Thymine Dimerization in DNA Is an Ultrafast Photoreaction

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Science  02 Feb 2007:
Vol. 315, Issue 5812, pp. 625-629
DOI: 10.1126/science.1135428


Femtosecond time-resolved infrared spectroscopy was used to study the formation of cyclobutane dimers in the all-thymine oligodeoxynucleotide (dT)18 by ultraviolet light at 272 nanometers. The appearance of marker bands in the time-resolved spectra indicates that the dimers are fully formed ∼1 picosecond after ultraviolet excitation. The ultrafast appearance of this mutagenic photolesion points to an excited-state reaction that is approximately barrierless for bases that are properly oriented at the instant of light absorption. The low quantum yield of this photoreaction is proposed to result from infrequent conformational states in the unexcited polymer, revealing a strong link between conformation before light absorption and photodamage.

The most abundant lesion in ultraviolet (UV)–irradiated DNA is the cyclobutane pyrimidine dimer (CPD) that is formed between adjacent thymine bases (Fig. 1) (1). This mutagenic photoproduct disrupts the normal cellular processing of DNA and leads to a complex web of biological responses, including apoptosis, immune suppression, and carcinogenesis (24). Organisms possess elaborate repair pathways to counter this constant threat to genomic integrity. Aside from their biological importance, CPDs are of interest as structural reporters. Thymine-dimer yields are not the same at all TT doublets in a given DNA sequence, but these yields depend, in poorly understood ways, on the identity of the flanking bases and on local conformation (1). By exposing DNA to UV light and then measuring the relative photoproduct yields with single-nucleotide resolution, it has been possible in favorable cases to obtain structural information (57). In order for this methodology to achieve its full potential, molecular-level understanding of the dimerization mechanism is essential. We report a dynamic study of thymine dimerization that provides insight into the coupling between DNA structure and DNA photodamage.

Fig. 1.

Schematic of the photodynamics of the DNA oligomer (dT)18. The DNA's sugar-phosphate backbone is shown as a gray ribbon in the partial structures. UV excitation populates a singlet ππ* state. This state decays overwhelmingly via internal conversion (IC) to the S0 ground state. To a smaller extent, the population of the ππ* state branches to a singlet nπ* state. Intersystem crossing to a triplet state has been detected in thymine but not in polymeric DNA. Finally, the ππ* state can decay to a dimer photoproduct (middle residues with new bonds shown in red) if a reactive conformation is present at the time of excitation.

CPD formation is a [2+2] photocycloaddition reaction in which the carbon-carbon double bonds of proximal pyrimidine bases react to form a cyclobutane ring. In the analogous reaction between two ethylene molecules, electronic excitation and the proper orientation of the reacting double bonds are needed for the reaction to occur (8). Unlikethe caseof freeethylene molecules, pyrimidine bases in DNA are tethered to the sugar-phosphate backbone, and this tethering restricts the achievable orientations. Some conformations are simply impossible because of backbone constraints. Thus, a single CPD isomer (the cis-syn isomer shown in Fig. 1) is formed in UV-irradiated oligo- and polynucleotides, whereas two thymine molecules diffusing freely in aqueous solution yield all six stereoisomers (1). Because DNA is moderately flexible, a vast number of conformations exist. Some of these have the bases positioned favorably for a reaction, whereas others do not. DNA is highly dynamic, and motions such as the stacking and unstacking of bases, base-pair breathing and opening, torsional oscillations, and helix bending will incessantly bring a given bipyrimidine doublet into and out of favorable geometries for dimerization. The impact of these motions on the reaction kinetics depends on how their rates compare to the rate of reaction by favorably oriented bases (9). Direct kinetic measurements of dimerization can thus elucidate the potentially complex interactions between conformational dynamics and photodamage.

In an excited-state reaction, motion along the reaction coordinate occurs in competition with energy-wasting steps such as fluorescence and internal conversion to the electronic ground state. In the past few years, it has become possible to directly observe the dynamics of excited electronic states in DNA model compounds by femtosecond spectroscopy (10, 11). It has been proposed that the very high rate of nonradiative decay by the singlet ππ*(1ππ*) states of single nucleobases can greatly restrict photodamage (10). However, recent work has revealed the presence of additional, rather long-lived singlet states in DNA (11) and single bases (12). In oligodeoxynucleotides, lifetimes of <1 ps to >100 ps have been observed, depending on base stacking and base sequence (11). Additionally, at least 10% of all singlet excitations in single pyrimidine bases such as thymidine 5′-monophosphate (TMP) decay to singlet nπ*(1nπ*) excited states with lifetimes in excess of 10 ps (11). Kinetic measurements can determine which of these diverse excited states is the dimer precursor.

Past efforts to observe dimerization kinetics have been unsuccessful. It has been shown by flash photolysis that photodimers are formed in the all-thymine oligodeoxynucleotide (dT)20 in <200 ns, the time resolution of the laser system that was used (13). Femtosecond transient electronic spectroscopy (11) has not provided direct evidence for dimer formation because CPDs do not absorb at wavelengths longer than ∼270 nm. Because of its chemical bond specificity, vibrational spectroscopy can often unambiguously identify transient species and stable photoproducts (14). We therefore recorded time-resolved infrared spectra of a DNA model compound that was excited by a femtosecond UV pump pulse (15). The system studied was single-stranded (dT)18, which was chosen in order to maximize the number of dimers that were formed with each laser pulse. In this DNA model system, every absorbed photon excites a residue that is capable of dimerization. Quantum yields in the closely related systems poly(dT) (0.033) (16) and (dT)20 (0.028) (13) are among the highest reported for any DNA compound. In contrast, the dimerization quantum yield is over 30 times lower in double-stranded genomic DNA (17). This large reduction is due to the low frequency of TT doublets and the absorption by nonthymine bases in mixed-sequence DNA. After presenting our results for (dT)18, we will discuss the implications for double-stranded nucleic acids.

Steady-state infrared (IR) absorption spectra of (dT)18 in D2O were recorded before and after UV irradiation at 266 nm, in order to locate IR marker bands that were indicative of dimerization. In the spectrum obtained before UV irradiation (black curve in Fig. 2A), three strong bands were observed at 1632, 1664, and 1693 cm–1. These bands, which arise from double-bond stretches associated with the two carbonyl groups and the C5=C6 double bond (18), bleached strongly after several minutes of UV exposure (Fig. 2A). Difference spectra were calculated by subtracting the steady-state IR spectrum from each spectrum of the UV-irradiated oligomer (Fig. 2B). Negative bleaching signals were apparent in the double-bond stretching region above 1600 cm–1. In addition, positive peaks between 1300 and 1500 cm–1 grew in with increasing exposure time. The IR absorption spectrum of the photoproduct (solid curve, Fig. 2C) was obtained from the difference spectra in Fig. 2B by target analysis (19), assuming that a single photoproduct is formed. In fact, a pyrimidine (6-4) pyrimidone photo-adduct is also generated at TT doublets, but it can be neglected because its quantum yield is 50 times lower in poly(dT) (20). The absorption spectrum (Fig. 2D) of a previously described model compound of the cis-syn thymine dimer (21) is in excellent agreement with the solid trace in Fig. 2C, showing that this is the only dominant photoproduct under these conditions. Bands in the dimer spectrum substantially overlap those of unirradiated (dT)18 above 1500 cm–1. In contrast, a trio of marker bands is evident at 1320, 1402, and 1465 cm–1 (Fig. 2C), and these bands became the focus of the time-resolved experiments.

Fig. 2.

(A) IR absorption spectra of (dT)18 (partial structure shown at right; Me, methyl) in D2O after exposure to UV laser pulses at 266 nm for the times indicated. (B) Difference IR spectra from the data in (A). (C) IR absorption spectrum of the photoproduct obtained from the data in (B) by target analysis (solid curve), showing three distinctive marker bands between 1300 and 1500 cm–1. The IR absorption spectrum of (dT)18 before UV irradiation is shown for comparison (dashed curve). (D) Steady-state IR absorption spectrum of the cis-syn dimer model compound in D2O (structure shown at right).

Broadband IR transient absorption signals were recorded between 1300 and 1550 cm–1 after excitation of (dT)18 by a femtosecond pump pulse at 272 nm (15). For comparison, measurements were carried out on TMP, which cannot dimerize on the time scales of interest here because of the slow rate of diffusional encounter by two TMP molecules. Transient spectra measured for both solutes are compared side-by-side in Fig. 3. Negative absorbance changes (bleaches) are colored blue, whereas positive signals are red. The bleaches monitor the repopulation of the starting material, whereas positive signals arise from the vibrational bands of excited states or photoproducts. At first glance, the transient IR spectra of TMP and (dT)18 are very similar. The quantum yield for dimerization in (dT)18 is just 2 to 3%, and most excitations in both systems decay nonradiatively on similar time scales (11).

Fig. 3.

Transient IR difference spectra at the indicated times after 272-nm excitation of TMP and (dT)18 in D2O solution in the photodimer marker-band region (typical errors are ∼10 μOD). Positive bands are shaded red, whereas negative signals are shaded blue. The green and purple curves at the top show the inverted steady-state IR spectra of each solute. Strong bands are indicated by the vertical dashed gray lines. The yellow dashed curve in the 3500-ps spectrum of TMP represents the steady-state difference IR spectrum obtained by raising the temperature of neat D2O (see SOM). After ultrafast internal conversion of the excited molecules, the transient spectra are dominated by the cooling dynamics of the hot ground states on a time scale of several picoseconds. Transient spectra at later delay times show the broad signature of the heated solvent. The residual bleach seen for TMP at 3 ns is assigned mainly to intersystem crossing to a triplet state [estimated quantum yield ≤0.02 (1)]. For (dT)18, one can see additional absorption due to the presence of thymine dimers.

Dynamic events revealed by the time-resolved spectra in Fig. 3 that are common to both solutes are discussed first. Initially, UV excitation populates the lowest-energy 1ππ* state, resulting in bleaches at frequencies corresponding to ground-state vibrations (dashed gray lines in Fig. 3). These bleaches have their maximum amplitudes near time zero, as seen in the spectra recorded 0.48 ps after the pump pulse. Positive signals are seen at this time at all frequencies where bleaching is not observed. These broad bands decay with a lifetime close to that of the 1ππ* state [540 fs for thymidine (10)] and are no longer present in the 3.3-ps spectra. The short lifetime of this state is limited by internal conversion, which moves the population nonradiatively from the 1ππ* state to the vibrationally excited electronic ground state. The photon energy is thus converted into sudden vibrational heating. This produces positive bands on the red edge of the negative bleach signals, resulting in distinctive sigmoidal line shapes (22) such as the one seen near 1480 cm–1 in the 3.3-ps spectra. These features disappear by vibrational energy transfer to the solvent (vibrational cooling), with a time constant of 2 to 4 ps (11, 14), and are no longer visible at 20 ps.

The bleach near the maximum of each vibrational mode recovers in multiexponential fashion with similar kinetics as those that were previously recorded by transient absorption signals at UV wavelengths (11). The decay is 85 to 90% complete within 10 ps, whereas the remainder of the bleach recovers with time constants that vary between 100 and 1000 ps because of the decay of the 1nπ* population (12). A broad positive band near 1350 cm–1 decays on a 100-ps time scale and is tentatively assigned to this 1nπ* state. The spectra at 3 ns (Fig. 3) are dominated by a broad sigmoidal line shape, extending from 1300 to 1800 cm–1. This distinctive signature arises from a temperature-jump effect, which is described in the supporting online material (SOM) in more detail. The hot-water contribution to the transient spectrum appears within a few picoseconds, but it then remains constant in our time window because of slow heat transport out of the laser focus (23).

There are subtle but significant differences between the time-resolved IR spectra in Fig. 3. Greater modulation in the 20-ps and 3.5-ns spectra for (dT)18 is due to absorption in the oligomer at each of the three marker-band frequencies that are identified in Fig. 2C. The difference is readily seen in a comparison of the transient spectra that are recorded for the two samples at a 3-ns delay time in the top panel of Fig. 4A. The water-heating signal is approximately the same for both samples because of the similar extent of ultrafast nonradiative decay. This signal can therefore be removed by subtracting the transient spectrum for TMP from the spectrum that was recorded at the same delay time for (dT)18. Difference spectra constructed in this manner are shown by the blue curves in Fig. 4A and as a contour plot between 1 and 25 ps in Fig. 4B. The subtraction procedure is discussed at length in the SOM.

Fig. 4.

Difference spectra formed by subtracting the transient spectra of TMP from those of (dT)18. (A) The top panel shows the transient spectra of TMP and (dT)18 at 3 ns. Their difference is plotted below as the blue curve, together with a difference spectrum at 15 ps. The red curve at the bottom represents the stationary IR difference spectrum of (dT)18 (dashed curve in Fig. 2C) and the dimer photoproduct (solid curve in Fig. 2C). It displays the absorption difference due to dimer formation. Vertical dashed gray lines indicate the position of the cyclobutane dimer marker bands. (B) Contour plot of the difference spectrum. Red and blue colors represent strong positive and negative differences, respectively. A time slice showing the difference spectrum at 15 ps (horizontal dashed line in bottom panel) is shown in the top panel. Positive signals due to dimer formation are visible from ∼1 ps onward for the bands at 1402 and 1320 cm–1, as indicated by the vertical dashed lines. Because vibrational cooling dynamics differ for (dT)18 and TMP, incomplete subtraction in the spectral region around the 1480 cm–1 ground-state band obscures the 1465 cm–1 photodimer band at early times.

The red trace in Fig. 4A is the difference spectrum calculated by subtracting the ground-state absorption spectrum of (dT)18 from the dimer spectrum of Fig. 2C. This trace represents the expected absorption changes induced by dimer formation. The transient difference spectra at 15 ps and 3 ns show positive peaks at each of the dimer marker-band frequencies and contributions from ground-state bleaching. The excellent agreement with the stationary spectrum shows unequivocally that thymine dimers are present ∼15 ps after excitation.

The dynamics of the marker bands at earlier times can be seen in a contour plot of the transient difference spectrum between 1 and 25 ps (Fig. 4B). The positive marker bands at 1402 and 1320 cm–1 are clearly visible over the entire time range. The marker band at 1465 cm–1 is visible down to 4 ps, but it is obscured by vibrational cooling of hot thymine molecules at earlier delay times. Because TMP and (dT)18 exhibit different cooling dynamics (11), the vibrational cooling signatures do not fully cancel each other and instead show up in the difference plot in the vicinity of intense ground-state bands. Thus, the cooling dynamics from the hot 1480 cm–1 band (Fig. 3) cover the 1465 cm–1 marker band at early delay times. Cooling is also seen at other wavenumbers during the first few picoseconds; e.g., around 1350 cm–1. For delay times <1 ps, the signals are dominated by ultrafast relaxation of the electronically excited state, which obscures direct observation of dimer formation at the shortest times. Nevertheless, the observation of the dimer marker bands 1 ps after light absorption indicates that the reaction occurs on a femto-second time scale.

The dimer yield can be estimated from the average amplitude of the marker bands in Fig. 4 of ∼30 μ–optical density (OD) units. This is 3% of the initial bleach of 1 mOD that was seen 1 ps after photoexcitation at 1480 cm–1. This band has a cross section comparable to that of the three marker bands, so the reported dimerization yield of 2 to 3% (13, 24) should produce asignalof 20 to 30 μOD, as observed. The dimer yield at ∼1 ps thus equals the value from steady-state experiments within experimental uncertainty, demonstrating that dimerization is an ultrafast photoreaction. The high speed of this bond-forming reaction is noteworthy but not unprecedented. Ultrafast reaction rates are seen for some bimolecular reactions when the reactants are suitably preoriented (25). Also, the closely related intramolecular [2+2] photocycloaddition reaction of norbornadiene occurs in the gas phase in <100 fs (26).

The ultrafast time scale of thymine dimerization suggests that an essentially barrierless path connects the initial 1ππ* state with the end product. This suggests that a conical intersection lies along this path as in computational studies of other pericyclic photoreactions (8). Dimerization in (dT)18 occurs more rapidly than many motions that could bring poorly oriented bases into a more favorable conformation for reaction. For example, base stacking and unstacking in thymine oligomers require tens of picoseconds, according to a molecular-dynamics study (27). Dimerization thus occurs only for thymine residues that are already in a reactive conformation at the instant of excitation (28, 29). Excited states of unfavorably oriented thymines are quenched before a change of conformation can occur. The extent of dimerization under steady-state irradiation thus depends on the fraction of time that a given doublet spends in reactive versus nonreactive conformations. Control of CPD formation by ground-state structure is fully consistent with the rapid saturation of CPD formation in poly(U) and poly(dT) in a rigid glass at 77 K as compared to room-temperature aqueous solution (30). This occurs because there is a finite number of reactive conformations in the low-temperature polymer, but the polymer in room-temperature solution is able to thermally fluctuate, allowing new reactive conformations to appear as exposure continues.

Because the rate of reaction by favorably aligned thymines is much faster than the rate of conformational change, the quantum yield is equal to the fraction of reactive conformations multiplied by the probability that a reactive conformation dimerizes upon excitation (9). The latter quantity is unknown, but it is likely to approach unity based on the high quantum yields of dimerization in molecular crystals of some pyrimidine bases (31) and in dimers split in rigid matrices (32). With this assumption, the quantum yield for dimerization is simply the fraction of favorably oriented conformations. The low yields for all-thymine oligomers thus reveal that only a few percent of the TT doublets are favorably positioned for reaction at the time of excitation. This finding is consistent with the disordered structure of this rather flexible oligomer (33).

Excited-state modeling is needed to fully characterize the reactive conformations, but some geometrical requirements are readily anticipated. Base stacking, which has been discussed in the past as a necessary criterion for reaction (30), reduces the distance between C5=C6 bonds as compared to an unstacked geometry. However, the dimer geometry suggests that a low value of the dihedral angle between the reacting double bonds may also be important. The conformational changes, such as partial helix unwinding and bending (34), that are observed near the site of a CPD are likely to be the same ones needed to make a conformation favorable for reaction (7).

We fully expect thymine dimerization to be ultrafast in double-stranded DNA, based on the speed of the reaction in single-stranded (dT)18. Base pairing could affect the rates of nonreactive decay steps such as internal conversion by the precursor excited state, but we consider this to be unlikely, because recent time-resolved measurements show no effects due to base pairing on the dynamics of the excited states in AT-containing oligodeoxynucleotides (11). We conclude that dimerization occurs with equal speed for bipyrimidine doublets in single- and double-stranded contexts, provided that the TT geometry is similar for both contexts. Base pairing, on the other hand, will greatly influence the quantum yields by altering the distribution of conformations. The structures of flexible all-thymine oligomers (27, 33) and double-stranded mixed-sequence DNA differ substantially, yet the quantum yields calculated per photon absorbed by a dimerizable thymine (see SOM) are the same to within a factor of ∼2 in room-temperature aqueous solution (17, 35). This means that a small percentage of TT doublets react in double-stranded DNA, even though virtually all doublets are well stacked. We propose that the winding of base pairs around the helix axis [the average twist angle is 36° in the B-type DNA (B-DNA) conformation (36)] keeps the C5=C6 double bonds too far apart. In contrast, although base stacking in single-stranded thymine oligomers is rare, the more flexible backbone does not prevent these rare stacks from adopting conformations that are suitable for dimerization.

A comparison of the literature that describes dimer yields in nucleic acids with A-type and B-type double-helical structures supports the hypothesis that dimerization in double-stranded DNA occurs as a result of uncommon conformations. The rate of dimer formation is decreased by up to a factor of 2 when double-stranded DNA is switched from the B-type to the A-type conformation (37). Even larger protective effects have been observed at TT steps in hairpins with A-type structure (38). The same base pairing is found in both structural classes, and the only difference is the distribution of accessible conformations. This evidence establishes that the conformation controls the reactivity in duplex DNA, just as in single-stranded (dT)18. The average twist angle between successive base pairs differs in A-DNA by only a few degrees when compared to B-DNA, suggesting that the ideal geometries in both helices are nonreactive. Instead, dimerization is proposed to take place at TT steps that deviate in just the right way from the average duplex structure. Thus, the smaller amount of conformational variation in A-type versus B-type structures (36) explains the greater resistance of A-DNA to CPD formation.

The model we have derived from our results implies that static TT conformation (7, 29), and not conformational motions after photoexcitation (6, 39), determines the outcome of a reaction. Flexibility does not help an excitation at a bipyrimidine doublet attain a better conformation within its lifetime, but a more flexible backbone can increase the fraction of reactive conformations that are present at the time of light absorption. With knowledge about the conformational criteria that make reaction inevitable, molecular-dynamics simulation can be used to identify damage hot spots.

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