Computational study of thymine dimer radical anion splitting in the self-repair process of duplex DNA

Formation of the thymine dimer is one of the most important types of photochemical damage in DNA, responsible for several biological pathologies. Though specifically designed proteins (photolyases) can efficiently repair this type of damage in living cells, an autocatalytic activity of the DNA itself was recently discovered, allowing for a self-repair mechanism. In this paper, we provide the first molecular dynamics study of the splitting of thymine dimer radical anions, using a quantum mechanical/molecular mechanics (QM/MM) approach based on density functional theory (DFT) to describe the quantum region. A set of seven statistically representative molecular dynamics trajectories is analyzed. Our calculations predict an asynchronously concerted process in which C5-C5' bond breaking is barrierless while C6-C6' bond breaking is characterized by a small free energy barrier. An upper bound of 2.5 kcal/mol for this barrier is estimated. Moreover, the molecular dynamics study and the low free energy barrier involved in C6-C6' bond breaking characterize the full process as being an ultrafast reaction.     

Ring opening of the cyclobutane in a thymine dimer radical anion

The reactions of hydrated electrons (e(aq) (-)) with thymine dimer 2 and thymidine have been investigated by radiolytic methods coupled with product studies, and addressed computationally by means of BB1K-HMDFT calculations. Pulse radiolysis revealed that one-electron reduction of the thymine dimer 2 affords the radical anion of thymidine (5) with t(1/2)<35 ns. Indeed, the theoretical study suggests that radical anion 3, in which the spin density and charge distribution are located in both thymine rings, undergoes a fast partially ionic splitting of the cyclobutane with a half-life of a few ps. This model fits with the in vivo observation of thymine dimer repair in DNA by photolyase. gamma-Radiolysis of thymine dimer 2 demonstrates that the one-electron reduction and the subsequent cleavage of the cyclobutane ring does not proceed by means of a radical chain mechanism, that is, in this model reaction the T(-)* is unable to transfer an electron to the thymine dimer 2.     

The triplet energy of thymine in DNA

Norfloxacin (NFX) photosensitizes formation of thymine dimers (T<>T) in DNA, while its N(4') acetyl derivative (ANFX) does not. This is evident from the observation of single-strand breaks after enzymatic treatment with T4 endonuclease V and subsequent gel electrophoresis. The triplet energies of NFX and ANFX are estimated at 273 and 268 kJ/mol, respectively, on the basis of triplet-triplet energy transfer quenching by a set of biphenyl and naphthalene derivatives. Hence, the triplet energy of thymine in DNA (i.e., the value for a photosensitizer to produce T<>T) can be estimated at 270 kJ/mol.     

Base flipping of the thymine dimer in duplex DNA

Exposure of two adjacent thymines in DNA to UV light of 260-320 nm can result in the formation of the cis,syn-cyclobutane pyrimidine dimer (CPD). The structure of DNA containing an intrahelical CPD lesion has been previously studied experimentally and computationally. However, the structure of the extrahelical, flipped-out, CPD lesion, which has been shown to be the structure that binds to the CPD repair enzyme, DNA photolyase, has yet to be reported. In this work the structure of both the flipped-in and the flipped-out CPD lesions in duplex DNA is reported. These structures were calculated using 8 ns molecular dynamics (MD) simulations. These structures are then used to define the starting and ending points for the base-flipping process for the CPD lesion. Using a complex, two-dimensional pseudodihedral coordinate, the potential of mean force (PMF) for the base-flipping process was calculcated using novel methodology. The free energy of the flipped-out CPD is roughly 6.5 kcal/mol higher than that of the flipped-in state, indicating that the barrier to flipping out is much lower for CPD than for undamaged DNA. This may indicate that the flipped-out CPD lesion may be recognized by its repair enzyme, DNA photolyase, whereas previous studies of other damaged, as well as nondamaged, bases indicate that they are recognized by enzymes in the intrahelical, flipped-in state.     

Valence anions of N-acetylproline in the gas phase: computational and anion photoelectron spectroscopic studies

We report the photoelectron spectrum of anionic N-acetylproline, (N-AcPro)(-), measured with 3.49 eV photons. This spectrum, which consists of a band centered at an electron binding energy of 1.4 eV and a higher energy spectral tail, confirms that N-acetylproline forms a valence anion in the gas phase. The neutrals and anions of N-AcPro were also studied computationally at the B3LYP∕6-31++G(d,p) level. Based on the calculations, we conclude that the photoelectron spectrum is due to anions which originated from proton transfer induced by electron attachment to the π* orbital localized at the acetyl group of N-AcPro. We also characterized the energetics of reaction paths leading to pyrrolidine ring opening in the anionic N-AcPro. These data suggest that electron induced decomposition of peptides/proteins comprising proline strongly depends on the presence of proton donors in the close vicinity to the proline residue.     

Synthetic functional pi-stack architecture in lipid bilayers

Neglected until recently, pi-stack architecture is rapidly emerging as a powerful strategy to create function in lipid bilayer membranes. Recent reports describe supramolecular rosettes acting as hosts of intercalating guests, to assemble in bilayer membranes and, in the case of stacked guanosine and folate quartets, to form ion channels. The introduction of rigid-rod pi-stack architecture allowed us to address one of the great challenges in the field, i.e. ligand gating. Inspiring pi-stack chemistry from related fields, covering rainbow coloration, conductivity, as well as the critical dependence of charge mobilities on the precision of supramolecular organization is summarized to zoom in on arguably the most promising application of functional pi-stack architecture in lipid bilayers, that is the creation of multifunctional photosystems.     

DNA base stacking: The stacked uracil/uracil and thymine/thymine minima

The potential energy surfaces of stacked uracil dimer (U/U) and stacked thymine dimer (T/T) have been explored at the counterpoise (CP)‐corrected M06‐2X/6‐31+G(d) level of theory, in the gas phase and in solution (with water and, for U/U, 1,4‐dioxane as the solvents) modeled by a continuum solvent using the polarizable continuum model. Potential energy scans were created by rotation of one monomer around its center‐of‐mass, whereas the other monomer remained still. Both face‐to‐back (one molecule exactly on top of the other) and face‐to‐face (one base molecule flipped by 180°) structures were considered. Five or six (dependent on whether CP correction is included or not) stacked uracil dimer minima and six stacked thymine dimer minima were located. A number of transition states on the U/U and T/T potential energy surfaces were likewise identified. The general effect of the continuum solvent is a flattening of the potential energy surface. Comparison of the gas‐phase M06‐2X/6‐31+G(d) U/U interaction energies with estimated CCSD(T)/complete basis set values (where available) show the excellent performance of this functional for stacking energies. © 2012 Wiley Periodicals, Inc.     

Kinetics and mechanism of protection of thymine from sulphate radical anion under anoxic conditions

The rates of photooxidation of thymine in presence of peroxydisulphate (PDS) have been determined by measuring the absorbance of thymine at 264 nm spectrophotometrically. The rates and the quantum yields (φ) of oxidation of thymine by sulphate radical anion have been determined in the presence of different concentrations of caffeic acid. Increase in [caffeic acid] is found to decrease the rate of oxidation of thymine suggesting that caffeic acid acts as an efficient scavenger of SO ₄ ^•- and protects thymine from it. Sulphate radical anion competes for thymine as well as for caffeic acid. The rate constant of sulphate radical anion with caffeic acid has been calculated to be 1.24 x 10¹⁰ dm³ mol^-1s^-1. The quantum yields of photooxidation of thymine have been calculated from the rates of oxidation of thymine and the light intensity absorbed by PDS at 254 nm, the wavelength at which PDS is activated to sulphate radical anion. From the results of experimentally determined quantum yields (φ_expt1) and the quantum yields calculated (φ_cl) assuming caffeic acid acting only as a scavenger of SO ₄ ^•- radicals show that φ_expt1 values are lower than φ_cl values. The φ ’ values, which are experimentally found quantum yield values at each caffeic acid concentration and corrected for SO ₄ ^•- scavenging by caffeic acid, are also found to be greater than φ_expt1 values. These observations suggest that the thymine radicals are repaired by caffeic acid in addition to scavenging of sulphate radical anions.     

Kinetics and mechanism of protection of thymine from phosphate radical anion under anoxic conditions

The rates of photooxidation of thymine in the presence of peroxydiphosphate (PDP) have been determined by measuring the absorbance of thymine at 264 nm spectrophotometrically. The rates and the quantum yields (φ) of oxidation of thymine by phosphate radical anion have been determined in the presence of different concentrations of dithiothreitol (DTT). An increase in DTT is found to decrease the rate of oxidation of thymine, suggesting that DTT acts as an efficient scavenger of PO₄^·2? and protects thymine from it. Phosphate radical anion competes for thymine as well as DTT; the rate constant for the phosphate radical anion with DTT has been calculated to be 2.21 × 10⁹ dm³ mol^?1 s^?1, assuming the rate constant of phosphate radical anion reaction with thymine as 9.6 × 10⁷ dm³ mol^?1 s^?1. The quantum yields of photooxidation of thymine have been calculated from the rates of oxidation of thymine and the light intensity absorbed by PDP at 254 nm, the wavelength at which PDP is activated to phosphate radical anion. From the results of experimentally determined quantum yields (φ_exptl) and the quantum yields calculated (φ_cl), assuming DTT acts only as a scavenger of PO₄^·2? radicals, show that φ_exptl values are lower than φ_cl values. The φ′ values, which are experimentally found quantum yield values at each DTT concentration and corrected for PO₄^·2? scavenging by DTT, are also found to be greater than φ_exptl values. These observations suggest that the thymine radicals are repaired by DTT in addition to scavenging of phosphate radical anions. © 2001 John Wiley & Sons, Inc. Int J Chem Kinet 33: 271–275, 2001     

Excess electron transfer in flavin-capped,thymine dimer-containing DNA hairpins

The transfer of an excess electron through DNA was investigated with DNA hairpins, which contain a flavin cap functioning as an electron donor. A thymine dimer with an open backbone acts as the electron acceptor. The dimer translates the electron capture into a strand break, which is readily detectable by HPLC. Analysis of four hairpins, in which the distance between the flavin donor and the dimer acceptor was systematically increased, revealed a flat distance dependence of the repair efficiency supporting the view that excess electrons hop through DNA using intermediate A-T base pairs as temporary charge carriers.     

Modeling thymine photodimerizations in DNA: mechanism and correlation diagrams

The basic mechanistic traits of the main photochemical reactions in DNA, the formation of the cyclobutane and oxetane thymine dimerization adducts, are established with the help of CASSCF and CASPT2 calculations for a gas-phase model of two stacked thymines. Both reactions go through conical intersections between the ground and the excited state that are connected through minimum energy paths to the corresponding products. This explains the ultrafast formation of the cyclobutane adduct detected experimentally, and it suggests that the oxetane formation also occurs on that time scale. Moreover, the states responsible for the photoproduct formation correlate with two high-lying states of the pair in its ideal B-DNA conformation. These states are different from the delocalized states resulting from coupling of the localized ones, which suggests that the origin of the reactive electronic states lies in the pi stacking. Formation of the photoproducts requires population of these states, by direct excitation of favorable conformations, or preceded by a localized excitation.     

Cylindrical projection of electrostatic potential and image analysis tools for damaged DNA: the substitution of thymine with thymine glycol

Changes of electrostatic potential around the DNA molecule resulting from chemical modifications of nucleotides may play a role in enzymatic recognition of damaged sites. Effects of chemical modifications of nucleotides on the structure of DNA have been characterized through electronic structure computations. Quantum mechanical structural optimizations of fragments of five pairs of nucleotides with thymine or thymine glycol were performed at the density functional level of theory with a B3LYP exchange-correlation functional and 6-31G(d,p) basis sets. The electrostatic potential (EP) around DNA fragments was projected on a cylindrical surface around the double helix. The 2D maps of EP of intact and damaged DNA fragments were compared using image analysis methods to identify and measure modifications of the EP that result from the occurrence of thymine glycol. It was found that distortions of phosphate groups and displacements of the accompanying countercations by up to approximately 0.5 angstroms along the axis of DNA are clearly reflected in the EP maps. Modifications of the EP in the major groove of DNA near the damaged site are also reported.     

The catalytic power of uracil DNA glycosylase in the opening of thymine base pairs

Uracil DNA glycosylase (UNG) locates uracil and its structural congener thymine in the context of duplex DNA using a base flipping mechanism. NMR imino proton exchange measurements were performed on free and UNG-bound DNA duplexes in which a single thymine (T) was paired with a series of adenine analogues (X) capable of forming one, two, or three hydrogen bonds. The base pair opening equilibrium for the free DNA increased 55-fold as the number of hydrogen bonds decreased, but the opening rate constants were nearly the same in the absence and presence of UNG. In contrast, UNG was found to slow the base pair closing rate constants (kcl) compared to each free duplex by a factor of 3- to 23-fold. These findings indicate that regardless of the inherent thermodynamic stability of the TX pair, UNG does not alter the spontaneous opening rate. Instead, the enzyme holds the spontaneously expelled thymine (or uracil) in a transient extrahelical sieving site where it may partition forward into the enzyme active site (uracil) or back into the DNA base stack (thymine).     

Electrochemical study of thymine dimer based on DNA charge transfer

Differential pulse voltammetry was used to study the formation and level of thymine dimer in DNA duplex modified on a gold electrode. The electrochemical signal of methylene blue coupled with ferricyanide can be obtained via DNA mediated electron transfer, which would be blocked during the formation of thymine dimer. DNA duplexes with different sequences differ in the level of thymine dimer under the same UV irradiation. Futhermore, the presence of guanine base directly preceding -TT- can effectively decrease the level of thymine dimer, possibly due to the self-repair process in which guanine participates. The proposed method can be further applied to DNA self-repair analysis.     

Direct measurement of the dynamics of excess electron transfer through consecutive thymine sequence in DNA

Charge transfer in DNA is an essential process in biological systems because of its close relation to DNA damage and repair. DNA is also an important material used in nanotechnology for wiring and constructing various nanomaterials. Although hole transfer in DNA has been investigated by various researchers and the dynamic properties of this process have been well established, the dynamics of a negative charge, that is, excess electron, in DNA have not been revealed until now. In the present paper, we directly measured the rate of excess electron transfer (EET) through a consecutive thymine (T) sequence in nicked-dumbbell DNAs conjugated with a tetrathiophene derivative (4T) as an electron donor and diphenylacetylene (DPA) as an electron acceptor at both ends. The selective excitation of 4T by a femtosecond laser pulse caused the excess electron injection into DNA, and led to EET in DNA by a consecutive T-hopping mechanism, which eventually formed the DPA radical anion (DPA(?-)). The rate constant for the process of EET through consecutive T was determined to be (4.4 ± 0.3) × 10(10) s(-1) from an analysis of the kinetic traces of the ΔO.D. during the laser flash photolysis. It should be emphasized that the EET rate constant for T-hopping is faster than the rate constants for oxidative hole transfers in DNA (10(4) to 10(10) s(-1) for A- and G-hopping).     

Molecular dynamics simulation of thymine glycol-lesioned DNA reveals specific hydration at the lesion

One nanosecond molecular dynamics (MD) simulation of a thymine glycol (TG)-lesioned part of human lymphoblast AG9387 was performed to determine structural changes in DNA molecule caused by the presence of a lesion. These changes can be significant for proper recognition of lesions by a repair enzyme. Thymine glycol is the DNA oxidative lesion formed by addition of OH radicals to C5 and C6 atoms of the thymine base. This lesion is known as causing Cockayne Syndrome-inherited genetic disorder. Distribution of water molecules in a hydration shell around the DNA molecule was analyzed for its contribution to the recognition of the TG lesion by the repair enzyme. The results of MD simulation show there is a specific DNA structural configuration formed at the lesion. After 500 ps the DNA is bent in a kink at the TG site. This change dislocates the glycosyl bond at C5' to a position closer to the DNA surface, and thus its atoms are more exposed to the surrounding water shell. The increased number of water molecules that are close to the TG site indicates that the glycosyl bond may be easily contacted by the repair enzyme. In addition, the higher number of water molecules at the TG site substantiates the importance of water-mediated hydrogen bonds created between the repair enzyme and the DNA upon formation of the complex. Copyright 2001 John Wiley & Sons, Inc. J Comput Chem 22: 1723-1731, 2001     

Analysis of ultrafast relaxation in photoexcited DNA base pairs of adenine and thymine

The photoinduced dynamics in base pairs of adenine and thymine were analyzed by femtosecond pump-probe spectroscopy. On the short-time scale up to a few picoseconds, the characteristic time constants for the dimers are quite similar to the corresponding values of the monomers. This leads to the conclusion that ultrafast intramolecular relaxation proceeds via ππ^* and nπ^* states of one component within the dimer. On the long-time scale, we obtained a novel time constant of roughly 40 ps for the thymine dimer and the adenine–thymine base pair. This time constant was never observed in the monomers and is tentatively assigned to an intermolecular relaxation process, possibly via a hydrogen transfer state.