Half-Life and DNA Strand Scission Products of 2-Deoxyribonolactone

Chem. Res. Toxicol. 2004, 17, 197-207

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Half-Life and DNA Strand Scission Products of 2-Deoxyribonolactone Oxidative DNA Damage Lesions Yan Zheng and Terry L. Sheppard* Department of Chemistry and The Robert H. Lurie Comprehensive Cancer Center, Northwestern University, 2145 Sheridan Road, Evanston, Illinois 60208-3113 Received September 26, 2003

Reactive oxygen species lead to oxidative damage of the nucleobase and sugar components of nucleotides in double-stranded DNA. The 2-deoxyribonolactone (or oxidized abasic site) lesion results from oxidation of the C-1′ position of DNA nucleotides and has been implicated in DNA strand scission, mutagenesis, and covalent cross-linking to DNA binding proteins. We previously described a strategy for the synthesis of DNA-containing deoxyribonolactone lesions. We now report an improved method for the site specific photochemical generation of deoxyribonolactone sites within DNA oligonucleotides and utilize these synthetic oligonucleotides to characterize the products and rates of DNA strand scission at the lactone lesion under simulated physiological conditions. A C-1′ nitroveratryl cyanohydrin phosphoramidite analogue was synthesized and used for the preparation of DNA containing a photochemically “caged” lactone precursor. Irradiation at 350 nm quantitatively converted the caged analogue into the deoxyribonolactone lesion. The methodology was validated by RP-HPLC and MALDI-TOF mass spectrometry. Incubation of deoxyribonolactone-containing DNA under simulated physiological conditions gave rise to DNA fragmentation by two consecutive elimination reactions. The DNAcontaining products resulting from DNA cleavage at the deoxyribonolactone site were isolated by PAGE and unambiguously characterized by MALDI-TOF MS and chemical fingerprinting assays. The rate of DNA strand scission at the deoxyribonolactone site was measured in singleand double-stranded DNA under simulated physiological conditions: DNA cleavage occurred with a half-life of ∼20 h in single-stranded DNA and 32-54 h in duplex DNA, dependent on the identity of the deoxynucleotide paired opposite the lesion site. The initial R,β-elimination reaction was shown to be the rate-determining step for the formation of methylene furanone and phosphorylated DNA products. These investigations demonstrated that the deoxyribonolactone site represents a labile lesion under simulated physiological conditions and forms the basis for further studies of the biological effects of this oxidative DNA damage lesion.

Introduction Oxidative DNA damage, mediated by free radicals and reactive oxygen species, represents an important mechanism for covalent modification of nucleobase and sugar components of DNA nucleotides (1-4). As shown in Scheme 1, oxidation of the C-1′ position of deoxyribose in DNA nucleotides (1) gives rise to the 2-deoxyribonolactone (or oxidized abasic site) lesion (2) within DNA. Numerous toxic agents have been shown to produce the deoxyribonolactone lesion, including enediyne antibiotics (5), bis(phenanthroline) copper(I) (6-8), cationic manganese porphyrins (9), oxoruthenium complexes (10), ultraviolet irradiation (11), and γ-radiolysis (12). The chemical mechanisms for oxidized abasic site generation by these agents have been the subject of intensive investigation. Although the details vary among damage reagents and conditions, central features of the damage process include hydrogen abstraction at the C-1′ position, nucleotide oxygenation, and extrusion of the coding nucleobase to produce the 2-deoxyribonolactone lesion in duplex DNA (7, 13-18). Building upon the mechanistic understanding of deoxyribonolactone site formation in DNA, recent investiga* To whom correspondence should be addressed. Tel: 847-467-7636. Fax: 847-491-7713. E-mail: [email protected].

Scheme 1. General DNA Damage Mechanism for Formation of Deoxyribonolactone Lesions and Subsequent DNA Strand Scission

tions have focused on the biochemical properties of the 2-deoxyribonolactone lesion. The C-1′ oxidized abasic site bears structural similarity to aldehyde abasic (apurinic or AP) sites (19) that are produced by depurination reactions (20) or by the action of DNA repair proteins (21). However, chemical reactivity differences between AP sites and deoxyribonolactone lesions have inspired chemical biologists to more closely examine the biochemical properties of the lesion. Specifically, the reported lability of the lactone lesion (22) as compared with AP

10.1021/tx034197v CCC: $27.50 © 2004 American Chemical Society Published on Web 01/17/2004

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sites (23) indicated that production and study of the lactone lesion through specific DNA damage reactions would prove challenging. To overcome these issues, several laboratories independently developed chemical methods for the specific generation of the deoxyribonolactone lesion within DNA oligonucleotides (14, 17, 24, 25). These approaches all involved the synthesis of a stable lactone precursor, its incorporation into DNA by solid phase synthesis, and conversion of the “caged” precursor into the lactone lesion by UV photolysis. These general methods for the synthesis of DNA oligonucleotides containing lactone lesions have proven invaluable for investigations of the biological effects of the deoxyribonolactone site. Using these approaches, several features of the toxicity, mutagenesis, and biochemical fate of the deoxyribonolactone lesion have been elucidated. Recent studies have demonstrated that DNA-containing deoxyribonolactone lesions form covalent adducts with DNA repair proteins that metabolize AP sites (26-29). The resulting covalent cross-links between the C-1′ carbonyl of the lactone site and the amino acid side chains of target proteins suggest potential toxicity mechanisms for the oxidized abasic lesion. Second, the lactone lesion was found to induce DNA polymerase-mediated mutagenesis in an in vitro system (30). NMR structural studies have illuminated aspects of the conformation of the DNA damage lesion in duplex DNA (31). Finally, recent work has provided support for the proposed mechanism for DNA strand scission at the deoxyribonolactone lesion (22, 32, 33). The general pathway for DNA strand scission at deoxyribonolactone lesions is shown in Scheme 1. Oxidation of the C-1′ position of DNA nucleotides (1) and production of a carbonyl group at C-1′ (2) lead to acidification of the C-2′ protons. As a result, the lactone lesion may undergo facile R,β-elimination of the 3′phosphate group to produce the 5′-phosphorylated DNA product (3) and a DNA product bearing an R,β-unsaturated lactone (4, ene-lactone) residue at its 3′ terminus. Intermediate 4 may decompose further by γ,δ-elimination to produce a 3′-phosphorylated DNA cleavage product (5), which releases the sugar residue as methylene furanone (6). Recently, the direct observation of the proposed DNA strand scission products, including the elusive intermediate (4), has been reported (33). Previous studies had inferred the existence of 4 by isolation of the cleavage products 3, 5, and 6 (22, 32) or by chemical fingerprinting of the lesion by nucleophilic reagents (32). Despite these careful mechanistic studies, key features of deoxyribonolactone lesion stability remain unclear. Notably, DNA strand scission occurs at the labile lactone lesion, but the rate of this process is highly dependent on the solution conditions (33). However, future studies of in vivo DNA repair, mutagenesis, and toxicity of the deoxyribonolactone lesion require the characterization of the chemical form of the lesion (full length vs cleaved) and the rate of DNA cleavage at the damage site under cellular conditions. This paper offers insights into these important issues. Our laboratory now reports the measurement of the half-life for DNA strand scission at the deoxyribonolactone lesion under simulated physiological conditions and the complete characterization of the cleavage products under these conditions. The work was aided by improvements to our previously reported method (25) for the photochemical generation of oxidized abasic sites at

Zheng and Sheppard

precise sites within DNA oligonucleotides. Utilizing synthetic oligonucleotides containing the lactone lesion, we have directly identified by MALDI-TOF mass spectrometry the ene-lactone intermediate (4) and other DNA products of strand scission at the lesion. The MALDITOF MS method was applied to establish the identity of cleavage products observed in gel electrophoresis experiments. In addition, the intermediacy of ene-lactone-DNA (4) was validated by chemical fingerprinting experiments. Finally, the half-life for DNA strand scission at the lactone lesion was measured under simulated physiological conditions in single- and double-stranded DNA. Kinetic measurements suggested that the half-life of the deoxyribonolactone lesion ranges from 20 to 54 h under simulated physiological conditions and is dependent on the base-pairing context of the lesion. The first R,βelimination step is the rate-determining step of the two step pathway, and the lesion undergoes DNA strand scission more slowly in duplex DNA as compared with single-stranded DNA. These half-life data provide a valuable starting point for the evaluation of the biological consequences of the lactone lesion in living cells.

Experimental Section General. All glassware was oven-dried (140 °C). Flash column chromatography was performed using silica gel (40-63 µm, EM Science). TLC utilized silica gel 60 glass plates (0.25 mm F254, EM science), and spots were visualized using a short wave UV lamp or by staining with anisaldehyde. All reagents were supplied by Aldrich and were used without further purification, unless stated otherwise. Anhydrous reagents were supplied by Aldrich in Sure-Seal bottles. N2 was used as an inert atmosphere. 1H, 13C, 31P NMR, and COSY data were recorded on a Varian Inova 500 or a Varian Mercury 400 spectrometer. Proton assignments were determined using 1H-1H COSY data. Mass spectral data for synthetic intermediates were obtained by FAB-MS. Matrix-assisted laser desorption ionization timeof-flight mass spectra (MALDI-TOF MS) were obtained on a Biosystems Voyager Pro DE spectrometer. All oligonucleotides were RP-HPLC purified and stored in 10 mM HEPES buffer, pH 7.5. Concentrations of oligonucleotide solutions were measured by UV absorption at 260 nm. Extinction coefficients of oligonucleotides were calculated by the nearest neighbors method (34). The nitroveratryl nucleoside analogue was assigned an extinction coefficient equal to a thymidine residue. T4 polynucleotide kinase was obtained from US Biochemical. Terminal deoxynucleotidyl transferase was from Promega. [γ-32P]ATP (7000 mCi/mmol) and [R-32P]dATP (3000 mCi/mmol) were purchased from ICN. Radioactive bands in polyacrylamide gels were visualized using a Molecular Dynamics Storm phosphorimager and quantitated using Molecular Dynamics ImageQuant software. Protocols are reported for the synthesis of analogue 7, necessary for the photochemical generation of deoxyribonolactone lesions within DNA oligonucleotides (Scheme 2). Compound numbers and a detailed synthetic scheme are provided in the Supporting Information. 2-Nitroveratryl 1′-Cyano-3′,5′-O-(di-4-chlorobenzoyl)-2′deoxy-D-ribofuranoside (20). To a stirring solution of 19 (539.2 mg, 1.080 mmol) in 10 mL of freshly distilled CH2Cl2 were added 2-nitroveratryl alcohol (739.9 mg, 3.471 mmol), AgOTf (841.3 mg, 3.300 mmol), and 2,6-lutidine (0.50 mL, 4.4 mmol). The reaction was stirred at room temperature, under N2, and in the dark for 3 h. The reaction mixture was filtered through 1 cm of Celite, and the Celite pad was washed with 200 mL of CH2Cl2. The combined filtrate was concentrated and purified by chromatography using 100% CHCl3 to afford 538.0 mg of 20 as a mixture of anomers (0.8521 mmol, 79%). 1H NMR (CDCl3): δ 8.00-7.81 (m, 8H, Ar-H), 7.68 and 7.62 (s, 2H, H-3), 7.45-

DNA Strand Scission at Deoxyribonolactone Lesions

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Scheme 2. New Method for the Photochemical Generation of Oxidized Abasic Sites in DNA Oligonuclotides

7.26 (m, 8H, Ar-H), 7.03 and 6.95 (s, 2H, H-6), 5.66 (m, 2H, H-3′), 5.57 (m, 2H, H-4′) 5.43 (d, 1H, J ) 12.0 Hz, Ar-CH2), 5.23 (m, 2H, Ar-CH2), 5.03 (d, 1H, J ) 12.0 Hz, Ar-CH2), 4.63 (m, 4H, H-5′), 3.96 (s, 3H, OCH3), 3.94 (s, 3H, OCH3), 3.93 (s, 3H, OCH3), 3.84 (s, 3H, OCH3), 3.00 (m, 4H, H-2′). 13C NMR (CDCl3): δ 165.14, 165.04, 153.46, 148.58, 140.67, 140.36, 140.23, 140.12, 139.96, 139.85, 131.36, 131.30, 131.22, 131.18, 131.13, 131.05, 129.20, 129.14, 129.02, 129.87, 127.87, 127.69, 127.29, 126.64, 115.94, 115.48, 111.58, 110.46, 108.43, 108.20, 100.99, 100.38, 84.84, 82.46, 74.68, 74.53, 72.13, 66.05, 65.55, 64.23, 64.16, 63.94, 56.77, 56.71, 45.79, 45.53. HRMS-FAB (m/ z): [M + Na]+ calcd for C29H24Cl2N2O10 + Na, 653.0706; found, 653.0706. 2-Nitroveratryl 1′-Cyano-2′-deoxy-D-ribofuranoside (21). To a solution of 20 (385.9 mg, 0.6112 mmol) in 0.6 mL of anhydrous THF was added 15 mL of saturated NH3/MeOH. The reaction was stirred at 35 °C, under N2, and in the darkness for 3.5 h. The reaction was concentrated and purified by chromatography using 100% EtOAc to afford 98.9 mg of 21 (0.279 mmol, 46%) as a mixture of anomers. CAUTION: This reaction generates pressure. 1H NMR (CDCl3): δ 7.67 (s, 1H, H-3), 7.64 (s, 1H, H-3), 7.16 (s, 1H, H-6), 7.04 (s, 1H, H-6), 5.32 (d, 1H, J ) 6.00 Hz, Ar-CH2), 5.29 (d, 1H, J ) 6.00 Hz, ArCH2), 5.02 (d, 1H, J ) 2.40 Hz, Ar-CH2), 4.98 (d, 1H, J ) 2.40 Hz, Ar-CH2), 4.50 (m, 2H, H-3′), 4.25 (m, 2H, H-4′), 3.98 (s, 6H, OCH3), 3.93 (s, 6H, OCH3), 3.75 (m, 4H, H-5′), 2.71 (m, 4H, H-2′). 13C NMR (CDCl ): δ 153.78, 153.62, 148.45, 148.37, 140.03, 3 139.91, 127.42, 126.99, 116.57, 115.85, 111.31, 108.29, 108.25, 100.56, 100.20, 90.13, 89.19, 72.08, 71.14, 65.66, 65.32, 62.58, 62.38, 56.94, 56.85, 56.74, 48.29, 47.85. HRMS-FAB (m/z): [M + Na]+ calcd for C15H18N2O8 + Na, 377.0961; found, 377.0963. 2-Nitroveratryl 1′-Cyano-5′-O-(4,4′-dimethoxytrityl)-2′deoxy-D-ribofuranoside (22). To a stirred solution of 21 (98.9 mg, 0.279 mmol) in 700 µL of anhydrous pyridine were added DMTCl (164.9 mg, 0.486 mmol, ChemGenes) and DMAP (33.6 mg, 0.275 mmol). The reaction was stirred at room temperature, under N2, and in the darkness for 18 h. Saturated aqueous NaHCO3 (2 mL) was added to quench the reaction, followed by 5 mL of 1:1 EtOAc:H2O. The aqueous layer was washed with 5 mL of EtOAc. The combined organic phase was washed with 6 mL of brine, dried over anhydrous Na2SO4, concentrated, and purified by chromatography using 1:1 hexane:EtOAc plus 1% triethylamine. The R- and β-anomers were separated by a second chromatography using 4% acetone in CHCl3 plus 1% triethylamine to yield 25.0 mg of R-anomer 22a and 80.7 mg of β-anomer 22b (0.161 mmol, 58% total yield). r-Anomer. 1H NMR (CD2Cl2): δ 7.74 (s, 1H, H-3), 7.50 (d, 2H, J ) 6.00 Hz, Ar-H), 7.40-7.27 (m, 8H, Ar-H), 6.91 (d, 4H, J ) 6.00 Hz, Ar-H), 5.40 (d, 1H, J ) 10.40 Hz, Ar-CH2), 5.16 (d, 1H, J ) 10.40 Hz, Ar-CH2), 4.38 (m, 1H, H-3′), 4.35 (m, 1H, H-4′), 4.00 (s, 3H, OCH3), 3.96 (s, 3H, OCH3), 3.83 (s, 6H, OCH3), 3.40 (dd, 1H, J5′a,4′ ) 2.40 Hz, J5′a,5′b ) 8.40 Hz, H-5a′), 3.20 (dd, 1H, J5′b,4′ ) 3.20 Hz, J5′a,5′b ) 8.40 Hz, H-5b′), 2.96 (dd, 1H, J2′a,3′ ) 4.80 Hz, J2′a,2′b ) 11.6 Hz, H-2a′), 2.62 (d, 1H, J2′a,2′b ) 11.6 Hz, H-2b′). 13C NMR (CD2Cl2): δ 158.93, 154.18, 148.55, 144.96, 139.92, 135.95, 135.75, 130.24, 128.26, 128.20, 128.05,

127.92, 127.10, 116.20, 113.42, 111.23, 108.31, 100.94, 89.82, 86.66, 72.54, 65.19, 63.68, 56.74, 56.52, 55.45, 48.35, 46.28. MSFAB (m/z): [M]+ calcd for C36H36N2O10, 656.2370; found, 656.2360. β Anomer. 1H NMR (CD2Cl2): δ 7.69 (s, 1H, H-3), 7.39 (d, 2H, J ) 6.00 Hz, Ar-H), 7.26 (d, 4H, J ) 7.20 Hz, Ar-H), 7.21 (m, 3H, Ar-H), 7.06 (s, 1H, H-6), 6.74 (d, 4H, J ) 9.00 Hz, ArH), 5.26 (d, 1H, J ) 9.00 Hz, Ar-CH2), 5.20 (d, 1H, J ) 9.00 Hz, Ar-CH2), 4.46 (m, 1H, H-3′), 4.29 (m, 1H, H-4′), 3.92 (s, 3H, OCH3), 3.77 (s, 3H, OCH3), 3.75 (s, 6H, OCH3), 3.29 (dd, 1H, J5′a,4′ ) 3.00 Hz, J5′a,5′b ) 10.0 Hz, H-5a′), 3.19 (dd, 1H, J5′b,4′ ) 4.50 Hz, J5′a,5′b ) 10.0 Hz, H-5b′), 2.83 (dd, 1H, J2′a,3′ ) 5.50 Hz, J2′a,2′b ) 13.0 Hz, H-2a′), 2.67 (dd, 1H, J2′b,3′ ) 5.50 Hz, J2′a,2′b ) 13.0 Hz, H-2b′). 13C NMR (CD2Cl2): δ 158.79, 153.99, 148.24, 144.86, 139.42, 135.85, 135.80, 130.08, 128.18, 127.99, 127.00, 116.77, 113.20, 109.92, 108.30, 100.19, 88.02, 86.40, 71.02, 65.03, 63.32, 56.45, 56.33, 55.34, 47.77, 46.16. MS-FAB (m/z): [M]+ calcd for C36H36N2O10, 656.2370; found, 656.2367. 2-Nitroveratryl 1′-Cyano-3′,5′-O-(2-cyanoethyl N,N-diisopropylphosphoramidityl)-5′-O-(4,4′-dimethoxytrityl)-2′deoxy-D-ribofuranoside (7). Both anomers were converted independently to phosphoramidites by the same protocol. To a solution of 22b (80.7 mg, 0.123 mmol) in 0.5 mL of freshly distilled CH2Cl2 were added [(iPr)2N]2 POCH2CH2CN (0.06 mL, 0.198 mmol) and diisopropylammonium tetrazolide (26.4 mg, 0.154 mmol). The reaction was stirred at room temperature, under N2, and in the darkness for 3 h. Then, 6 mL of H2O and 6 mL of CH2Cl2 were added. The resulting aqueous layer was back extracted with 2 × 5 mL of CH2Cl2, and the combined organic phase was washed with 2 × 6 mL of saturated NaHCO3 and 2 × 6 mL of brine. The resulting material was dried over anhydrous Na2SO4, concentrated, and purified by chromatography using 40% EtOAc in hexane plus 1% triethylamine to afford 61.9 mg of 7b (0.0722 mmol, 59%). Compound 7b. 1H NMR (CD3CN): δ 7.68 (1H, s, H-3), 7.38 (m, 2H, Ar-H), 7.25-7.14 (m, 8H, Ar-H), 6.74 (m, 4H, Ar-H), 5.25 (m, 1H, Ar-CH2), 5.10 (m, 1H, Ar-CH2), 4.72 (m, 1H, H-3′), 4.39 (m, 1H, H-4′), 3.87 (s, 3H, OCH3), 3.78 (s, 3H, OCH3), 3.73 (s, 6H, OCH3), 3.61 (m, 2H, N(CH)2), 3.30 (m, 1H, H-5a′), 3.12 (m, 1H, H-5b′), 2.96 (m, 1H, H-2a′), 2.77 (m, 1H, H-2b′), 2.68 (m, 1H, POCH2), 2.53 (m, 1H, POCH2), 2.49 (m, 2H, CH2CN), 1.20 (m, 12H, CH(CH3)2). 13C NMR (CD3CN): δ 158.83, 154.11, 148.33, 144.91, 139.45, 135.96, 135.87, 135.78, 130.07, 128.18, 128.02, 128.84, 127.08, 118.82, 118.59, 117.57, 116.57, 113.18, 110.09, 108.36, 108.28, 100.34, 100.22, 87.36, 87.26, 87.21, 86.26, 72.75, 72.62, 71.95, 71.81, 64.84, 64.74, 62.68, 62.37, 58.88, 58.74, 58.66, 58.51, 56.23, 56.12, 55.07, 46.27, 43.37, 43.32, 43.22, 24.20, 24.13, 20.34, 20.29, 20.22, 20.17. 31P NMR (CD3CN): δ 149.03, 148.80. MS-FAB (m/z): [M + H]+ calcd for C45H53N4O11P + H, 857.3527; found, 857.3526. The R-anomer 22a was converted to 7a using the same protocol (59%). Compound 7a: 1H NMR (CD3CN): δ 7.75 (1H, s, H-3), 7.50 (m, 2H, Ar-H), 7.40-7.28 (m, 8H, Ar-H), 6.93 (m, 4H, Ar-H), 5.29 (m, 1H, Ar-CH2), 5.18 (m, 1H, Ar-CH2), 4.58 (m, 1H, H-3′), 4.38 (m, 1H, H-4′), 3.99 (d, 3H, J ) 2.40 Hz, OCH3), 3.93 (s, 3H, OCH3), 3.82 (s, 6H, OCH3), 3.62 (m, 2H, POCH2), 3.54 (m, 2H, N(CH)2), 3.40 (m, 1H, H-2a′), 3.15 (m, 1H, H-2b′), 3.03 (m, 1H, H-5a′), 2.76 (m, 1H, H-5b′), 2.46 (m, 2H, CH2CN), 1.14 (m, 12H, CH(CH3)2). 13C NMR (CD3CN): δ 159.01,

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153.96, 148.58, 145.24, 140.15, 136.06, 135.88, 135.80, 130.26, 128.17, 127.85, 127.63, 127.16, 118.70, 118.52, 117.59, 116.55, 113.39, 111.39, 111.15, 108.45, 100.98, 100.88, 88.13, 87.87, 87.82, 86.48, 73.40, 73.26, 73.00, 72.87, 64.68, 64.57, 63.22, 58.90, 58.84, 58.75, 58.68, 56.52, 56.44, 56.21, 55.19, 46.18, 44.45, 43.32, 43.23, 24.14, 24.08, 24.01, 23.97, 23.24, 22.96. 31P NMR (CD3CN): δ 148.99, 148.80. MS-FAB (m/z): [M + H]+ calcd for C45H53N4O11P + H, 857.3527; found, 857.3532. DNA Synthesis, Deprotection, and Purification. Standard unmodified DNA oligonucleotides were synthesized by Integrated DNA Technologies and purified by RP-HPLC. Oligonucleotides containing 7b were synthesized on a Pharmacia Gene Assembler Plus at the 1.3 µmol scale using phosphoramidites with “Ultramild” protecting groups (Glen Research). The synthesized phosphoramidite, 7b, was prepared as 0.10 M solution in anhydrous CH3CN and was incorporated into DNA strands with a coupling time of 6 min. Coupling yields for each nucleotide addition were calculated by UV/visible spectrometric analysis of the DMT cation effluents produced at the beginning of each cycle. Average coupling yields for 7b were ∼90%, with an average coupling yield of ∼94% for addition of the next nucleotide. DNA strands containing 7a were synthesized by the same protocol and similar efficiency. However, all mechanistic and kinetic studies described herein were performed with DNA strands synthesized from 7b. After synthesis, the cassettes were dissembled, and the resins were treated with 2 M NH3/MeOH at 37 °C for 5 h. Following deprotection, the NH3/MeOH was removed using a Speedvac concentrator, and the resulting material was redissolved in 10 mM HEPES, pH 7.5, and purified using RP-HPLC. RP-HPLC oligonucleotide purifications were performed using a Waters series 600 HPLC system. Strand 8 was eluted using a gradient as 10-30% solvent B (A: 0.1 M triethylamine acetate in H2O, pH 7.2; B: 0.1 M triethylamine acetate in 60% CH3CN, pH 7.2) over 40 min. Peaks were monitored at 254 nm. The fractions were collected, frozen, and lyophilized to dryness using a FreeZone 4.5 L benchtop Freeze-Dry system (Labconco). The dried oligonucleotides 8 were redissolved in 10 mM HEPES buffer, pH 7.5, and stored at -20 °C. Photolytic Generation of 2′-Deoxyribonolactone Lesions. Strand 8 was prepared as a 50 µM solution in 10 mM HEPES and 0.1 mM EDTA at pH 8.0. This sample was irradiated in a Rayonet photochemical minireactor (Southern New England) using eight 350 nm bulbs (4 W each). After 10 min, the sample was removed and the decaged oligonucleotides were immediately isolated by RP-HPLC using a gradient of 1035% solvent B over 20 min. The product peak was collected, divided into 50 µL fractions, frozen on dry ice, and dried in a Speedvac concentrator that was prechilled with dry ice. The pure lactone oligonucleotides then were redissolved in 10 mM HEPES, pH 7.5, and stored at -20 °C. MALDI-TOF Mass Spectrometric Analysis of Oligonucleotides. The MALDI-TOF mass spectrometry matrix was a 8:1 mixture of 0.2 M 2′,4′,6′-trihydroxyacetophenone in 1:1 CH3CN:H2O and 0.3 M aqueous ammonium citrate solution (35). DNA samples were mixed with the matrix in a 1:1 (v/v) ratio. The mixture (1 µL) was spotted on the MALDI-TOF MS sample plate and another 0.5 µL of matrix was applied on top of the mixture. The sample plate was placed on top of an ice bath, dried under ambient conditions, and analyzed by MALDI-TOF mass spectrometry. Mass Spectrometry Analysis of the Cleavage Reaction. A 100 µL cleavage reaction was prepared containing 50 µM decaged strand 2 in 10 mM HEPES, 150 mM NaCl, and 2 mM MgCl2, pH 7.5. The reaction was incubated at 37 °C for 25 h. DNA samples were purified by ZipTip (Millipore) desalting, using a modification of the manufacturer’s protocol. A ZipTip was washed consecutively with 3 × 10 µL of 1:1 CH3CN:H2O and 3 × 10 µL of H2O. Then, a 10 µL sample from the cleavage reaction was loaded onto the tip, and the tip was washed with 5 × 10 µL of H2O. The reaction mixture loaded on the tip was

Zheng and Sheppard eluted consecutively with 1 µL each of 6, 8, 10, 12, 14, and 50% CH3CN in H2O. The eluents were kept on ice and immediately subjected to MALDI-TOF MS sample preparation. 5′-Phosphorylation of Lactone-Containing Oligonucleotides. Strand 8 (4 nmol) was incubated at 37 °C for 3 h with 50 units of polynucleotide kinase (USB) in a 100 µL reaction containing 10 mM Tris-acetate, pH 7.6, 10 mM magnesium acetate, 50 mM potassium acetate, and 0.4 mM ATP. The labeling reaction was submitted to 350 nm UV irradiation for 10 min. The decaged product 2p was purified by RP-HPLC using a 10-35% gradient of solvent B over 20 min. The fractions were collected, divided into 50 µL fractions, frozen on dry ice, and dried in a Speedvac concentrator that had been precooled with dry ice. The resulting material was redissolved in 10 mM HEPES, pH 7.5, as 50 µmol solution and stored at -20 °C. 5′-Radiolabeling of Lactone-Containing Oligonucleotides. Oligonucleotide 2 (20 pmol) was incubated at 37 °C for 30 min with 24.5 units of polynucleotide kinase (USB) in a 20 µL reaction containing 10 mM Tris-acetate, pH 7.6, 10 mM magnesium acetate, and 50 mM potassium acetate and 24 pmol of [γ-32P]ATP (7000 Ci/mmol, 8.4 Ci/µL). The reaction was purified using a QIAquick Nucleotide Removal Kit (QIAGEN), and the product strand was eluted with 80 µL of ddH2O. Radiolabel-Assisted Isolation of Cleavage Products. A nonradioactive reaction was prepared including 0.4 nmol of strand 2p in a 10 µL reaction buffered with 10 mM HEPES, 150 mM NaCl, and 10 mM MgCl2 (pH 7.5 at 37 °C). In parallel, a radioactive reaction was prepared in the same manner except that ∼100 fmol of radiolabeled 2 was included with nonradioactive 2p strand. The two reactions were incubated at 37 °C for 20 h, mixed with an equal volume of 2 × GLB (20% w/v sucrose, 0.05 M EDTA, 0.5% w/v SDS, 0.05% w/v bromophenol blue, and 0.05% w/v xylene cyanol), and then applied to a 20% denatured polyacrylamide gel (dPAGE, 0.75 mm × 400 mm) in two parallel lanes. After electrophoresis, the radiolabeled DNA bands were imaged on BioMax film, and the corresponding nonradioactive bands in the adjacent lane were excised with a sterilized razor blade using adjacent radioactive bands as markers. The bands were crushed into small pieces, and the oligonucleotides were eluted into 10 mM HEPES, 200 mM NaCl, and 1 mM EDTA, pH 7.7, for 4 h at 0 °C. The gel extraction solutions were desalted using a C18 resin ZipTip (Millipore) and submitted to MALDITOF MS. Chemical Trapping of the Ene-lactone Intermediate. Radiolabeled strand 2 was buffered with 10 mM HEPES, 150 mM NaCl, and 10 mM MgCl2 (pH 7.5 at 37 °C) and incubated at 37 °C for 20 h. The reaction was quenched with an equal volume of 2 × GLB and applied to 20% PAGE (0.75 mm × 400 mm). After electrophoresis, bands were located by exposing the gel to Biomax film. Both the full length DNA (2) and the enelactone intermediate (4) bands were excised with sterilized razor blades and soaked at 37 °C for 4 h with the trapping reagents: (i) 100 mM piperidine (pip); (ii) 100 mM pip, 50 mM β-mercaptoethanol (BME); (iii) 100 mM N,N′-dimethylethylenediamine (DMED), 50 mM BME; (iv) 50 mM BME; (5) 100 mM DMED; (vi) 200 mM NaCl, 10 mM Tris, 1 mM EDTA; and (vii) H2O. The products were desalted using a ZipTip, eluting with 1 µL of 20% CH3CN in H2O. The eluents were mixed with an equal volume of 2 × GLB and analyzed by 20% PAGE. Preparation of Radiolabeled Marker 5p. DNA sequence 14, 5′-TGT GCC YAA CCT ACC GT-3′, where Y ) dU, was radiolabeled as described. The product was incubated in a 80 µL reaction containing 70 mM HEPES, pH 8.0, 1 mM EDTA, and 1 mM DTT and treated with 3.5 units of uracil DNA glycosylase (USB) for 1.5 h at 37 °C to produce 14 containing an AP site (Y ) deoxyribose). A 100 mM aqueous solution of spermine (20 µL) was added, and the reaction was incubated at 37 °C for another 30 min. The reaction was quenched with 100 µL of 2 × GLB and purified using 20% dPAGE as described. The marker 5p was identified by exposure to film and was isolated from the gel slice by crush and soak

DNA Strand Scission at Deoxyribonolactone Lesions elution. Oligonucleotide 14, where Y ) deoxyribose, also was used for the determination of the half-life of DNA strand cleavage at AP sites under simulated physiological conditions. Half-Life Measurement under Simulated Physiological Conditions. Five 50 µL cleavage reactions were prepared each containing 2.3 µM of strand 2 with a trace amount of radiolabeled 2. Reaction 1 contained single-stranded 2. Reactions 2-5 contained 2.3 µM 2 with an equimolar amount of the four different complementary strands (5′-ACGGTAAGTT-N-GGCACA3′; 15, N ) A; 16, N ) C; 17, N ) G; and 18, N ) T). The reactions were incubated at 37 °C in 10 mM HEPES, 150 mM NaCl, and 2 mM MgCl2, pH 7.50, and 5 µL aliquots were taken every 4-12 h over 3 days. Native PAGE analysis demonstrated rapid and complete duplex formation under the reaction conditions. The aliquots were quenched with 2 × GLB and stored at -20 °C. After all aliquots had been removed, samples were applied to a 20% dPAGE and electrophoresed at 37 °C for 1 h. The gel was imaged and quantified to calculate the half-life data. Half-Life Measurements with 3′-Radiolabeled Oligonucleotides. Strand 8 (120 pmol) was incubated at 37 °C for 50 min with 60.0 units of TDT enzyme (Promega) in a 20 µL reaction containing 100 mM cacodylate, pH 6.8, 1 mM CoCl2, and 0.1 mM DTT and 40 pmol [R-32P] dATP (3000 Ci/mmol, 30 mCi/µL). The 3′-labeled oligonucleotide was purified by 20% dPAGE and imaged on Biomax film. Radioactive bands were excised with a sterilized razor blade. The gel pieces were crushed and eluted into 10 mM HEPES, 200 mM NaCl, and 1 mM EDTA, pH 7.7, at 0 °C for 10 h. The radioactive oligonucleotide was isolated using a QIAquick Nucleotide Removal Kit and eluted with ddH2O. The eluents were buffered with 10 mM HEPES and 1 mM EDTA, pH 7.5, subjected to UV irradiation at 350 nm for 10 min, and purified a second time using QIAquick Nucleotide Removal Kit to remove the buffer salts and photolytic byproducts. The pure decaged 3′-radiolabeled strand 2 was eluted with 10 mM HEPES buffer and included in cleavage reactions as described in the experiments with 5′-radiolabeled material. Data Processing for Half-Life Measurements. ImageQuant software was used to process the analytical gel data from the half-life measurements. Intensities of radioactive bands were measured relative to standard background correction. The decay ratio at the lesion site at each time point was calculated as the intensity of products over the summed intensity of both cleavage products and full length strand. The percentage of remaining full length strands was plotted vs time and fit to an exponential decay curve: y ) (1 - A) + A exp [-(x - x0)k], where A represents the amount of full length DNA at equilibrium, x0 represents the amount of full length DNA that was cleaved at time zero, and k represents the decay rate constant. The parameters A, x0, and k were fit using Origin 7SR1 (Originlab). Both 5′- and 3′-radiolabeled measurements were repeated four times, and error is reported (Figure 6B) as the standard deviation of these four replicates.

Results and Discussion Synthesis of the Photocaged Analogue. Strategies for the site specific introduction of DNA damage lesions into DNA oligonucleotides by solid phase synthesis have provided powerful methods for the study of DNA damage and repair mechanisms (16). In the context of C-1′ oxidation, we previously reported a method for the independent synthesis of deoxyribonolactone lesions in DNA oligonucleotides (25). The strategy involved the photochemical conversion of a stable nitrobenzyl-protected C-1′ cyanohydrin nucleotide analogue in the lactone lesion. We now report an improved method for deoxyribonolactone generation in DNA, which is outlined in Scheme 2. The approach involves the synthesis of a caged phosphoramidite analogue 7, its introduction into DNA (8), and conversion to the lactone lesion (2) by photolysis


at 350 nm. The enhanced method takes advantage of two structural features of 7. First, the 5′-hydroxyl of 7 was protected with the more standard dimethoxytrityl (DMT) group instead of the monomethoxytrityl group, used previously. This substitution eliminated a manual detritylation step and provided access to a fully automated method for modified DNA synthesis. Second, the nitroveratryl ether, which has a greater absorption at 350 nm (36), was expected to provide more efficient photolytic deprotection than the previously used o-nitrobenzyl group. The synthetic route to 7 paralleled our previously published route (25) and is detailed in the Supporting Information. The nitroveratryl group was installed by glycosylation of 2-nitroveratryl alcohol with a bromocyanide donor (19) using silver triflate and 2,6-lutidine in dichloromethane (79% yield). The 3′- and 5′-O-p-chlorobenzoyl protecting groups were removed using methanolic ammonia at 35 °C. The resulting nucleoside anomers (22a,b) were separated after conversion to their DMT ether derivatives by treatment with DMTCl and DMAP in pyridine. The R- and β-isomers independently were converted into the corresponding phosphoramidite derivative (7a,b). Incorporation of the Caged Analogue into DNA Oligonucleotides. Both anomers of phosphoramidite 7 were successfully incorporated into DNA oligonucleotides 8 using standard solid phase DNA synthesis, using an increased coupling time of 6 min for 7. The coupling efficiency of 7b was ∼90%, and the addition of subsequent nucleotides proceeded with an average coupling yield of 94%. Following DNA synthesis, DNA deprotection by ammonia treatment was required. As we reported previously (25), the cyano group at the C1′ position of protected 8 was vulnerable to modification by the 28% aqueous ammonia solution typically employed for DNA deprotection. In fact, quantitative modification of the cyano group was observed when synthetic strand 8 was deprotected with this reagent. Other deprotection conditions, including methylamine or potassium carbonate/ MeOH, also failed to produce 8 as the major product. However, on resin deprotection using a 2 M solution of ammonia in methanol at 37 °C for 5 h yielded the desired caged oligonucleotide, 8. A small amount of modification product was observed under these conditions. However, it was easily separated from 8 by RP-HPLC. To support DNA deprotection under these milder conditions, nucleoside phosphoramidites with labile base-protecting groups were used for the four natural nucleotides. The caged DNA 8 was purified by RP-HPLC, and the identity of the oligonucleotide was validated by MALDI-TOF MS: calculated m/z for 8, 5248.3; observed, 5248.2. Photolytic Production of the Deoxyribonolactone Lesion within DNA. As shown in Scheme 2, the conversion of caged DNA 8 to the deoxyribonolactone lesion was accomplished by UV irradiation. In our earlier work using the o-nitrobenzyl ether protecting strategy (25), complete conversion of the caged nucleotide into the lactone lesion was accomplished in 45 min of irradiation. Use of the nitroveratryl protecting group in 8 significantly improved the efficiency for deoxyribonolactone lesion production in DNA oligonucleotides. A representative RP-HPLC trace for the photolytic decaging reaction of oligonucleotide 8 (50 µM oligonucleotide, 10 mM HEPES, 1 mM EDTA, pH 7.5) is shown in Figure 1. Figure 1A demonstrates that ∼30% of 8 (retention time

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Figure 2. Gel electrophoresis analysis of DNA strand scission at the deoxyribonolactone lesion 2 under simulated physiological conditions (10 mM HEPES, pH 7.5, 150 mM NaCl, 2 mM MgCl2, 37 °C). Reaction aliquots were removed at the times listed and analyzed by 20% PAGE. Lane 1, untreated 2; lane 2, 4 h; lane 3, 8 h; lane 4, 20 h; lane 5, 24 h; lane 6, 28 h; lane 7, 32 h; and lane 8, 44 h.

Figure 1. Efficient photolytic generation of deoxyribonolactone lesions in DNA oligonucleotides. (A) RP-HPLC profile of photolysis of 50 µM 8 in 10 mM HEPES, pH 7.5, 1 mM EDTA after 1 min of irradiation; 30% conversion of 8 to 2 was observed. (B) RP-HPLC profile of the same reaction after 10 min shows complete conversion of 8 to 2.

(tR) ) 19.1 min) underwent conversion to a new product (tR ) 14.0 min, 2, vide infra) within 1 min of irradiation at 350 nm. Complete photolysis was achieved within 10 min of irradiation at 350 nm (Figure 1B). To characterize the observed product of the photolysis reaction, the product peak at tR ) 14.0 min was isolated by RP-HPLC and subjected to MALDI-TOF MS. The mass spectrometric data revealed that the reaction product was the expected lactone-modified oligonucleotide 2 (calculated m/z for 2, 5026.3; observed, 5025.0). Thus, the methodology based on 8 offers a significant improvement in the rate of photolytic conversion to 2, as compared with our previously described approach. The shorter irradiation time (10 vs 45 min) may offer an advantage for production of the lesion within larger DNA sequences, which may be more prone to damage by UV irradiation. Isolation and MALDI-TOF MS Characterization of the Products of DNA Strand Scission at the Deoxyribonolactone Lesion under Simulated Physiological Conditions. Since the early observations of deoxyribonolactone lesions in damaged DNA, it has been recognized that DNA strand scission occurs readily at sites of C-1′ oxidation (5, 22). By analogy to AP sites in DNA, rates of DNA strand cleavage at oxidized abasic sites are highly dependent on solution conditions and are facilitated by alkaline and heat treatment (32, 33). On the basis of the mechanism outlined in Scheme 1, four products are expected for DNA decomposition at the deoxyribonolactone lesion. The sugar moiety is extruded as methylene furanone (6). Each elimination reaction produces a DNA product with a terminal phosphate: 3, produced in the first step, and 5, produced in the second step. The first step also yields the intermediate 4, which bears an ene-lactone moiety at the 3′-end of the 5′cleavage product. On the basis of earlier studies and our experience with the lactone lesion, we set out to measure the rate of DNA strand scission (Scheme 1) under physiological conditions. To facilitate this effort, two preliminary steps were required as follows: (i) the isolation and unambiguous

characterization of the products of DNA strand scission at the lesion and (ii) the correlation of the identity of the cleavage products with bands observed in PAGE experiments. To address these issues, we developed isolation techniques for strand scission intermediates and products and utilized MALDI-TOF MS to characterize these materials. To simulate physiological conditions, we performed DNA strand cleavage assays in aqueous buffer containing 10 mM HEPES, 150 mM NaCl, and 2 mM MgCl2 at pH 7.5 at 37 °C, which is widely used as a model of physiological salt, pH, and temperature conditions (37). Our first goal was to inventory the DNA components of the DNA strand scission reaction by gel electrophoresis. Single-stranded radiolabeled oligonucleotide 2 was incubated under simulated physiological conditions. Aliquots of the reaction were removed at specified times, and the products were analyzed by denaturing PAGE, as shown in the reaction time course in Figure 2. During incubation, the starting lactone-containing DNA 2 (lane 1) was converted into two products (lanes 2-8). A lower mobility DNA band accumulated at a low level over the course of the reaction and eventually disappeared over 48 h. The second DNA product demonstrated a higher mobility and accumulated over time as the major product. Because 5′-radiolabeled 2 was used for these experiments, the data were consistent with a mechanism in which a labile intermediate 4 (lower mobility) was generated before it degraded to a more stable fragment 5 (higher mobility). Further experiments showed that the higher mobility band migrated at the same rate in a gel as a synthetic oligonucleotide 5, which had been prepared by an independent method (see Figure 5). Having demonstrated the presence of two DNA products containing the DNA sequence upstream of the lactone lesion, we next sought to identify the products of DNA strand cleavage of 2 through a rapid isolation/ MALDI-TOF MS protocol. Initial studies, which focused on characterization of the crude reaction mixture, required a DNA isolation method that enriched all DNA products, including low abundance materials such as the proposed intermediate 4 (Figure 2). Second, because of the instability of the lactone lesion, the isolation protocol required rapid manipulations under mild conditions. A reaction in which the lactone lesion was allowed to decompose at 37 °C under simulated physiological conditions for 24 h was sampled directly by a pipet tip packed with a C18 matrix. After binding of the reaction mixture to the tip, buffer and salts were removed by aqueous washes. Subsequently, DNA strands were eluted with a gradient of acetonitrile in H2O. Eluents from the tip at


Figure 3. Analysis of the DNA product mixture resulting from strand scission at 2 by MALDI-TOF MS. (A) MALDI-TOF MS of DNA strand scission products from m/z ) 1000-5500. Strand 2 was incubated under simulated physiological conditions for 24 h. DNA products were recovered as a mixture using a C18packed pipet tip. Products observed included the following: 2 (calcd, 5026.2; found, 5020.5), 3′-terminal DNA 3 (calcd, 3067.0; found, 3064.9), ene-lactone-DNA 4 (calcd, 1959.3; found, 1958.6), and 5′-DNA fragment 5 (calcd, 1863.2; found, 1863.1). Nonproduct peaks were assigned to double ionized of 4 (1533.7) and the T10 molecular weight standard (1490.8). (B) Expanded view (m/z ) 1750-2200) of the MALDI-TOF MS shown in panel A with the ene-lactone intermediates 4 and 5.

12% acetonitrile were subjected to MALDI-TOF MS, as shown in Figure 3. All DNA components involved in the cleavage mechanism were observed as follows: full length DNA 2 (calcd, 5026.2; found, 5020.5), the ene-lactone intermediate 4 (calcd, 1959.3; found, 1958.6), and both 3′- and 5′-DNA fragments 3 (calcd, 3067.0; found, 3064.9) and 5 (calcd, 1863.2; found, 1863.1). Ether extraction of a DNA cleavage reaction and subsequent GC-MS analysis revealed the presence of methylene furanone 6 (not shown). Thus, our inventory of the reaction components was consistent with the mechanism of DNA strand cleavage at the lactone lesion, as outlined in Scheme 1. After demonstrating the presence of the expected DNA fragmentation products under simulated physiological conditions, it became necessary to correlate the identities of the DNA products with product bands visible in polyacrylamide gels. In particular, because 5′-radiolabeled DNA typically is used for gel electrophoresis experiments, it was critical to assign the gel mobilities of the two DNA cleavage products that contained the DNA sequence upstream of the lactone lesion, intermediate 4 and product 5. To isolate and characterize 4 and 5, two parallel DNA cleavage reactions were performed under standard conditions (10 mM HEPES, 150 mM NaCl, and 2 mM MgCl2 at pH 7.5 and 37 °C): one that contained radioactive 2 and a second that contained 0.4 nmol of 5′-phosphorylated nonradioactive 2p (p indicates that the DNA is labeled at its 5′-end with a nonradioactive phosphate group). These two reactions were applied to the same polyacrylamide gel. After electrophoresis, the positions of the products from the radioactive reaction were located by exposure of the gel to X-ray film. The corresponding product bands from the nonradioactive reaction were isolated using the radiolabeled materials


Figure 4. MALDI-TOF MS analysis of the cleavage products after PAGE isolation. (A) Mass spectrum of the lower mobility product band (Figure 2). Two peaks, corresponding to the 5′DNA fragment 5p (calcd, 1943.2; found, 1942.9) and the enelactone 4p (calcd, 2039.2; found, 2039.0), were observed. (B) MALDI-TOF MS of the higher mobility product band (Figure 2). Only 5p was observed (m/z ) 1942.9).

as markers. The two unlabeled DNA products were eluted from the gel slices under low temperature conditions and subjected to MALDI-TOF MS analysis. Figure 4A shows the results of MALDI-TOF MS analysis of the lower mobility product of DNA strand scission. Two peaks, corresponding to the intermediate 4p (m/z calcd, 2039.2; found, 2039.0) and the final cleavage product 5p (m/z calcd, 1943.2; found, 1942.9), were observed. In contrast, MALDI-TOF MS spectra of the higher mobility band (Figure 4B) revealed only the final phosphorylated DNA fragment 5p (m/z calcd, 1943.2; found, 1942.9). Therefore, the upper band observed in PAGE analysis (Figure 2) was assigned as the ene-lactone intermediate 4. The appearance of 4p with a peak corresponding to 5p was attributed to spontaneous decomposition of 4p during sample preparation and mass spectrometry. The lower band observed in PAGE analysis (Figure 2) was confirmed as the 5′-DNA fragment 5. Independent Characterization of the Ene-lactone Intermediate by Reaction with Nucleophilic Reagents. Our gel electrophoresis and MALDI-TOF MS experiments validated the identities of the intermediate and products of DNA strand scission at the deoxyribonolactone lesion. To further confirm the identity of the enelactone intermediate (4), we utilized a method developed by Greenberg and co-workers (32) for analysis of DNA containing deoxyribonolactone lesions. This “chemical fingerprinting” approach relies on the susceptibility of the ene-lactone intermediate 4, which contains an R,βunsaturated lactone group, to reaction with nucleophilic reagents. Greenberg and co-workers treated DNA-containing deoxyribonolactone with reagents, including pip, BME, and DMED, either alone or in combination as shown in Scheme 3. DNA strand scission at the lactone lesion resulted in the in situ production of the ene-lactone intermediate, which was intercepted by the nucleophilic reagents to produce adducts with distinct gel mobilities (32).

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Scheme 3. Adducts Formed by Chemical Trapping of Ene-Lactone Intermediate (4) by Nucleophilic Reagents

Figure 5. Chemical fingerprinting of lactone-DNA 2 and enelactone intermediate 4. Lanes 1: 5′-TGTGCC-p, 5′-TGTGCCOH, markers; 2: 4, 100 mM pip; 3: 4, 100 mM pip, 50 mM BME; 4: 4, 100 mM DMED, 50 mM BME; 5: 4, 50 mM BME; 6: 4, 100 mM DMED; 7: 4, 200 mM NaCl, 10 mM Tris, 1 mM EDTA; 8: 4, H2O; 9: 2, 100 mM DMED; 10: 2, 100 mM pip, 50 mM BME. Abbreviations: pip ) piperidine; BME ) β-mercaptoethanol; DMED ) N,N′-dimethylethylenediamine. DNA products 2 and 4 were subjected to the reagents listed above in the gel slice at 37 °C for 2 h.

We applied the “chemical fingerprinting” method to the demonstrate that ene-lactone intermediate 4, isolated from the decomposition of lactone-containing DNA 2, generated a reactivity profile that paralleled previous observations (32). Radiolabeled DNA 2 was incubated under simulated physiological conditions for 24 h, and the reaction products were separated by PAGE. Bands corresponding to full length DNA 2 and the ene-lactone intermediate 4 were excised from gel and soaked into solutions containing trapping reagents shown in Scheme 3. Two oligonucleotides, 5′-TGTGCC-OH-3′ and 5′-TGTGCC-p-3′ (5), were prepared for use as markers for the assay (Figure 5, lane 1). The upper marker was derived from a synthetic oligonucleotide that was radioactively labeled. An authentic sample of radiolabeled 5 was not accessible by standard radiolabeling due to the 3′phosphatase activity of T4 polynucleotide kinase (38).

Thus, authentic 5 was produced by strand cleavage of a DNA oligonucleotide (14, see Experimental Section) containing an AP site (39, 40). The analytical gel showing the products of nucleophilic adduction reactions is shown in Figure 5. Treatment of lactone-containing DNA 2 with nucleophilic reagents reproduced the results previously seen by Greenberg and co-workers (32). For example, reaction of full length DNA 2 with DMED (lane 9) produced adduct 10 and full cleavage product (DNA 5, compare lane 1). Similarly, reaction of 2 with pip and BME resulted in the formation of adducts 11 and 13 (lane 10). The same gel patterns were observed when the isolated ene-lactone-DNA 4 was eluted from gel slices in the presence of DMED (lane 6) or in with pip/BME (lane 3). Reaction of isolated 4 (lanes 2 and 4) gave adduct profiles that paralleled the results for 2 (32). For comparison, the elution of 4 from gels in the presence of Tris buffer (lane 7) or deionized water (lane 8) at 37 °C led to complete decomposition to product 5. These data suggest that ene-lactone 4 is the reactive species in the chemical fingerprinting reactions and independently confirms the assignment of 4 as the central intermediate of DNA cleavage at oxidized abasic sites. Half-Life Measurements of DNA Strand Scission under Simulated Physiological Conditions. The biochemical consequences of the deoxyribonolactone lesion in a living cell depend on the abundance of the lesion, the precise chemical form of the lesion under physiological conditions, and therefore the rate of DNA strand scission at the damage site. An understanding of the halflife of this lesion and a comparison of this lifetime to cell cycle rates are imperative for a detailed understanding of the properties of this lesion. Having characterized the products of DNA strand scission at the deoxyribonoloactone site and correlated the products with bands in polyacrylamide gels, our attention turned to the measurement of the half-life of the DNA fragmentation process at the lactone abasic site. The rate of DNA strand scission at the deoxyribonolactone lesion was determined by incubation of the lactone-containing oligonucleotide 2 in buffered aqueous solutions designed to mimic physiological conditions (10 mM HEPES, pH 7.5, 150 mM NaCl, and 2 mM MgCl2) (37). The rate of decomposition of single-stranded 2 was measured and was compared to the rate for DNA strand cleavage in the context of a DNA duplex. DNA duplexes


Figure 6. Half-life for DNA strand scission at the deoxyribonolactone lesion under simulated physiological conditions (10 mM HEPES, 150 mM NaCl, 2 mM MgCl2, pH 7.5, at 37 °C). (A) Exponential decay curves for the deoxyribonolactone lesion in single strand and duplex contexts. (B) Comparison of half-life data obtained by 5′- and 3′-labeling experiments.

were prepared by annealing 2 with one of four complementary oligonucleotides (5′-ACGGTAAGTT-N-GGCACA3′, 15, N ) A; 16, N ) C; 17, N ) G; 18, N ) T, as indicated in Figure 6A) that incorporated one of the natural deoxynucleotides opposite the lesion site. Reactions were performed with 2.3 µM strand concentrations (each strand) in the presence of a trace amount of radiolabeled 2 at 37 °C under standard buffer conditions. Reaction aliquots were removed and analyzed by PAGE (see Figure 2). Cleavage data were fit to an exponential decay model, which was used to calculate the half-life for DNA strand scission at the damage site. Figure 6A shows the data obtained for analysis of single-stranded 2 DNA cleavage as compared with 2 hybridized with a complementary strand (15-18). The half-life of the lactone lesion in single-stranded 2 was 20 ( 1 h. The halflife for strand scission at the lactone lesion in duplex DNA differed in two important ways from the singlestranded form. First, cleavage at the lactone lesion occurred more slowly in duplex DNA. Second, the rate of DNA strand scission was dependent on the identity of the natural base paired opposite the damage site: t1/2 ) 54 ( 5 h for A, 32 ( 4 h for T, 44 ( 5 h for C, and 38 ( 2 h for G. Thus, in a duplex context, with one of the four natural deoxynucleotides inserted opposite the lesion, the strand scission reaction exhibited a progressive decrease in half-life according to the order A > C > G > T. Although the data in Figure 6A gave preliminary insight into the rates of cleavage at deoxyribonolactone lesions, the results were complicated by the fact that two DNA products were observed (Figure 2) and used to calculate the decay rates. Thus, it was critical to determine the rate of the first R,β-elimination step as compared with the second step. The first elimination reaction was kinetically isolated from the overall reaction by repeating the half-life determination experiments using DNA 2 that had been 3′-radiolabeled. Because DNA fragment 3 is involved only in the first step of the


mechanism, it offered an unambiguous probe for the rate of the first β-elimination reaction. Figure 6B shows a comparison of data obtained from 5′-radiolabeling experiments (Figure 6A) with data derived from 3′-radiolabeling experiments for single-stranded and double-stranded DNA containing the lactone lesion. The half-life data obtained with 3′-labeled 2 were in excellent agreement with those obtained in the 5′-labeling experiments: 23 ( 2 h for single-stranded 2 and 53 ( 1, 46 ( 3, 30 ( 5, and 30 ( 4 h when the lesion was paired with A, C, G, and T, respectively. These data suggest that the first R,βelimination reaction is the rate-determining step for the formation of methylene furanone (6), the final product of DNA strand cleavage at the deoxyribonolactone lesion. The observation that the intermediate 4 accumulates only to low levels under simulated physiological conditions is consistent with this model. A useful comparison can be made between DNA strand scission at deoxyribonolactone sites and AP sites produced by depurination or base excision repair. The rate of DNA strand cleavage at AP sites was measured in the same sequence context under identical conditions. At pH 7.5, in the presence of 150 mM NaCl and 2 mM MgCl2, cleavage at an AP site in single-stranded DNA occurred with a t1/2 of 284 ( 23 h at 37 °C. In a duplex context, the half-life of the AP site ranged from 273 to 974 h, dependent on the identity of the nucleotide opposite the deoxyribose abasic site lesion: A, 273 ( 23 h; T, 426 ( 47 h; C, 974 ( 124 h; and G, 758 ( 57 h. These data are consistent with previously reported half-lives for DNA cleavage at AP sites under physiological conditions (23) and suggest that deoxyribonolactone lesions are ∼1255 times more labile than AP sites.

Conclusions and Biological Implications In summary, we have reported an improved synthetic strategy for the introduction of deoxyribonolactone lesions inside DNA oligonucleotides. The technique was employed for the independent synthesis of lactone-damaged DNA designed to characterize the products and rates of DNA strand cleavage of deoxyribonolactone lesions. A combination of MALDI-TOF MS, gel electrophoresis, and chemical fingerprinting experiments was used to unambiguously characterize the identities and the gel mobilities of the DNA strand scission products at oxidized abasic sites. The data were consistent with the mechanism outlined in Scheme 1. We also reported the first measurement of the half-life of this lesion in both singlestranded and duplex DNA under simulated physiological conditions. DNA cleavage occurred more rapidly in singlestranded DNA (t1/2 ∼ 20 h) as compared with doublestranded DNA (t1/2 ) 32-54 h). Finally, we showed that the deoxyribonolactone lesion is more labile to DNA strand scission than the structurally related aldehyde AP site. Several laboratories have made significant contributions to our understanding of the biochemical fate of the lesion. Recently, Kotera and co-workers (33) reported elegant NMR characterization of pathway intermediates (4, Scheme 1). Furthermore, the studies of Greenberg and colleagues provided early mass spectral data for the lactone lesion and demonstrated the intermediacy of enelactone (4) by covalent trapping with nucleophilic reagents (32). Our MALDI-TOF MS, gel electrophoresis, and chemical fingerprinting data are consistent with results from these laboratories and support the mechanism for DNA cleavage at deoxyribonolactone lesions.

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In addition to validating the cleavage products of oxidized abasic site decomposition, our studies have provided the first data on the rates of DNA strand cleavage at the deoxyribonolactone lesion in both singlestranded and duplex DNA under conditions similar to cellular environments. Our results differ from those reported by Kotera et al. in several important ways. The half-life for cleavage at the deoxyribonolactone lesion in single-stranded DNA obtained from our system was ∼20 h, as compared with the ∼100 h value obtained under similar but distinct buffer conditions (33). These differences highlight the dramatic effect that reaction conditions and buffers have on DNA cleavage rates at the lactone lesion. For example, the earlier assay conditions (33) included a range of pH (7.5-10), temperature (3770 °C), and salt concentration (0.1-0.6 mM of Na+) and focused on single-stranded forms of the lesion. These experiments were designed to define the landscape of deoxyribonolactone cleavage chemistry, not to address the stability of the lesion in a biological context. However, the conditions used in our investigations may offer a better mimic for cellular environments (37). Our unpublished observations further suggest that divalent ions may significantly alter the rates of the cleavage reaction. The sequence context and duplex form of deoxyribonolactone lesions also have a significant effect on DNA strand scission rates. In general, lactone lesion sites in duplex DNA undergo cleavage more slowly as compared with single-stranded damaged DNA. These observations suggest that future studies of DNA cleavage at the deoxyribonolactone lesion should focus on double-stranded DNA, because of its greater relevance to genomic DNA. Furthermore, the cleavage rates are dependent on the identity of the deoxynucleotide incorporated opposite the damage site in the duplex. Although the chemical basis for these observations is not understood, the differences may be attributed to enhanced religation rates within a DNA duplex or to sequence-dependent effects such as base stacking or nucleotide flipping. We also compared the rate of DNA strand scission at lactone sites to the aldehyde AP site and found that cleavage at the deoxyribonolactone lesion is ∼12-55 times faster than at AP sites under simulated physiological conditions, which is consistent with previous reports (33). The pronounced difference in cleavage rates between the two abasic sites, deoxyribonolactone and AP sites, may be explained by the differing reactivities of the cyclic forms of the sugars. Standard AP sites exist predominantly in the cyclic hemiacetal form, with the ring-opened aldehyde form representing

Half-Life and DNA Strand Scission Products of 2-Deoxyribonolactone

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