Repair and Coding Properties of 5-Hydroxy-5-methylhydantoin

Chem. Res. Toxicol. 2000, 13, 575-584

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Repair and Coding Properties of 5-Hydroxy-5-methylhydantoin Nucleosides Inserted into DNA Oligomers Didier Gasparutto,† Mourad Ait-Abbas,† Michel Jaquinod,‡ Serge Boiteux,§ and Jean Cadet*,† Laboratoire des Le´ sions des Acides Nucle´ iques, Service de Chimie Inorganique et Biologique, UMR 5046, De´ partement de Recherche Fondamentale sur la Matie` re Condense´ e, CEA-Grenoble, F-38054 Grenoble Cedex 9, France, Laboratoire de Spectrome´ trie de Masse des Prote´ ines, Institut de Biologie Structurale, F-38027 Grenoble Cedex, France, and Laboratoire de Radiobiologie du DNA, CEA/DSV/DRR, UMR217 CNRS-CEA, 92265 Fontenay-aux-Roses, France Received January 18, 2000

1-(2-Deoxy-β-D-erythro-pentofuranosyl)-5-hydroxy-5-methylhydantoin (5-OH-5-Me-dHyd) (3) has been shown to be a major oxidation product of thymidine formed upon exposure of DNA to •OH-radical and excited photosensitizers. To investigate the biological and structural significance of the 5-OH-5-Me-dHyd residue to DNA, the latter modified 2′-deoxyribonucleoside was chemically prepared and then site-specifically incorporated into oligodeoxyribonucleotides. This was efficiently achieved using the phosphoramidite approach that involved mild deprotection conditions. The purity and the integrity of the modified synthetic DNA fragments were checked using different complementary techniques such as HPLC and polyacrylamide gel electrophoresis, together with electrospray ionization and MALDI-TOF mass spectrometry. The piperidine test applied to 5-OH-5-Me-dHyd containing oligonucleotides showed a weak instability of hydantoin nucleoside inserted into the oligonucleotide chain. Several enzymatic experiments aimed at determining the biochemical features of such a DNA lesion were carried out. Thus, processing of 5-OH-5-Me-dHyd by nuclease P1, snake venom phosphodiesterase, and calf spleen phosphodiesterase was investigated. The specificity and the mechanism of excision of the lesion by several bacterial and yeast DNA N-glycosylases, namely, endonuclease III (endo III), endonuclease VIII (endo VIII), formamidopyrimidine DNA N-glycosylase (Fpg), Ntg1 protein (Ntg1), Ntg2 protein (Ntg2), and Ogg1 protein (yOgg1), were also determined. These repair studies clearly showed that all these enzymes, with the exception of the yOgg1 protein, are able to recognize and remove 5-hydroxy-5-methylhydantoin from the doublestranded DNA fragment. Finally, a 22-mer DNA oligomer bearing a 5-OH-5-Me-dHyd residue was used as a template to study the in vitro nucleotide incorporation opposite the damage by the Klenow fragment of Escherichia coli polymerase I, Taq DNA polymerase, and DNA polymerase β. Thus, it may be concluded that the oxidized thymine residue is a strongly blocking lesion for the three studied DNA polymerases.

Introduction Deoxyribonucleic acid is a critical cellular target for several oxidation reactions, which are associated with aerobic cellular metabolism or to exposure to physical and chemical agents. The resulting oxidative damage may contribute to cancer and aging processes and also be implicated in a number of neurological disorders (1-4). Thus, one-electron oxidation of nucleobases is a major reaction associated with the direct effect of ionizing radiation and the type I photosensitization mechanism. Moreover, it was shown that various reactive oxygen and nitrogen species, including hydroxyl radical (OH•), ozone (O3), and peroxynitrite (ONOO-), are able to react with both pyrimidine and purine bases to afford a wide set of lesions (see refs 5-9 for reviews). Among the four normal * To whom correspondence should be addressed. Telephone: (33)4-76-88-49-87. Fax: (33)-4-76-88-50-90. E-mail: [email protected]. † CEA-Grenoble. ‡ Institut de Biologie Structurale. § UMR217 CNRS-CEA.

nucleosides, many studies were performed to characterize the bulk of the oxidation-induced decomposition products of thymidine. A large body of information is now available on the structure and the mechanism of formation of the latter nucleoside modifications (10-16). Among these, 1-(2-deoxy-β-D-erythro-pentofuranosyl)-5-hydroxy-5-methylhydantoin [5-OH-5-Me-hydantoin nucleoside (5-OH-5Me-dHyd)1 (3)] was shown to be a major degradation product of the thymidine moiety since it can be generated upon several oxidative processes (Scheme 1). Thus, this 1 Abbreviations: 5-OH-5-Me-Hyd, 5-hydroxy-5-methylhydantoin; 5-OH-5-Me-dHyd, 1-(2-deoxy-β-D-erythro-pentofuranosyl)-5-hydroxy5-methylhydantoin; MMTrCl, 4-monomethoxytrityl chloride; TFA, trifluoroacetic acid; FAB-MS, fast atom bombardment mass spectrometry; ESI-MS, electrospray ionization mass spectrometry; MALDI-TOF MS, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry; PAGE, polyacrylamide gel electrophoresis; endo III, endonuclease III; endo VIII, endonuclease VIII; Fpg, formamidopyrimidine DNA N-glycosylase; Ntg1, Ntg1 protein of Saccharomyces cerevisiae; Ntg2, Ntg2 protein of S. cerevisiae; yOgg1, 8-oxoguanine glycosylase 1 of S. cerevisiae; Kf, Klenow fragment of E. coli polymerase I; Taq pol, Taq DNA polymerase; pol β, DNA polymerase β.

10.1021/tx000005+ CCC: $19.00 © 2000 American Chemical Society Published on Web 06/27/2000

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Chem. Res. Toxicol., Vol. 13, No. 7, 2000

Scheme 1. Structure of the 5R and 5S Diastereomers of 5-OH-5-Me-dHyd 3, Generated by Oxidation of Thymidine

lesion was isolated both within the free nucleoside and at the DNA level. However, the exact biological role of 3, in terms of repair specificity and coding property, remains to be established. In this respect, oligonucleotides bearing the DNA damage at a specific site appear to be powerful probes for investigating the substrate specificity and the mechanism of action of repair enzymes, and for assessing the mutagenic potential of the lesion during DNA synthesis by DNA polymerases. In this paper, we report the synthesis of 5-OH-5-MedHyd (3) and its site-specific insertion into several oligodeoxyribonucleotides using the solid-phase phosphoramidite chemistry. As previously reported, no protection of the additional tertiary hydroxyl group of the hydantoin base was necessary (17). The modified DNA oligomers were used to study the properties of 3 with respect to several endonucleases and exonucleases. Information is provided on the specificity and the mechanism of excision of this damaged pyrimidinic residue by DNA repair enzymes. The coding properties of 3 during the copy of the modified oligonucleotides by several DNA polymerases were also investigated.

Experimental Procedures General Procedures and Materials. The silica gel (70200 µm) used for the low-pressure column chromatography was purchased from SDS (Peypin, France). TLC was carried out on Merck DC Kieselgel 60 F-254 plastic sheets (Darmstadt, Germany). All reagents that were used were of the highest available purity. Anhydrous solvents were purchased from SDS. Acetonitrile and methanol (HPLC grade) were obtained from Carlo Erba (Milan, Italy). Buffers for HPLC were prepared using water purified with a Milli-Q system (Milford, MA). The porous graphitized Hypercarb carbon column (98.5% carbon, particle size of 5 µm, porosity of 250 Å, 100 mm × 3 mm i.d.) was from Hypersil (Runcorn, Cheschire, U.K.), whereas the Hypersil ODS column (5 µm, 4.6 mm × 250 mm i.d.) was purchased from Interchim (Montlucon, France). Functionalized CPG supports and unmodified 2′-deoxyribonucleoside 3′-phosphoramidites, protected with phenoxyacetyl for dAdo, isopropyl-phenoxyacetyl for dGuo, and acetyl for dCyd, were from Glen Research (Sterling, VA). Enzymes. Ntg1, Ntg2, and yOgg1 were prepared as previously described (18, 19). Calf spleen phosphodiesterase, snake venom phophodiesterase, and Taq polymerase were purchased from Boehringer Mannheim (Mannheim, Germany). Endo VIII and DNA polymerase β were from Trevigen-Interchim (Montluç on, France). Nuclease P1 (Penicillium citrium) and alkaline

Gasparutto et al. phosphatase were purchased from Sigma (St. Louis, MO). T4 polynucleotide kinase and the Klenow fragment (exo-) of Escherichia coli DNA polymerase I were obtained from Amersham Pharmacia Biotech (Uppsala, Sweden). Mass Spectrometry Measurements. FAB (fast atom bombardment) mass spectrometry analyses were carried out on a VG ZAB 2-EQ apparatus (Manchester, U.K.). The samples were dissolved in either a glycerol or a NBA matrix prior to being analyzed. All modified and unmodified oligonucleotides were characterized by electrospray ionization mass spectrometry measurements (ESI-MS) on a Micromass Platform 3000 model spectrophotometer (Manchester, U.K.). Typically, 0.1 AU260 of the sample (approximately 3 µg) was dissolved in a solution of acetonitrile and water (50/50, v/v) containing 1% triethylamine prior to being analyzed in the negative mode. The modified nucleoside 5-OH-5-Me-dHyd (3) and its 5′-MMTr protected derivative were analyzed by ESI-MS in both the negative and positive modes. For the positive mode analysis, the sample was dissolved in a solution of acetonitrile and water (50/50, v/v) that contained 0.5% formic acid. MALDI mass spectra were obtained with a commercially available time-of-flight mass spectrometer (Voyager-DE, Perseptive Biosystems, Framingham, MA) equipped with a 337 nm nitrogen laser and pulsed delay source extraction. Spectra were recorded from 256 laser shots with an accelerating voltage of 25 kV in the linear and positive modes. For the matrix, a mixture of 3-hydroxypicolinic acid and picolinic acid in a 4/1 (w/w) ratio was dissolved in an aqueous acetonitrile solution (50%) that contained 0.1% TFA and a small amount of Dowex50W 50X8-200 cation exchange resin (Sigma). Typically, 1 µL of the sample was added to 1 µL of the matrix, and the resulting mixture was stirred. Then, the solution was then placed on the top of the target plate and allowed to dry by itself. The spectra were calibrated with a 1 pmol/µL solution of myoglobin (m/z 16 952), using the same conditions that were described for the analysis of oligonucleotides. Labeling of Oligonucleotides. Oligonucleotides (10 pmol) were labeled at the 5′-terminus with 10 µCi of [γ-32P]ATP (2 pmol, 10 mCi/mL) which was from Amersham Pharmacia Biotech (Uppsala, Sweden) upon incubation with T4 polynucleotide kinase (9.5 units) in 10 µL of supplied buffer at 37 °C for 30 min. The reaction was quenched by addition of 1 µL of a 0.5 M EDTA solution (pH 8). Unincorporated [γ-32P]ATP was removed by purification of the oligonucleotide on a MicroSpin column (Amersham Pharmacia Biotech). Synthetic Procedures. (1) 1-(2-Deoxy-β-D-erythro-pentofuranosyl)-5-hydroxy-5-methylhydantoin (3) and Its 5′Monomethoxytrityl Derivative (2). The 5′-monomethoxytrityl derivative 2 of 5-OH-5-Me-dHyd was prepared by chemical oxidation of the 5′-monomethoxytrityl thymidine (1) using KMnO4 followed by Pb(OAc)4 treatment as previously described (11, 17, 19). After purification by flash chromatography on a silica gel column, using a step gradient of 2-propanol (0 to 7%) in CHCl3 as the mobile phase, the title compound 2 was obtained as a white foam in 35% yield (360 mg). Compound 2 (5R and 5S): ESI-MS (negative mode) m/z 517.18 [M - H]- (calcd mass, 518.21); 1H NMR (200.13 MHz, CD3COCD3) δ 7.15-7.68 (m, 14H, H arom.), 6.05 (t, 1H, H-1′), 4.64 (m, 1H, H-3′), 4.04 (m, 1H, H-4′), 3.91 (s, 3H, CH3O trityl), 3.40-3.45 (m, 2H, H-5′ and H-5′′), 2.86-3.02 (m, 2H, H-2′ and H-2′′), 1.61 (s, 3H, CH3). 5-OH-5-Me-dHyd 3 was prepared by leaving 10 mg of compound 2 in 1 mL of a 80% acetic acid aqueous solution at room temperature for 30 min. Then, the solvents were evaporated to dryness. The residue was dissolved in 1 mL of water and extracted twice with 1 mL of diethyl ether. The deprotected nucleosides 3a and 3b were then separated by HPLC on a Hypercarb column, using a linear gradient of acetonitrile (from 0 to 10%) in a 25 mM ammonium formiate solution (UV detection at 230 nm): λmax (H2O, pH 7) 214 nm (shoulder); ESIMS (negative mode) m/z 245.19 [M - H]- (calcd mass, 246.22).

5-Hydroxy-5-methylhydantoin-Containing Oligomers Compound 3a: 1H NMR (400.13 MHz, D2O) δ 5.63 (t, 1H, H-1′), 4.60 (m, 1H, H-3′), 4.07 (m, 1H, H-4′), 3.85 (m, 2H, H-5′ and H-5′′), 3.02 (m, 1H, H-2′′), 2.27 (m, 1H, H-2′), 1.75 (s, 3H, CH3); 13C NMR (100.61 MHz, DMSO-d6) δ 176.1 (1C, C-4), 155.6 (1C, C-2), 86.7 (1C, C-4′), 85.4 (1C, C-5), 81.7 (1C, C-1′), 71.3 (1C, C-3′), 62.5 (1C, C-5′), 36.4 (1C, C-2′), 22.4 (1C, CH3). Compound 3b: 1H NMR (400.13 MHz, D2O) δ 5.80 (t, 1H, H-1′), 4.55 (m, 1H, H-3′), 4.03 (m, 1H, H-4′), 3.86 (m, 2H, H-5′ and H-5′′), 2.93 (m, 1H, H-2′′), 2.26 (m, 1H, H-2′), 1.74 (s, 3H, CH3); 13C NMR (100.61 MHz, DMSO-d6) δ 173.9 (1C, C-4), 154.7 (1C, C-2), 86.1 (1C, C-4′), 85.2 (1C, C-5), 81.1 (1C, C-1′), 70.7 (1C, C-3′), 62.2 (1C, C-5′), 36.5 (1C, C-2′), 22.9 (1C, CH3). (2) 1-{2-Deoxy-3-O-[2-cyanoethoxy(diisopropylamino)phosphino]-5-(4-monomethoxytrityl)-β-D-erythro-pentofuranosyl}-5-hydroxy-5-methylhydantoin (4). Compound 2 (300 mg, 0.58 mmol) was coevaporated twice with dry pyridine and subsequently dissolved in anhydrous CH2Cl2 (15 mL) under an argon atmosphere. Diisopropylammonium tetrazolate (50 mg, 0.29 mmol) and 2-cyanoethyl N,N,N′,N′-tetraisopropyldiamidite (190 µL, 0.64 mmol) were added to the solution while it was being stirred. The course of the reaction was monitored by TLC (95/5/1 CH2Cl2/MeOH/TEA). After 2 h at room temperature, the mixture was diluted with ethyl acetate (25 mL) and washed with a saturated NaHCO3 aqueous solution (30 mL). The organic layer was dried over Na2SO4 and concentrated under vacuum. The resulting residue was purified by flash chromatography on a silica gel column with a step gradient of methanol (0 to 3%) in CHCl3/TEA (99/1, v/v) as the mobile phase. Then, the collected fractions corresponding to the four diastereomers were dried to afford compound 4 as a white foam (yield of 67%, 0.39 mmol, 280 mg): Rf (95/5/1 CH2Cl2/MeOH/TEA) ) 0.5; 31P NMR (101.21 MHz, CD3COCD3) δ 151.14; FAB-MS (positive mode) m/z 719 [M + H]+, 273 [MMTr]+. Stability Studies of the 5R and 5S Diastereomers of 1-(2-Deoxy-β-D-erythro-pentofuranosyl)-5-hydroxy-5-methylhydantoin (3a and 3b) under the Alkaline Conditions Used for Oligonucleotide Chemical Synthesis. Aqueous ammonia (32%, 500 µL) was added to 0.5 AU230 of compounds 3a and 3b in sealed tubes. The solutions were placed at either room temperature or 55 °C. Then, the reactions were stopped at increasing time intervals (0, 1, 2, 4, 8, 16, and 24 h) by freezing the mixtures in liquid nitrogen and subsequent lyophilization. Samples were analyzed by HPLC using a Hypercarb column. The elution was achieved with a 0 to 10% linear gradient of acetonitrile in 25 mM ammonium formiate buffer over the course of 30 min (flow rate of 0.5 mL/min, UV detection at 230 nm). Stability Studies of the 5R and 5S Diastereomers of 1-(2-Deoxy-β-D-erythro-pentofuranosyl)-5-hydroxy-5-methylhydantoin (3a and 3b) under the Acidic Conditions Used for Oligonucleotide Chemical Synthesis. A similar procedure, as described above for the alkali-stability assay, was used. This involved incubation of compounds 3a and 3b in an 80% acetic acid aqueous solution for 0, 1, 2, 4, 8, 16, and 24 h at room temperature. Stability Studies of the 5R and 5S Diastereomers of 1-(2-Deoxy-β-D-erythro-pentofuranosyl)-5-hydroxy-5-methylhydantoin (3a and 3b) under Oxidizing Conditions Used for Oligonucleotide Chemical Synthesis. Similarly, compounds 3a and 3b were incubated in a 0.02 M oxidizing solution of iodine for 0, 1, 2, 4, 8, 16, and 24 h at room temperature. Solid-Phase Synthesis of Oligodeoxyribonucleotides. The synthesis of 5-OH-5-Me-dHyd 3 containing oligodeoxyribonucleotides was performed at the 1 µmol scale using the “Pac phosphoramidite” chemistry (21), with retention of the 5′terminal DMTr group (trityl-on mode). The standard 1 µmol DNA cycle was used, on an Applied Biosystems 392 DNA synthesizer, with slight modifications. The duration of the condensation was increased by a factor of 4 for the modified nucleoside phosphoramidite 4 (120 s instead of 30 s for normal nucleoside phosphoramidites). Under these conditions, a coupling efficiency of more than 90% for the modified monomer 4

Chem. Res. Toxicol., Vol. 13, No. 7, 2000 577 was achieved. A 0.3 M solution of phenoxyacetic anhydride in tetrahydrofuran and a 0.02 M solution of iodine in a water/ pyridine/tetrahydrofuran mixture were used for the capping and the oxidation steps, respectively. Deprotection and Purification of Oligodeoxyribonucleotides. Upon completion of the synthesis, the alkali-labile protecting groups of the oligodeoxyribonucleotides were removed by treatment with concentrated aqueous ammonia (32%) at room temperature for 4 h. Solvents were removed by evaporation under vacuum. Then, the crude 5′-DMTr oligomers were purified and deprotected on-line by reverse-phase HPLC using a polymeric support, as previously described (22). The modified 22mer oligonucleotide 7, used in repair and replication studies, was further purified by preparative polyacrylamide gel electrophoresis and, then, was desalted using a NAP-25 Sephadex column (Amersham Pharmacia Biotech). Piperidine Treatment of the 5-OH-5-Me-dHyd-Containing Oligodeoxyribonucleotides. Oligonucleotides were treated with a freshly made 1 M piperidine aqueous solution at 90 °C for 30 and 60 min. The reactions were carried out on 0.01 AU260 of 5′-32P-labeled modified oligodeoxyribonucleotides in 100 µL of a piperidine solution in sealed tubes. After cooling, the samples were coevaporated twice with water and then loaded onto a 20% polyacrylamide denaturing gel. The electrophoresis was carried out at 1300 V for 3 h, and subsequently, the gel was exposed to X-ray films. Digestion of Modified Oligonucleotides 5 and 6 by Nuclease P1 and Alkaline Phosphatase. The 5-OH-5-MedHyd-containing oligonucleotides 5 and 6 (1 AU260 - approximately 30 µg) were digested into nucleotides upon incubation for 2 h at 37 °C with 5 EU (enzyme units) of nuclease P1 in a 30 mM NaOAc and 0.1 mM ZnSO4 aqueous solution (pH 5.5), in a total volume of 50 µL. Then, 10 EU (5 µL) of calf intestinal alkaline phosphatase in 500 mM Tris and 1 mM EDTA (pH 8.5) was added, and the resulting mixture was incubated for a further 1 h. The digestion mixture of 2′deoxyribonucleosides was then analyzed by HPLC onto a Hypercarb column. The elution was performed with a 0 to 30% linear gradient of CH3CN in 25 mM ammonium formiate buffer, over 40 min at a flow rate of 0.5 mL/min (UV detection at 230 nm). The different collected products were analyzed by electrospray ionization MS in the negative mode. Enzymatic Digestion of Modified Oligonucleotides 5 and 6 by 3′- or 5′-Exonuclease Followed by MALDI-TOF Mass Spectrometry Analysis. Oligonucleotides were precipitated twice in a 0.3 M ammonium acetate/ethanol (1/3, v/v) solution prior to enzymatic digestions. (1) Digestion by Calf Spleen Phosphodiesterase (5′-exo). Oligonucleotides (0.1 AU260) were incubated at 37 °C with 10-3 EU of calf spleen phosphodiesterase (2 units/mL) in 0.02 M ammonium citrate (pH 5) in a total volume of 30 µL. Aliquots (1.5 µL) were withdrawn at increasing periods of time, and the reactions were quenched by addition of 50 µL of water and subsequent freezing in liquid nitrogen. Then, the samples were lyophilized and analyzed by MALDI-TOF mass spectrometry using the conditions described above. (2) Digestion by Snake Venom Phosphodiesterase (3′exo). Oligonucleotides (0.1 AU260) and 3 × 10-4 EU of snake venom phosphodiesterase (3 units/mL) were added to 30 µL of 0.02 M ammonium citrate (pH 9). The resulting solution was incubated at 37 °C, and aliquots (1.5 µL) were withdrawn at increasing periods of time. The reactions were quenched by addition of 50 µL of water, and the resulting solutions were frozen in liquid nitrogen. The samples were lyophilized before being analyzed by MALDI-TOF mass spectrometry using the conditions described above. Excision of 5-OH-5-Me-dHyd by DNA N-Glycosylases. (1) Specificity Studies by PAGE Analyses. The modified 22mer oligonucleotide 7 (10 pmol) was 5′-end labeled using [γ-32P]ATP and then purified using MicroSpin G-25 columns. The complementary strand 5′-d(AGA TCA GTC ACG ATC CGA AGT G)-3′ (12 pmol) was added to afford a double-stranded DNA

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fragment that contained the lesion. The hybridization was performed in 10 µL of buffer, consisting of 20 mM Tris-HCl, 1 mM EDTA, and 100 mM KCl (pH 7.5) for Fpg and endo III. The buffer included 25 mM Tris-HCl, 50 mM NaCl, and 2 mM EDTA (pH 7.6) for Ntg1, Ntg2, and Ogg1 proteins. The buffer for endo VIII digestion consisted of 10 mM Hepes, 1 mM EDTA, and 50 mM NaCl (pH 7.5). The hybridization was achieved by heating the respective solutions for 5 min at 80 °C and cooling slowly to 4 °C. Water (40 µL) was added, and each solution was aliquoted (5 µL per reaction). Then, 5 µL of repair enzyme at a final concentration of 2-200 ng/µL in a 2× buffer was added to the aliquoted solutions of double-stranded DNA. Reactions using increasing amounts of enzyme were performed at 37 °C for 30 min and stopped by addition of formamide (15 µL). Samples were denatured by heating at 85 °C for 3 min and then electrophoresed on a 20% polyacrylamide-7 M urea gel at 1500 V in TBE buffer [50 mM Tris, 50 mM boric acid, and 50 mM EDTA (pH 8)]. The reaction products were visualized by autoradiography after exposure of the gel to X-ray films. (2) Mechanistic Studies by MALDI-TOF MS Analyses. The same procedure as described above for the PAGE analyses was applied by using 20 pmol of double-stranded modified oligonucleotide and 500-1000 ng of repair enzymes. Resulting enzymatic digestions were precipitated twice in a 0.3 M ammonium acetate/ethanol (1/3, v/v) solution prior to the MALDITOF MS analyses. Modified Oligonucleotide Replication Assays. Typically, 10 pmol of modified DNA template 7 was annealed with 10 pmol of the complementary 5′-end-32P-labeled 12-mer primer [5′d(AGA TCA GTC ACG)-3′] in 10 µL of 50 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 1 mM DTT, 0.02 mg/mL BSA buffer. The primer-template solution was incubated at 80 °C for 5 min, and then cooled slowly to 4 °C, for at least 2 h. Subsequently, primer elongation by the Klenow fragment was carried out in the same buffer for 1 h at 25 °C, while an incubation at 37 °C for 1 h in 10 mM Tris-HCl (pH 8.3), 15 mM MgCl2, and 50 mM KCl was used with Taq polymerase. A similar extension experiment was performed at 25 °C for 1 h with DNA polymerase β by using 50 mM Tris-HCl (pH 8.8), 10 mM MgCl2, 100 mM KCl, 0.4 mg/mL BSA, and 1 mM DTT as the buffer. Each sample contained 0.5 pmol of the labeled primer-template duplex, 1 unit of polymerase, and the four dNTPs (each at 100 µM) in a final volume of 25 µL. The reactions were stopped by addition of formamide (15 µL). Samples were denatured by heating at 85 °C for 3 min and then electrophoresed on a 20% polyacrylamide-7 M urea gel at 1500 V in TBE buffer. The reaction products were visualized by exposing the gel to X-ray films. Similar experiments using the unmodified 22-mer template, containing the thymidine parent nucleoside [5′-d(CAC TTC GGA TCG TGA CTG ATC T)-3′], were performed as positive controls for each DNA polymerase.

Results and Discussion Synthesis of the Modified Phosphoramidite Building Block and Its Insertion into Defined Sequence Oligonucleotides. (1) Preparation and Stability Studies of 5-OH-5-Me-dHyd Derivatives 2 and 3. The modified nucleoside 3, as a mixture of 5R and 5S diastereoisomers, can be efficiently prepared by oxidation of thymidine in two successive steps, including potassium permanganate first and then lead tetraacetate, as previously described by Cadet and Teóule (11, 20). In fact, a 5′-monomethoxytritylated derivative of thymidine (1) was used as the starting material (17) to incorporate the hydantoin lesion 3 into oligonucleotides by solid-phase condensation with the phosphoramidite chemistry (Scheme 2). This approach, by introducing the 4-methoxytrityl group in the first step of the synthesis, greatly facilitates the detection and purification of the oxidation products. After purification by chromatography on a silica gel

Gasparutto et al. Scheme 2. Synthetic Pathway of the Preparation of the Modified DNA Oligomers That Contain the 5-OH-5-Me-dHyd 3

column, the structure of the 5′-MMTr-protected Hyd (2), obtained as a 5R and 5S diastereoisomeric mixture in a 35% yield, was confirmed by 1H and 13C NMR analyses together with ESI mass spectrometry measurements. To perform structural analyses and stability studies with the unprotected modified nucleoside 3, a small amount of the 5′-monomethoxytrityl derivative 2 was detritylated by acid treatment and purified by HPLC on a Hypercarb graphitized column. The latter chomatographic system allowed an efficient resolution of the mixture of diastereomers 3a and 3b. Their characterization was achieved by 1H and 13C NMR analyses and ESI mass spectrometry measurements. Then, the stability of each diastereomer under the various conditions met during the chemical synthesis and the deprotection of the oligonucleotides was investigated. Thus, it clearly appeared that neither degradation nor isomerization occurred under the usual acidic and oxidizing conditions applied during the chemical preparation of DNA fragments. On the other hand, the alkali-lability studies showed that 3 was partly decomposed in a 30% ammonium hydroxide aqueous solution at high temperature while no detectable degradation was detected after 4 h at room temperature. However, an efficient epimerization of each diastereomer was found to occur, and a 60/40 mixture of diastereomers 3a and 3b was observed upon incubation of 3b under the latter alkaline conditions for 4 h (Figure 1). This alkali-mediated isomerization of 3 is ascribed to a ring-chain tautomerism mechanism involving the 1,5 bond. It should be added that approximately 30% of such a C5 epimerization was observed by leaving 3a or 3b for 24 h at pH 7 in water or buffers (data not shown). It may be concluded that the mixture of 5R and 5S diastereomers of 5-OH-5-Me-dHyd (3a and 3b) can be used in standard automated oligonucleotide synthesis. However, this required the application of mild ammoniacal deprotection conditions associated with the Pacprotected phosphoramidite chemistry (21). The selective insertion into oligonucleotides of each separated diastereomer would be of interest only if an alternative strategy of synthesis which avoids the final alkali deprotection step were available. (2) Preparation of a Modified Phosphoramidite Building Block. Then, the monomethoxytritylated derivative 2 was treated with the 2-cyanoethyl N,N,N′,N′tetraisopropyldiamidite phosphitylating reagent, affording the expected phosphoramidite building block 4 in

5-Hydroxy-5-methylhydantoin-Containing Oligomers

Figure 1. HPLC analysis of the epimerization reaction of 3b in a 30% ammoniacal aqueous solution: (A) without alkali treatment and (B) after 4 h at room temperature (detection at 230 nm; the chromatographic conditions are reported in Experimental Procedures).

good yield (67%) after silica gel column chromatography. The characterization of the isolated product, obtained as a mixture of four diastereomers, was performed by 31P NMR and mass spectrometry. Interestingly, the protection of the tertiary hydroxyl group of the base was found not to be necessary due to the lack of reactivity. This observation is in agreement with previous fundings (17). (3) Synthesis and Characterization of the Oligonucleotides. Several oligodeoxyribonucleotides (Table 1) bearing a 5-OH-5-Me-dHyd residue 3 were synthesized on a solid support using the phosphoramidite chemistry, with the modifications previously described in Experimental Procedures. This allowed a coupling efficiency of more than 90% for the modified monomer. After ammonia deprotection at room temperature for 4 h, the crude 5′tritylated oligonucleotides were purified by RP-HPLC on a polymeric support using an on-line detritylationpurification procedure (22). The purity and homogeneity of the material were assessed by several approaches, including analytical HPLC, polyacrylamide gel electrophoresis of 5′-32P-labeled fragments, and mass spectrometry. The molecular weights of the oligonucleotides were inferred from electrospray ionization mass spectrometry measurements in the negative mode (Table 1). These results confirmed the incorporation into and the integrity of 5-OH-5-Me-dHyd 3 in the oligomers. Piperidine Stability of 5-OH-5-Me-dHyd 3 Inserted into Oligodeoxyribonucleotides. The preparation of modified oligonucleotides that contained the 5-OH5Me-dHyd lesion allowed the determination of the stability of 3 under the piperidine conditions used to reveal alkalilabile DNA modifications in oxidized oligonucleotides. Then, the study of the stability of 3 was performed by

Chem. Res. Toxicol., Vol. 13, No. 7, 2000 579

Figure 2. PAGE analysis of the 5′-end-labeled 14- and 22-mer oligonucleotides containing 5-OH-5-Me-dHyd, after heating in a 1 M piperidine aqueous solution at 90 °C for 0, 30, and 60 min.

treating the 5′-32P-labeled oligonucleotides 5′-d[ATC GTG AC(Hyd) GAT CT]-3′ (6) and 5′-d[CAC TTC GGA (Hyd)CG TGA CTG ATC T]-3′ (7), with piperidine at 90 °C for 30 and 60 min. In a subsequent step, the resulting DNA fragments were analyzed by denaturing polyacrylamide gel electrophoresis (Figure 2). It was found that 5-OH5-Me-dHyd-containing oligonucleotides 6 and 7 were stable after the usual piperidine treatment which requires a 30 min heating. However, approximately 20% of strand breaks was detected after treatment for 60 min. Thus, 5-OH-5-Me-dHyd 3 can be considered a weakly alkali-labile lesion. Nuclease-Mediated Digestions of Modified Oligonucleotides That Contained 5-OH-5Me-dHyd. The ability of several nucleases to cleave the phosphodiester linkages between 3 and the vicinal normal 2′-deoxyribonucleosides was assessed using 5-OH-5-Me-dHyd-containing oligodeoxyribonucleotides 5 and 6. First, the trimer d(T[Hyd]T) 5 and the 14-mer 6 were submitted to the action of nuclease P1 and bacterial alkaline phosphatase. The resulting mixtures of 2′-deoxyribonucleosides arising from the two sucessive digestions were then analyzed by reverse-phase HPLC (data not shown). Two peaks were observed in the HPLC elution profiles in addition to those of normal 2′-deoxyribonucleosides as detected by UV absorption at 230 nm. Interestingly, the two HPLC peaks were found to contain the 5R and 5S diastereomers of 3 as inferred from cochromatography with authentic samples and electrospray ionization mass spectrometry analysis. This clearly shows the ability of nuclease P1 to cleave 5-OH-5-Me-dHyd 3 from DNA. The latter observation provides additional support for the presence and the integrity of 3 in the synthetic oligomers. Additional enzymatic digestion experiments were performed with the 14-mer 6 using exonucleases, including

Table 1. Sequences and Molecular Weights of the Modified Oligodeoxyribonucleotides Synthesized and Used in the Study Presented Here (Hyd ) 5-hydroxy-5-methylhydantoin)a

a

name

sequence (5′-3′)

length

calcd weight

found weight

5 6 7

T(Hyd)T ATC GTG AC(Hyd) GAT CT CAC TTC GGA (Hyd)CG TGA CTG ATC T

3 14 22

854.6 4257.8 6705.4

854.4 4257.7 6704.6

All the oligonucleotide masses were obtained by electrospray ionization mass spectrometry measurements in the negative mode.

580


snake venom phosphodiesterase (3′-exo) and calf spleen phosphodiesterase (5′-exo). Thus, the course of the hydrolysis of the DNA strand by the nucleases was followed by withdrawing aliquots from the digestion mixtures at increasing periods of time. The DNA fragments were then analyzed by MALDI-TOF mass spectrometry (23, 24). Using the latter powerful technique, the different molecular ions observed correspond to the digested DNA fragments which differ in mass by the successive loss of nucleotides. The difference in mass between two sucessive fragments allowed us to identify the released nucleotide and then to determine the overall sequence of the oligomer. Thus, a MALDI-TOF MS analysis allows the determination of the location of the lesion within the DNA strand. In addition, relevant information is provided about the integrity of the modified nucleoside and the processing of the damage by the exonucleases. The thymidine-containing unmodified 14-mer oligodeoxyribonucleotide 5′-d(ATC GTG ACT GAT CT), used first as a control, was totally hydrolyzed by both 5′- and 3′-exonucleases in less than 15 min, allowing the determination of the complete sequence (data not shown). In contrast, the presence of nucleoside 3 in oligonucleotide 6 induces a total resistance to digestion by both snake venom phosphodiesterase (3′-exo) and calf spleen phosphodiesterase (5′-exo). It was found that snake venom phosphodiesterase induced the release of the first five nucleotides at the 3′-end of the oligonucleotide sequence but failed to cleave the phosphodiester linkage between 3 and 2′-deoxycytidine, even after a prolonged treatment (data not shown). This was inferred from the observation of a single peak at m/z 2719.7 Da that corresponds to the positive ion [M + H]+ of the 9-mer {5′-d[ATC GTG AC(Hyd)]-3′} (calcd mass ) 2717.8 Da). The digestion of oligonucleotide 6 by calf spleen phosphodiesterase and the MALDI-MS analyses were also performed. The enzymatic hydrolysis after 4 min shows a sequential cleavage of the height normal nucleosides (data not shown). The spectrum obtained after 120 min is indicative of a mixture of the 7-mer {5′-d[C (Hyd)GA TCT]-3′} (calcd mass ) 2075.4 Da; mass found for [M + H]+ ) 2076.9 Da) and the 6-mer {5′-d[(Hyd)GA TCT]-3′} (calcd mass ) 1786.2 Da; mass found for [M + H]+ ) 1788.1 Da). This shows that the calf spleen phosphodiesterase enzyme is able to digest sequentially the oligonucleotide from the 5′-end until it reaches the 2′-deoxycytidine nucleoside before the hydantoin lesion 3, whose phosphodiester bond was resistant to cleavage. Finally, the calf spleen phosphodiesterase failed to release the hydantoin nucleoside, even after longer enzymatic treaments. Repair Assays of 5-OH-5-Me-dHyd-Containing Oligonucleotides with DNA Repair Proteins. Sitespecifically modified oligonucleotides are suitable tools for studies aimed at determining the substrate specificity of DNA repair enzymes. In this respect, attempts were made to assess whether 3 may be a substrate for several base excision repair enzymes, including formamidopyrimidine DNA N-glycosylase (Fpg), endonuclease III (endo III), endonuclease VIII (endo VIII), Ntg1 protein, Ntg2 protein, and yOgg1 protein. E. coli Fpg protein is a well-known repair enzyme which is able to excise several modified purine bases from DNA duplexes, through both a N-glycosylase and AP endonuclease activities (25, 26). Substrates recognized and excised by Fpg include 8-oxo7,8-dihydroguanine (8-oxoG), 2,6-diamino-4-hydroxy-5-

Gasparutto et al.

formamidopyrimidine (Fapy-guanine), 2,6-diamino-4-hydroxy-5-N-methylformamidopyrimidine (Me-Fapy-guanine), 4,6-diamino-5-formamidopyridine (Fapy-adenine) (27-32), and oxazolone (33). It was also found that 5-hydroxycytosine (5-OHC), 5,6-dihydrothymine (DHT), 5,6-dihydroxy-5,6-dihydrothymine (thymine glycol), formylamine, and N-3-(2-hydroxyisobutyric acid)urea, with five modified pyrimidine bases, are also recognized and excised by Fpg (34-38). On the other hand, previous studies have shown that endo III and endo VIII process a wide variety of pyrimidine lesions. Thus, endo III recognizes 5-hydroxy-5methylhydantoin (5-OH-5-Me-Hyd), 5,6-dihydrothymine (DHT), 5,6-dihydroxy-5,6-dihydrothymine (thymine glycol), 5-hydroxy-5,6-dihydrothymine (5-OH-5,6-HT), 5,6dihydrouracil (DHU), 5,6-dihydroxy-5,6-dihydrouracil (uracil glycol), 5-hydroxy-5,6-dihydrouracil (5-OH-5,6HU), 5-hydroxyuracil (5-OHU), 6-hydroxy-5,6-dihydrocytosine (6-OH-5,6-HC), 5-hydroxycytosine (5-OHC), urea, formylamine, methyltartronyl-N-urea, and alloxan (35, 37-44). Recently, we have reported that oxazolone, which results from an oxidative rearrangement of the guanine moiety, was efficiently recognized and removed by endo III (33). The latter result provided the first evidence for the removal of an oxidized purine base by endo III. E. coli endo VIII protein exhibits a substrate specificity similar to that of endo III (45, 46) and may act in complement to the latter enzyme when the level of damage to DNA increases significantly within the cell. With regard to the yeast Ntg1 and Ntg2 enzymes, which were more recently isolated and characterized, more limited information about their specificity and action mechanism is available. The two genes NTG1 and NTG2 encode proteins whose sequences are significantly homologous to that of E. coli endo III, as well as a similar substrate specificity (47-51). Thus, Ntg1 and Ntg2 proteins have been shown to remove several modified pyrimidine nucleobases, namely, 5-OH-5-Me-Hyd, Tg, DHU, 5-OHC, 5-OHU, 5-OH-5,6-HT, and 5-OH-5,6-HU, as well as abasic sites (18, 48, 49, 51). Interestingly, Ntg1 and Ntg2 are able to excise modified purine bases such as FapyA, FapyG, and Me-FapyG, whereas Ntg1 exhibits an additional capacity to release 8-oxoG mispaired with guanine (18). The yOgg1 protein is a DNA N-glycosylase/ AP lyase which is functionally homologous with the E. coli Fpg enzyme and excises guanine lesions such as 8-oxoG, Fapy-G, and Me-FapyG (19, 47, 52-55). A recent study has clearly shown that yOgg1 efficiently removed 8-oxo-7,8-dihydroadenine (8-oxoA) when the latter damaged base was placed opposite cytosine (56). To obtain further information about the substrate specificity of these six DNA N-glycosylases toward the 5-hydroxy-5-methylhydantoin, the modified 22-mer oligonucleotide 7 that contained 3 at a central position was used as the substrate. This involved 5′-32P-end labeling and subsequent hydridization of modified oligonucleotide 7 with its complementary sequence 5′-d(AGA TCA GTC ACG ATC CGA AGT G)-3′, which contains an adenine in front of the lesion. Then, 5-OH-5-Me-dHyd excision by the repair enzymes was assessed by monitoring the oligonucleotide strand breakage using polyacrylamide gel electrophoresis. Thus, it was shown that endo III, Ntg1, and Ntg2 proteins, which act primarily at modified pyrimidine bases, are able to cleave the modified DNA duplex at the site of 3 (Figure 3). These results are in agreement with previous investigations, involving glo-



Figure 3. PAGE analysis of the cleavage products upon incubation of 7 with Fpg, endo III, endo VIII, Ntg1, and Ntg2.

bally chemically oxidized or γ-irradiated DNA as the substrate, which concluded with the recognition and excision of the 5-OH-5-methylhydantoin base by the three latter repair enzymes (18, 41, 42, 44). In the study presented here, it appears clearly that endo VIII is able to remove 3 from the double-stranded DNA chain, confirming the high degree of functionnal similarity between this enzyme and endo III. More surprisingly, it was found that 5-OH-5-Me-dHyd was also a substrate for the Fpg protein (Figure 3) since the oligonucleotide 7 was quantitatively cleaved at the modified site using 100 ng of the latter repair enzyme. This provides further support for some recent observations, which indicate that the Fpg protein exhibits a much broarder substrate specificity that was initially expected, being able to remove several fragmented and rearranged purine and pyrimidine lesions (33-35, 37, 38). In contrast, the yeast homologue of Fpg, namely, the yOgg1 protein, was not able to excise 3 from the DNA oligomer (data not shown). To confirm the mechanisms of action and to obtain additional information about the processing of the 5-OH5-Me-hydantoin lesion by these different repair enzymes, MALDI-TOF MS analyses of the cleavage reaction mixtures were carried out. As was already shown (vide supra), MALDI-TOF mass spectrometry, when coupled with a sequential digestion by exonucleases, is a powerful tool for locating and identifying DNA lesions within DNA fragments. In addition, when used to investigate lesion processing by repair enzymes, the latter analytical tools permit us to gain insights into mechanistic aspects of oligonucleotide cleavage. It should be remembered that earlier studies concluded with a β-δ-elimination mechanism for Fpg and endo VIII (45, 57), whereas endo III, Ntg1, and Ntg2 are processing base damage via a β-elimination mechanism (47, 58-62). Under the experimental conditions of cleavage and analysis used here, Fpg and endo VIII, when acting on 5-OH-5-Me-hydantoincontaining oligonucleotides, gave rise to fragments with molecular weights corresponding to those of the expected products of a β-δ-elimination mechanism. Figure 4 shows the MALDI-TOF mass spectrum of an oligonucleotide mixture arising from the incubation of oligonucleotide 7 with Fpg protein. The fragment at m/z 2781.2 ascribed to the 9-mer oligonucleotide released 5′ to the lesion (5′-CAC TTC GGAp-3′, calcd mass for [M + H]+ ) 2779.8), while those at m/z 3718.6 corresponds to the 12mer oligonucleotide released 3′ to the damage (5′-pCGT

Figure 4. MALDI-MS analyses of the mixture of oligonucleotides arising from the incubation of 7 with Fpg (a similar spectrum is obtained with endo VIII, not shown).

GAC TGA TCT-3′, calcd mass for [M + H]+ ) 3717.4). The same spectrum was obtained for the endo VIII processing (data not shown). In contrast, the results of the endo III-, Ntg1-, and Ntg2-mediated cleavage of the latter modified oligonucleotide 7 were apparently not consistent with the previous proposed mechanism of action of these enzymes. Thus, no peak corresponding to the molecular weight of the β-elimination product (calcd mass for [M + H]+ ) 2878.8) was observed (data not shown). On the other hand, the main observed peaks (m/z 2897.9 and 3720) were assigned to the products of hydrolysis of the phosphodiester bond 3′ to the lesion (5′CAC TTC GGAp-deoxyribose-3′, calcd mass for [M + H]+ ) 2896.8; 5′-pCGT GAC TGA TCT-3′, calcd mass for [M + H]+ ) 3717.4). It should be added that similar observations were recently made for the endo III-mediated process of several pyrimidine base lesions, including DHT, Tg, and 5-OHC (34). These results may suggest that endo III, Ntg1, and Ntg2 act as AP endonucleases, as was originally proposed by Linn for the endo III protein (63), and not as AP lyases (64, 65). However, the possibility of hydration of the alkenal, which is the likely product of the β-elimination reaction, after the cleavage step cannot be ruled out at the present time. In particular, the latter alkenal has been shown to be highly reactive toward several nucleophile species (66). Work is currently in progress to resolve this apparent discrepancy by using isotopic labeling experiments coupled to mass spectrometry analyses. In Vitro Replication Experiments by DNA Polymerase. The ability of three different DNA polymerases, namely, the Klenow fragment of E. coli polymerase I, Taq

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Gasparutto et al.

Figure 5. Primer extension reactions catalyzed by the Klenow fragment (A), Taq pol (B), and pol β (C). A modified 22-mer template {5′-d[CAC TTC GGA (Hyd)CG TGA CTG ATC T]-3′} annealed with a 32P-labeled 12-mer [5′-d(AGA TCA GTC ACG)-3′] (lane 1) was used; primer extension reactions were carried out by adding 1 unit of polymerase in the presence of 100 µM dNTP (lane 2), dATP (lane 3), dGTP (lane 4), dCTP (lane 5), and dTTP (lane 6), as described in Experimental Procedures. Part D shows the control primer extension reactions of an unmodified 22-mer template under the same conditions using no enzyme (lane c1), Kf (lane c2), Taq pol (lane c3), and pol β (lane c4). Then, the reaction mixtures were subjected to denaturing 20% PAGE, and then the extended products were visualized by exposing the gel to X-ray film.

DNA polymerase, and DNA polymerase β, to extend a primer annealed with a template bearing a 5-OH-5-MedHyd residue 3 was investigated. The primer was 32Plabeled at its 5′ end so that extension by nucleotide incorporation could be observed by sequencing PAGE using either a single dNTP or a mixture of all four dNTPs. Figure 5 shows the autoradiographies of the denaturing PAGE bands obtained by elongation of the 12-mer primer 5′-d(AGA TCA GTC ACG)-3′ annealed with 5′-d[CAC TTC GGA (Hyd)CG TGA CTG ATC T]-3′ (template that contained a 5-OH-5-Me-dHyd 3 in its sequence) in the presence of DNA polymerases. In addition, similar replication assays were performed using the unmodified thymine-containing 22-mer oligonucleotide as the template, to assess the activity and specificity of the polymerases (Figure 5D). It was found that Kfmediated polymerization incorporates mainly dAMP opposite 5-OH-5-Me-dHyd (Figure 5A, lane 3). In addition, a small amount of dGMP was also incorporated (Figure 5A, lane 4). Moreover, the enzyme was not able to extend the 5-OH-5-Me-dHyd/dA pair beyond the damage in the presence of the four dNTPs (Figure 5A, lane 2). Thus, 3 appeared to strongly inhibit Klenow fragment but did not significantly alter the fidelity of the enzyme. Using Taq polymerase, the primer extension reactions led to the same dAMP incorporation opposite the lesion (Figure 5B, lane 3), dGMP also being inserted to a lower extent (Figure 5B, lane 4). As observed with Kf, Taq polymerase was blocked in front of 3 since only small amounts of nucleotides, likely to be dAMP and dGMP, were inserted opposite 5-OH-5-Me-dHyd 3 with no fully extended primer (Figure 5B, lane 2). In vitro DNA synthesis catalyzed by pol β was also performed with modified oligonucleotides that contain 3 (Figure 5C, lanes 1-6). Under the latter conditions, no dNTP incorporation opposite the hydantoin residue was observed, leading to a complete polymerase block at the damaged site. The primer extension results reported here suggest that the hydantoin damage 3 acts as a potential stop point for DNA polymerases, inducing cell lethality. These mutational and lethal properties appear to be similar to those determined for two other major oxidative thymine lesions, namely, thymine glycol (67-72) and formylamine (37).

Conclusion and Perspectives The synthesis of 5-OH-5-Me-dHyd (3) and its incorporation into several oligonucleotides by the phosphora-

midite approach were achieved using mild alkali deprotection conditions. The synthetic oligomers were isolated in good yields and characterized by various complementary techniques, showing the integrity of the incorporated modified nucleoside. The piperidine stability experiment performed on the modified oligonucleotides indicates a slight instability of the hydantoin lesion 3 inserted into DNA strands. The processing of 3 by different nucleases was studied. Thus, it was shown that nuclease P1 is able to cleave the hydantoin residue from the oligonucleotides, while both snake venom phosphodiesterase and calf spleen phosphodiesterase failed to release the latter modification from the DNA fragments. On the other hand, the ability of several repair enzymes, including Fpg, endo III, endo VIII, Ntg1, Ntg2, and Ogg1 proteins, to excise 5-OH-5-Me-Hyd was investigated. A series of experiments was performed which clearly indicated that 3 was a substrate for all the repair enzymes that were tested, with the exception of the Ogg1 protein. Interestingly, the ability for Fpg protein to efficiently remove 5-OH-5-Me-Hyd constitutes a new example of the broad spectrum of action of this repair enzyme. On the other hand, yOgg1, its yeast functional homologue, shows a higher specificity. Information about the coding properties of 5-OH-5-Me-dHyd is also provided. This involved the determination of the base-specific incorporation directed by the latter oxidized thymidine residue during primer extension assays by three DNA polymerases. Thus, 3 acts in vitro as a block for the three tested polymerases; therefore, 5-OH-5-Me-dHyd may represent a potential lethal lesion within the cell. Investigations are currently in progress to compare the kinetic constants (Vm and Km) of the excision reactions of 5-OH-5-Me-Hyd with those of other oxidized thymine lesions, such as 5,6-dihydroxy-5,6-dihydrothymine, which show the same ring-chain tautomerism. Finally, the 5-OH-5-Me-dHyd 3 and the different modified oligonucleotides prepared in this work could be used as tools for investigations aimed at optimizing assays for the assessment of 3 within isolated and cellular oxidized DNA.

Acknowledgment. The contributions of Colette Lebrun (LRI/SCIB/CEA-Grenoble) and Daniel Ruffieux (Biochimie C/CHRU-Grenoble) to the FAB and electrospray ionization mass spectrometry measurements are gratefully acknowledged. We thank Dr. Victor Duarte for helpful discussions about the replication experiments.


Financial support from the Comite´ de Radioprotection (Electricite´ de France) is acknowledged. Supporting Information Available: ESI-MS spectra of compounds 3a and 3b (in the negative mode), reverse-phase HPLC elution profile and MALDI-MS spectra of enzymatic digestion mixtures of modified 14-mer ODN 6, and MALDI-MS analysis of the DNA fragments released upon incubation of ODN 7 with endo III. This material is available free of charge via the Internet at http://pubs.acs.org.


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