LETTER pubs.acs.org/JPCL
Conformational Control of Thymine Photodimerization in Purine-Containing Trinucleotides Zhengzheng Pan, Martin McCullagh, George C. Schatz,* and Frederick D. Lewis* Department of Chemistry, Northwestern University, Evanston, Illinois 60208-3113, United States
bS Supporting Information ABSTRACT: The effects of a flanking purine base on the thymine-thymine photodimerization efficiencies and product distributions have been determined for the trinucleotides 50 ATT, TTA, GTT, and TTG and the results compared to those for the dinucleotide TT. The highest quantum yield and selectivity for formation of the major cissyn (2 þ 2) dimer was observed for ATT. The relative yields of (2 þ 2) and (6 4) adduct formation from the four trinucleotides are well-correlated with ground-state conformational populations using the single-parameter model developed in our previous studies of TT dimerization. The effect of the flanking purine base is determined mainly by its influence on the extent of TT groundstate stacking, which is more extensive for A versus G. The low probability of stacking of all three nucleotides and the fast decay times of the nucleotide excited states can account for the apparent absence of electronic interactions between thymines and the flanking purine base. SECTION: Biophysical Chemistry
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hotodimerization of adjacent thymines is an important cause of mutagenesis of DNA.1,2 Femtosecond time-resolved IR studies of TT dimerization in the dinucleotide TT and the oligonucleotide (dT)18 have indicated that the dimerization reaction is complete within 1 ps.3,4 Ultrafast dimerization is consistent with theoretical studies that show that entry to the conical intersection for dimerization from a pair of stacked thymines is barrier free.5 However, the quantum yield of formation of the major product, the cissyn dimer (Scheme 1), in both single-strand and duplex DNA is quite low, typically 60% conversion) by HPLC, which provided clear separation from the transsyn dimer and (6 4) adduct. Aliquots were taken from the irradiated solution at 5 min intervals and analyzed by HPLC using a Microsorb-MV C18 reversed-phase column (250 4.6 mm) with a column temperature of 25 °C. The optimal eluting condition was found to be a 20 min gradient increase of acetonitrile from 5 to 15% in 20 mM NH4OAc solution at a flow rate of 1 mL min1. The composition of each aliquot was determined by the HPLC peak areas monitored at 260 (for the trinucleotides) and 240 nm (for the dinucleotide). The quantum yields of photodimerization were calculated from the initial photochemical reaction yields using the least-squares-fit slope from HPLC quantification traces. Molecular dynamics simulations were employed for the study of ground-state conformations. Starting structures for the trinucleotide systems in B-DNA geometries were generated using the Nucgen program in Amber 8. The systems were then neutralized with sodium ions and solvated with 8 Å buffers of TIP3 water. Short minimizations were carried out followed by NPT MD simulations. The simulations were all done using the NAMD program with the CHARMM27 force field.3234 A Langevin barostat was employed to keep the pressure at 1 bar with a piston period of 200 fs and a decay of 100 fs. The temperature was also kept constant with a Langevin thermostat that had a damping coefficient of 5.0 ps1. Periodic boundary conditions were 1436
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The Journal of Physical Chemistry Letters employed with a real space nonbonded cutoff of 12 Å and particle mesh Ewald summation for the long-range electrostatics. An initial simulation of each system at 350 K was carried out in order to generate 10 independent starting geometries for simulations at 300 K. Each of these 10 runs consisted of a short minimization followed by 2 ns of equilibration and 12 ns of production time. This procedure yielded a total of 120 ns of simulation time for each system and provided a thorough sampling of conformational space. Geometries were sampled every 2 ps.
’ ASSOCIATED CONTENT
bS
Supporting Information. MALDI-TOF analysis of the trinucleotides and corresponding (2 þ 2) cissyn dimers, representative UVvisible, 1H NMR, and HPLC traces, dihedral angel distributions, and a figure for irradiation time versus conversions. This material is available free of charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION Corresponding Author
*E-mail:
[email protected] (F.D.L.); schatz@chem. northwestern.edu (G.C.S.).
’ ACKNOWLEDGMENT Funding for this project was provided by the National Science Foundation (NSF-CRC Grant CHE-0628130). ’ REFERENCES (1) Beukers, R.; Eker, A. P. M.; Lohman, P. H. M. 50 Years Thymine Dimer. DNA Repair 2008, 7, 530–543. (2) Cadet, J.; Vigny, P. In The Photochemistry of Nucleic Acids; Morrison, H., Ed.; Wiley: New York, 1990. (3) Schreier, W. J.; Schrader, T. E.; Koller, F. O.; Gilch, P.; CrespoHernandez, C. E.; Swaminathan, V. N.; Carell, T.; Zinth, W.; Kohler, B. Thymine Dimerization in DNA Is an Ultrafast Photoreaction. Science 2007, 315, 625–629. (4) Schreier, W. J.; Kubon, J.; Regner, N.; Haiser, K.; Schrader, T. E.; Zinth, W.; Clivio, P.; Gilch, P. Thymine Dimerization in DNA Model Systems: Cyclobutane Photolesion Is Predominantly Formed via the Singlet Channel. J. Am. Chem. Soc. 2009, 131, 5038–5039. (5) Boggio-Pasqua, M.; Groenhof, G.; Schaefer, L. V.; Grubmueller, H.; Robb, M. A. Ultrafast Deactivation Channel for Thymine Dimerization. J. Am. Chem. Soc. 2007, 129, 10996–10997. (6) Johns, H. E.; Pearson, M. L.; Helleiner, C. W.; Leblanc, J. C. Ultraviolet Photochemistry of Thymidylyl-(30 -50 )-Thymidine. J. Mol. Biol. 1964, 9, 503–524. (7) Douki, T.; Court, M.; Sauvaigo, S.; Odin, F.; Cadet, J. Formation of the Main UV-Induced Thymine Dimeric Lesions within Isolated and Cellular DNA as Measured by High Performance Liquid ChromatographyTandem Mass Spectrometry. J. Biol. Chem. 2000, 275, 11678– 11685. (8) Marguet, S.; Markovitsi, D. Time-Resolved Study of Thymine Dimer Formation. J. Am. Chem. Soc. 2005, 127, 5780–5781. (9) McCullagh, M.; Hariharan, M.; Lewis, F. D.; Markovitsi, D.; Douki, T.; Schatz, G. C. Conformational Control of TT Dimerization in DNA Conjugates. A Molecular Dynamics Study. J. Phys. Chem. B 2010, 114, 5215–5221. (10) Ramamurthy, V.; Venkatesan, K. Photochemical Reactions of Organic Crystals. Chem. Rev. 1987, 87, 433–481. (11) Johnson, A. T.; Wiest, O. Structure and Dynamics of Poly(T) Single-Strand DNA: Implications toward CPD Formation. J. Phys. Chem. B 2007, 111, 14398–14404.
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