental absorption spectra obtained upon photo‐ionization of A and P are different. This is explained by the different topology of the two G4, as well as by hole delocalization between two stacked guanines, possible only in P+. The spectral signature of delocalized hole in guanine‐rich regions is provided and the chemical physical effects which rule the hole delocalization are discussed

UV-induced damage to DNA: effect of cytosine methylation on pyrimidine dimerization

International audienceMethylation/demethylation of cytosine plays an important role in epigenetic signaling, the reversibility of epigenetic modifications offering important opportunities for targeted therapies. Actually, methylated sites have been correlated with mutational hotspots detected in skin cancers. The present brief review discusses the physicochemical parameters underlying the specific ultraviolet-induced reactivity of methylated cytosine. It focuses on dimerization reactions giving rise to cyclobutane pyrimidine dimers and pyrimidine (6–4) pyrimidone adducts. According to recent studies, four conformational and electronic factors that are affected by cytosine methylation may control these reactions: the red-shift of the absorption spectrum, the lengthening of the excited state lifetime, changes in the sugar puckering modifying the stacking between reactive pyrimidines and an increase in the rigidity of duplexes favoring excitation energy transfer toward methylated pyrimidine

Effect of C5-Methylation of Cytosine on the Photoreactivity of DNA: A Joint Experimental and Computational Study of TCG Trinucleotides

International audienceDNA methylation, occurring at the 5 position of cytosine, is a natural process associated with mutational hotspots in skin tumors. By combining experimental techniques (optical spectroscopy, HPLC coupled to mass spectrometry) with theoretical methods (molecular dynamics, DFT/TD-DFT calculations in solution), we study trinucleotides with key sequences (TCG/T5mCG) in the UV-induced DNA damage. We show how the extra methyl, affecting the conformational equilibria and, hence, the electronic excited states, increases the quantum yield for the formation of cyclobutane dimers while reducing that of (6-4) adducts