TT Step Research Articles

Thymine photodimer formation in DNA hairpins. Unusual conformations favor (6 − 4) vs. (2 + 2) adducts

The photochemical reactions of eleven synthetic DNA hairpins possessing a single TT step either in a base-paired stem or in a hexanucleotide linker have been investigated. The major reaction products have been identified as the cis-syn (2 + 2) adduct and the (6 - 4) adduct on the basis of their spectroscopic properties including 1D and 2D NMR spectra, UV spectra and stability or instability to photochemical cleavage. Product quantum yields and ratios determined by HPLC analysis allow the behaviour of the eleven hairpins to be placed into three groups: Group I in which the (2 + 2) adduct is the major product, as is usually the case for DNA, Group II in which comparable amounts of (2 + 2) and (6 - 4) adducts are formed, and Group III in which the major product is the (6 - 4) adduct. The latter behaviour is without precedent in natural or synthetic DNA and appears to be related to the highly fluxional structures of the hairpin reactants. Molecular dynamics simulation of ground state conformations provides quantum yields and product ratios calculated using a single parameter model that are in reasonable agreement with most of the experimental results. Factors which may influence the observed product ratios are discussed.

Sequence-dependent thymine dimer formation and photoreversal rates in double-stranded DNA

The kinetics of thymine-thymine cyclobutane pyrimidine dimer (TT-CPD) formation was studied at 23 thymine-thymine base steps in two 247-base pair DNA sequences irradiated at 254 nm. Damage was assayed site-specifically and simultaneously on both the forward and reverse strands by detecting emission from distinguishable fluorescent labels at the 5'-termini of fragments cleaved at CPD sites by T4 pyrimidine dimer glycosylase and separated by gel electrophoresis. The total DNA strand length of nearly 1000 bases made it possible to monitor damage at all 9 tetrads of the type XTTY, where X and Y are non-thymine bases. TT-CPD yields for different tetrads were found to vary by as much as an order of magnitude, but similar yields were observed at all instances of a given tetrad. Kinetic analysis of CPD formation at 23 distinct sites reveals that both the formation and reversal photoreactions depend sensitively on the identity of the nearest-neighbour bases on the 5' and the 3' side of a photoreactive TT base step. The lowest formation and reversal rates occur when two purine bases flank a TT step, while the highest formation and reversal rates are observed for tetrads with at least one flanking C. Overall, the results show that the probabilities of CPD formation and photoreversal depend principally on interactions with nearest-neighbour bases.

Electron Donor–Acceptor Interactions with Flanking Purines Influence the Efficiency of Thymine Photodimerization

Quantum yields for thymine photodimerization (Φ(TT)) have been determined for a series of short DNA single-strand and base-paired hairpin structures possessing a single thymine-thymine step with flanking purines. Values of Φ(TT) are strongly dependent upon the oxidation potential of the flanking purine, decreasing in the order: inosine > adenine > guanine > deazaguanine. The dependence of Φ(TT) on the ionization potential of the flanking purine is more pronounced when the purine of lower oxidation potential is located at the 5'- versus 3'-position in either a single strand or a hairpin. Molecular dynamics simulations for hairpin structures indicate that the TT step is π-stacked with both the 5' and 3' purine, but that there is little π-stacking with either purine in single-strand structures. The observation of moderately intense long-wavelength UV absorption features for hairpins having 5'-Z or G flanking purines suggests that excitation of ground state donor-acceptor complexes may account for more extensive quenching of dimerization by 5'- versus 3'-purines. The "purine effect" on Φ(TT) is attributed to a combination of ground state conformation, ground state electron donor-acceptor interactions, and excited state exciplex formation.

Conformational Control of Thymine Photodimerization in Single-Strand and Duplex DNA Containing Locked Nucleic Acid TT Steps

Conformational Control of Thymine Photodimerization in Single-Strand and Duplex DNA Containing Locked Nucleic Acid TT Steps

Structure and Stability of Alkane-Linked DNA Hairpin Conjugates

The synthesis and properties of three related families of alkane-linked DNA hairpins are reported. The first possesses a dodecane linker (C12) and 2-8 AT base pairs. The second possesses six AT base pairs and straight chain alkane linkers having 8-16 methylenes. The third has three alkane linkers of different length and a constant six base-pair stem with alternating A-T bases and a single TT step. The spectroscopic properties (UV, CD, and (1)H NMR) and molecular modeling are consistent with the formation of base-paired B-DNA structures for all hairpins having four or more AT base pairs. The thermal stability of hairpins having a C12 linker is greater than that of the commonly used hexa(ethylene glycol) linker but less than that of the stilbenediether linker having the same AT base-pair domain. Hairpin stability is related to both hydrophobic interactions between the linker and the adjacent base pair (stilbene > alkane > glycol) and the overall length of the linker. The stability of the alkane-linked hairpins having six AT base pairs is greater for a tetradecane linker than for either shorter or longer linkers. The good thermal stability of alkane-linked hairpins and absence of a chromophore which absorbs in the UV region makes them well-suited for studies of the electronic spectra and photochemistry of short hairpins having variable base-pair sequences.

Conformational Control of Thymine Photodimerization in Single-Strand and Duplex DNA Containing Locked Nucleic Acid TT Steps

The results of an integrated experimental and theoretical study of thymine-thymine photodimerization in short single-strand and duplex DNA structures possessing a single locked nucleic acid TT step are reported. Control of ground-state conformation by the locked nucleic acids results in a marked increase in both the quantum yield and the selectivity of photoproduct formation.

Conformational Control of TT Dimerization in DNA Conjugates. A Molecular Dynamics Study

The paper presents quantum yield results for the [2+2] and 6-4 photodimerization of TT steps in several DNA structures, including hairpins where the context dependence of the photodimerization yield is determined, and it develops a theoretical model that correctly describes the trends in dimerization yield with DNA structure. The DNA conjugates considered include dT(20), dA(20)dT(20), and three alkane-linked hairpins that contain a single TT step. The theoretical modeling of the [2+2] process is based on CASSCF electronic structure calculations for ethylene + ethylene, which show that photoexcitation of low-lying excited states leads to potential surfaces that correlate without significant barriers to a conical intersection with the ground state surface at geometries close to the dimer structure. The primary constraint on dimerization is the distance d between the two double bonds, and it is found that d < 3.52 A leads to quantum yield trends that match the observed trends within a factor of 3. Constraints on the dihedral angle between the two double bonds are not as important, and although it is possible to generate better dimerization yield predictions for some structures by including these constraints, the best overall picture is obtained with no constraint. For 6-4 dimerization, a distance g < 2.87 A and no constraint on dihedral angle provide an accurate description of the yield.

One-electron oxidation of DNA: thymine versus guanine reactivity

One-electron oxidation of anthraquinone (AQ)-linked DNA oligonucleotides containing A/T base pairs with repeating TT steps results in the distance-dependent reaction of the resulting radical cation and base damage at the TT steps that is revealed by subsequent reaction as strand cleavage. However, the inclusion of a remote guanine or GG step inhibits the reaction at thymine and results in predominant reaction at the guanine bases. For the oligomers examined in this work, the results reveal that the specific sequence of nucleobases determines the distance dependence, location of reaction and the efficiency of radical cation migration. In particular, a sequence of A/T base pairs can behave either as a trap, shuttle or barrier, depending on the context of the entire oligomer. The A/T sequences act as a shuttle when reaction occurs at a remote G or GG step and the same sequence of A/T bases acts as a barrier when there is more than one GG step in the sequence. In contrast, the A/T steps act as a trap in sequences that lack guanines.

Oxidation of DNA: Damage to Nucleobases

All organisms store the information necessary to maintain life in their DNA. Any process that damages DNA, causing a loss or corruption of that information, jeopardizes the viability of the organism. One-electron oxidation is such a process. In this Account, we address three of the central features of one-electron oxidation of DNA: (i) the migration of the radical cation away from the site of its formation; (ii) the electronic and structural factors that determine the nucleobases at which irreversible reactions most readily occur; (iii) the mechanism of reaction for nucleobase radical cations. The loss of an electron (ionization) from DNA generates an electron "hole" (a radical cation), located most often on its nucleobases, that migrates reversibly through duplex DNA by hopping until it is trapped in an irreversible chemical reaction. The particular sequence of nucleobases in a DNA oligomer determines both the efficiency of hopping and the specific location and nature of the damaging chemical reaction. In aqueous solution, DNA is a polyanion because of the negative charge carried by its phosphate groups. Counterions to the phosphate groups (typically Na(+)) play an important role in facilitating both hopping and the eventual reaction of the radical cation with H(2)O. Irreversible reaction of a radical cation with H(2)O in duplex DNA occurs preferentially at the most reactive site. In normal DNA, comprising the four common DNA nucleobases G, C, A, and T, reaction occurs most commonly at a guanine, resulting in its conversion primarily to 8-oxo-7,8-dihydroguanine (8-OxoG). Both electronic and steric effects control the outcome of this process. If the DNA oligomer does not contain a suitable guanine, then reaction of the radical cation occurs at the thymine of a TT step, primarily by a tandem process. The oxidative damage of DNA is a complex process, influenced by charge transport and reactions that are controlled by a combination of enthalpic, entropic, steric, and compositional factors. These processes occur over a broad distribution of energies, times, and spatial scales. The emergence of a complete picture of DNA oxidation will require additional exploration of the structural, kinetic, and dynamic properties of DNA, but this Account offers insight into key elements of this challenge.

Oxidatively Damaged Nucleobases in Duplex DNA Oligomers: Reaction at Thymine−Thymine Mispairs

Thymine-thymine mispairs are barriers to long-distance radical cation migration and are high reactivity sites in duplex DNA oligomers. A DNA oligomer was prepared that contains only A/T base pairs, arranged into a regularly repeating series of TT steps, and a covalently linked anthraquinone photosensitizer. Its UV irradiation causes the one-electron oxidation of the DNA introducing a radical cation that reacts predominantly at the TT steps as revealed by subsequent strand cleavage. When a remote GG step is introduced into the DNA oligomer, there is little reaction at any of the TT steps and strand cleavage is detected almost exclusively at the GG step. However, when a TT step contains a thymine-thymine mispair, one electron oxidation of the oligomer results in strand cleavage at the mispair and at TT steps preceding it with little reaction at the remote GG step. Experiments in which a thymine in the mispair is replaced by uracil show that the mispair is both a highly reactive site and a barrier to radical cation hopping. These effects of the thymine-thymine mispairs may be associated with its wobble base pair structure.

One-electron oxidation of DNA: mechanism and consequences

All living organisms store the information necessary to maintain life in their DNA. Any process that damages DNA and causes loss or corruption of that information threatens the viability of the organism. One-electron oxidation is such a process. Loss of an electron from DNA generates a radical cation that is located primarily on its nucleobases. The radical cation migrates reversibly through duplex DNA by hopping until it is eventually trapped in an irreversible chemical reaction. The particular sequence of nucleobases in a DNA oligomer determines both the efficiency of hopping and the specific location and nature of the damaging chemical reaction. In its normal aqueous solutions, DNA is a polyanion because of the negative charge carried by its phosphate groups. Counter ions (typically Na(+)) to the phosphate groups play an important role in facilitating both the migration of the radical cation and in its eventual reaction with H(2)O. Irreversible reaction of a radical cation with H(2)O in duplex DNA occurs preferentially at the most reactive site. In normal DNA that is comprised of the four common DNA nucleobases, reaction occurs most commonly at a guanine and results in its conversion primarily to 8-oxo-7,8-dihydroguanine (8-OxoG). Both electronic and steric effects control the outcome of this process. If the DNA oligomer does not contain a suitable guanine, then reaction of the radical cation occurs at the thymine of a TT step primarily by a tandem process. The general outcomes revealed in the one-electron oxidation of DNA oligomers in solution appear to be generally valid also for more complex DNA structures and for the cellular DNA of living organisms.

Predicting Thymine Dimerization Yields from Molecular Dynamics Simulations

It was recently shown that thymine dimers in the all-thymine oligonucleotide (dT) 18 are fully formed in <1 ps after ultraviolet excitation. The speed and low quantum yield of this reaction suggest that only a small fraction of the conformers of this structurally disordered oligonucleotide are in a position to react at the instant of photon absorption. In this work, we explore the hypothesis that conventional molecular dynamics simulations can be used to predict the yield of cyclobutane pyrimidine dimers in DNA. Conformations obtained from simulations of thymidylyl-(3′-5′)-thymidine in various cosolvents were classified as dimerizable or nondimerizable depending on the distance between the C5-C6 double bonds of the adjacent thymine bases and the torsion angle between them. The quantum yield of cyclobutane pyrimidine dimer formation was calculated as the number of dimerizable conformations divided by the total number of conformations. The experimental quantum yields measured in the different solvents were satisfactorily reproduced using physically reasonable values for the two parameters. The mean dimerizable structure computed by averaging all of the dimerizable cis-syn conformations is structurally similar to the actual cis-syn dimer. Compared to the canonical B-form TT step, the most important structural property of a dimerizable conformation is its reduced helical twist angle of 22°.

Selective Binding of the TATA Box-Binding Protein to the TATA Box-Containing Promoter: Analysis of Structural and Energetic Factors

We report the results of an energy-based exploration of the components of selective recognition of the TATA box-binding protein (TBP) to a TATA box sequence that includes 1) the interaction between the hydrophobic Leu, Pro, and Phe residues of TBP with the TA, AT, AA, TT, and CG steps, by ab initio quantum mechanical calculations; and 2) the free energy penalty, calculated from molecular dynamics/potential of mean force simulations, for the conformational transition from A-DNA and B-DNA into the TA-DNA form of DNA observed in a complex with TBP. The GTAT, GATT, GAAT, and GTTT tetramers were explored. The results show that 1) the discrimination of TA, AT, AA, TT, or CG steps by TBP cannot rest on their interaction with the inserting Phe side chains; 2) the steric clash between the bulky and hydrophobic Pro and Leu residues and the protruding —NH2 group of guanine is responsible for the observed selectivity against any Gua-containing basepair; 3) the Pro and Leu residues cannot selectively discriminate among TA, AT, AA, or TT steps; and 4) the calculated energy required to achieve the TA-DNA conformation of DNA that is observed in the complex with TBP appears to be a key determinant for the observed selectivity against the AT, AA, and TT steps. The simulations also indicate that only the TA step can form a very efficient interbase hydrogen bond network in the TA-DNA conformation. Such an energetically stabilizing network is not achievable in the AA and TT steps. While it is viable in the AT step, structural constraints render the hydrogen bonding network energetically ineffective there.

Genomes and DNA conformation.

Genomic DNA sequences code not only for protein sequences but also for their own physical characteristics. The composition of a genome for creating particular physical characteristics is reviewed by first summarizing the conformational characteristics of short DNA fragments, and second by summarizing the combination of fragments in a genome. In this analysis the pyrimidine (Y)/purine (R) classification of the four bases is important. In genomes, there is a tendency to create YY and RR combinations rather than YR and RY combinations, with a resulting minimization of the deviation of the local DNA structure from the standard B-conformation. Among the YY combinations, TT steps are positioned with a preference for the periodicity of approximately 10 bases, which is close to the helical periodicity of the DNA. The coding regions possess another periodicity of 3 bases in addition to the approximately 10 base periodicity, which differentiates these regions from the non-coding regions in terms of the physical characteristics. It is likely that the above characteristics of genomes are designed as part of their efficient transcription mechanisms.

Role of base-backbone and base-base interactions in alternating DNA conformations

Sequence-specific conformational differences between dinucleotide steps are characterised using published crystal coordinates with special attention to steric hindrance of the methyl group of a T base to the neighbouring base, and, more importantly, to the sugar-phosphate backbone. The TT step is inflexible and B-like, as it has two methyl groups which interlock with each other and with the sugar-phosphate backbones. AT slides, or overtwists, so that the methyl groups move away from the backbones, both lead the step towards the A-conformation. TA is most flexible as it does not have such restriction. These characteristics are observed with other pyrimidine-pyrimidine, pyrimidine-purine, purine-pyrimidine steps, respectively, but to less extent, depending on the number of non-A: T basepairs in the steps.

Computational Simulations of DNA Distortions by a cis,syn-Cyclobutane Thymine Dimer Lesion

Results are presented from 500 ps molecular dynamics simulations on the native dodecamer d(CGCGAATTCGCG)2 and the lesioned dodecamer containing a cis,syn-thymine cyclobutane dimer at the TT step. The computations, performed with AMBER4.1, included explicitly represented solvent with periodic boundary conditions applied within the constant temperature and pressure algorithm. Electrostatic interactions were calculated with the particle-mesh Ewald method. Distortions to DNA structure produced by the lesion were found to be localized at the dimer site and include mainly a substantial kink in the helical axis, rolled and tilted base pairs, and weakened hydrogen bonding at the 5‘ base pair of the lesion. A slight change in orientation around the glycosyl bond for the 5‘ thymine of the lesion and highly stiffened deoxyribose rings for both thymine bases were also observed. The global curvature of DNA is increased by about 10° by dimer incorporation. Calculations of H(1‘)-H(6)(pyrimidine) and H(1‘)-H(8)(purine) interproton distances from the performed simulations agree very well with the pattern of NMR NOE signals reported in various dimer containing oligonucleotides, where an interruption of NOE connectivities is found on the 5‘ side of the lesion. Comparison of the pattern of distortions observed at the dimer site with the crystal structure of a complex between dimer-containing DNA and repair enzyme endonuclease V (Cell 1995, 83, 773−782) leads to the hypothesis that dimer recognition may involve a whole pattern of small distortion at the lesion site rather than one particular structural/dynamical feature associated with the lesion.

The intrinsic curvature of DNA in solution

We propose a detailed quantitative scheme for explaining the anomalous electrophoretic mobility in polyacrylamide gels of repeating sequence DNA. We assume that such DNA adopts a superhelical configuration in these circumstances, and migrates less quickly than straight DNA of the same length because it can only pass through larger holes. The retardation is maximal when the length of the DNA reaches one superhelical turn, but is less for shorter pieces. We attribute the curvature of the superhelix to different angles of roll at each kind of dinucleotide step, i.e. an opening up of an angle by an increased separation on the minor-groove side. The main effect is due to a difference of about 3 ° in roll values between AA TT and other steps, together with a difference of about 1 ° in the angle of helical twist: we deduce these values explicitly from some of the available data on gelrunning. The scheme involves a simple calculation of the superhelical parameters for any given repeating sequence, and it gives a good correlation with all of the available data. We argue that these same base-step angular parameters are also consistent with observations from X-ray diffraction of crystallized oligomers, and particularly with the recent data on CGCA 6GCG from Nelson et al. We are concerned here with the intrinsic curvature of unconstrained DNA, as distinct from the curvature of DNA in association with protein molecules; and this paper represents a first attempt at an absolute determination.