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J. Biol. Chem., Vol. 275, Issue 26, 19482-19489, June 30, 2000

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Butadiene-induced Intrastrand DNA Cross-links: A Possible Role in Deletion Mutagenesis^*

J. Russ Carmical^§¶, Agnieszka Kowalczyk, Yue Zou^§, Bennett Van Houten^§**, Lubomir V. Nechev, Constance M. Harris, Thomas M. Harris, and R. Stephen Lloyd^§

From the  Department of Preventive Medicine and Community Health, the ^§ Sealy Center for Molecular Science, The University of Texas Medical Branch, Galveston, Texas 77555, and the  Department of Chemistry and the Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, Tennessee 37235

Received for publication, March 10, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

To initiate studies designed to identify the mutagenic spectrum associated with butadiene diepoxide-induced N²-N² guanine intrastrand cross-links, site specifically adducted oligodeoxynucleotides were synthesized in which the adducted bases were centrally located within the context of the human ras 12 codon. The two stereospecifically modified DNAs and the corresponding unmodified DNA were ligated into a single-stranded M13mp7L2 vector and transfected into Escherichia coli. Both stereoisomeric forms (R,R and S,S) of the DNA cross-links resulted in very severely decreased plaque-forming ability, along with an increased mutagenic frequency for both single base substitutions and deletions compared with unadducted DNAs, with the S,S stereoisomer being the most mutagenic. Consistent with decreased plaque formation, in vitro replication of DNA templates containing the cross-links by the three major E. coli polymerases revealed replication blockage by both stereoisomeric forms of the cross-links. The same DNAs that were used for replication studies were also assembled into duplex DNAs and tested as substrates for the initiation of nucleotide excision repair by the E. coli UvrABC complex. UvrABC incised linear substrates containing these intrastrand cross-links with low efficiency, suggesting that these lesions may be inefficiently repaired by the nucleotide excision repair system.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Metabolic bioactivation of 1,3-butadiene results in a diepoxide. As a bifunctional electrophile, butadiene diepoxide is theoretically capable of producing inter- and intrastrand DNA-DNA cross-links. Cross-linked adducts are thought to be responsible for the observation that the diepoxide is considerably more mutagenic in mice than the monoepoxide under identical exposure conditions (1) and for the fact that butadiene is more genotoxic to mice than rats. The latter observation is attributed to the greater effectiveness of mice at metabolizing butadiene to the diepoxide (2). Both species appear to be equally susceptible to cytogenetic damage inflicted by butadiene diepoxide when the epoxide is introduced directly into isolated rat or mouse lymphocytes (splenic or peripheral blood) (3).

There are a number of studies supporting the existence of butadiene diepoxide-induced interstrand cross-links (4-9). Evidence for such cross-links is based largely on denaturation/renaturation experiments in which interstrand-cross-linked DNA renatures more rapidly than noncross-linked. The only cross-linked species thus far identified, a guanine N7-guanine N7 cross-link, was isolated from salmon sperm DNA by Lawley and Brookes (8). In 1993, Millard and White (10) reported that synthetic oligonucleotide duplexes of varying sequences reacted rather diffusely with butadiene diepoxide but showed preference for interstrand cross-linking at 5'-GNC sites. As expected for guanine N7 cross-links most of the bands that migrated in denaturing gels in the region expected for dimeric structures were cleavable by piperidine at 90 °C. However, some cross-linked material persisted after the alkaline treatment indicating that stable cross-links (of unknown structure) were also formed.

Interstrand cross-links are known to be highly cytotoxic, whereas intrastrand cross-links tend to be more mutagenic (11-13). Although certain types of intrastrand cross-links such as those arising from pyrimidine photodimerization and the chemotherapeutic agents, cisplatin and mitomycin, have been well studied, the question of intrastrand cross-link formation by butadiene diepoxide has not been examined; in theory the guanine N7-guanine N7 cross-link that has been isolated could arise from an intrastrand as well as an interstrand cross-link. We decided to focus on possible stable intrastrand cross-links, because little is known about the replication and repair of aliphatic intrastrand cross-links. Inasmuch as we had data on replication and mutagenicity of butadiene diolepoxide monoadducts on guanine N² for comparison (14), we chose guanine N²-guanine N² cross-links as our first target. Furthermore, we knew from other experiments¹ that a cross-link involving adjacent guanines connected N² to N² by an unsubstituted 4-carbon alkyl chain introduces very little distortion into the double helix. Such a lesion might escape detection by repair enzymes and lead to mutations if it were replicated. Hence 8-mer oligonucleotides were synthesized containing site-specific guanine N²-guanine N² cross-links of (R,R) and (S,S) butadiene diepoxide in the N-ras codon 12 (-GGT-) sequence. To better understand what roles such adducts might play in molecular mechanisms responsible for butadiene-induced carcinogenesis, replication efficiency and mutagenic spectra have been investigated, as well as the initiation of repair by the UvrABC exinuclease complex. These data suggest that intrastrand butadiene cross-links may contribute significantly to the mutagenic spectrum observed in butadiene-exposed animals.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Synthesis and Characterization of Cross-linked Oligonucleotides

Materials and Methods-- Oligonucleotides were prepared on an Expedite^TM 8909 Nucleic Acid Synthesizer using tert-butyl-phenoxyacetyl 2-cyanoethyl phosphoramidites and the modified phosphoramidite of 2-fluoro-O⁶-trimethylsilylethyl (TMSE)²-5'-O-dimethoxytrityl 2'-deoxyinosine on a 1 µmol scale. Modified oligonucleotides were deprotected and purified as described previously (15). HPLC purifications were done on a Beckman HPLC (System Gold software, model 125 pump, model 168 photodiode array detector). Oligonucleotides were desalted on Sephadex G-25 using a Bio-Rad FPLC system. Enzymatic digestion mixtures (0.2-0.5 A₂₆₀ units of oligonucleotide, 20 µl of buffer (0.01 M Tris-HCl and 0.01 M MgCl₂, pH 7), and 3.2 units of nuclease P1 (Sigma N-8630) were incubated at 37 °C for 4 h, and then 20 µl of 0.1 M Tris-HCl buffer, pH 9.0, 0.04 unit of snake venom phosphodiesterase (Sigma 5785), and 0.4 unit of alkaline phosphatase (Sigma P-4282) were added and incubation at 37 °C continued overnight) were analyzed by HPLC (4.6 × 250-mm YMC ODS-AQ column) with the following gradient: (A) 0.1 M ammonium formate and (B) CH₃CN, 1-10% B over 15 min, 10-20% B over 5 min, hold for 5 min, and then to 100% B over 10 min at a flow rate of 1.5 ml/min. ¹H NMR spectra were recorded at 300.13 and 400.13 MHz on Bruker AC300 and AM400 NMR spectrometers in MeOD-d₄ or Me₂SO-d₆. High resolution mass spectra were obtained in positive fast atom bombardment (FAB) mode on a VG 70-250 instrument or at the Mass Spectrometry Facility at the University of Notre Dame, Notre Dame, Indiana. Electrospray ionization mass spectroscopy was carried out on a Finnigan TSQ-7000 mass spectrometer. Melting profiles were recorded on a Varian Cary 04E UV spectrophotometer.

(2R,3R)-2,3-O-Benzylidenetartramide-- (-)-Dimethyl 2,3-O-benzylidene-L-tartrate (5 g) was dissolved in 150 ml of methanol. A slow stream of ammonia (gas) was bubbled through this solution for 3-4 h. TLC (ethyl acetate:hexane, 2:1) showed complete disappearance of the starting material. The solvents were evaporated to give a clear oil, which crystallized under vacuum. The product (4.3 g, 97%) was used in the next step without purification. ¹H NMR (methanol-d₄)  (ppm) 4.77 (m, 2H, 2xCH), 6.05 (s, 1H, CH-benzyl), 7.41 (m, 3H, m-, p-aromatic), 7.57 (m, 2H, o- aromatic).). HRMS (FAB⁺) m/z calculated for [M+H]⁺ 237.0875 found 237.0882. [ ]_D²⁰ was + 3.9° (c = 2, ethanol).

(2S,3S)-2,3-O-Benzylidenetartramide-- Starting from (+)-dimethyl 2,3-O-benzylidene-D-tartrate (3 g) and following the procedure described above afforded 2.55 g (96%) of the (2S,3S)-isomer. ¹H NMR (methanol-d₄)  (ppm): 4.77 (m, 2H, 2xCH), 6.05 (s, 1H, CH-benzyl), 7.41 (m, 3H, m-, p-aromatic), 7.57 (m, 2H, o- aromatic). HRMS (FAB⁺) m/z calculated for [M+H]⁺ 237.0875 found 237.0879. []_D²⁰ was  3.1° (c = 2, ethanol).

(2S,3S)-2,3-O-Benzylidene-1,4-Diamino-2,3-Butanediol-- The (2R, 3R) 2,3-O-benzylidenetartramide from the previous step was dissolved in 15 ml of anhydrous tetrahydrofuran and added dropwise (10 min) to a stirring mixture of LiAlH₄ (2 g) and anhydrous tetrahydrofuran (150 ml). The reaction was carried out under argon. After stirring for 1 h at room temperature and under reflux for 5 h, complete disappearance of the starting material was observed. The reaction was followed by TLC (CH₃CN:H₂O:NH₄OH, 85:8:7). Each aliquot was treated with water before loading on a TLC plate. The reaction was cooled (ice/water bath), and water was carefully added until the color of the precipitate became white. The mixture was filtered, diluted with ether, and dried over sodium sulfate. Solvents were evaporated, and the crude yellowish oil was purified (silica gel column; CH₃CN:H₂O:NH₄OH, 85:8:7) to give 1.68 g (95%) of pure benzylidene-protected diamine. ¹H NMR (methanol-d₄),  (ppm) 2.88 (m, 4H, 2x CH₂), 3.92 (m, 2H, 2x CH), 5.93 (s, 1H, CH-benzyl), 7.37 (m, 3H, m-, p-aromatic), 7.48 (m, 2H, o-aromatic). HRMS (FAB⁺) m/z calculated for [M+H]⁺ 209.1290 found 209.1291. []_D²⁰ was 14.9° (c = 2, ethanol).

(2R,3R)-2,3-O-Benzylidene-1,4-Diamino-2,3-Butanediol-- The above procedure with the (2S,3S)-2,3-O-benzylidenetartramide (2.5 g) as a starting material led to 2.0 g (91%) of the corresponding (2R,3R)-isomer. ¹H NMR (methanol-d₄),  (ppm): 2.87 (m, 4H, 2x CH₂), 3.92 (m, 2H, 2x CH), 5.93 (s, 1H, CH-benzyl), 7.37 (m, 3H, m-, p-aromatic), 7.49 (m, 2H, o-aromatic). HRMS (FAB⁺) m/z calculated for [M+H]⁺ 209.1290 found 209.1312. []_D²⁰ was + 16.1° (c = 2, ethanol).

(2S,3S)-1,4-Diamino-2,3-Butanediol-- (2S,3S)-2,3-O-Benzylidene-1,4-diamino-2,3-butanediol from the previous step (1 g) was neutralized with 1 N HCl to pH 6.0. The solution was evaporated to dryness (rotary evaporator, 40 °C). Sulfuric acid (0.01 N, 15 ml) was added, and the mixture was stirred at 100 °C for 3 h. The solution was evaporated to dryness. Water was then added and evaporated three times (to remove benzaldehyde). The product was dissolved in a small amount of water, and the pH was adjusted (1 N NaOH) to 12.0. The solution was evaporated to dryness. The mixture was dissolved in a small amount of boiling 80% ethanol. The insoluble inorganic salts were filtered out, and the filtrate was kept in a freezer for 1 h. The additional amounts of inorganic salts, which crystallized, were filtered out again, and the solvents were evaporated to give 0.46 g (80%) of the product, a clear oil, which crystallized upon standing. ¹H NMR (methanol-d₄),  (ppm): 2.70 (m, 4H, 2xCH₂), 3.47 (m, 2H, 2xCH). HRMS (FAB⁺) m/z calculated for [M+H]⁺ 121.0977 found 121.0992. []_D²⁰ was 15.4° (c = 2, ethanol).

(2R,3R)-1,4-Diamino-2,3-Butanediol-- The above procedure using (2R,3R)-2,3-O-benzylidene-1,4-diamino-2,3-butanediol (1.8 g) as a starting material led to 0.85 g (82%) of (2R,3R)-1,4-diamino-2,3-butanediol. ¹H NMR (methanol-d₄),  (ppm) 2.71 (m, 4H, 2xCH₂), 3.47 (m, 2H, 2xCH). HRMS (FAB⁺) m/z calculated for [M+H]⁺ 121.0977 found 121.0987. []_D²⁰ was + 15.9° (c = 2, ethanol). A sample of this isomer was converted to its HBr salt. []_D²⁰ +20.1° (c = 2, water) (literature value: []_D²⁰ +20.3° (c = 2, water) (16)).

Synthesis and Purification of Intrastrand Cross-links (3a and 3b)-- The general procedure was as follows. The starting material 5'-d(CATXXTCC)-3' (1) (X = 2-fluoro-O⁶-TMSE 2'-deoxyinosine) was reacted with the appropriate diaminediol in Me₂SO in the presence of diisopropylethylamine (DIEA) for 2-3 days at 55 °C. The reactions were monitored by HPLC on a C18 column (4.5 × 250 mm, YMC ODS-AQ) with the following gradient: (A) 0.1 M ammonium formate and (B) CH₃CN, 1-35% B over 20 min, 35-90% B over 3 min, hold for 2 min, and then to 1% B over 2 min at a flow rate of 1.5 ml/min. Starting material eluted at 21-22 min, and cross-linked products 2a and 2b eluted at ~18-19 min (fully TMSE-protected), partially TMSE-protected products at ~12-13 min, and final products 3a and 3b at ~9-10 min (fully deprotected).

(R,R)-Butadiene Cross-link (3a)-- For synthesis of this cross-link, 18 A₂₆₀ units of (1), 1 drop of DIEA, and 67 µl (1.5 equivalents) of a solution of (2R,3R)-1,4-diaminobutanediol (0.65 µg/µl) in Me₂SO were used. After 3 days the starting material was consumed, and Me₂SO was removed in vacuo. The crude reaction mixture was purified by HPLC on a C18 column (250 × 10 mm, YMC ODS-AQ) with the following gradient: (A) 0.1 M ammonium formate and (B) CH₃CN, 1-15% B over 21 min, 15-35% B over 12 min, 35-90%B over 8 min, hold for 2 min, and then to 1% B over 2 min at a flow rate of 3 ml/min. Three cross-linked species were collected: fully TMSE-protected cross-link 2a (isolated 4.2 A₂₆₀ units) eluted at ~34 min, partially TMSE-protected (isolated 1.8 A₂₆₀ units) at ~28 min, and fully deprotected 3a (isolated 1.2 A₂₆₀ units) at ~19 min. Partially and fully TMSE-protected cross-linked products were combined and after lyophilization the TMSE-protecting groups were removed by treatment with aqueous acetic acid (pH 3) for 1 h at room temperature; the product coeluted with fully deprotected cross-link 3a. The reaction mixture was then neutralized and combined with 3a isolated previously.

(S,S)-Butadiene Cross-link (3b)-- For synthesis of this cross-link, 13 A₂₆₀ units of (1), 1 drop of DIEA and 80 µl (2.5 equivalents) of a solution of (2S,3S)-1,4-diaminobutanediol (0.65 µg/µl) in Me₂SO were used. The diaminediol solution was added in two increments over 24 h. After 2 days the starting material was consumed, and Me₂SO was removed in vacuo. The crude reaction mixture was purified by HPLC as described above for 3a. Three cross-linked species were collected: fully TMSE-protected cross-link 2b (isolated 3.6 A₂₆₀ units) eluted at ~34 min, partially TMSE-protected (isolated 1.2 A₂₆₀ units) at ~28 min, and fully deprotected 3b (isolated 0.6 A₂₆₀ units) at ~19 min. Partially and fully TMSE-protected cross-linked products were combined, and after lyophilization the TMSE-protecting groups were removed by treatment with aqueous acetic acid (pH 3) for 2 h at room temperature; the product coeluted with fully deprotected cross-link 3b. The reaction mixture was then neutralized, combined with 3b, lyophilized, and repurified by HPLC on a C18 column (250 × 10 mm, YMC ODS-AQ) with the following gradient: (A) 0.1 M ammonium formate and (B) CH₃CN, 3-13% B over 25 min, 13-90% B over 2 min, hold for 2 min, and then to 3% B over 2 min at a flow rate of 3 ml/min; the product (3b) eluted at ~22 min.

The HPLC-purified cross-linked oligonucleotides 3a and 3b were desalted (Sephadex G-25) and analyzed by mass spectroscopy: electrospray ionization-mass spectroscopy: (3a) calculated M_r of 2471.7; measured mass based on [M+Na-2H]/2z 1246.0, [M+Na-3H]/3z 830.6, [M-3H]/3z 822.8 = 2471.4; (3b) calculated M_r of 2471.7; measured mass based on [M+Na-2H]/2z 1245.7; [M-3H]/3z 822.8 = 2470.9. Capillary gel electrophoresis and enzymatic digestion to the constituent nucleosides were also used to confirm the purity and composition of 3a and 3b. Bisnucleoside standards for use in HPLC analysis of enzymatic digests were synthesized as described in the following section.

Synthesis of Bisnucleoside Standards-- A solution of the appropriate diamine, 2-fluoro-O⁶-TMSE 2'-deoxyinosine, and DIEA (molar ratio in order mentioned 1:3:2.5) in Me₂SO was stirred at 55 °C for 30 h. The resulting solution was acidified with aqueous acetic acid to pH 4 to remove the TMSE groups and stirred at room temperature for 8 h. The reaction mixture was neutralized, lyophilized, and purified by reverse-phase HPLC on a C8(2) column (250 × 10 mm, Phenomenex) with the following gradient: (A) H₂O and (B) CH₃CN, 5-8% B over 5 min, 8-11% B over 11 min, 11-90% B over 2 min, hold for 3 min, and then 90-5% B over 2 min at a flow rate of 3 ml/min, both products eluted at 11.5-12.5 min.

1,4-Bis(2'-Deoxyguanosin-N²-yl)-2R,3R-Butanediol-- 2.7 mg (0.022 mmol) of (2R,3R)-1,4-diaminobutanediol, 25 mg (0.067 mmol) of 2-fluoro-O⁶-TMSE 2'-deoxyinosine, and 7.1 mg (0.055 mmol) of DIEA in 50 µl of Me₂SO afforded after purification 9.5 mg (68% yield) of the cross-linked 2R,3R nucleoside. ¹H NMR (Me₂SO-d₆, 300 MHz)  (ppm) 10.58 (br, 2H, NH-ring), 7.90 (s, 2H, H8), 6.46 (br, 2H, NH-exocyclic), 6.15 (m, 2H, H1'), 5.24 (d, 2H, 3'-OH, J = 3.9 Hz), 5.04 (d, 2H, CH-OH, J = 5.4 Hz), 4.85 (t, 2H, 5'-OH, J = 5.7 Hz), 4.33 (br, 2H, H3'), 3.79 (m, 2H, H4'), 3.63 (br, 2H, CH-OH), 3.54 (m, 4H, H5', H5", 2H CH₂-N), 3.27 (hidden under H₂O peak in Me₂SO, visible in HH-COSY), 2.54 (m, 2H, H2", partially hidden under Me₂SO peak, confirmed with HH-COSY), 2.18 (m, 2H, H2'). HRMS (FAB⁺) calculated for C₂₄H₃₃N₁₀O₁₀ (M+H)⁺ 621.2384 found 621.2381.

1,4-Bis(2'-Deoxyguanosin-N²-yl)-2S,3S-Butanediol-- 2.7 mg (0.022 mmol) of (2S, 3S)-1,4-diaminobutanediol, 25 mg (0.067 mmol) of 2-fluoro-O⁶-TMSE 2'-deoxyinosine, and 7.1 mg (0.055 mmol) of DIEA in 50 µl Me₂SO afforded after purification 9.0 mg (65% yield) of the 2S,3S nucleoside. ¹H NMR (Me₂SO-d₆, 400 MHz)  (ppm) 10.54 (br, 2H, NH-ring), 7.90 (s, 2H, H8), 6.47 (br, 2H, NH-exocyclic), 6.14 (m, 2H, H1'), 5.26 (d, 2H, 3'-OH, J = 4.0 Hz), 5.05 (br, 2H, CH-OH), 4.87 (t, 2H, 5'-OH, J = 5.6 Hz), 4.33 (br, 2H, H3'), 3.79 (m, 2H, H4'), 3.62 (br, 2H, CH-OH), 3.49 (m, 4H, H5', H5", 2H CH₂-N), 3.29 (hidden under H₂O peak in Me₂SO, visible in HH-COSY), 2.56 (m, 2H, H2"), 2.19 (m, 2H, H2'). HRMS (FAB⁺) calculated for C₂₄H₃₃N₁₀O₁₀ (M+H)⁺ 621.2384 found 621.2377.

Melting Studies-- Adducted oligonucleotides 3a and 3b and the complementary strand (0.5 A₂₆₀ units each) were dissolved in 1 ml of melting buffer (10 mM Na₂HPO₄/NaH₂PO₄, 1.0 M NaCl, 50 mM Na₂EDTA, pH 7.0). The sample vials were heated to 100 °C, maintained at that temperature for 3 min, and allowed to cool to room temperature. UV measurements were taken at 1-min intervals with a 1 °C/min temperature gradient with observation at 260 nm. The temperature was raised from 5 °C to 85 °C. The (R, R)-butadiene cross-link had T_m = 44 °C, (S,S)-butadiene cross-link had T_m = 30 °C, wild type duplex had T_m = 40 °C.

Construction of 1,3-Butadiene-modified Circular Template

Single-stranded M13mp7L2 vector was isolated according to the procedures described by Sambrook et al. (17). Subsequently, site specifically modified or the corresponding unadducted oligodeoxynucleo-tides were ligated into the cloning site of the M13 vectors under the conditions previously reported by our group (14, 18-21). To visualize the efficiency of this reaction, 10 µl of the modified M13mp7L2 were separated on a 1.4% agarose gel via electrophoresis and monitored by ethidium bromide staining. The efficiency of these reactions was then quantitated using an Appligene Bioimager.

In Vivo Replication of 1,3-Butadiene-modified Circular DNA

Modified M13mp7L2 was used to transfect repair-deficient AB2480 (uvrA, recA) Escherichia coli cells via electroporation as previously reported (14, 21). Subsequently, the electroporation mixture was plated on prewarmed LB broth agarose plates in the presence of 500 µl of AB2480 E. coli and 5 ml of top agar (LB + 0.7% agarose). Each plate was inverted and incubated overnight at 37 °C. The resulting plaques were then transferred to nitrocellulose filters in four successive lifts for each plate. These filters were then processed as described previously (14, 21). The plaques were subsequently screened for possible base substitutions at position 2 of the ras 12 codon via differential hybridization techniques (18-21). Four oligodeoxynucleotide probes (17-mers) were synthesized to be directly complementary both 5' and 3' to the DNA surrounding and including the 8-nucleotide insert. The 17-mers were varied in the sequence identity of the nucleotide opposite the 3'-guanine of the cross-link, where each of the four possible nucleotides were incorporated. These 17-mer probes were radioactively labeled by incubating 1 µg of DNA with 0.30 mCi of [-³²P]ATP and 20 units of T4 polynucleotide kinase according to the supplier's protocol. One of each of the four nitrocellulose filters were labeled accordingly and subsequently hybridized with the radioactively labeled probes overnight. The hybridization conditions were such that only the perfectly hybridized complement would anneal. Radiolabeled probes were washed as described previously, and filters were exposed to autoradiographic film overnight.

Construction of 1,3-Butadiene-modified Linear Template

The adducted 8-mer oligodeoxynucleotides utilized in the construction of the modified M13 vector were also used to construct a 50-mer linear template. The template was constructed such that the adducted guanines were approximately centrally located, therefore providing a sufficient template for primer extensions. To construct the template, the unadducted or adducted 8-mers, a 22-mer 5'-flanking DNA, and a 20-mer 3'-flanking DNA were annealed to a 45-mer scaffold that was complementary to the 8-mer internally and to the respective ends of the flanking DNAs; this DNA served as a bridge to facilitate ligation of the individual oligodeoxynucleotides. Prior to oligonucleotide annealing, the 8-mers and 20-mer flanking sequences were phosphorylated at the 5'-end. To visualize the 50-mer ligation product and to aid in the purification, the 22-mer flank was phosphorylated with a 1:10 mixture of [-³²P]ATP/ATP. Each component was then added in approximately equal molar concentrations and heated to 70 °C for 5 min. The mixture was cooled to room temperature and then incubated in ice slurry for 15 min. T4 DNA ligase (2000 units) was added, and the reaction was allowed to proceed overnight at 16 °C. The 50-mer ligation product was then gel-purified to remove the 45-mer scaffold, as described previously.

In Vitro Replication of 1,3-Butadiene-modified Linear Template

Oligodeoxynucleotides were synthesized to serve as primers for replication of the 50-mer template. Three primers were designed such that they would anneal to specific sites on the templates, thus providing a 3'-hydroxyl at various distances relative to the adduct. In effect, the primers would simulate scenarios that a polymerase might encounter in vivo. The first positioned the 3'-hydroxyl one base prior to the adduct, which would simulate a "standing" start. The second positioned the 3'-hydroxyl four bases prior to the adduct, which would simulate a "running" start. Finally, the third primer placed the 3'-hydroxyl five bases beyond the adduct to determine any downstream effects. Each primer was phosphorylated by T4 polynucleotide kinase to affix a 5'--³²P label. Subsequently, each was diluted to a concentration of 50 fmol/µl and added to the 50-mer template in a ratio of 1:3 in the presence of the appropriate reaction salts. To promote proper annealing, the mixture was heated to 90 °C for 2 min and slowly cooled to room temperature. This reaction was carried out in triplicate for each template/primer combination. The polymerases assayed and suppliers were as follows: large fragment of polymerase I (Klenow exo) was purchased from New England Biolabs, Beverly, MA; polymerase II was provided by Drs. M. F. Goodman and L. Bloom, University of Southern California, Los Angeles, CA; and polymerase III was supplied by Dr. Mike O'Donnell, Rockefeller University, New York, NY. Finally, the appropriate salts, 1 µM dNTPs, and the buffer specific for the polymerase being assayed were added to the template/primer complex in a total reaction volume of 9 µl. Individually, the polymerases were added at 2-fold molar excess of enzyme to DNA and allowed to proceed at room temperature for 10 min. The reaction was stopped by adding an equal volume of loading buffer, consisting of formamide, xylene cyanol, and bromphenol blue. The extension products were then analyzed by electrophoresis through a 15% polyacrylamide sequencing gel and visualized by exposing an autoradiographic film overnight.

Construction of 1,3-Butadiene-modified Duplex Template for UvrABC Assays

The 50-mer templates containing the butadiene cross-linked DNAs were constructed as above except the 5'-end was not labeled until immediately prior to use in incision and binding assays. A complementary 50-mer oligodeoxynucleotide was synthesized and subsequently gel-purified as described previously and used as the substrate for UvrABC binding and incision assays. Prior to annealing, each template was phosphorylated by T4 polynucleotide kinase incorporating a -³²P label on the 5'-end. The labeled 50-mer templates were then annealed to the complement in individual reactions. A reaction mixture containing a 5-fold molar excess of complement (500 nmol:2500 nmol) and 10 mM Tris-HCl, pH 7.5, 0.1 µM EDTA was heated to 85 °C and allowed to slowly cool to room temperature. The duplex formation was then gel-purified on a 10% native polyacrylamide gel.

Nucleotide Incision of 1,3-Butadiene-modified Linear Template

The aforementioned, double-stranded 50-mer substrates (5 nM) were incubated with E. coli UvrABC proteins (10 nM UvrA, 250 nM UvrB, and 50 nM UvrC) at 37 °C for 30 min in the presence of UvrABC reaction buffer (50 mM Tris-HCl, pH 7.8, 50 mM KCl, 10 mM MgCl₂, 5 mM dithiothreitol, and 1 mM ATP). Prior to the addition of the DNA substrate, the Uvr subunits were diluted with storage buffer. Finally, the reactions were terminated by adding EDTA (20 mM) or heating to 90 °C for 3 min. The samples were denatured with formamide and heated to 90 °C for 5 min and then quick-chilled on ice. The digested products were analyzed by electrophoresis through a 12% polyacrylamide sequencing gel under denaturing conditions with UvrABC buffer. The gel was dried and exposed to an x-ray film and a PhosphorImaging screen (Molecular Dynamics) for quantification.

Gel Mobility Shift Assays

Binding of the DNA substrates by the E. coli UvrA protein was determined by gel mobility shift assays. Typically, the substrate (4 nM) was incubated with the UvrA with the indicated concentrations in 20 µl of UvrABC buffer without ATP at 37 °C for 15 min. After the incubation, 2 µl of 80% (v/v) glycerol was added, and the mixture was immediately loaded onto a 3.5% native polyacrylamide gel in TBE running buffer and separated by electrophoresis at room temperature.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Synthesis and Characterization of Butadiene Diepoxide Cross-linked Oligonucleotides 3a and 3b-- The oligonucleotides utilized in these studies were synthesized by a novel application of the postoligomerization strategy developed by Harris et al. (15, 22, 23) (Fig. 1). For the intrastrand cross-link, two adjacent halopurines were introduced into the 8-mer oligonucleotide using the phosphoramidite of 2-F-O⁶-trimethylsilylethyl 2'-deoxyinosine. The halopurine-containing oligonucleotide was removed from the matrix, deprotected, and purified before reaction with the chiral diaminobutanediol. The stoichiometry of the reaction was carefully controlled to avoid the risk of introduction of two diaminodiol residues. Oligonucleotides 3a and 3b were purified by HPLC and characterized by electrospray mass spectrometry, which showed them to be of the expected mass. Enzymatic digestion showed the constituent nucleosides in the expected ratios (3dC, 2T, 1dA, and 1 bisnucleoside). To rigorously minimize the possibility of contaminants that could alter the mutagenic spectrum, the ras 12² 8-mers were extensively purified and analyzed. Each 8-mer was labeled by T4 DNA kinase, using [-³²P]ATP, and the final purity was determined by separating the labeled 8-mers on a denaturing polyacrylamide gel and examining for the presence of any contaminating bands. The DNA adducts greatly perturb base stacking and resulted in a retarded mobility with respect to the unmodified 8-mer (Fig. 2). In an attempt to visualize even the minutest contaminant, the autoradiographic film was overexposed. However, no contaminants were detected. These data reinforce that a single species was present in each of the adducted samples and that the correct chemical composition was obtained. Ultimately, capillary gel electrophoresis analysis of the synthesis confirmed the purity of each sample.

View larger version (15K): [in this window] [in a new window]   Fig. 1.   Structures of 1,3-butadiene-induced N2-N2 guanine stereoisomers.

View larger version (48K): [in this window] [in a new window]   Fig. 2.   End-labeled 8-mer oligodeoxynucleotides (nonadducted and adducted). The purity of the adducted oligodeoxynucleotides was determined by end-labeling and polyacrylamide gel electrophoresis. 1, nonadducted; 2, R,R N2-N2 guanine; 3, S,S N2-N2 guanine.

In Vivo Replication of 1,3-Butadiene-modified Circular DNA-- The in vivo replication fate for each of the R,R and S,S stereo-specific butadiene-induced guanine cross-link-containing DNAs (Fig. 1), as well as for unmodified counterpart, was determined by inserting the individual oligodeoxynucleotides into the EcoRI site of a single-stranded M13 vector (see "Experimental Procedures"). Each 8-mer was ligated into the EcoRI cloning site of M13mp7L2 and quantitated. The modified M13 vectors were replicated in E. coli and evaluated for the inhibition of phage replication and mutagenesis (Tables I and II, respectively). Using the M13mp7L2 single-stranded DNA system, the E. coli replicative polymerases are forced to synthesize past the cross-linked site to generate an intact minus strand from which to catalyze rolling circle replication. Thus, the efficiency of plaque formation is indicative of the efficiency of lesion bypass. The data shown in Table I reveal that both R,R and S,S N²-guanine cross-links were extremely inhibitory to replication bypass, with plaque formation decreased by more than four orders of magnitude in both cases. These data are suggestive that in the absence of an efficient DNA repair mechanism, these butadiene lesions could produce severe or permanent blocks to replicative synthesis.

Even though replication efficiencies were severely compromised, the plaques that were formed were subsequently analyzed for point mutations and deletions (Table II and III). These experiments were carried out as three separate and independent processes from construction of the adducted M13mp7L2 vector through the screening of plaques for mutations. The data represented in Tables I-III are the culmination of these experiments. As expected, screening of the unmodified DNAs resulted in no mutations of any type. In contrast, analyses of the plaques that were formed using the modified DNAs revealed a multitude of mutations (Table II). Each putative mutant was screened a second time to confirm it as a positive, and subsequently, the sequences of a subset of plaques were determined. Representatives of each type of base substitution are depicted in Fig. 3. On average, the mutation frequencies corresponding to the cross-links exceeded those of the monobase guanine adducts by an order of magnitude (14, 21). However, it should be emphasized that the plaque-forming ability was down by more than four orders of magnitude relative to the unadducted DNAs. The mutation frequencies were calculated and determined to be statistically significant by calculating the binomial confidence intervals to a 0.995 degree of certainty. The confidence intervals for each stereo-specific adduct were then plotted against those for the unmodified DNAs (Fig. 4).

View this table: [in this window] [in a new window]   Table II In vivo replication of ras 122-N2-N2guanine1,3-butadiene-modified M13mp7L2 in AB2480 (uvrA, recA) E. coli

View larger version (41K): [in this window] [in a new window]   Fig. 3.   Ras 122 base substitutions as determined by differential hybridization. Top panel, G  A transition; middle panel, G  T transversion; bottom panel, G  C transversion.

View larger version (18K): [in this window] [in a new window]   Fig. 4.   Confidence intervals of mutagenic spectrum. The confidence intervals for the mutagenic spectrum of each adduct was calculated via binomial analysis to a 0.995 degree of certainty.

In addition to those plaques that hybridized to one of the specific mutant probes (Table II), a portion of resulting plaques either did not hybridize or only lightly hybridized (Table III) to any of the four probes, an observation that could be indicative of deletions. To address this issue, a subset of both "nonhybridizing" and "lightly hybridizing" plaques was selected and sequenced in the appropriate portion of the M13L2 genome to screen for possible deletions. DNA sequence analysis of those plaques that did not hybridize to any of the mutant probes revealed that these were M13mp7L2 molecules that had not been linearized at the EcoRI site. Although the EcoRI-restricted M13L2 DNAs were analyzed by electrophoresis through an agarose gel and visualized by ethidium bromide staining, an undetectable amount of circular M13 may have been present in the input DNA. These plaques were not included in the final analysis. Conversely, the plaques that only lightly hybridized to the four probes were sequenced and revealed various mutations at other sites within the probed region (Table III). Interestingly, the majority of those sequenced (R,R cross-link, 83% and S,S cross-link, 92%) contained a G  C transversion 8 base pairs downstream from the cross-link. The remainder of the plaques contained a variety of deletions, insertions, and base substitutions (Table III). These deletions accounted for about the same frequency of changes as did the point mutations.

In Vitro Replication of 1,3-Butadiene-modified Linear Template-- The severity of the replication blockage in vivo suggested that both the R,R and S,S cross-linked DNAs would pose severe blocks to in vitro replication bypass using purified E. coli polymerases I, II, and III. DNA templates (50-mers) containing the lesions or an unadducted site were constructed (Fig. 5) and primed for synthesis using 1, 4, and +5 primer DNAs (Fig. 6). As expected, the 1, 4, and +5 primers were successfully extended by all three E. coli polymerases to full-length product when utilizing the unadducted 50-mer template. In the case of the +5 primer, where the 3'-hydroxyl is beyond the site of the adduct, all three polymerases were able to extend the primers to full-length products (Fig. 6). These data confirmed the integrity of each of the DNA templates. In contrast, when the templates containing the guanine-guanine cross-links were utilized and primed by the 1 primer, no incorporation could be detected. However, at millimolar concentrations of dNTPs, the Klenow fragment of polymerase I was able to incorporate a guanine opposite the first adducted guanine using the 1 primer (data not shown). Extension assays using the 4 primer showed blockage of all three polymerases one nucleotide prior to the first guanine of the cross-link; however, upon extended incubation with Klenow, it was able to incorporate a base opposite the first guanine of the cross-link (data not shown).

View larger version (22K): [in this window] [in a new window]   Fig. 5.   Construction of 1,3-butadiene cross-link-adducted 50-mer template.

View larger version (44K): [in this window] [in a new window]   Fig. 6.   Primer extensions of the butadiene-cross-link adducted templates. Each of the templates (unadducted, R,R N2-N2 guanine, and S,S N2-N2 guanine) was annealed to 1 of 3 primers (1 primer, 4 primer, and +5 primer). Each possible template-primer combination was extended with each of three polymerases (E. coli Pol I, II, and III), and the products were separated by electrophoresis through a polyacrylamide gel. 1 primer (lanes 1-4) lane 1, primer (P) alone; lane 2, nonadducted (U); lane 3, R,R N2-N2 guanine (GGR); lane 4, S,S N2-N2 guanine (GGS); 5 primer (lanes 5-8) lane 5, primer alone; lane 6, nonadducted; lane 7, R,R N2-N2 guanine; lane 8, S,S N2-N2 guanine; +5 primer (lanes 9-12) lane 9, primer alone; lane 10, nonadducted; lane 11, R,R N2-N2 guanine; lane 12, S,S N2-N2 guanine.

UvrABC Incision of 1,3-Butadiene-modified Linear Templates-- Nucleotide excision repair is one of the major cellular defense systems that removes a large variety of structurally diverse DNA lesions. It is generally believed that DNA repair is an important modulating factor in mutagenesis, because a rapid removal of DNA lesions in cells could lead to the reduction of their mutagenic potentials. Therefore, the incision of the cross-linked DNAs was examined by the E. coli UvrABC nuclease (Fig. 7A). To serve as a positive control, a 50-mer DNA containing a (+)-cis-BPDE DNA adduct was tested.

View larger version (29K): [in this window] [in a new window]   Fig. 7.   Incision and binding of butadiene cross-link and BPDE-DNA adducts by Uvr proteins. A, the 5'-labeled 50-mer DNA substrates were incised by UvrABC (10 nM UvrA, 250 nM UvrB, and 50 nM UvrC) in UvrABC buffer at 37 °C for 20 min and analyzed on a 12% polyacrylamide sequencing gel. The ND, R, S, and BPDE represent unadducted, butadiene R,R and S,S guanine-guanine cross-linked, and (+)-cis-BPDE-DNA adducts. Quantification of the incision products gives the order of incision efficiencies: R,R (0.024) < S,S (0.044) BPDE (0.31 pmol DNA incised/min). B, binding of the UvrA protein to the DNA substrates was conducted by incubating the substrates with the indicated concentrations of protein in the UvrABC buffer in the absence of ATP at 37 °C for 15 min and then subjected to analysis on a 3.5% native polyacrylamide gel. The UvrA-DNA complex formed as indicated. At 50 nM of UvrA concentration, the binding follows the order: R,R (1.0) < S,S (1.4) BPDE (14.2% DNA bound).

As can be observed in Fig. 7A, the UvrABC incised the R,R and S,S guanine-guanine cross-linked DNAs with a very poor efficiency relative to a linear oligonucleotide containing a site-specific (+) cis-BPDE lesions. Comparison of the extent of cleavage after the 15-min reaction revealed relative cleavage rates of 0.024, 0.044, and 0.31 pmols of DNA incised/min for the R,R and S,S guanine-guanine cross-links and BPDE-containing substrates, respectively. No specific incision was detected for the control-unadducted DNA template.

To investigate whether this relatively inefficient cleavage reaction of the cross-linked DNAs could be attributed to reduced specific DNA binding by UvrA, gel shift assays were performed using increasing concentrations of UvrA. As revealed in Fig. 7B, UvrA displayed a significantly reduced ability to bind to either of the cross-linked DNAs relative to the BPDE-adducted DNA. Comparisons at a 50 nM concentration of UvrA revealed that 1.0, 1.4, and 14.2% of the DNA was bound to the R,R, S,S, and BPDE lesions, respectively.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Previously, we reported the mutagenic frequencies of butadiene-induced N²-guanine and N⁶-adenine adducts using the same system described herein (14, 21). Whereas the mono-adducted guanine lesions were much more blocking to replication and an order of magnitude more mutagenic, both adducts resulted in a <1% mutagenic frequency with no evidence for deletions. However, this degree of mutagenic potency does not account for the mutagenic frequencies observed in rodent studies (24-29).

Like the guanine monobase adducts, the N²-N² guanine cross-links served as a major block to DNA replication (Fig. 6). Each of the three E. coli polymerases was blocked one base prior to the first adducted guanine. These data are consistent with the in vivo replication studies, which showed a very large decrease in plaque-forming ability (Table I). The severity of replication blockage in vitro corresponds well with that observed in vivo. The fact that some replication bypass can occur in vivo but could not be visualized in vitro may suggest that the intracellular environment possesses other accessory factors lacking in the in vitro reaction, which may aid in replication bypass. Bypass of these lesions may also result in a more error prone replication. Alternatively, the in vitro assays may simply not be sufficiently robust to detect this minor bypass mechanism.

Of the few DNA molecules that could be replicated, the screening of these plaques revealed a mutagenic frequency that was on average an order of magnitude higher than that produced by the guanine monobase adducts. Consistent with the monobase adducts, the S,S stereoisomer was more mutagenic than the R,R stereoisomer. Interestingly, the melting temperatures determined for the adducted 8-mers were somewhat lower for the S stereoisomers compared with the corresponding R stereoisomers for both the monobase adducts and cross-links. These data are indicative of the S stereoisomer being more disruptive to the DNA helix compared with the R stereoisomers and may result in a lesion more prone to mutagenic bypass. The mutagenic spectrum for both stereoisomeric forms of the cross-link consists of an array of base substitutions at position 2 of ras codon 12. Each of the possible base substitutions was well represented, and these data are generally consistent with the mono- and diolepoxide N²-guanine adducts. However, the severity of the replication blockage is likely to create in vivo scenarios in which direct bypass cannot be achieved, but rather deletions are created. Previous studies in human lymphocytes and Drosophila indicate that butadiene-induced deletions may be responsible for the carcinogenesis associated with butadiene exposure (30-32). Thus, these data may suggest that the intrastrand G-G cross-links could be a primary butadiene lesion that results in genomic deletions.

In cells that are exposed to activated forms of butadiene, it might be predicted that intrastrand cross-links would be repaired via a mechanism that repairs other intrastrand cross-links such as cis,syn-cyclobutane dimers, 6-4 photoproducts, Dewar photoproducts, and bifunctional psoralen cross-linking agents; that system is the nucleotide excision repair pathway. To test whether the cross-linked lesions were subject to this type of repair, these lesions were assayed for both binding and incision by E. coli UvrABC. The R,R and S,S guanine-guanine intrastrand cross-linked DNAs were inefficiently recognized and incised by UvrABC (Fig. 7). These data may imply that intrastrand DNA cross-links arising from butadiene exposure may elude or be poorly detected by the nucleotide excision pathway, thus allowing them to persist in the cell. This may then lead to increased problems for the cell, because neither of the lesions could be bypassed, as measured in vitro. In addition, the in vivo assays revealed a bypass efficiency of less than one in ten thousand. Thus, an increased cellular half-life may ultimately result in an increased mutagenic potential, both for point mutations and deletions, for these lesions.

	ACKNOWLEDGEMENTS

We acknowledge the work of the staff in the NIEHS Center Molecular Biology Core, The University of Texas Medical Branch, for the synthesis of nonadducted templates and DNA sequence analyses. We also thank Lisa Pipper Stephenson and Rosemary Martinez for preparation of the manuscript and figures. In addition, we thank Drs. M. F. Goodman and L. Bloom, University of Southern California, Los Angeles, CA, and Mike O'Donnell, Rockefeller University, New York, NY, for providing polymerase II and III respectively. Finally, we are grateful to Dr. Judah Rosenblatt, The University of Texas Medical Branch, for sharing expertise in the area of statistical analysis.

	FOOTNOTES

^* This work was supported by National Institutes of Health Grants P30-ES06676, ES05355, ES07781, P30-ES00267, and T32-ES07253.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ Current address: Phase-1 Molecular Toxicology, Inc., Santa Fe, NM 87505.

^** Current address: NIEHS, National Institutes of Health, Research Triangle Park, NC 27709.

Mary Gibbs Jones Distinguished Chair in Environmental Toxicology from the Houston Endowment. To whom correspondence should be addressed: University of Texas Medical Branch, 5.144 MRB, 301 University Blvd., Galveston, TX 77555. Tel.: 409-772-2179; Fax: 409-772-1790; E-mail: rslloyd@utmb.edu.

Published, JBC Papers in Press, April 13, 2000, DOI 10.1074/jbc.M002037200

¹ A. Kowalczyk, unpublished data.

	ABBREVIATIONS

The abbreviations used are: TMSE, 2-fluoro-O⁶-trimethylsilylethyl; HPLC, high pressure liquid chromatography; FAB, fast atom bombardment; DIEA, diisopropylethylamine; BPDE, benzo[a]pyrene diolepoxide.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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