Theoretical Insight into the Intrinsic Ultrafast Formation of Cyclobutane

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2008, 112, 14096–14098 Published on Web 10/18/2008

Theoretical Insight into the Intrinsic Ultrafast Formation of Cyclobutane Pyrimidine Dimers in UV-Irradiated DNA: Thymine versus Cytosine Juan Jose´ Serrano-Pe´rez, Israel Gonza´lez-Ramı´rez, Pedro B. Coto, Manuela Mercha´n, and Luis Serrano-Andre´s* Instituto de Ciencia Molecular, UniVersitat de Vale`ncia, Apartado 22085, ES-46071 Valencia, Spain ReceiVed: July 30, 2008

The higher formation yields measured in the ultrafast photoinduced formation of cyclobutane thymine dimers (TT) with respect to those of cytosine (CC) are explained, on the basis of ab initio CASPT2 results, by the existence in thymine of more reactive orientations and a less efficient photoreversibility, whereas in cytosine the funnel toward the photolesion becomes competitive with that mediating the internal conversion of the excited-cytosine monomer. Among the possible photoreactions that pyrimidine bases of nucleic acids may undergo on ultraviolet (UV) irradiation, cyclobutane thymine dimers (TT) formed by intrastrand adjacent thymine bases constitute one of the major photoinduced lesions, particularly in cellular DNA.1 Despite the fact that there are repair mechanisms for photodamaged sections of the DNA sequence, the UV irradiation of cells can result in mutation or death. In contrast to thymine-thymine (TT) sites, which are not actual mutational hot spots, cytosine-cytosine (CC) sequences are sources of relatively frequent CC-to-TT tandem mutations, although the corresponding photoproducts (CC) are produced with relatively lower yields.1 Femtosecond spectroscopy has proved that thymine dimerization is an ultrafast photoreaction in which TT dimers are fully formed ∼1 ps after UV illumination, pointing to an excited-state reaction that is approximately barrierless for bases that are properly oriented at the instant of light absorption.2 From a theoretical standpoint, relevant aspects of the [2 + 2] cycloaddition photoreaction forming the respective cyclobutane pyrimidine dimers have been analyzed for both thymine3,4 and cytosine.5 The concerted nonadiabatic photoreaction is mediated by a conical intersection (CI) involving the lowest singlet excited and the ground state, hereafter (S1/S0)CI, which is related to the expected funnel for ultrafast nonradiative decay leading to TT and CC. There is, however, an elusive question still open. Why is the photoinduced formation of TT globally more efficient than that producing CC? Since the efficiency of the photodimerization markedly depends on the experimental conditions, the sequence of nucleotides, and the type (A-, B-like) of DNA conformation, the full response to this question is truly challenging. In order to get further insight into this complex issue, in the present contribution, we focus on whether the distinct photochemical behavior of TT and CC sites can be understood on the basis of the intrinsic molecular characteristics of the systems. The present research anticipates that the relatiVe stability of the formed excimers with respect to the placement of (S1/S0)CI is the main * Author to whom correspondence should be addressed. E-mail: [email protected].

10.1021/jp806794x CCC: $40.75

effect responsible at the molecular leVel for the different efficiency obserVed in the production of TT Versus CC. The results discussed next were obtained by using the CASPT2 method with the active space of 12 π active electrons/ 12 π active orbitals, including the basis set superposition error (BSSE) through the counterpoise (CP) correction, CASPT2(12,12)+BSSE results. The ANO-S basis set with the contraction scheme C,N,O[3s2p1d]/H[2s1p] was employed throughout. Geometry optimizations were carried out for the ground state of the TT dimer, for a delocalized excimer 1(TT) , and the crossing (S /S ) (see the Supporting Informaexc 1 0 CI tion for details). In addition, the lowest-lying excited states were computed at the geometrical arrangements of the B-form DNA, (TT)B. All of the calculations were performed using the MOLCAS-6.0 package.6-8 Figure 1 compiles the main findings for TT. For proper comparison, results on CC at the same level are also included.5 The CP-corrected binding energy (CP-Eb) for the 1(TT)B state is computed to be 0.29 eV, about 3 times larger than that obtained for 1(CC)B.5 At the TT ground-state B-form DNA, the transition to the lowest excited singlet state (4.60 eV) becomes, as expected, slightly red-shifted as compared with the lowest singlet-singlet transition of the monomer (4.89 eV).9 On the other hand, the S2-S1 gap is 0.1 eV larger for TT than that for CC, reflecting a more efficient coupling between the two states in the former. If the TT system is in the B-form DNA at the time of irradiation, the pathway from 1(TT)B toward the funnel (S1/S0)CI (path I in Figure 1) can be related to the actual decay path taking place in the biopolymer, which is predicted to be barrierless on the basis of the energy calculations derived from the linearly interpolated structures between those two geometries.4 This also holds true for B-like arrangements energetically close to the B-form. Since DNA has a highly flexible backbone, motions such as the rise of stacking, torsional oscillation, and helix bending will continuously bring a given bipyrimidine pair into a favorable geometry for dimerization. Clearly, π-stacking facilitates formation of excimer states. In particular, the most favorable structure for producing a fully stabilized excimer corresponds to the idealized sandwich geometry.5 The relaxed  2008 American Chemical Society

Letters

J. Phys. Chem. B, Vol. 112, No. 45, 2008 14097

Figure 1. Relative energies (in eV) computed at the CASPT2(12,12)+BSSE level, with respect to two noninteracting ground-state thymine monomers, for the lowest excited state (S1) at the ground-state B-form DNA, 1(TT)B, at the relaxed geometry of the delocalized excimer, 1(TT)exc, and at the ground-state structure of the TT dimer (left). The conical intersection (S1/S0)CI, the ground state of the dimer, and the lowest transition of the monomer are also included. The main intermolecular geometric parameters (C-C distances in Å) are given in italics. The corresponding scheme for cytosine is on the right. The Qx coordinate is mainly related to the average intermolecular distance, whereas Qy is associated to the remaining degrees of freedom.

Figure 2. Scheme of the photodimerization process for π-stacked cytosine (top) and thymine (bottom) dimers along the singlet manifold. The shadowed volumes in the sphere represent regions of the DNA strand with reactive orientations, in which the decay path lies above the conical intersection (S1/S0)CI.

delocalized excimer 1(TT)exc bears the largest overlap between the monomers; consequently, the computed CP-Eb, 1.25 eV, considerably increases with respect to 1(TT)B. Independently from how the 1(TT)exc state is achieved, it can be directly deactivated through the funnel in a barrierless fashion (path II in Figure 1). The situation for CC is somewhat different. In order to reach the CI, the 1(CC)exc state has to surmount a barrier of 0.2 eV. The presence of a barrier does not imply, however, that the overall process of CC formation is forbidden. It simply predicts that the existence of stable excimer-like states below (S1/S0)CI decreases the effectiveness of photoproduct yield in the singlet manifold, since an excess of vibrational energy is required to overcome the barrier. As a result, production of CC becomes as a whole comparatively less effective than that of TT (see Figure 2). The lack of stable excimers represents an intrinsic potential of the TT system to lead to photoproducts with a higher formation yield. For TT, no structure could be indeed determined whose S1 was placed below the CI. This finding is supported by experimental evidence. Excimers were not observed in (dT)18,

that is, the single-stranded DNA 18-mer containing consecutive thymidine (dT) residues.10 The strongest fluorescence for the T-containing oligonucleotide can just be attributed to monomer fluorescence. Accordingly, we conclude that in TT any possible structural arrangement becomes in principle prone to be a reactiVe orientation at the time of light irradiation, which is defined as those energetically aboVe the shearing-type CI. In contrast, for CC sites, a certain percentage of arrangements shall not be so reactive due to the existence of the 1(CC) excimers. On the basis of the red-shifted emission (the so-called excimer fluorescence) seen in the base multimers 15-mer, it was concluded that C and adenine (A), whose intrastrand excimer states were found in high yields whether stacked with itself or with T,10 have a similar tendency to form excimers.11 In principle, a similar kinetic model can then be used for C- and A-containing oligonucleotides; namely, every excitation in a base stack decays to an excimer, while every excitation of an unstacked base decays by internal conversion to the ground state of the monomer.10 CC sites have, however, a striking uniqueness: the (S1/S0)CI leading to the CC formation at 3.51 eV is in the same energy range as that of the monomer, 3.6 eV.9 It means that both decays may be competitive, making the production of CC less effective. For TT, the (S1/S0)CI and the monomer funnels are energetically far apart: 3.26 (cf. Figure 1) and 4.0 eV,9 respectively, and no competition is established. In addition, the S1 state is anticipated to play a more relevant role in the direct photoreversibility of CC sites than in TT sequences, since the associated oscillator strength for the transition of the former is somewhat larger (0.070 vs 0.024), with the 1(CC) and 1(TT) states located vertically at 4.57 and 5.48 eV, respectively. In this manner, the 1(CC)exc state may be repopulated within the middle UV range. In summary, three distinct features have been deduced which account at a molecular level for the higher formation yield of TT with respect to CC observed experimentally, that provide an expansion of our understanding of this type of DNA photolesions: (i) TT has more reactive orientations than CC; (ii) photoreversibility by direct absorption to S1 is expected to be less efficient for TT; (iii) in CC, the funnel toward CC production becomes competitive with the funnel that mediates the internal conversion of the excited-cytosine monomer,

14098 J. Phys. Chem. B, Vol. 112, No. 45, 2008 whereas, for TT, the decay of the excited monomer becomes relevant only for unstacked thymine bases. By following these general guidelines, a number of designed derivatives can be envisaged with potential use in different areas of interest, from health care phototherapeutic treatments to industrial technological oriented purposes. Acknowledgment. The research reported has been supported by the MEC-FEDER projects CTQ2007-61260 and CSD20070010 Consolider-Ingenio, and Juan de la Cierva programme (PBC). Supporting Information Available: Computational details and Cartesian coordinates (PDF). This material is available free of charge via the Internet at http://pubs.acs.org. References and Notes (1) Douki, T.; Cadet, J. Biochemistry 2001, 40, 2495. (2) Schreier, W. J.; Schrader, T. E.; Soller, F. O.; Gilch, P.; CrespoHerna´ndez, C. E.; Swaminathan, V. N.; Carell, T.; Zinth, W.; Kohler, B. Science 2007, 315, 625.

Letters (3) Boggio-Pasqua, M.; Groenhof, G.; Scha¨fer, L. V.; Grubmu¨ller, H.; Robb, M. A. J. Am. Chem. Soc. 2007, 129, 10996. (4) Blancafort, L.; Migani, A. J. Am. Chem. Soc. 2007, 129, 14540. (5) Roca-Sanjua´n, D.; Olaso-Gonza´lez, G.; Gonza´lez-Ramı´rez, I.; Serrano-Andre´s, L.; Mercha´n, M. J. Am. Chem. Soc. 2008, 130, 10768. (6) Andersson, K.; Barysz, M.; Bernhardsson, A.; Blomberg, M. R. A.; Carissan, Y.; Cooper, D. L.; Cossi, M.; Fu¨lscher, M. P.; Gagliardi, L.; de Graaf, C.; Hess, B.; Hagberg, G.; Karlstro¨m, G.; Lindh, R.; Malmqvist, P.-Å.; Nakajima, T.; Neogra´dy, P.; Olsen, J.; Raab, J.; Roos, B. O.; Ryde, U.; Schimmelpfennig, B.; Schu¨tz, M.; Seijo, L.; Serrano-Andre´s, L.; Siegbahn, P. E. M.; Stålring, J.; Thorsteinsson, T.; Veryazov, V.; Widmark, P.-O. MOLCAS, Version 6.4; Department of Theoretical Chemistry, Chemical Centre, University of Lund: Lund, Sweden, 2006. (7) Karlstro¨m, G.; Lindh, R.; Malmqvist, P.-Å.; Roos, B. O.; Ryde, U.; Veryazov, V.; Widmark, P.-O.; Cossi, M.; Schimmelpfennig, B.; Neogrady, P.; Seijo, L. Comput. Mater. Sci. 2003, 28, 222. (8) Veryazov, V.; Widmark, P. O.; Serrano-Andre´s, L.; Lindh, R.; Roos, B. O. Int. J. Quantum Chem. 2004, 100, 626. (9) Mercha´n, M.; Gonza´lez-Luque, R.; Climent, T.; Serrano-Andre´s, L.; Rodrı´guez, E.; Reguero, M.; Pela´ez, D. J. Phys. Chem. B 2006, 110, 26471. (10) Crespo-Herna´ndez, C. E.; Cohen, B.; Kohler, B. Nature 2005, 436, 1141. (11) Plessow, R.; Brockhinke, A.; Eimer, W.; Kohse-Ho¨inghaus, K. J. Phys. Chem. B 2000, 104, 3695.

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