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• W2138655924 abstract "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 intoEscherichia 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,Sstereoisomer 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. coliUvrABC 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. 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 intoEscherichia 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,Sstereoisomer 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. coliUvrABC 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. 2-fluoro-O 6-trimethylsilylethyl high pressure liquid chromatography fast atom bombardment diisopropylethylamine benzo[a]pyrene diolepoxide 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.de Meester C. Mutat. Res. 1988; 195: 273-281Crossref PubMed Scopus (60) Google Scholar) 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.Henderson R.F. Thornton-Manning J.R. Bechtold W.E. Dahl A.R. Toxicology. 1996; 113: 17-22Crossref PubMed Scopus (60) Google Scholar). 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.Kligerman A.D. DeMarini D.M. Doerr C.L. Hanley N.M. Milholland V.S. Tennant A.H. Mutat. Res. 1999; 439: 13-23Crossref PubMed Scopus (30) Google Scholar). There are a number of studies supporting the existence of butadiene diepoxide-induced interstrand cross-links (4.Verly W.G. Brakier L. Feit P.W. Biochim. Biophys. Acta. 1971; 228: 400-406Crossref PubMed Scopus (19) Google Scholar, 5.Verly W.G. Brakier L. Biochim. Biophys. Acta. 1969; 174: 674-685Crossref PubMed Scopus (53) Google Scholar, 6.Jelitto B. Vangala R.R. Laib R.J. Arch. Toxicol. 1989; 13: 246-249Crossref Google Scholar, 7.Lawley P.D. Brookes P. Nature. 1965; 206: 480-483Crossref PubMed Scopus (155) Google Scholar, 8.Lawley P.D. Brookes P. J. Mol. Biol. 1967; 25: 143-160Crossref PubMed Scopus (220) Google Scholar, 9.Vangala R.R. Laib R.J. Bolt H.M. Arch. Toxicol. 1993; 67: 34-38Crossref PubMed Scopus (34) Google Scholar). 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.Lawley P.D. Brookes P. J. Mol. Biol. 1967; 25: 143-160Crossref PubMed Scopus (220) Google Scholar). In 1993, Millard and White (10.Millard J.T. White M.M. Biochemistry. 1993; 32: 2120-2124Crossref PubMed Scopus (68) Google Scholar) 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.Roberts J.J. Thomson A.J. Prog Nucleic Acids Res. Mol. Biol. 1979; 22: 71-133Crossref PubMed Scopus (501) Google Scholar, 12.Skladanowski A. Konopa J. Biochem. Pharmacol. 1994; 47: 2269-2278Crossref PubMed Scopus (78) Google Scholar, 13.Yarema K.J. Lippard S.J. Essigmann J.M. Nucleic Acids Res. 1995; 23: 4066-4072Crossref PubMed Scopus (55) Google Scholar). 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.Carmical J.R. Zhang M. Nechev L. Harris C.M. Harris T.M. Lloyd R.S. Chem. Res. Toxicol. 2000; 13: 18-25Crossref PubMed Scopus (38) Google Scholar), we chose guanine N2-guanine N2 cross-links as our first target. Furthermore, we knew from other experiments 1A. Kowalczyk, unpublished data. 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 theN-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. Oligonucleotides were prepared on an Expedite™ 8909 Nucleic Acid Synthesizer usingtert-butyl-phenoxyacetyl 2-cyanoethyl phosphoramidites and the modified phosphoramidite of 2-fluoro-O 6-trimethylsilylethyl (TMSE)2-5′-O-dimethoxytrityl 2′-deoxyinosine on a 1 μmol scale. Modified oligonucleotides were deprotected and purified as described previously (15.DeCorte B.L. Tsarouhtsis D. Kuchimanchi S. Cooper M.D. Horton P. Harris C.M. Harris T.M. Chem. Res. Toxicol. 1996; 9: 630-637Crossref PubMed Scopus (70) Google Scholar). 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 A 260 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 mammonium 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. (-)-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) δ (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. [α ]D20 was + 3.9° (c = 2, ethanol). 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) δ (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/zcalculated for [M+H]+ 237.0875 found 237.0879. [α]D20 was − 3.1° (c = 2, ethanol). 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), δ (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/zcalculated for [M+H]+ 209.1290 found 209.1291. [α]D20 was −14.9° (c = 2, ethanol). 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), δ (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/zcalculated for [M+H]+ 209.1290 found 209.1312. [α]D20 was + 16.1° (c = 2, ethanol). (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), δ (ppm): 2.70 (m, 4H, 2xCH 2), 3.47 (m, 2H, 2xCH). HRMS (FAB+) m/z calculated for [M+H]+121.0977 found 121.0992. [α]D20 was −15.4° (c = 2, ethanol). 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), δ (ppm) 2.71 (m, 4H, 2xCH 2), 3.47 (m, 2H, 2xCH). HRMS (FAB+) m/z calculated for [M+H]+121.0977 found 121.0987. [α]D20 was + 15.9° (c = 2, ethanol). A sample of this isomer was converted to its HBr salt. [α]D20 +20.1° (c = 2, water) (literature value: [α]D20 +20.3° (c = 2, water) (16.Feit P.W. Nielsen O.T. J. Med. Chem. 1970; 13: 447-452Crossref PubMed Scopus (10) Google Scholar)). The general procedure was as follows. The starting material 5′-d(CATXXTCC)-3′ (1) (X = 2-fluoro-O 6-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). For synthesis of this cross-link, 18 A 260 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) 0.1m 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-link2a (isolated 4.2 A 260 units) eluted at ∼34 min, partially TMSE-protected (isolated 1.8A 260 units) at ∼28 min, and fully deprotected3a (isolated 1.2 A 260 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. For synthesis of this cross-link, 13 A 260 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 for3a. Three cross-linked species were collected: fully TMSE-protected cross-link 2b (isolated 3.6A 260 units) eluted at ∼34 min, partially TMSE-protected (isolated 1.2 A 260 units) at ∼28 min, and fully deprotected 3b (isolated 0.6A 260 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 and3b 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 3aand 3b. Bisnucleoside standards for use in HPLC analysis of enzymatic digests were synthesized as described in the following section. 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. 2.7 mg (0.022 mmol) of (2R,3R)-1,4-diaminobutanediol, 25 mg (0.067 mmol) of 2-fluoro-O 6-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) δ (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 CH 2-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. 2.7 mg (0.022 mmol) of (2S, 3S)-1,4-diaminobutanediol, 25 mg (0.067 mmol) of 2-fluoro-O 6-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) δ (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 CH 2-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. Adducted oligonucleotides 3a and3b and the complementary strand (0.5A 260 units each) were dissolved in 1 ml of melting buffer (10 mmNa2HPO4/NaH2PO4, 1.0m 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 hadTm = 44 °C, (S,S)-butadiene cross-link hadTm = 30 °C, wild type duplex hadTm = 40 °C. Single-stranded M13mp7L2 vector was isolated according to the procedures described by Sambrook et al. (17.Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Labratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). 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.Carmical J.R. Zhang M. Nechev L. Harris C.M. Harris T.M. Lloyd R.S. Chem. Res. Toxicol. 2000; 13: 18-25Crossref PubMed Scopus (38) Google Scholar,18.Latham G.J. Zhou L. Harris C.M. Harris T.M. Lloyd R.S. J. Biol. Chem. 1993; 268: 23427-23434Abstract Full Text PDF PubMed Google Scholar, 19.McNees A.G. O'Donnell M. Horton P.H. Kim H.Y. Kim S.J. Harris C.M. Harris T.M. Lloyd R.S. J. Biol. Chem. 1997; 272: 33211-33219Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar, 20.Chary P. Latham G.J. Robberson D.L. Kim S.J. Han S. Harris C.M. Harris T.M. Lloyd R.S. J. Biol. Chem. 1995; 270: 4990-5000Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar, 21.Carmical J.R. Nechev L.V. Harris C.M. Harris T.M. Lloyd R.S. Environ. Mol. Mutagen. 2000; 35: 48-56Crossref PubMed Scopus (57) Google Scholar). 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. Modified M13mp7L2 was used to transfect repair-deficient AB2480 (uvrA −, recA−)Escherichia coli cells via electroporation as previously reported (14.Carmical J.R. Zhang M. Nechev L. Harris C.M. Harris T.M. Lloyd R.S. Chem. Res. Toxicol. 2000; 13: 18-25Crossref PubMed Scopus (38) Google Scholar, 21.Carmical J.R. Nechev L.V. Harris C.M. Harris T.M. Lloyd R.S. Environ. Mol. Mutagen. 2000; 35: 48-56Crossref PubMed Scopus (57) Google Scholar). 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.Carmical J.R. Zhang M. Nechev L. Harris C.M. Harris T.M. Lloyd R.S. Chem. Res. Toxicol. 2000; 13: 18-25Crossref PubMed Scopus (38) Google Scholar, 21.Carmical J.R. Nechev L.V. Harris C.M. Harris T.M. Lloyd R.S. Environ. Mol. Mutagen. 2000; 35: 48-56Crossref PubMed Scopus (57) Google Scholar). The plaques were subsequently screened for possible base substitutions at position 2 of the ras 12 codon via differential hybridization techniques (18.Latham G.J. Zhou L. Harris C.M. Harris T.M. Lloyd R.S. J. Biol. Chem. 1993; 268: 23427-23434Abstract Full Text PDF PubMed Google Scholar, 19.McNees A.G. O'Donnell M. Horton P.H. Kim H.Y. Kim S.J. Harris C.M. Harris T.M. Lloyd R.S. J. Biol. Chem. 1997; 272: 33211-33219Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar, 20.Chary P. Latham G.J. Robberson D.L. Kim S.J. Han S. Harris C.M. Harris T.M. Lloyd R.S. J. Biol. Chem. 1995; 270: 4990-5000Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar, 21.Carmical J.R. Nechev L.V. Harris C.M. Harris T.M. Lloyd R.S. Environ. Mol. Mutagen. 2000; 35: 48-56Crossref PubMed Scopus (57) Google Scholar). 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 [γ-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. 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 [γ-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. 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′-γ-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. 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 γ-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. The aforementioned, double-stranded 50-mer substrates (5 nm) were incubated with E. coli UvrABC proteins (10 nm UvrA, 250 nm UvrB, and 50 nmUvrC) 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 wer" @default.
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• W2138655924 title "Butadiene-induced Intrastrand DNA Cross-links: A Possible Role in Deletion Mutagenesis" @default.
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