Chem. Res. Toxicol. 2002, 15, 671-676
671
HPLC Isolation and Mass Spectrometric Characterization of Two Isomers of Thymine Glycols in Oligodeoxynucleotides Yinsheng Wang* Department of Chemistry-027, University of California at Riverside, Riverside, California 92521-0403 Received November 21, 2001
Thymine glycol, or 5,6-dihydroxy-5,6-dihydrothymine, is the major oxidation product of thymine. Herein we report the isolation of both the (5S, 6R) and (5R, 6S) isomers of cis thymine glycols from several synthetic oligodeoxynucleotides (ODNs) upon oxidation with osmium tetraoxide. Our results show that tandem mass spectrometry can determine the sites of thymine glycol in ODNs by producing characteristic fragment ions, [a - 143] and its complementary w ions at the modification site. We further demonstrate that the [M + H]+ and [M + Na]+ ions of the two cis stereoisomers of thymine glycol in the dinucleotides, which are extricated from the ODNs by nuclease P1, gave distinctive product-ion spectra.
Introduction Reactive oxygen species (ROS),1 such as hydroxyl radical, superoxide anion, and hydrogen peroxide, are generated by both exogenous and endogenous processes and they can damage nucleic acids. Those nucleic acid damages have been implicated in cancer, neurological disorders, and natural processes of aging (1-3). Although our ultimate goal is the identification and quantification of those DNA adducts generated in vitro or in vivo, a more immediate one is to characterize modified oligodeoxynucleotides (ODNs)1 that are prepared by chemical synthesis or chromatographic purification and used to investigate the effects of damage on DNA replication and repair (4). Because MS is sensitive and tandem MS (MS/MS)1 provides structure information, modified ODNs have been characterized by MS/MS. For example, electrospray ionization (ESI)1-MS/MS has been used for characterizing ODNs bearing aflatoxin B1guanine adducts (5), DNA photoproducts (6, 7), hedamycin-DNA adducts (8), and methylated cytosine and adenine adducts (9). In addition, enzymatic digestion combined with MS has been used for characterizing the structures of a number of different modifications in ODNs. Exonuclease digestion combined with matrixassisted laser desorption/ionization (MALDI)1 time-offlight (TOF)1 MS has been used for locating the site of modifications (10-13). On the other hand, MS/MS of the digestion product of an endonuclease, nuclease P1, has been used for determining the structures of modifications in ODNs (7, 14, 15). Thymine glycol, or 5,6-dihydroxy-5, 6-dihydrothymine (mostly cis isomers, structures shown in Scheme 1), is * To whom correspondence should be addressed. E-mail:
[email protected]. Fax: (909) 787-4713. Phone: (909) 787-2700. 1 Abbreviations: ROS, reactive oxygen species; ODN, oligodeoxynucleotide; Tg, thymine glycol; SM, starting material; MS/MS, tandem mass spectrometry; ESI, electrospray ionization, MALDI, matrixassisted laser desorption/ionization; TOF, time-of-flight; HOMO, highest occupied molecular orbital.
Scheme 1
the major stable product of thymine modification in vitro and in vivo (16, 17). For example, thymine glycol and thymidine glycol have been detected in the urine of laboratory animals and human (17, 18). It can also form from the deamination of the 5-methylcytosine glycol. The deamination process may be involved in the C to T transition mutation in p53 tumor suppressor gene, which is the most frequent mutation in p53 (19). Previously, only one cis isomer of thymine glycol has been isolated from ODNs after potassium permanganate (KMnO4) treatment (20-22). As a result, NMR structure of only one cis isomer of thymine glycol has been determined (22). Teebor et al. (23) showed that both cis isomers of thymine glycols, however, were produced in DNA upon in vitro ionizing radiation; Rieger and coworkers (24) recently found that both cis isomers of thymine glycols are produced in ODNs upon osmium tetraoxide (OsO4) treatment. The two cis isomers of thymidine glycols are known to have different chemical stability upon alkali treatment (25), and they may affect the DNA helix structure in different ways. To study the effects of different isomers on the structures and chemical stabilities of ODNs or duplex DNA bearing the lesion, we need to prepare and characterize ODN substrates with defined lesions. Herein, we report the HPLC isolation and detailed mass spectrometric characterization of two cis isomers of thymine glycols in several different ODNs. We will demonstrate that MS/ MS can not only locate the site of thymine glycol in an
10.1021/tx0155855 CCC: $22.00 © 2002 American Chemical Society Published on Web 04/03/2002
672
Chem. Res. Toxicol., Vol. 15, No. 5, 2002
Wang
ODN, but also distinguish the two stereoisomers of cis thymine glycol in ODNs.
Experimental Section All ODNs in this study were obtained from Integrated DNA Technologies, Inc. (Coralville, IA) and used without further purification. For oxidation, 30 nmol of ODN was dissolved in 100 µL of 2% aqueous solution of OsO4 and incubated at room temperature for 6-48 h. The residual OsO4 was extracted three times with an equal volume of carbon tetrachloride, and the aqueous layer was dried by using a Savant Speed-Vac (Savant Instruments Inc., Holbrook, New York). The dried residue was redissolved in water and injected directly for HPLC analysis. The HPLC separation was carried out on a Surveyor system with a photodiode array detector (ThermoFinnigan, San Jose, CA), and a 4.6-mm i.d. reversed-phase C18 column (25 cm in length, 5 µm in particle size, and 300 Å in pore size, Varian, Walnut Creek, CA) was used. The flow rate was 1.0 mL/min, and a 35-min gradient of 6 to 12% CH3CN in 50-mM triethylammonium acetate (pH 6.8) was used. The photodiode array detector was set at 260 nm for monitoring the eluents. The HPLC fractions were then dried by using the Speed-Vac. ESI-MS and MS/MS experiments were carried out on an LCQ Deca XP ion-trap mass spectrometer (ThermoFinnigan, San Jose, CA). A solution of 50/50 (v/v) of acetonitrile/H2O was used as the carrier and electrospray solvent. A 2-µL aliquot of a 3-µM sample solution was injected in each run. The spray voltage was 4.6 kV, and the capillary temperature was maintained at 220 °C. MS/MS was done by selecting the [M - 2H]2-, [M - H]-, or other ions as indicated for collisional activation; the mass width for precursor selection was set at 3 m/z units, and the collision gas was helium. Each spectrum was an average of approximately 30 scans, and the time for each scan was 0.3 s. Nuclease P1 was purchased from Sigma-Aldrich (St. Louis, MO) and used without further purification. For all the digestion reactions, no additional buffer was used except that present in the commercial preparation of the enzyme. In a typical digestion reaction, 0.5 µL of 1 unit/µL nuclease P1 solution was added to 25 µL of a 3-µM ODN solution, and the mixture was incubated at room temperature for 10 min. The resulting digestion sample was subject to mass spectrometric analysis by direct flow injection, and 1-2 µL of sample aliquot was injected for each data acquisition. Quantum calculations were carried out on a computer with a 1 GHz Pentium III processor using PC Spartan Pro (Wavefunction, Inc., Irvine, CA). The geometries of the two isomers of N1-methyl thymine glycols were optimized by using the Hartree-Fock method with the 6-31G* basis set.
Results and Discussion 1. HPLC Isolation of Thymine Glycol-Containing ODNs. Following treatment of d(AATGAAA), d(CGCGATACGCC) and d(GAGTTGAG) in a 2% OsO4 aqueous solution at room temperature for 6-48 h, we were able to isolate single thymine glycol-containing ODNs by HPLC (chromatograms and the respective oxidation times are shown in Figure 1). We separated the two cis thymine glycols from each other and from the starting ODN except for d(CGCGATACGCC), where the (5S, 6R) isomer coelutes with the starting ODN. Our assignments of the stereoisomers of those ODNs are based on the following lines of evidence. First, only cis isomers of thymine glycols are generated upon osmium tetraoxide or potassium permanganate oxidation (26, 27). Second, the (5S, 6R) isomer of thymidine glycol elutes earlier than the (5R, 6S) isomer on a reversedphase HPLC column (21,22,28). Furthermore, our assignment is consistent with distinctive gas-phase frag-
Figure 1. HPLC chromatograms for the separation of oxidation products of ODNs upon 2% OsO4 treatment: (a) d(GAGTTGAG), 6 h; (b) d(AATGAAA), 48 h; (c) d(CGCGATACGCC), 48 h (SM is the starting material).
mentations of the two isomers as discussed in the latter section of this article. Our results show that the oxidation of d(GAGTTGAG) is significantly faster than that of the other two ODNs. We estimated that, from relative peak areas in the chromatograms, the yield of thymine glycol formation in d(GAGTTGAG) upon a 6-h OsO4 treatment is close to that in d(AATGAAA) and d(CGCGATACGCC) upon a similar treatment for 48 h. Furthermore, both cis isomers of thymine glycols are isolated. In contrast, only one isomer of cis thymine glycol in ODNs was isolated when potassium permanganate was used for oxidation (21, 22, 29). Interestingly, we found that the 5′ thymine in d(GAGTTGAG) was preferentially oxidized over the 3′ thymine. Upon longer treatment (24 h), we also obtained ODNs where both thymines are modified to thymine glycols. We, however, were not able to isolate any ODN where only the 3′ thymine is modified to thymine glycol. 2. ESI-MS/MS for the Site Identification of Thymine Glycols in ODNs. The identity of thymine glycol modification is determined by ESI-MS, which shows a molecular weight increase of 34 units (data not shown). The site of thymine glycol modification can be located by MS/MS. MS/MS of 29.1 and 35.6 min fractions from the oxidation mixture of d(GAGTTGAG) show that the thymine on the fourth position is modified to thymine glycol. Product-ion spectra of the [M - 2H] 2- ions of unmodified d(GAGTTGAG) and the (5S, 6R) isomer of d(GAGTgTGAG) are shown in Figure 2, and Tg1 is thymine glycol [the nomenclature for the fragmentation of ODNs follows
Isolation and MS of Thymine Glycols
Chem. Res. Toxicol., Vol. 15, No. 5, 2002 673 Scheme 2
Scheme 3
Figure 3. Negative-ion ESI mass spectra of nuclease P1 digestion products of d(GAGTgTGAG).
Figure 2. Product-ion spectra of ESI-produced [M - 2H]2- ions of (a) d(GAGTTGAG) and (b) d(GAGTgTGAG).
that of McLuckey et al. (30)]. Comparing the two production spectra, we found that the masses of the [a3 - G], w2, and w4 ions of the (5S, 6R) isomer remain the same upon thymine glycol formation, whereas those of [a6 G], [a7 - A], w5, w6, and w7 ions increase by 34 units, indicating that thymine at the fourth position is modified. It is important to note that the w4 ion in the MS/MS of the [M - 2H]2- ions of the (5S, 6R) isomer is significantly more abundant than that of the unmodified ODN. Furthermore, we observe an ion corresponding to the loss of a 143 unit fragment from the a4 ion, which is due to the loss of a neutral moiety from the thymine glycol and is not present in the MS/MS of the starting ODN. Scheme 2 shows the cleavage leading to the formation of this ion and its complementary wN-n ion. The [an - 143] ion appears unusual because its formation involves cleavages of two bonds. Cleavages of two bonds within modified bases, however, have been observed in ODNs containing adenine photoproducts (7) and in trinucleotides bearing a pyrimidine[6-4]pyrimidone thymine dimer or its Dewar valence isomer (14). We proposed a mechanism for the formation of the [an - 143] ion, and
the mechanism involves two steps of hydrogen transfer (Scheme 3). The first hydrogen transfer involves the breaking of the N1-C6 bond, which has been observed for thymidine glycol in solution (31). The second hydrogen transfer in the proposed mechanism is very similar to that involved in the fragmentation of trinucleotides containing a pyrimidine (6-4)pyrimidone thymine dimer (14). The latter process gives rise to a neutral loss of a 113 unit fragment (14). The formation of both the [an 143] ion and its complementary wN-n ion involves cleavage 3′ to the thymine glycol modification and those two ions provide a diagnosis for the site of thymine glycol modification in ODNs. Similarly we were able to obtain both cis isomers of thymine glycols in two other ODNs, d(AATGAAA) and d(CGCGATACGCC), and ESI-MS/MS can determine the site of the thymine glycol modification (data not shown). 3. Coupled Nuclease P1 Digestion-MS/MS for the Structure Determination of Stereoisomers of Thymine Glycols. We examined whether the coupled nuclease P1 digestion-MS/MS method (14) can distinguish the two cis isomers of the thymine glycol. To this end, we digested the (5S, 6R) and (5R, 6S) isomer-containing d(GAGTgTGAG) with nuclease P1 and acquired the negative-ion ESI-MS [Figure 3 gives the ESI-MS of the nuclease P1 digestion product of the (5S, 6R) isomer of d(GAGTgTGAG)]. The results show that the digestion products of both isomers give abundant ions of m/z 659 and 681, which are the [M - H]- and [M + Na - 2H]- ions of dinucleotide d(pTgpT). The identities of the dinucleotides resulting from nuclease P1 digestion provide further support for our assignment of the site of thymine glycol.
674
Chem. Res. Toxicol., Vol. 15, No. 5, 2002
Wang
Figure 5. Product-ion spectra of ESI-produced [M + Na]+ ions of (a) the (5S, 6R) and (b) the (5R, 6S) isomers of d(pTgpT) resulting from nuclease P1 digestion of d(GAGTgTGAG).
Figure 4. Product-ion spectra of ESI-produced (a) [M (b) [M + Na - 2H]-, and (c) [M + 2Na - 3H] - ions of d(pTgpT) resulting from nuclease P1 digestion of d(GAGTgTGAG).
H]-,
The retaining of the adjacent nucleotide 3′ to Tg after nuclease P1 digestion has been observed by many investigators (32-35), though the origin of which has not been identified. We find that the results are consistent with the mechanism of nuclease P1 digestion as revealed by the X-ray cocrystal structure of the enzyme with its bound substrate (36). The X-ray structure shows that the base 5′ to the phosphodiester bond to be cleaved must fit into a small binding pocket in the active site and forms a π stacking interaction with a phenylalanine residue in the active site. Because of lack of aromaticity and its bulky nature, thymine glycol may not fit into the active site and form a stacking interaction with the phenylalanine residue. Therefore, the adjacent 3′ phosphodiester bond cannot be hydrolyzed, resulting in the formation of a dinucleotide. Product-ion spectra of the [M - H]-, [M + Na - 2H]-, and [M + 2Na - 3H]- ions of the two cis isomers of d(pTgpT), which were from the nuclease P1 digestion of d(GAGTgTGAG), show no difference. MS/MS of the [M - H]- ions of d(pTgpT) shows a facile loss of a familiar neutral moiety C5H5NO4 (- 143 units), which is from the base portion of the thymidine glycol (Figure 4). The assignment of this fragment is consistent with a further stage MS/MS of this fragment ion, which shows that the deoxythymidine moiety is retained in this fragment ion (MS3, data not shown). Other fragment ions with m/z 321 and 401 were also produced. In addition to the loss of 143 unit ion, MS/MS of the [M + Na - 2H]- ion shows an ion with loss of 117 unit fragment (m/z 564), whereas those fragment ions originate from strand cleavages are not produced any more. Product-ion spectrum of the [M + 2Na - 3H]- ion, however, shows that the loss of the 117 unit ion is predominant. To explain the effect of Na+ ion on frag-
mentation, we proposed that the Na+ ion on the terminal phosphate may coordinate with O2 of thymine glycol thereby stabilizing the N1-C2 bond. Therefore, instead of losing the 143 unit moiety, it loses the 117 unit moiety (Scheme 4). In contrast, such stabilization does not exist in the [M - H]- ion. The product-ion spectra of the [M + Na]+ ions of the two dinucleotides, however, are distinctive (spectra shown in Figure 5). The MS/MS of the [M + Na]+ ion of dinucleotide containing the (5S, 6R) isomer of thymine glycol shows that the water loss is in parallel with many other cleavage pathways, whereas the same water loss is the predominant cleavage for the (5R, 6S) isomer. The product-ion spectra of the [M + H]+ ions of the two isomers also show that the water loss occurs more readily for the (5R, 6S) isomer than for the (5S, 6R) isomer (data not shown). Similar results were obtained for dinucleotides d(pTgpA) and d(pTgpG) that were extricated from d(CGCGATgACGCC) and d(AATgGAAA) by nuclease P1 (data not shown). To explain the different susceptibility of water loss of the two isomers, we calculated the energy of the [M + H]+ and [M + Na]+ ions of a model compound, N1methylthymine glycol. We replaced the sugar and phosphate moieties with a methyl group and we used the Hartree-Fock method with the 6-31 G* basis set (Scheme 5). For the protonated or sodiated 1-methylthymine glycol, the ab initio energy for the (5S, 6R) isomer is 5-12 kcal/mol lower than that of the (5R, 6S) isomer (Table 1). The water loss from the (5R, 6S) isomer, therefore, is expected to be a thermodynamically more favorable process than that from the (5S, 6R) isomer because the water loss from the base moiety of the two isomers of thymine glycol leads to the formation of the same product (Scheme 5). It is important to point out that the coupled enzymatic digestion tandem mass spectrometry method is not only structurally informative, but also very sensitive. The amount of material that we injected for each MS analysis was less than 10 pmol. In addition, because samples can
Isolation and MS of Thymine Glycols
Chem. Res. Toxicol., Vol. 15, No. 5, 2002 675 Scheme 4
OsO4. It remains to be seen whether the preferential oxidation of the 5′ thymine in stacked TT also occurs in the formation of other types of thymine damage, i.e., 5-hydroxymethyluracil and 5-formyluracil. Second, the HPLC retention times of the two isomers are dramatically different, suggesting that the two isomers may affect duplex DNA structure in a quite different way. It is important to note that only one isomer of thymine glycol was previously isolated in ODNs by KMnO4 oxidation (20-22).
Scheme 5
Conclusions
Table 1. Absolute (in a.u.) and Relative (in kcal/mol, shown in italics) Energies for the N1-Methylthymine Glycol Computed at the 6-31 G* Level H+
O4 O2
Na+
O4 O2
(5R, 6S)
(5S, 6R)
-641.744 741 11.7 -641.754 024 5.8 -803.138 378 8.9 -803.132 147 12.8
-641.763 334 0 -641.762 393 0.6 -803.152 575 0 -803.144 242 5.2
be analyzed immediately after the enzymatic digestion without any workup procedure, the total analysis time from the enzymatic digestion to the end of the data acquisition is less than 20 min. The method, therefore, is also rapid. 4. Biological Implications. The results of OsO4 oxidation of d(GAGTTGAG) are significant in several ways. First, the 5′ thymine in a stacked TT is significantly more susceptible to oxidation than its stacked 3′ thymine or an isolated thymine. It has been known for many years that the 5′ guanine of stacked GG is more susceptible to one electron photooxidation than the 3′ stacked guanine or an isolated guanine, which has been attributed to the localization of highest occupied molecular orbital (HOMO)1 on the 5′ base (37, 38). Saito and co-workers also showed that the HOMO of stacked TT localizes on the 5′ thymine (37), which is consistent with the preferential oxidation of the 5′ thymine in TT by
After OsO4 oxidation, we were able to isolate two stereoisomers of cis thymine glycols in several different ODNs by HPLC. Collisional activation of the [M - 2H]2ions of the thymine glycol-containing ODNs leads to characteristic cleavage 3′ to the thymine glycol site, resulting in the formation of [an - 143] and of wN-n ions. The production of those ions and the mass shifts of certain fragment ions facilitate the location of the sites of thymine glycols in ODNs. Another conclusion can be drawn from this study is that the thymine on the 5′ of a stacked TT is more susceptible to oxidation by OsO4, which is in accordance with the preferential localization of HOMO on the 5′ base in TT sequence (37) and is also analogous to many observations of the preferential damage of the 5′ guanine in stacked GG (37, 38). Further studies are under way to determine whether this is also valid for other types of oxidative thymine damage. We further demonstrated that the coupled nuclease P1 digestion-MS/MS method, which was developed for the differentiation of photomodified ODN isomers (7, 14), is also useful for distinguishing the two stereoisomers of thymine glycols. Upon collisional activation, the [M + H]+ and [M + Na]+ ions of the (5R, 6S) isomer of d(pTgpN) (N is an unmodified nucleoside) show more facile water loss than those ions of the (5S, 6R) isomer. This result is consistent with predictions from molecular orbital calculations.
Acknowledgment. The author acknowledges the University of California at Riverside for supporting this research.
References (1) Lindahl, T. (1993) Instability and decay of the primary structure of DNA. Nature 362, 709-15.
676
Chem. Res. Toxicol., Vol. 15, No. 5, 2002
(2) Finkel, T., and Holbrook, N. J. (2000) Oxidants, oxidative stress and the biology of ageing. Nature 408, 239-47. (3) Rolig, R. L., and McKinnon, P. J. (2000) Linking DNA damage and neurodegeneration. Trends Neurosci. 23, 417-24. (4) Wang, D., Kreutzer, D. A., and Essigmann, J. M. (1998) Mutagenicity and repair of oxidative DNA damage: insights from studies using defined lesions. Mutat. Res. 400, 99-115. (5) Marzilli, L. A., Wang, D., Kobertz, W. R., Essigmann, J. M., and Vouros, P. (1998) Mass spectral identification and positional mapping of aflatoxin B1- guanine adducts in oligonucleotides. J. Am. Soc. Mass Spectrom. 9, 676-82. (6) Wang, Y., Taylor, J. S., and Gross, M. L. (1999) Differentiation of isomeric photomodified oligodeoxynucleotides by fragmentation of ions produced by matrix-assisted laser desorption ionization and electrospray ionization. J. Am. Soc. Mass Spectrom. 10, 32938. (7) Wang, Y., Taylor, J. S., and Gross, M. L. (2001) Isolation and mass spectrometric characterization of dimeric adenine photoproducts in oligodeoxynucleotides. Chem. Res. Toxicol. 14, 738-45. (8) Iannitti, P., Sheil, M. M., and Wickham, G. (1997) High Sensitivity and Fragmentation Specificity in the Analysis of Drug-DNA Adducts by Electrospray Tandem Mass Spectrometry. J. Am. Chem. Soc. 119, 1490-1. (9) McLuckey, S. A., and Habibi-Goudarzi, S. (1994) Ion trap tandem mass spectrometry applied to small multiply charged oligodeoxyribonucleotides with a modified base. J. Am. Soc. Mass Spectrom. 5, 740-7. (10) Wu, H., Chan, C., and Aboleneen, H. (1998) Sequencing regular and labeled oligonucleotides using enzymatic digestion and ionspray mass spectrometry. Anal. Biochem. 263, 129-38. (11) Tretyakova, N., Matter, B., Ogdie, A., Wishnok, J. S., and Tannenbaum, S. R. (2001) Locating nucleobase lesions within DNA sequences by MALDI-TOF mass spectral analysis of exonuclease ladders. Chem. Res. Toxicol. 14, 1058-70. (12) Zhang, L. K., and Gross, M. L. (2000) Matrix-assisted laser desorption/ionization mass spectrometry methods for oligodeoxynucleotides: improvements in matrix, detection limits, quantification, and sequencing. J. Am. Soc. Mass Spectrom. 11, 854-65. (13) Zhang, L. K., Rempel, D., and Gross, M. L. (2001) Matrix-assisted laser desorption/ionization mass spectrometry for locating abasic sites and determining the rates of enzymatic hydrolysis of model oligodeoxynucleotides. Anal. Chem. 73, 3263-73. (14) Wang, Y., Taylor, J. S., and Gross, M. L. (1999) Nuclease P1 digestion combined with tandem mass spectrometry for the structure determination of DNA photoproducts. Chem. Res. Toxicol. 12, 1077-82. (15) Wu, H., Morgan, R. L., and Aboleneen, H. (1998) Characterization of labelled oligonucleotides using enzymatic digestion and tandem mass spectrometry. J. Am. Soc. Mass Spectrom. 9, 660-7. (16) Teoule, R., Bonicel, A., Bert, C., Cadet, J., and Polverelli, M. (1974) Identification of radioproducts resulting from the breakage of thymine moiety by gamma irradiation of E. coli DNA in an aerated aqueous solution. Radiat. Res. 57, 46-58. (17) Cathcart, R., Schwiers, E., Saul, R. L., and Ames, B. N. (1984) Thymine glycol and thymidine glycol in human and rat urine: a possible assay for oxidative DNA damage. Proc. Natl. Acad. Sci. U.S.A. 81, 5633-7. (18) Adelman, R., Saul, R. L., and Ames, B. N. (1988) Oxidative damage to DNA: relation to species metabolic rate and life span. Proc. Natl. Acad. Sci. U.S.A. 85, 2706-8. (19) Pfeifer, G. P. (2000) p53 mutational spectra and the role of methylated CpG sequences. Mutat. Res. 450, 155-66. (20) Basu, A. K., Loechler, E. L., Leadon, S. A., and Essigmann, J. M. (1989) Genetic effects of thymine glycol: site-specific mutagenesis and molecular modeling studies. Proc. Natl. Acad. Sci. U.S.A. 86, 7677-81.
Wang (21) Kao, J. Y., Goljer, I., Phan, T. A., and Bolton, P. H. (1993) Characterization of the effects of a thymine glycol residue on the structure, dynamics, and stability of duplex DNA by NMR. J. Biol. Chem. 268, 17787-93. (22) Kung, H. C., and Bolton, P. H. (1997) Structure of a duplex DNA containing a thymine glycol residue in solution. J. Biol. Chem. 272, 9227-36. (23) Teebor, G. et al. (1987) Quantitative measurement of the diastereoisomers of cis thymidine glycol in gamma-irradiated DNA. Free Radical Res. Commun. 2, 303-9. (24) Rieger, R., and Iden, C. (2001) Introduction of thymine glycol stereoisomers into oligomeric DNA. 49th ASMS Conference and Mass Spectrometry and Allied Topics, ThPK 273, Chicago. (25) Lustig, M. J., Cadet, J., Boorstein, R. J., and Teebor, G. W. (1992) Synthesis of the diastereomers of thymidine glycol, determination of concentrations and rates of interconversion of their cis-trans epimers at equilibrium and demonstration of differential alkali lability within DNA. Nucleic Acids Res 20, 4839-45. (26) Burton, K., and Riley, W. T. (1966) Selective degradation of thymidine and thymine deoxynucleotides. Biochem. J. 98, 70-7. (27) Iida, S., and Hayatsu, H. (1971) The permanganate oxidation of thymidine and thymidylic acid. Biochim. Biophys. Acta 228, 1-8. (28) Iwai, S. (2001) Synthesis and thermodynamic studies of oligonucleotides containing the two isomers of thymine glycol. Chem.-A Eur. J. 7, 4343-51. (29) D’Ham, C., Romieu, A., Jaquinod, M., Gasparutto, D., and Cadet, J. (1999) Excision of 5,6-dihydroxy-5,6-dihydrothymine, 5,6dihydrothymine, and 5-hydroxycytosine from defined sequence oligonucleotides by Escherichia coli endonuclease III and Fpg proteins: kinetic and mechanistic aspects. Biochemistry 38, 3335-44. (30) Habibi-Goudarzi, S., and McLuckey, S. A. (1995) Ion trap collisional activation of the deprotonated deoxymononucleoside and deoxydinucleoside monophosphates. J. Am. Soc. Mass Spectrom. 6, 102-13. (31) Cadet, J., Ulrich, J., and Teoule, R. (1975) Isomerization and new specific synthesis of thymine glycol. Tetrahedron 31, 2057-61. (32) Weinfeld, M., and Soderlind, K. J. M. (1991) Phosphorus-32postlabeling detection of radiation-induced DNA damage: identification and estimation of thymine glycols and phosphoglycolate termini. Biochemistry 30, 1091-7. (33) Reddy, M. V., Bleicher, W. T., and Blackburn, G. R. (1991) Phosphorus-32 postlabeling detection of thymine glycols: evaluation of adduct recoveries after enhancement with affinity chromatography, nuclease P1, nuclease S1, and polynucleotide kinase. Cancer Commun. 3, 109-17. (34) Falcone, J. M., and Box, H. C. (1997) Selective hydrolysis of damaged DNA by nuclease P1. Biochim. Biophys. Acta 1337, 26775. (35) Maccubbin, A. E., et al. (1999) Assay for reactive oxygen speciesinduced DNA damage: measurement of the formamido and thymine glycol lesions. Biochim. Biophys. Acta 1454, 80-88. (36) Romier, C., Dominguez, R., Lahm, A., Dahl, O., and Suck, D. (1998) Recognition of single-stranded DNA by nuclease P1: highresolution crystal structures of complexes with substrate analogs. Proteins 32, 414-24. (37) Sugiyama, H., and Saito, I. (1996) Theoretical Studies of GGSpecific Photocleavage of DNA via Electron Transfer: Significant Lowering of Ionization Potential and 5′-Localization of HOMO of Stacked GG Bases in B-Form DNA. J. Am. Chem. Soc. 118, 7063-8. (38) Saito, I., Nakamura, T., and Nakatani, K. (2000) Mapping of Highest Occupied Molecular Orbitals of Duplex DNA by CobaltMediated Guanine Oxidation. J. Am. Chem. Soc. 122, 3001-3006.
TX0155855
