Chem. Res. Toxicol. 1993,6, 681-689
68 1
Construction of Escherichia coli Vectors Containing Deoxyadenosine and Deoxyguanosine Adducts from (+)-anti-Dibenz[a,jlanthraceneDiol Epoxide at a Defined Site Rosalynn
D. Gill,+ Zhao Min,+ Cecilia Cortez,$ Ronald G. Harvey,$ Edward L. Loechler,! and J o h n DiGiovanni’9t
Science Park-Research Division, The University of Texas M. D. Anderson Cancer Center, Smithville, Texas 78957, The Ben May Institute, University of Chicago, Chicago, Illinois 60637, and Department of Biology (ELL),Boston University, Boston, Massachusetts 02215 Received April 30, 199P
Dibenz[aj]anthracene (DB[a j l A) is a carcinogenic polycyclic aromatic hydrocarbon, which is metabolically activated through the formation of bay region diol epoxides. Site-specifically modified M13mpl9-based vectors containing a single (+)-anti-dibenz[aj] anthracene diol epoxide I(+)-anti-DB[a j]A-DE)-deoxyguanosine (dGuo) or -deoxyadenosine (dAdo) adduct were constructed. Four-base oligonucleotides, 5’-~oTGcA-3’and 5’-~ocATG-3’,corresponding to the central four base pairs in the PstI and SphI restriction endonuclease sites, respectively, in the multiple cloning region of M13mp19, were reacted in solution with (&)-anti-DB[ a i ]A-DE. The resulting adducted oligonucleotides were separated and purified using reverse-phase HPLC. Several different singly adducted oligonucleotides were isolated, consisting of the various cis and trans addition products of the (+) and (-) enantiomers of the diol epoxide bound to dGuo or dAdo in the oligonucleotides. 5’-~oTGcA-3’containing the (+)-anti-DB[ajlA-trans-N2dGuo adduct [T(DB[aj]A-N2)GCA] and 5’-~ocATG-3’containing the (+)-anti-DB[aj]Atrans-Ne-dAdo adduct [C(DB [ajlA-N6)ATG) were selected for subsequent ligation into M13mp19 vectors that had been constructed with a corresponding four base gap in the minus strand. Both unmodified and adducted oligonucleotides were successfully ligated into the M13mp19 vectors, [yields: unmodified -TGCA-M13mpl9 (-32 % ) and -CATG- M13mp19 (-42%); adducted T(DB[ajlA-N2)GCA-M13mp19 (-13%) and C(DB[aj]A-N6)ATGM13mp19 (-12%)1. The dAdo adduct-containing vector was characterized. The presence of a dAdo-DNA adduct a t the recognition site of SphI inhibited restriction by SphI. The dAdo adduct-containing vector was cleaved with HindIIIIXbaI to release the adduct in a 24-mer, which migrated slower than the corresponding 24-mer released from the unmodified vector. The dGuo adduct-containing vector was characterized similarly. While the results were more complicated, approximately 25 % of the vectors contained the dGuo adduct. These sitespecifically modified vectors will be used in future studies in vitro and in vivo to compare the effects of dAdo vs dGuo adducts on replication, repair, and mutagenesis.
Introduction Polycyclic aromatic hydrocarbons (PAH)l are widespread environmental contaminants and have been implicated in human cancer ( I ) . In general, a strong correlation between carcinogenesis and mutagenesis by various chemical carcinogens, including PAH, has been observed (2,3). The observation that certain oncogenes arise from their normal counterparts via mutations has indicated the importance of mutagenic events in carcinogenesis (4-6),especially when considering examples such as c-Ha-ras,in which specific point mutations are observed ~~
~~
~
* Author to whom correspondence should be addressed.
t The University of Texas M. D. Anderson Cancer Center.
t The Ben May Institute. SBoston University. * Abstract published in Advance ACS Abstracts, September 1,1993. 1 Abbreviations: PAH, polycyclic aromatic hydrocarbons; DB[ajl A, dibenz[aj]anthracene; (+)-anti-DB[ajlA-DE, (+)-anti-dibenz[ajlanthracene diol epoxide; B[alP, benzo[alpyrene;dGuo, deoxyguanosine; dAdo, deoxyadenosine; nt, nucleotide;TEAA, 0.1 M triethylammonium acetate, pH 7.0; ACN, acetonitrile;PAGE, polyacrylamide gel electrophoresis; AF, aminofluorene; AAF,(acety1amino)fluorene;TE, 10 mM Tris, 1 mM EDTA (pH 8.0); DTT,dithiothreitol; BSA, bovine serum albumin.
after treatment with a particular carcinogen (7). The mechanisms of mutagenesis by bulky carcinogens such as PAH are not well understood; however, various studies have led to the speculation that deoxyadenosine (dAdo) residues may be important in terms of carcinogenesis for some (8,9) while deoxyguanosine (dGuo) residues may be important for others (9, 10). DNA molecules contain many potential sites for reaction with electrophiliccarcinogenicmetabolites (11,12). This makes the question of determining which DNA adduct is actually a critical premutagenic lesion quite difficult. Investigators have developed site-specific methods in which a single DNA adduct is incorporated into a vector that is inserted into a cell, such that cellular repair and replication processes can act upon a single lesion (13,14). Mutations can then be unambiguously assigned to the DNA adduct used for the site-specific experiments. This approach has been used with successin studyingmutations induced by the DNA lesions of several compounds in bacterial systems, including the following: Os-methylguanine (15),04-alkylthymines (16-18),Os-alkylguanines(19, 201,8-oxodeoxyguanosine (211, thymine glycol (22), cis-
0893-228~/93/2706-0681$04.00/0 0 1993 American Chemical Society
682 Chem. Res. Toxicol., Vol. 6, No. 5, 1993
Gill et al. specific DNA adducts, especially those at dAdo residues, in tumor initiation by D B [ a j l A and other PAH.
Experimental Procedures 8
7 DB[aj]A
6 anti-DB[aj]A-diol-epoxide
Figure 1. Chemical structures of DB[aj]A and (+)-antiDB [ a i ]A-DE. platinum-(d(GpG)) crosslinks (23),cis-platinum-{d(ApG) j cross-links (24), aminofluorene (AF)-CE-dGuo (25, 26), (acety1amino)fluorene (AAF)-CB-dGuo (25-29), and (+)anti-B[alP-N2-dGuo (30,31). Some of these DNA lesions have also been examined after replication in mammalian cells: 0 4 - e t h y l t h p i n e ( 17), 06-methyl- and 06-ethylguanine (19,32),and AAF-C8-dGuo (29),and in general, the mutations observed are consistent with those previously detected using bacteria. Studies with bulky DNA lesions, specifically placed in replication vectors, have shown that t h e types of mutations observed are generally consistent with those expected from the type of DNA damage (24, 26, 29, 31). However, to date, most studies using sitespecifically placed bulky DNA adducts have not utilized dAdo adducts despite their presumed importance in chemical carcinogenesis by certain P A H as noted above. Our laboratorieshave been interested in t h e role of dAdo adducts in tumor initiation and chemical carcinogenesis by PAH (10,33). Recently, these studies have focused on the PAH dibenz[ajlanthracene ( D B [ a j l A ) (34-37). DB[ajl A (Figure 1) is an effective tumor initiator in mouse skin (34-36), and further analyses revealed that the (&Ianti-diol epoxide of DB[aj]A (Figure 1) exhibited approximately 3 times more initiating activity than the parent compound (35,361. The major DNA adducts formed in mouse epidermal cells in vitro or i n vivo exposed t o DB[ajlA arise from reaction of t h e bay region diol epoxides, primarily the (+)-anti-diol epoxide, which bind t o t h e exocyclic amino groups of dGuo a n d dAdo (38-401, although minor adducts are also formed with t h e syn-diol epoxide and t h e K-region oxide. Studies of DNA reacted with either t h e pure (+I- or (&)-anti-DB[ajlA-DE in vitro revealed that approximately 25% of the adducts were formed through trans addition of t h e exocyclic amino (N6) group of dAdo and approximately 75% were formed through tram addition df the exocyclic amino (N2) group of dGuo (38, 39). Recent studies of t h e mutagenic specificity of (+)-anti-DB[aj]A-DE in Escherichia coli have indicated a preference for mutations at dGuo residues, where G A transitions dominate (37). However, analysis of DNA from skin tumors initiated by D B [ a j l A or (&Ianti-DB[aj] A-DE indicated a high proportion of AlE2 T transversions in codon 61 of c-Ha-ras (41). Thus, dAdo adducts derived from D B [ a j l A - D E may have an important role in t h e carcinogenic activity of D B [ a j l A and its derivatives in vivo. T h e present paper reports the construction of M13mp19 vectors containing individual dAdo and dGuo adducts derived from t h e (+)-anti-diol epoxide of DB[ajlA. I n particular, we have placed (+)-anti-DB[ajl A-DE-transN6-dAdo and (+)-anti-DB[aj]A-DEtrans-N2-dGuointo a defined location in M13mp19. T h e use of these sitespecific vectors will now allow direct comparisons of t h e effects of a carcinogen bound to two different DNA bases and may lead to a better understanding of the role of
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Chemicals and Enzymes. (*)-anti-DB[aj]A-diol epoxide was synthesizedas previously described (36,38).The diol epoxide stocks were stored at -20 "C as dried crystal and dissolved in spectral-gradeacetone immediatelyprior to use. Oligonucleotides were commercially synthesized (National Biosciences, Inc., Plymouth, MN). [-ps2P1ATPwas obtained from NEN/DuPont (Wilmington, DE) at a specific activity of 6000 Ci/mmol. Snake venom phosphodiesterase (Crotalus atrox, type 11)and alkaline phosphatase were purchased from Sigma Chemical Co. (St.Louis, MO). PstI, SphI,PuuII,XbaI,HindIII,T4 polynucleotide kinase, T4 DNA polymerase, and T4 DNA ligase were obtained from New England Biolabs (Beverly,MA). Double-strandedM13mp19 (RFI) was isolated from E. coli JMlOl K12 cells by alkaline lysis followed by cesium chloride density ultracentrifugation (42). Single-stranded M13mp19was prepared by poly(ethy1eneglycol) precipitation of the supernatant remaining from the broth used for RFI isolation (42). All other chemicals and enzymes used were of the highest quality deemed necessary. The hydrocarbons used in the present study should be considered carcinogens and should be handled with extreme care. Synthesis of Adducted Oligonucleotides. Four-base oligonucleotides,5'-~oTGcA-3'or W-HOCATG-~', corresponding to the four internal base pairs of the PstI or the SphI restriction endonuclease recognition sites of M13mp19, respectively, were purified by HPLC prior to use. The oligonucleotides (trityl-off, 10-pmol scale) were dissolved in 10 mL of 0.01 M triethylammonium acetate (TEAA) buffer (pH 7.0) and then purified by reverse-phase chromatography on an Altex Ultrasphere octadecysilane (ODS) column (10 mm X 25 cm) using a Shimadzu LC-6A HPLC equipped with a SPD-6A UV detector (293 nm). The mobile phases used were 0.1 M TEAA, pH 7.0, and acetonitrile (ACN) at a flow rate of 4.7 mL/min. The program used to purify the oligonucleotides was as follows: 100% TEAA (0% ACN) for 10 min, 0-10% ACN in TEAA for 10 min, and 10% ACN in TEAA for 10 min. The purified oligonucletides eluted after approximately 24 min (data not shown). The oligonucleotides were subsequently evaporated to dryness in a Speed-Vac (Savant Instruments, Farmingdale, NY), dissolved in 50 mM potassium acetate (pH 5.2), and quantitated using the absorbance at 260 nm (42). The average yield after purification was approximately 5 mg of each oligonucleotide. One milligram (800 nmol) of -TGCA- or -CATG- was reacted with 500 pg of (f)-anti-DB[aj]A-diol epoxide (640 nmol) in 4 mL of 50 mM potassium acetate (pH 5.2) for 24 h. The diol epoxide was dissolved in 500 pL of acetone and added in 100-pL aliquots over an 8 hour period. This reaction mixture was maintained at room temperature overnight with gentle mixing. The reactions were stopped by extracting 5 times with ethyl acetate and then 2 times with anhydrous ether to remove any unreacted diol epoxide or hydrolysis products. Nitrogen gas was then briefly blown over the surface of the remaining solution to remove residual organic solvent. Analysis and Identification of Adducted Oligonucleotides. The adducted oligonucleotide products obtained above were separated by 2 (-CATG)-or 3 (-TGCA-)rounds of reversephase HPLC with the addition of an RF-535fluorescencedetector (excitation: 293 nm; emission: 405 nm). The gradients used to separate the adducted oligonucleotideswere as follows: -TGCA-, 10-15% ACN in TEAA over 60 min; and -CATG-, l0-15% ACN in TEAA over 15 min followed by 1 5 2 0 % ACN in TEAA over 75 min. Individual peaks corresponding to adducted oligonucleotides were collected and digested to mononucleosides using snake venom phosphodiesterase and alkaline phosphatase as described (38).Adducted mononucleosideswere separated from unmodified mononucleosides using C l Sep-Paks ~ (Waters, Milford, MA) as follows: digested samples were loaded onto CIS Sep-Paks equilibrated with water, the column was washed with
Chem. Res. Toxicol., Vol. 6, No. 5, 1993 683
Construction of Escherichia coli vectors B.
A.
5'-CAlG-3' (*)anti-DB[a,j]A-DE 5 ' - T g L A - 3 ' y A 0%OH
())M13d;P19 W
TBCA TGCA HPLC to separate adducted oligonucletides
identify specific adducts by HPLC
'0'
blunt end with T4 DNA polymerase
purified oligonucleotides enzymatically digest to mononucleosides
,o
5'32P End label
,o, 0 '
@- &G @-&A
Figure 2. Diagram of the protocol used for the construction of site-specific vectors of M13mp19 containing single (+)-anti-DB[aj]Atrans-NZ-dGuo or (+)-anti-DB[afA-trans-NB-dAdo adducts. Panel A Four-base oligonucleotides, 5'-~ocATG-3'or V-HOTGCA-~', corresponding to the four internal base pairs of the recognition sequence for SphI or PstI restriction endonucleases, respectively, were reacted with (&)-anti-DB[aj]A-DE in solution. The resulting adducted oligonucleotides were then separated and purified using HPLC. A portion of the oligonucleotide collected was then digested to mononucleosides and analyzed by HPLC to determine which DNA adduct was present in each adducted oligonucleotideon the basis of cochromotography with authentic DNA adduct markers. The remaining adducted oligonucleotide preparation was 5'-end-labeled using szP.Panel B: Gapped heteroduplexes were constructed by digesting double-stranded M13mp19 with SphI or PstI, resulting in the formation of 3' four-base overhangs, which were removed by T4 DNA polymerase in a blunt-ending reaction. The linear DNA, missing four bases, was then denatured and renatured with singlestranded M13mp19 [(+) strand], forming circular structures with a four-base gap [in the (-) strand] spanning either the SphI or PstI recognition site in the multiple cloning region of M13mp19. water (10 mL), and then the hydrocarbon-DNA adducts were eluted with 100% methanol (5 mL). The DNA adducts were identified by cochromatography with the authentic (&)-antiDB[aj]A-DE-DNA adduct markers using HPLC as previously described (38). Because radioactively labeled diol epoxides of DB[aj]A were not available, a method was utilized to quantitate DNA adduct levels using HPLC coupled with fluorescence detection as recently described (37). Oligonucleotidescontaining the (+)-anti-DB[ajlA-DetransN2-dGuoor (+)-anti-DB[ajlA-DE-trans-N6-dAdoadduct at the internal purine of -TGCA- and -CATG-, respectively, were selected for construction into site-specific vectors. Sufficient quantities of the above adducted oligonucleotideswere obtained in additional reactions and purified as described above. From each reaction, a portion of the desired peak was taken, digested, and runon the HPLC to verify ita authenticity and also tomeasure the reaction yield. V-End-Labeling of Oligonucleotides. The adducted oligonucleotides of interest were 5'-end-labeled using 10 unita of T4 polynucleotide kinase and 10 nmol of [ys2P]ATP diluted with cold ATP to a specific activity of 100 Ci/mmol. Unmodified oligonucleotideswere also 5'-end-lahled and included as controls. End-labeled products were subsequently separated from ATP using CISSep-Paks equilibrated with water. The samples were loaded on the Sep-Paks, washed with water (20 mL) to remove ATP, and then eluted with 30% ACN in water (5 mL) followed by 50% ACN (5 mL). The ACN washes were pooled and evaporated to dryness in a Speed-Vac. Approximately 10 000 cpm of the labeled oligonucleotides were then run on a 20% native polyacrylamide gel, followed by autoradiography using Hyperfilm-MP (Amersham, Arlington Heights, IL). Relative intensities of bands on the autoradiograms were quantitated using a Bio-Image Visage 60.
Gapped Heteroduplex Construction. Gapped heteroduplex molecules of M13mp19 were prepared essentially as described byBenasuttiet al. (30). One hundred-twentypgofRFIM13mp19 was cut with either PstI or SphI, leaving a 3' four-base overhang (see Figure 2 ). The overhangs were removed in a blunt-ending reaction with T4 DNA polymerase (New England Biolabs). The blunt-ended molecules were then denatured and renatured with 600 pg of the (+) strand of M13mp19, leading to the formation of a double-stranded molecule with a four-base gap in the (-) strand at either the PstI or the SphI site (Figure 2). Singlestranded circular DNA was separated from the double-stranded DNA (gapped heteroduplexes) using hydroxyapatite chromatography (3.6 mL, Bio-ad, Hercules, CA). Single-stranded DNA was eluted with 0.18 M sodium phosphate (pH 7.0) collecting 7 X 1.0-mL fractions; then double-stranded DNA was eluted with 0.4 M sodium phosphate, pH 7.0, collecting14 X 0.5-mL fractions. Aliquota of each fraction were run on an agarose gel including ethidium bromide (0.25pg/mL), and fractions containing gapped heteroduplexes were pooled, desalted using a Centricon-30 concentrator (Amicon,Danvers, MA), and then precipitated. The gapped heteroduplex DNA was then dissolved in 10 mM Tris and 1mM EDTA (pH 8.0)(TE), quantitated using absorbance at 260 nm (421, and stored in 20% glycerol in T E at -20 OC. Ligation of Oligonucleotides into Gapped Heteroduplexes. 5'-Phosphorylated oligonucleotideswere ligated intogapped heteroduplexes of M13mp19. Typical ligation reaction mixtures contained the following in a totalvolume of 100 pL: 50 mM Tris (pH 7.Q 10 mM MgCl2, 20 mM dithiothreitol (DTT), 1 mM ATP, 50 pg/mL bovine serum albumin (BSA), 4 pg (0.83 pmol) gapped duplex, 2000 units of T4 DNA ligase, and the oligonucleotides present at a 100-fold molar excess relative to gapped duplex. After 3 h, an additional 2000 units of T4 DNA ligase was added as well as 0.5 mM ATP; this step was repeated after 5 h.
684 Chem. Res. Toxicol., Vol. 6, No. 5, 1993
The M13 genome was separated from unligated oligonucleotides using Sepharose CL-4B gel exclusion chromatography (3.6 mL, Bio-Rad) eluting with T E (pH 8.0). Prior to loading on the CL4B column the samples were freeze/thawed several times. Without this step, no products appeared to enter the gels during electrophoresis such as that shown in Figure 6. It is presumed that freeze/thawing eliminated DNA-protein aggregates which may have formed during the ligation step. Fractions (200 pL) were collected from CL-4B columns, and a small (1-2 pL) aliquot was taken for scintillation counting. The ligation yield was calculated as previously described (30). The ligation products were initially characterized by digestion with a series of restriction endonucleases. Vectors were treated with PstI, which cleaves at the site of the dGuo adduct; with SphI, which cleaves DNA at the site of the dAdo adduct; and with PvuII, which cleaves a 322 base pair region surrounding the ligation site. Reaction conditions were essentially as described by the manufacturer. Unadducted, unlabeled M13mp19 (0.1pg) was added to each restriction digest as a control to ensure that the digestions had gone to completion. The products of the PstI, SphI, and PVuII digestionswere run on 1% agarosegels containing 0.25 pg/mL ethidium bromide. The gels were photographed, dried, and autoradiographed using Hyperfilm-MP. The vectors were also treated with XbaI and Hind111to liberate the adduct in 24-nucleotide fragments for analysis. Following 23 % denaturing polyacrylamide gel electrophoresis (PAGE) and autoradiography, the resulting bands indicated whether ligation was complete on both the 5’ and 3’ ends of the oligonucleotide as follows: (i) complete ligation will yield a 24-nucleotide (nt) fragment; (ii) failure to ligate the 5’side or the 3‘side of -TGCAwill yield a 12-nt or a 16-nt fragment, respectively; (iii) failure to ligate the 5’ side or the 3’ side of -CATG- will yield a 6-nt or an 18-nt fragment, respectively. This assay also allowed determination of whether the adduct was still present in the modified samples due to decreased migration rate in the 24-nt fragment relative to unmodified control.
Gill et al.
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Figure 3. Separation of adducted oligonucleotides by reversephase HPLC. The peaks were monitored by fluorescence. Subsequent analysis (e.g., Figure 4) revealed that the peaks in panel A correspond to -CATG- containing the following DNA adducts C1, (+)-cis-N2-dGuo;C2, unknown; C3, (-)trans-NBdAdo; C4, (+)-trans-N2-dGuo; C5 (-)-trans-N2dGuo; C6, (+)trans-N6-dAdo; C7, unknown; C8, unknown; and C9,(-)&-N6dAdo. The peaks in panel B correspond to -TGCA- containing the following DNA adducts of (*)-anti-DB[aj]A-diol epoxide: T1, unknown; T2, (+)-cis-N2-dGuoand (-)-tram-N’-dAdo; T3, (-)-trans-N2-dGuo;T4, (+)-cis-N2-dGuo;T5, (+)-trans-N-dGuo; T6, unknown; and T7, unknown.
HPLC Separation and Identification of Adducted Oligonucleotides. The four-base olignucleotides-TGCAand -CATG- were reacted with (*)-anti-DB[aj]A-DE, and adducted oligonucleotides were separated using reverse-phase HPLC (Figure 3A, -CATG- reaction products; Figure 3B, -TGCA- reaction products). Excellent separation of the various adducted oligonucleotidereaction products was obtained. Individual peaks from the HPLC were collected and digested to monucleosides, and the DNA adducts present were identified as previously described (38,40). The peaks in Figure 3A correspond to -CATGmodified with the following DNA adducts of (*)-antiDB[ajl A-diol epoxide: C1, (+)-cis-N2-dGuo; C2, unknown; C3, (-)-trans-NG-dAdo; C4, (+)-tram-N2-dGuo; C5 (-)-tram -N2dGuo; C6, (+)-tram-NG-dAdo;C7, unknown; C8, unknown; and C9, (-)cis-N6-dAdo. The peaks in Figure 3B correspond to -TGCA- containing the following DNA adducts: T1, unknown; T2, (+)-cis-N2dGuo and (-)-trans-NB-dAdo; T3, (-)-trans-N2-dGuo; T4, (+)-cis-N2-dGuo;T5, (+)-trans-N2-dGuo; T6, unknown; and T7, unknown. The reaction products corresponding to the other three possible dAdo adducts were also bound to -TGCA-; however, these singly adducted oligonucleotides eluted much later in the HPLC chromatogram (data not shown). Evidence that peaks C6 and T5 contained oligonucleotideswith (+)-anti-DB[aj]A-DE-trans adduct at the central dAdo [C(DB[ajlA-N6)ATGl and dGuo [T(DB[ajlA-N2)GCAl,respectively,is presented in Figure 4. These products were repurified prior to further use as described in the Experimental Procedures section.
32P-End-Labelingand Characterization of EndLabeled Oligonucleotides. The unadducted oligonucleotides or those containing the specific (+)-antiDBrajIA-DE adducts of interest were collected and 5’32P-end-labeled as described in the Experimental Procedures section. A portion of the end-labeled product was then analyzed on a 20% polyacrylamide gel (Figure 5) . In each case the products were >99.9% pure on the basis of the appearance of a single band in an autoradiogram of the same gel as in Figure 5 following prolonged exposure and densitometric analysis. The presence of a DNA adduct decreased migration in the gel. It is interesting to note that the presence of a dGuo adduct retarded migration to a greater extent than the dAdo adduct. The Rf for 5’pC(DB[ajlA-N6)ATG-3’relative to unmodified 5’-pCATG3’was 0.58, comparedto 0.44 for 5’-pT(DB[aj]A-N2)GCA3’ relative to unmodified 5’-pTGCA-3’. The unmodified controls, either 5’-pTGCA-3’ or 5’-pCATG-3’, migrated a t approximately the same rate. An identical pattern of migration was also observed on 20% denaturing PAGE gels containing 8 M urea (datanot shown). HPLC analysis of the end-labeled oligonucleotides containing (+)-antiDB[ajl A-DE adducts indicated >95% conversion to the phosphorylated derivative (data not shown). Construction of M13mp19 Vectors Containing (+)anti-DB[aj’JA-DE-N2-dGuoand -N6-dAdo Adducts at a Defined Site. Oligonucleotides containing (+)-antiDB[ajlA-DE-trans-N2-dGuo, (+)-anti-DB[aj]A-DEtram-N6-dAdo, or unmodified control oligonucleotides were ligated into M13mp19 gapped heteroduplex mole-
Constructionof Escherichia coli vectors
Ia.
Chem. Res. Toxicol., Vol. 6, No. 5, 1993 685
(&))anti -DB[a,j]A-DE + Calf Thymus DNA U
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1 2 3 4 Figure 5. Characterization of adducted 5’-32P-end-labeled oligonucleotides using 20 % polyacrylamide gel eletrophoresis. Lane 1is unmodified pCATG, lane 2 is pC(DB[aj]A-N6)ATG, lane 3is unmodified pTGCA, and lane 4 is pT(DB[aj]A-N2)GCA.
EO
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Figure 4. Identification of (+)-or (-)-anti-DB[aj]A-DE-DNA adducts by cochromatography with authentic DNA adduct markers using reverse-phase HPLC. The oligonucleotides of interest were digested to mononucleosides, passed through CU Sep-Paks, and run on the following program: 47 % methanol in water for 50 min, 4 7 4 0 % methanol in water for 50 min, and 60-100% methanol in water for 15 min. The flow rate was 1 mL/min. Column effluent was monitored by fluorescence as described in Experimental Procedures. (a) Marker DNA adducts prepared from calfthymus DNAreacted with (f)-anti-DB[aj]ADE (38); (b) the (+)-anti-DB[ajlA-trans-N6-dAdo adduct of peak C6 in Figure 3A; (c) the (+)-anti-DB[aj]A-trans-N2-dGuo adduct of peak T5 in Figure 3B. Note that B[a]P-9,10-diol was included as a marker in all three HPLC chromatograms shown.
cules (Figure 2). The unligated oligonucleotides were separated from the double-stranded molecules using gel exclusionchromatography (see ExperimentalProcedures). The overallyield of ligation of oligonucleotides into gapped heteroduplex molecules was calculated to be -32 f 8% for unmodified -TGCA-, -42 f 21% for unmodified -CATG-, -13 f 7% for T(DB[aj]A-N2)GCA, and -12 f 5% for C(DB[aj]A-N6)ATG. These figures are based on the average of at least 8 separate experiments. Once inserted into the vector, the N2-dGuoadduct was located at position 6249 in the (-) strand of the M13mp19 genome,while the N6-dAdoadduct was located at position 6243 in the (-) strand (43). Successful ligation was confirmed by agarose gel electrophoresis followed by autoradiography of the gel. Although the nicked, open circular form of M13mp19 dominated, some covalently closed circular DNA was produced (-25%), and there was a small fraction of linear product (Figure 6 ,lanes 1 and 5). Both control and site-specific vectors were characterized following restriction endonuclease digestion.
The presence of an (+)-anti-DB[aj]A-DE-N6-dAdo adduct at the SphI restriction site (5’-GCATGC-3’) inhibited cleavage by SphI, where only 6% of this material was linearized (Figure 6A, lane 7). This is not the result of partial digestion because unlabeled, unadducted M13mp19 included in the same digestion was completely linearized as revealed by ethidium bromide staining (data not shown). The dAdo adduct at the SphI site did not inhibit cleavage by PstI, which is 4 bases away (Figure 6A, lane 6). The presence of a (+)-anti-DB[aj] A-DE-N2-dGuo adduct at the PstI recognition site (5’-CTGCAG-3’) partially inhibited cleavage by PstI, where 25 % remained nonlinear (Figure 6B, lane 6). Once again, unlabeled, unadducted M13mp19 in the same reaction tube was cleaved to completion. The dGuo adduct at the PstI site did not inhibit cleavage by SphI, which is 5 bases away (Figure 6B, lane 7). PuuII digestion localized the ligated oligonucleotides in a single radioactive fragment of 322 base pairs as expected (lanes 4 and 8; Figure 6). The ligation products were analyzed further by digestion with Hind111 and XbaI, isolating the adducts in 24-mers, which were analyzed on a 23 % denaturing PAGE gel. As shown in Figure 7 ,unmodified 5’-pCATG-3’ appeared to be ligated on both the 5’ and 3’ sides, on the basis of the presence of a single band 24 nucleotides (nt) in length. The 5’-pC(DB[ a i ]A-N6)ATG-3oligonucleotide also appeared to be ligated on both the 5’ and 3’ sides because no bands of less than 24 nt were present. A major band (95%) migrated more slowly than that observed in the lane correspondingto the unadducted control, suggesting it contained the dAdo adduct (compare lanes 1and 2 of Figure 7). A minor band (-5%) comigrated with the control band: this percentage correlated with the proportion of C(DB[aj]A-N6)ATGM13mp19that was sensitive to SphI cleavage (-6%) (Figure 6A, lane 7); therefore, the SphI sensitivity of C(DB[aj]A-N6)ATG
686 Chem. Res. Toxicol., Vol. 6, No.5, 1993
Gill et at.
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C(DS[a,flA-NG)ATG-M13mpl9
TGCA-Ml3mpl9
T(DB[a,flA-N*)GCA-M13mpl9
ocL-
LCCC-
-322bp CCC-
1
2
3
4
5
6
7
0
-322bp
1 2 3 4 5 6 7 8 Figure 6. (A) Characterization of CATG-M13mpl9and C(DB[aj]A-N6)ATGM13mpl9. (B)Characterization of TGCA-M13mpl9 and T(DB[ajc1A-N2)GCA-M13mp19. The abbreviations stand for the following: OC, open circular DNA; L, linear DNA; CCC, covalently closed circular DNA. Lanes 1 and 5 are controls showing the ligation products without further manipulations;lanes 2 and 6 were digestedwith PstI; lanes 3 and 7 were digested with SphI; and lanes 4 and 8 were digested with PuuII,which releases a single radioactive fragment of 322 base pairs. CATGM13mpl9 was completely linearized by the restriction enzymes, while -6% of C(DB[aj]A-N6)ATGM13mp19was linearizedby SphI. TGCA-Ml3mpl9 was completelylinearized by the restrictionenzymes;while T(DB[aj]A-N2)GCA-M13mpl9was partially linearized (-75%) by PstI.
M13mp19 could be accounted for by the presence of a small amount of successfully ligated but unadducted 5'pCATG-3'. A similar set of experiments was also conducted with T(DB[aj]A-N2)GCA-M13mp19 and TGCA-M13mpl9. The latter consistently had -25% of the material incompletely ligated on the 3' side as evidenced by the appearance of a 16-nt fragment following digestion with Hind111 and XbaI (Figure 7, lane 4). In contrast, 5'-T( DB[aj]A-N2)GCA-M13mp19 was ligated efficiently on both sides because no bands of less than 24 nt were apparent (Figure 7, lane 5). Approximately 20% of this material migrated slower than the control and presumably contained an adduct. However, -80% comigrated with the unadducted control band at 24 nt, which was approximately the same as the proportion of T(DB[a,j]A-N2)GCA-M13mpl9 that was sensitive to PstI cleavage (Figure 6B, lane 6).
Discussion The present study has demonstrated the feasibility of isolating four-base oligonucleotides, ~'-HoTGCAor 5'HoCATG,containing single adducts of (+)-anti-DB [aj]A-DE at the central purine. ~'-HoTGCA with (+)-trans-, (-)-trans-, and (+)-cis-N2-dGuo adducts were isolated. Similarly ~'-H&ATG with (-)trans-, (-)-cis-, and (+)trans-N6-dAdo adducts were also isolated. Although reactions with racemic anti-DB [aj]A-DE complicated these efforts, the use of the pure (+) or (-) enantiomers (38)in the future may facilitate separation and reaction yields for specific adducted oligonucleotides of interest.
Oligonucleotides containing DNA adducts at the 3' terminus were not of interest because ligation into a gapped heteroduplex would likely be difficult. The overall yields for reaction of both oligonucleotides with (A)-anti-DB[aj]A-DE were similar (-25%) on the basis of fluorescence and UV absorbance of the peaks in the chromatograms (Figure 3, and data not shown). The reaction yield for T(DB [aj]A-N2)GCAwas -9 % and for C(DB[aj]A-N6)ATGwas -3.6%. The variety of singlyadducted oligonucleotides that were obtained suggeststhat it may be possible to eventually study several different types of adducts, e.g., cis vs trans addition products, within the same sequence context. The relatively high reaction yield (and apparent stability) of adducted oligonucleotides is advantageous. The stability of (+)- or (&)-antiDB [a j ]A-DE and its DNA reaction products was indicated in previous studiesanalyzing reaction of these diol epoxides with calf thymus DNA or deoxyribonucleotides in vitro (38).2 The chemical stablility of (&)-anti-DB[aj]A-DE may explain the observation that this diol epoxide was approximately 3 times more potent as a mouse skin tumor initiator than the parent hydrocarbon (35). For the purposes of the present study, we were primarily interested in using the (+)-transaddition products at the N2 of dGuo or N6 of dAdo because these predominate in vivo and in vitro (38-40). The adducted oligonucleotides of interest were shown to contain a single trans-N2-dGuo or trans-N6-dAdo adduct on the basis of HPLC chromatograms, in which a single deoxyribonucleoside adduct peak appeared in each of the corresponding chromato2 Unpublished
observation.
Construction of Escherichia coli vectors
m
m
Chem. Res. Toxicol., Vol. 6, No.5, 1993 687 [aj]A-N2)GCA] exhibited substantial differences in migration through a polyacrylamide gel. This finding indicates possible conformational differences between the N2-dGuo and N6-dAdo adducts. Several older studies indirectly suggested possible structural differences between N2-dGuoand N6-dAdoadducts of bulky carcinogens
7
E m 7
(45, 46).
n
S J m
n
W
I-
-24nt
-1 6nt
1 2 3 4 5 Figure 7. Determination of the ligation efficiency of CATG M13mp19, C(DB[aj]A-N6)ATGM13mp19,TGCA-M13mpl9, and T(DB[aj] A-N2)GCA-M13mpl9. Ligation products were cleaved with XbaI and HindIII, and then separated on a 23% denaturing polyacrylamide gel. Band size was determinedusing an oligo dT(4-22) ladder (lane 3). Lane 1 shows the digestion of CATGM13mpl9; the upper band of 24 nt indicates complete ligation on both 5' and 3' ends. Lane 2 shows the digestion of C(DB[aj]A-N6)ATGM13mplS; the upper band indicates a completely ligatedfragment of 24 nt that is retarded in migration due to the presence of an N6-dAdoadduct, while the lower band that comigrates with the 24-nt fragment of CATGM13mpl9 indicates complete ligation of an oligonucleotide apparently lacking the DNA adduct. Lane 4 shows the digestion of TGCAM13mp19, with a band of 24 nt indicatingcomplete ligation and a lower band of 16 nt indicating that 25 % of the oligonucleotide was incompletelyligatedon the 3' end. Lane 5 shows the digestion of T(DB[aj]A-N2)GCA-M13mpl9, which has a pattern similar to that of C(DB[aj]A-N6)ATGM13mp19,
-
grams after enzymatic digestion (Figure 4). Electrophoresis of 32P-end-labeledoligonucleotideon 20 % PAGE gels indicated that they were pure (>99.9 % ) and contained 95% of the radioactivity was associated with the modified oligonucleotide (data not shown). Thus, it is unlikely that there were significant amounts of unadducted oligonucleotides present during the ligation reactions used to incorporate the adducted oligonucleotides. As shown in Figure 5, interesting differences were observed in the migration of the two adducted oligonucleotides in 20 % polyacrylamide gels. While others have noted a slower migration of oligonucleotides containing DNA adducts (25, 30, 44), to our knowledge the data presented in Figure 5 are the first instance where two modified oligonucleotides of identical size and similar base composition [i.e., pC(DB[aj]A-N6)ATG and pT(DB-
The relative proportion of M13mp19 with no adduct that contaminated preparations of C(DB[aj]A-NG)ATG M13mp19 as revealed by SphI sensitivity (Figure 6A, lane 7) and XbaI/HindIII double digest (Figure 7, lane 2) was comparable to that reported for a benzo [a]pyrene-containing vector constructed similarly (30). In contrast, the T(DB[aj]A-N2)GCA-M13mp19 vector appeared to have a much higher fraction of contamination by unadducted M13mp19. Several explanations for this finding are possible: (i) T(DB[aj]A-N2)GCAis contaminated with unmodified -TGCA-. The presence of the latter at the 1%level could be sufficient to explain the presence of a fraction of contaminating TGCA-M13mpl9, if contaminating TGCA is preferentially ligated into the gapped duplex, because the ratio [oligonucleotide: gapped duplex] in the ligation was [100:1] (see Experimental Procedures). To test this possibility, the [oligonucleotide:gapped duplex] ratio was dropped to 1O:l in the ligation; the results were similar (data not shown) where 80 % of the resulting ligation product was still cleaved by PstI. In addition, T(DB[aj]A-N2)GCAwas repurified, phosphorylated, and ligated; this did not decrease the fraction of PstI-cleavable material, implying that contaminating -TGCA- in the ligation mixture is not the source of the PstI cleavable material. (ii) The dGuo adduct in T(DB[aj]A-N2)GCA is unstable. This seems unlikely because previous studies have not revealed differences in stability of the (+)-antiDB[aj] A-dGuo vs -dAdo adducts (38-40).2 In addition, analysis of end-labeled oligonucleotides using 20 % PAGE gels at various steps in the procedure has not revealed evidence of adduct instability. Finally, no evidence of breakdown of pT(DB[aj]A-N2)GCA and pC(DB[aj]A-N6)ATG was obtained when either was exposed to 0.1 N NaOH or to 80 "C (pH 7.0) for 15min (data not shown). (iii) The adduct is unstable either during the ligation or during the subsequentcharacterizationassays. This would only be sensible if the dGuo adduct, which is stable as the nucleoside adduct and in a single-stranded oligonucleotide, were unstable in double-stranded DNA. Although intuitively unreasonable, evidence for enhanced instability in double-stranded vs single-stranded DNA has been obtained for (+)-anti-B [a]P-N2-dGuo recently,3 where breakdown liberates (+)-anti-B[a]P-tetraol. However, it should be stressed that the fraction of vectors containing adducts once prepared appeared to be stable when stored at -20 "C for up to 4 weeks as determined by reexamination of PstI sensitivity. The fact that the relative proportion of material insensitive to PstI cleavage is very similar to the proportion of material that migrated more slowly in the 23% PAGE gel strongly indicates that the PstIinsensitive vectors do contain an intact (+)-anti-DB[aj]A-DE-dGuo adduct. Preliminary results from DNA polymerase stop assays in which the vectors were first treated with PstI and then Ba131 nuclease to remove any PstI-sensitive material indicate that there is a strong block to polymerization by several different DNA polymerases at the site of the dGuo adduct, further indicating the 3 Drouin
and Loechler, personal communication.
688 Chem. Res. Toxicol., Vol. 6, No. 5, 1993
presence of the adduct in the vector (47). Therefore, it seems likely that adduct instability occurs during the ligation step and/or during the subsequent analyses but not during routine manipulation. Fortunately, the presence of some unadducted material in T(DB[a,jlA-N2)GCA-M13mpl9 will be of little concern in mutagenesis studies, because it can be cleaved with PstI and effectively rendered inconsequential biologically (31). Alternatively, this cleaved material can be removed from the preparations by cesium chloride centrifugation (48). In summary, we have constructed site-specific DNA adducts derived from reaction of the (+)-anti-DB[ajlA-diol epoxide with the N6 of dAdo or the N2 of dGuo in an M13mp19 replication vector. These site-specificvectors should be very useful in comparative studies of both in vitro and in vivo effects of dAdo vs dGuo (+)-antiDB[ajlA-DE adducts. Preliminary studies of the M13mp19 vector containing the (+)-anti-DB[ajlA-N6dAdo adduct have shown that this adduct is an effective block to in vitro DNA synthesis by T4 DNA polymerase, T7 DNA polymerase Version 2.0 (Sequenase), and E. coli polymerase I (Klenow fragment), and it appears that in each case the polymerase is blocked after incorporating a base opposite the adduct (47). Future studies will also examine the comparative mutagenicity of these sitespecificvectors in repair-proficient and -deficient bacteria, with the ultimate goal of understanding the role of specific DNA adducts in the carcinogenic process.
Acknowledgment. The authors wish to thank Carrie McKinley and Yolanda Valderrama for their help in preparing this manuscript and John Riley and Judy Ing for their help with graphics and photography. Research was supported by U.S. Department of Health and Human Services Grants CA 36979 (J.D.), ES 03775 (E.L.L.), and ES 04732 (R.G.H.), and American Cancer Society Grants CN-22 (R.G.H.), CN-54 (E.L.L.), and FRA-375 (J.D.). A portion of this work was presented a t the 82nd American Association for Cancer Research (Abstract 549) held May 1991 in Houston, Texas.
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