19714
J. Phys. Chem. 1996, 100, 19714-19718
Electron Addition to Thymine Dimers and Related Compounds: A Mimic of Natural Repair Abbas Pezeshk* Department of Chemistry, Moorhead State UniVersity, Moorhead, Minnesota 56563
Ian D. Podmore Department of Chemistry, UniVersity of Leicester, Leicester LE1 7RH, U.K.
Paul F. Heelis* North East Wales Institute, Mold Road, Clwyd LL11 2AW, U.K.
Martyn C. R. Symons Department of Applied Science, De Montfort UniVersity, Leicester LE 19BH, U.K. ReceiVed: July 22, 1996; In Final Form: September 30, 1996X
Solar UV light can be dangerous to cellular systems. One manifestation of this is the formation of thymine dimers, containing cyclobutane rings, in suitable regions of duplex DNA. Fortunately, these dangerous defects are rapidly recognized by a number of DNA repair enzymes, including the photolyase enzymes, whose activity is also triggered by the sun’s rays. This is thought to result in rapid electron transfer from the excited chromophores therein, to the pyrimidine dimer, resulting in opening of the cyclobutane ring. The electron then moves back, reconstituting the enzyme and the two pyrimidine monomer units. In an attempt to mimic this process in the absence of the enzyme, we have added radiation-generated electrons to thymine and uracil dimers at 77 K and used EPR spectroscopy to study the initial products. The results show that for transsyn-1,3-dimethyluracil dimer, a long-lived dimer radical anion is formed, the electron being confined to one of the uracil units. On annealing, this was converted irreversibly into the monomer radical anion. In contrast, the cis-syn thymine dimer, and its structural analogue, the cis-syn-1,3-dimethyluracil derivative, gave the monomer anions directly at 77 K. These results confirm that electron addition is facile and that the dimer anions are very unstable. The results also show that both bonds break rapidly, rather than just one. We have also studied the structurally related dihydro derivatives in order to extend and confirm these conclusions.
Introduction Perhaps the most significant damage center induced by ultraviolet light on cells is the cyclobutane pyrimidine type dimers, PyrPyr.1-3 This is formed by direct absorption of light quanta in the 250-300 nm range by DNA. Probably there is some migration of excitation within the stacked bases, and it seems that thymine (T) centers are favored. These may emit light and return to the ground state or may form various photoproducts. Of these, the thymine dimer centers, which are cyclobutane derivatives, dominate. The duplex structure of DNA favors the cis-syn diastereomer only, as shown in Figure 1. This lesion is potentially very dangerous, but nature has developed a beautifully simple method for removing the damage in an unusually direct manner.4,5 This is achieved by various DNA photolyase enzymes that simply break the cyclobutane structure, giving back the two adjacent, undamaged, thymine units (Figure 1). This requires photoexcitation (300-400 nm) of the enzyme, which is then thought to transfer one electron to the PyrPyr dimer.6,7 The resulting radical anion then breaks open, and the electron is returned to the enzyme. (See below.) Despite the wide occurrence of this repair process in nature, it is not clear whether or not it is used in the human body. This X
Abstract published in AdVance ACS Abstracts, November 15, 1996.
S0022-3654(96)02196-X CCC: $12.00
Figure 1. Thymine dimer formation by stacked thymine nucleotides in the same strand of double-strand DNA induced by ultraviolet light (UV-B, 280-315 nm). The reverse reaction, i.e., repair, is catalyzed by DNA photolyase, which uses near UV and visible light.
is a highly controversial area at present, with strong views on both sides.8,9 In view of the remarkable efficiency, beauty, and simplicity of the process involved, it would seem strange if mankind has not developed it in his own fight against such damage. There are a range of different photolyases known and probably others yet to be discovered. The active site of cyclobutane pyrimidine dimer photolyase seems to comprise © 1996 American Chemical Society
Electron Addition to Thymine Dimers
J. Phys. Chem., Vol. 100, No. 50, 1996 19715 dine radical intermediate in the reaction, revealing that photorepair is initiated by a photoinduced electron transfer between the pyrimidine dimer and the cofactor. Pouwels et al.,12 using photochemically induced dynamic nuclear polarization spectroscopy and the cis-syn-2′-deoxythymidylyl-(3′f5′)-2′-deoxythymidine cyclobutane dimer and reduced flavin as sensitizer, have shown that thymine radical anion is the intermediate that is involved in the splitting of the dimer. The crucial requirement of this electron-transfer process is the rapid breakdown of the dimer following electron addition before electron return to the sensitizer can occur. Our aim was to explore this process using “temperature-resolved” spectroscopy. The results strongly support this simple process and show that the dimer anion gives the monomer anion rapidly, even at 77 K.8 Experimental Section Pyrimidine dimers were kindly provided by S. D. Rose and R. F. Hartman, Arizona State University. The preparation of cis-syn-dimethyluracil dimer and trans-syn-dimethyluracil dimer was carried out by irradiation of 1,3-dimethyluracil in 40% aqueous acetone as sensitizer (N2 purging) with a 450 W Hanovia mercury lamp with a Pyrex filter, followed by separation of isomers by silica gel column chromatography.13 The preparation of cis-syn thymine dimer was carried out by irradiation of a frozen solution of thymine in water, followed by purification by recrystallization from water.14 Sample Preparation. Various aqueous (H2O or D2O) glasses were used, the most satisfactory being aqueous lithium chloride, which we have favored previously in studies of electron addition to DNA.15 Glasses containing ca. 50 mg/mL of sample were frozen as small pellets in liquid nitrogen and exposed to ionizing radiation at 77 K using a Vickrad 60Co γ-ray source. Doses up to ca. 1 Mrad were used, the results being independent of dose. EPR spectra were recorded on a Varian E-109 X-band spectrometer, interfaced with an Archimedes computer. Samples were annealed in situ in the insert dewar after decanting the liquid nitrogen, with continuous monitoring of spectra and rapid recooling when significant changes were detected. Also, samples were annealed using a copper block cryostat up to the required temperatures and held at this temperature for ca. 5 min before recording at 77 K. Results and Discussion
Figure 2. Structures of the electron-donating cofactor FADH2 and the two light-gathering antenna cofactors MTHF and 8-HDF.
two units, a folate and a reduced flavin (see Figure 2). The former is best at harvesting near UV light, while the latter is best at rapid electron donation. Thus the folates have an intense, broad band in the 390 nm region (max ca. 25 000), while that for the flavin is at ca. 350 nm and of about one-quarter the intensity. It has therefore been suggested that the light is largely absorbed by the folate and that the electronic energy moves across to the flavin. This can then transfer the outer electron to the dimer. This has been specially recognized by the protein and is presumably held close to the flavin during the ringopening process. Recently, Park et al. have reported the threedimensional crystallographic structure of DNA photolyase from Escherichia coli.10 The enzyme has two domains, an aminoterminal alpha/beta domain and a carboxyl-terminal helical domain. The cofactor folate binds in a cleft between the two domains. The energy transfer from folate to the cofactor occurs over a distance of 16.8 A.10 Okamura et al.,11 using picosecond flash photolysis and the deoxyuridine dinucleotide photodimer, have detected a pyrimi-
In this study, we used the crucial cis-syn thymine dimer (I) which is the major species formed in cells, but we also studied the cis- and trans-syn uracil derivatives for comparative purposes. In addition, we used our procedure to generate
electron adducts of the “parent” dihydro derivatives of thymine and uracil. For comparison, we also studied duplex DNA, using ionizing radiation together with EPR spectroscopy, by simply freezing aqueous solutions.16 However, if this method is used for small molecules, phase separation occurs, which can greatly modify the results, relative to those for true dilute solutions.
19716 J. Phys. Chem., Vol. 100, No. 50, 1996
Pezeshk et al.
TABLE 1: 1H Hyperfine Coupling Constants for the Radical Anions Prepared in This Study and for Related Radicals 1
H hyperfine coupling/G
source
radical
T c/sa T
c/s U2 TH2 UH2
T•T•TH• U•U2•U•U•TH2•UH2•-
UH2b
UH2•-
U t/s U2
a
RH
βH
12.5 (av) 12.5 (av) 12.5 (av) 20.7 12.5 (av) 12.5 (av)
α
26 (1) 29 (1) 9 (1) 31.7 (1) 10.7
c/s and t/s are cis-syn and trans-syn. b See ref 14.
To retain the use of largely aqueous solvent systems, it is necessary to use additives that interfere with the rapid growth of pure ice crystals. In the present study we used, primarily, lithium chloride, with H2O or D2O as solvent, since these gave the best spectra. When rapidly frozen, these solvents give good glasses, with negligible phase separation. (This was confirmed using the Mn2+ test reported previously.17) Therefore, on exposure of dilute solutions of organic substrates in these aqueous solvents to ionizing radiation at 77 K, electron capture is the only primary process of the substrate to occur significantly. This arises because under these conditions electron loss is almost statistical, so that direct loss from the substrate can be ignored. However, only the substrate molecules can readily add electrons, so there are high yields of their electron adducts. In this way we can be sure that EPR spectra from organic radicals must be assignable to these electron adducts or to breakdown products thereof. The EPR spectra show features for these electron adducts, together with broad features of Cl2•- radicals and probably ClOH•- anions, which are the major electron loss centers. These features do not interfere significantly with those of interest herein and are not considered further. Our results for the electron adducts are summarized in Table 1, and typical EPR spectra are shown in Figures 3 and 4. Spectra for the anions of the related dihydrouracil and -thymine are shown in Figure 5. The Cis-Syn Dimers. Both the thymine and uracil cis-syn dimers gave only EPR features assignable to the monomer radical anions. That for the uracil anion was identical to the spectrum shown in Figure 4b, and that for the thymine anion is shown in Figure 3a. The monomer radical anions will protonate irreversibly at the C(6) position upon annealing from 77 to 163 K, forming the characteristic octet assigned to TH• radicals (Figure 3b, VI). For the uracil dimer it could possibly be argued
Figure 3. First-derivative X-band EPR spectra for a dilute solution of thymine dimer in 10 M aqueous lithium chloride after exposure to ionizing radiation at 77 K, (a) recorded at 77 K, showing the characteristic doublet assigned to monomeric thymine radical anions, and (b) after annealing to ca. 163 K and recooling to 77 K, showing the characteristic octet from TH• radicals. (Feature R is a part of the spectrum for Cl2•- radicals.)
Figure 4. As for Figure 3, for trans-syn-1,3-dimethyluracil dimer, (a) at 77 K, showing the 20 G assigned to the parent radical anions, and (b) after annealing to 145 K, showing features assigned to monoemric uracil radical anions.
that the proton splitting for the parent dimer anion is fortuitously equal to that for the monomer anion. We reject this on the basis that the form of the spectra is as expected for coupling to an R-proton, as for the monomer and not for a β-proton. More compelling is the crucial case of the thymine dimer; the parent radical anions should have relatively narrow singlet spectra,
since the β-proton has been replaced by a methyl group. Since γ-protons always give very small splittings, they are not expected to contribute. Thus the observed doublet cannot be due to the parent species and must surely be assigned to the monomer anions. The Trans-Syn Dimer of 1,3-Dimethyluracil. This compound gave a species having a well-defined 20 G EPR doublet (Figure 4), which we assign to the parent adduct (see below). On annealing from 77 K to ca. 145 K this doublet was lost irreversibly, giving a species with a poorly defined doublet of average splitting, ca. 12 G (Figure 4b). The former is a radical
Electron Addition to Thymine Dimers
Figure 5. As for Figure 3, but for (a) 5,6-dihydrothymine, showing features assigned to the radical anions, and (b) for 5,6-dihydrouracil, showing features also assigned to the radical anions.
Figure 6. Two possible modes of breakdown of the U•- pyrimidine dimer radical anions.
with very strong coupling to a single β-proton, while the latter shows a smaller coupling to an R-proton. In fact, the second species has a spectrum that is identical to that of a normal uracil radical anion (U•-).18 Since the 20 G doublet center is a precursor to the formation of U•-, it must either be the parent cyclobutane radical anion or one with one bond retained (Figure 6). The former is expected to have the spin confined to one ring and thus could well give rise to the 20 G doublet. However, the latter should show coupling to one RH and one βH, giving a quartet, which was not observed. We conclude that the 20 G species is the
J. Phys. Chem., Vol. 100, No. 50, 1996 19717 parent anion. Therefore we also conclude that the monobonded intermediate is very unstable and breaks to give U•- + U directly when it is formed, as shown in Figure 6. These results require that there is a high spin-density on C(4) of the parent anion. Also, the lack of major asymmetry in the 20 G doublet shows that there is very little g-shift, (i.e. gx ≈ gy ≈ gz), showing that delocalization onto oxygen is quite small and also that the hydrogen is indeed β to the unpaired electron. Begley et al.,19,20 using secondary deuterium isotope effects, have studied the mechanism of dimer splitting with both photolyase and model systems. Following electron addition to the dimer, they have concluded that the fragmentation occurs by a stepwise mechanism for the dimer radical cation, whereas for the dimer radical anion a nonsynchronous concerted fragmentation mechanism was suggested.19,20 Dihydro Derivatives. To confirm the above results, we have prepared the electron adducts of 5,6-dihydrothymine and -uracil, using exactly the same procedures. The results tie in most satisfactorily with that for the uracil dimer anion (Figure 3 and Table 1). Thus, for the dihydrothymine anion, which has only one C(5) β-hydrogen, the hyperfine splitting is ca. 26 G. This should be compared with ca. 20 G splitting from the uracil dimer anion. For the dihydrouracil anion, the two β-protons gave 29 and 7 G, with an average of 18 G. These results confirm that, when C (5) and C (6) are saturated, the added electron is quite strongly localized on C (4). In fact, we can use the dihydrouracil data to estimate a spin-density of ca. 49% using the average value for θ ) 30°, together with the usual cos2 θ law. (θ is the angle between the direction of the p(π) orbital on C(4) and the average C-H (β) direction.) Alternatively, setting 29 G as the value for β ) 0, we get ca. 56%. We conclude that about 52% of the SOMO is on this single carbon. The fact that the features are narrow, with ∆HMS ≈ 5 G, shows that, indeed, the spin-densities on the two nitrogen atoms must be very low. Hence, the remaining spin-density is presumably associated with the C(4) carbonyl oxygen and the C(2) carbonyl group. We also conclude that there is a considerable degree of twist for the dihydrouracil anion, which must therefore be far from a “planar” structure. This is less marked for the dihydrothymine derivative. However, for the diuracil anion the β-proton splitting is very close to the “average” required for β ≈ 30°, suggesting that the ring containing the excess electron is in this case close to being planar. This seems to accord with the rigid requirements of the cyclobutane ring. The “radical anion” of dihydrothymine was probably detected by Henrikson and Snipes using a sulfuric acid matrix,21 although no EPR data were given for this species. Also, the EPR spectrum of the dihydrouracil derivative was first reported by Egtvedt et al.,22 in irradiated crystals of the parent compound. They obtained 31.7 G for the two β-protons, but in this case the C(4) carbonyl oxygen was protonated. This fact nicely explains the slightly larger βH coupling constants found in this case. Alternatively, we can conclude that, in the present study, carbonyl protonation does not occur at 77 K. Acknowledgment. This work was supported by NIH Grant R15-CA60045 (A.P.) and by Fogarty Grant F06-TW02095 (A.P.). References and Notes (1) Heelis, P. F.; Hartman, R. F.; Rose, S. D. Chem. Soc. ReV. 1995, 285. (2) Rupert, C. S.; Enzymatic Photoreactivation: Overview in Molecular Mechanisms for Repair of DNA, Part A; Hanawalt, P. C., Setlow, R. B., Eds.; Plenum Press: New York, 1975; p 73.
19718 J. Phys. Chem., Vol. 100, No. 50, 1996 (3) Sancar, A. Biochemistry 1994, 33, 2. (4) Kim, S.-T.; Sancar, A. Photochem. Photobiol. 1993, 57, 895. (5) Eker, A. P. M.; Yajima, J.; Yasui, A. Photochem. Photobiol. 1994, 60, 125. (6) Kim, S.-T.; Sancar, A.; Essenmacher, C.; Babcok, G. T. J. Am. Chem. Soc. 1992, 114, 4442. (7) Heelis, P. F.; Parsons, B. J. J. Chem. Soc., Chem. Commun. 1994, 793. (8) Podmore, I. D.; Heelis, F.; Symons, M. C. R.; Pezeshk, A. Chem. Commun. 1994, 1005. (9) deGrujil, F. R.; Roza, L. J. Photochem. Photobiol. B: Biol. 1991, 10, 367. (10) Park, H.-W.; Kim, S.-T.; Sancar, A.; Deisenhofer, J. Science 1995, 268, 1866. (11) Okamura, T.; Sancar, A.; Heelis, P. F.; Hirata, Y.; Mataga, N. J. Am. Chem. Soc. 1989, 111, 5967. (12) Pouwels, P. J. W.; Kaptein, R. Applied Magn. Reson. 1994, 7, 107. (13) Elad, D.; Rosenthal, I.; Sasson, S. J. Chem. Soc. C 1971, 2053. (14) Wulff, D. L.; Fraenkel, G. Biochim. Biophys. Acta 1961, 51, 332.
Pezeshk et al. (15) See, for example: Cullis, P. M.; McClymont, J. D.; Malone, M. E.; Mathew, A. N.; Podmore, I. D.; Sweeney, M. C.; Symons, M. C. R. J. Chem. Soc., Perkin Trans. 2 1992, 1695. (16) See, for example: Cullis, P. M.; Jones, G. D. D.; Lea, J.; Symons, M. C. R.; Sweeney, M. J. Chem. Soc., Perkin Trans. 2 1987, 1907, and references therein. (17) Ginns, I. S.; Symons, M. C. R. J. Chem. Soc. (A) 1972, 143. (18) See, for example: Boon, P. J.; O’Connell, A.; Podmore, I. D.; Symons, M. C. R. J. Chem. Soc., Perkin Trans. 2 1993, 2061, and references therein. (19) Austin, R.; McMordie, S.; Begley, T. P. J. Am. Chem. Soc. 1992, 114, 1886. (20) Begley, T. P. Acc. Chem. Res. 1994, 27, 394. (21) Henriksen, T.; Snipes, W. J. Chem. Phys. 1970, 52, 1997. (22) Egtvedr, L.; Sagstuen, E.; Bergene, R.; Henriksen, T. Radiat. Res. 1978, 75, 252.
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