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Chem. Res. Toxicol. 1999, 12, 258-263
Mutational Consequences of Replication of M13mp7L2 Constructs Containing Cis-Opened Benzo[a]pyrene 7,8-Diol 9,10-Epoxide-Deoxyadenosine Adducts John E. Page,† Anthony S. Pilcher,‡ Haruhiko Yagi,‡ Jane M. Sayer,‡ Donald M. Jerina,‡ and Anthony Dipple*,† Chemistry of Carcinogenesis Laboratory, ABL-Basic Research Program, NCI-FCRDC, Frederick, Maryland 21702, and Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892 Received November 9, 1998
The four adducts that arise by cis ring opening of the four optically active benzo[a]pyrene diol epoxides by the exocyclic N6-amino group of deoxyadenosine were incorporated synthetically into each of two different oligonucleotide 16-mers, 5′-TTTXGAGTCTGCTCCC-3′ [context I(A)] and 5′-CAGXTTTAGAGTCTGC-3′ [context II(A)], at the X position. The eight resultant oligonucleotides were separately ligated into bacteriophage M13mp7L2 and replicated in Escherichia coli that had been SOS-induced, and the progeny were analyzed to evaluate the consequences of replication past these adducts. The presence of these adducts reduced plaque yields substantially. However, the progeny obtained exhibited high frequencies of base substitution mutation ranging from 9 to 68%, depending upon the individual adduct and the sequence context in which it was placed. For most of the adducts, A f T transversion was the mutation found most frequently in either sequence context, and mutation frequencies in context I(A) were always substantially greater than those in context II(A). In context I(A), adducts with an R configuration at the site of nucleoside attachment were more mutagenic than those with an S configuration. In both sequence contexts that were studied, the cis adduct arising from the (7S,8R)-diol (9S,10R)-epoxide was the most mutagenic adduct. These findings clearly show that individual mutation frequencies are determined by the combined effects of both adduct structure and sequence context.
Introduction Interactions between reactive metabolites of chemical carcinogens and cellular DNA, leading to the generation of mutations in oncogenes and tumor suppressor genes, are believed to constitute key events in the initiation of the carcinogenic process (1). Reactive metabolites usually induce the formation of more than one type of product (adduct) in DNA as a consequence of reaction with more than one site on a given nucleotide and/or reaction on more than one nucleotide. Thus, studies of the mutagenic effects of reactive metabolites do not allow the adduct responsible for specific mutations to be determined. Consequently, site-specific mutation analyses have been developed to allow specific biological effects to be attributed to specific adducts (2, 3). Either double-stranded or single-stranded vectors have been used in site-specific mutation studies, and each has advantages and disadvantages (3). In single-stranded vectors, the absence of strand bias effects (4) and of adduct repair (5) allows relatively high mutation frequencies to be obtained. For this reason, several laboratories selected a single-stranded vector for a study of the intrinsic mutagenicity of polycyclic aromatic hydrocarbon diol epoxide-deoxyribonucleoside adducts (6-11). Previously, we have used the M13mp7L2 vector (12) to examine the intrinsic mutagenicities of the isomeric † ‡
NCI-FCRDC. National Institutes of Health.
adducts that arise from trans opening of the four optically active benzo[a]pyrene (B[a]P)1 diol epoxides by the exocyclic amino groups of both dGuo and dAdo residues in DNA (11). Different isomeric dGuo and dAdo adducts gave differing mutagenic responses, but the most dramatic effect found was that the mutation frequency for a given adduct varied considerably with sequence context. In attempts to understand the effects of DNA adduct structure and sequence context on the intrinsic mutagenicity of polycyclic aromatic hydrocarbon-DNA adducts, we have extended our earlier work on trans adducts and now report site-specific mutation studies of isomeric adducts that arise from cis opening of the four optically active B[a]P diol epoxides (DEs) by the exocyclic N6amino group of dAdo residues in DNA. Structures of the B[a]P DE-adducts studied and the sequences into which they were incorporated are shown in Figure 1. The sequence contexts used in these studies are derived from the Escherichia coli supF gene and were selected because they were either relatively susceptible or resistant [context I(A) and context II(A), respectively] to mutation when a shuttle vector containing this gene was exposed to reactive diol epoxides (11). In the two different sequence contexts, mutation frequencies ranged from 9 1 Abbreviations: B[a]P, benzo[a]pyrene; DE, diol epoxide; DE-1, diol epoxide in which the epoxide oxygen and the benzylic hydroxyl group are cis; DE-2, diol epoxide in which the epoxide oxygen and benzylic hydroxyl group are trans; CPG, controlled-pore glass; DMT, 5′dimethoxytrityl; CD, circular dichroism.
10.1021/tx980244l CCC: $18.00 © 1999 American Chemical Society Published on Web 02/09/1999
Context Effects on Mutation by BPDE-dAdo Adducts
Chem. Res. Toxicol., Vol. 12, No. 3, 1999 259 Table 1. HPLC Retention Timesa and Configurational Assignmentsb for the Oligonucleotides Containing Cis-Opened B[a]P DE-dAdo Adducts parent diol epoxide
context
DE-1
I(A)
DE-1
II(A)
DE-2
I(A)
DE-2
II(A)
configuration at C-10
retention time (min)
R S Rc Sc R S R S
19 22 17 19 18 19 15 16
a On Hamilton PRP-1 columns eluted at 3 mL/min with a linear gradient of acetonitrile in 0.1 M ammonium carbonate buffer (pH ∼7.5) which increased the acetonitrile concentration from zero to 17.5% over the course of 20 min. The column size was 7 mm × 305 mm (10 µm) for the DE-1 adducts and 10 mm × 250 mm (7 µm) for the DE-2 adducts. b Configurational assignments were based on CD spectra of the oligonucleotides (see the text) except as otherwise noted. c Configurational assignment based on enzymatic digestion to the adducted nucleosides.
Figure 1. Structures of the adducts formed on cis opening at C-10 of the four isomeric B[a]P diol epoxides by the exocyclic N6-amino group of deoxyadenosine. Adducts are labeled on the basis of the diol epoxide diastereomer from which they are derived (in DE-2, the epoxide oxygen and benzylic hydroxyl group are trans, whereas in DE-1, these groups are cis) and with an indication of the configuration of the carbon to which the dAdo amino group is bound. Thus, for example, cis DE-2/10S is the adduct derived from cis opening of the epoxide ring at the 10-position of the (7S,8R)-diol (9R,10S)-epoxide of B[a]P. Each of the four adducts was placed at position X in each of the two sequence contexts shown in this figure.
to 68% for the cis adducts [5-58% was reported earlier for the trans adducts in the same contexts (11)]. Though A f T base substitutions predominated in most cases, one context [context II(A)] supported higher frequencies of A f G mutations than another [context I(A)], and for one adduct isomer, in context II(A), A f G transition was the most prominent mutation found.
Experimental Section EcoRI restriction enzyme, T4 DNA ligase, T4 polynucleotide kinase, and [γ-32P]ATP were obtained from Amersham Corp. (Arlington Heights, IL). Uracil DNA glycosylase, 2x prehybridization/hybridization solution, and some unmodified oligonucleotides and 56-mer scaffold oligonucleotides were from Gibco/ BRL (Gaithersburg, MD). Most unmodified oligonucleotides were provided by M. Powers (SAIC, NCI-FCRDC, Frederick, MD). For DNA sequencing, ABI-PRISM Dye Terminator Cycle Sequencing Ready Reaction kits were obtained from PerkinElmer (Norwalk, CT). Qiagen (Santa Clarita, CA) QIAprep M13 kits were used for purification of the M13 DNA. E. coli SMH77 [F′lacZ, ∆M15, pro+, ∆(pro-lac), leu+], a derivative of AB1157, and bacteriophage M13mp7L2 were generous gifts from C. W. Lawrence (University of Rochester, Rochester, NY). Synthesis of Oligonucleotides. Oligonucleotides containing cis-opened N6-dAdo adducts of B[a]P DE-1 were prepared (13) by postoligomerization modification (14) of support-bound 16mer oligonucleotides containing a 6-fluoro-9-(2-deoxy-β-D-erythropentofuranosyl)purine residue at the desired modification site. Typically, 43 mg of controlled-pore glass (CPG) support, bearing 2 µmol of the fluorinated oligonucleotide, was allowed to react with 9 mg of racemic 7β,8R,9β-trihydroxy-10β-amino-7,8,9,10tetrahydrobenzo[a]pyrene (cis-opened B[a]P DE-1) (15) in 200 µL of DMSO containing 13 µL of triethylamine at 60 °C for 6 days. Workup of the reaction mixtures was carried out as described previously (13). The oligonucleotides were initially purified by HPLC as their 5′-dimethoxytrityl (DMT) derivatives [Hamilton PRP-1 7 mm × 305 mm column, eluted at 3 mL/min
with a gradient that increased the percentage of acetonitrile in 0.1 M ammonium carbonate buffer (pH 7.5) from 15 to 32.5% over the course of 35 min]. Retention times for the diastereomeric, adducted 5′-DMT-oligomers of context I(A) (5′-TTTXGAGTCTGCTCCC-3′), where X indicates the cis-opened B[a]P DE-1 adduct diastereomers, were 23.0 and 24.9 min; for context II(A) (5′-CAGXTTTAGAGTCTTGC-3′), the retention times were 18.2 and 20.1 min. After cleavage of the DMT group (80% acetic acid in water for 30-45 min at room temperature), the oligonucleotides were chromatographed on the Hamilton column (see Table 1 for details). Approximately 2-3 A260 units of each diastereomer was obtained. The late-eluting (cis DE-1/10S) diastereomer of context II(A), although apparently homogeneous in the chromatographic system used, contained approximately 20% of an impurity that was removed by gel electrophoresis (see below). For synthesis of oligonucleotides containing cis-opened N6dAdo adducts of B[a]P DE-2, a mixture of 10R and 10S diastereomers of the 5′-DMT-3′-phosphoramidites of N6-[10(7,8,9-triacetoxy-7,8,9,10-tetrahydrobenzo[a]pyrenyl)]-2′-deoxyadenosine was prepared as described (15). Oligonucleotides were synthesized (16) on a 1.5 µmol scale using 170 Å CPG loaded with N4-benzoyl-5′-O-(4,4′-dimethoxytrityl)-2′-deoxycytidine-3′succinic acid (95 µmol/g). The support-bound oligonucleotides containing the appropriate 12-base sequence 3′ to the adduct were synthesized on an automated DNA synthesizer, with modifications to the standard protocol as described (17). In a typical reaction for the preparation of context I(A), CPG beads with the bound 12-mer 5′-GAGTCTGCTCCC were removed from the column and treated with 4.4 µmol of the diastereomeric phosphoramidite mixture and 0.5 M 1H-tetrazole in acetonitrile (50 µL) for 16 h. The yield on manual coupling of the modified phosphoramidite was 37%. End-capping was omitted following the manual step. The support-bound oligonucleotide was returned to the column, and the three remaining residues were added by reaction on the synthesizer. The oligonucleotide was cleaved from the support by the standard procedure, and protective groups were removed by ammonolysis (16 h, 58 °C). The reaction mixture was evaporated to dryness, and the crude residue was detritylated (see above). After chromatographic purification on a Hamilton PRP-1 column (Table 1), 9.7 A260 units of the early-eluting (cis DE-2/10R) and 7.9 A260 units of the late-eluting (cis DE-2/10S) adducted oligonucleotides were obtained. The context II(A) 16-mers were prepared by essentially the same procedure, except that they were subjected to a preliminary purification as their 5′-DMT derivatives [Hamilton PRP-1 10 mm × 250 mm column, eluted at 3 mL/min with a gradient that increased the percentage of acetonitrile in 0.1 M ammonium carbonate buffer (pH 7.5) from 15 to 32.5% over the course of 35 min; retention times of 15.8 and 17.9 min]. The
260 Chem. Res. Toxicol., Vol. 12, No. 3, 1999
Figure 2. Circular dichroism spectra of the synthesized oligonucleotides containing cis-opened B[a]P DE adducts in 0.02 M phosphate buffer (pH 7) with the ionic strength adjusted to 0.1 M with NaCl. Spectra are normalized to 1.0 absorbance unit at 260 nm. The pyrene bands at 300-350 nm in the oligonucleotides are positive for those containing 10R adducts (broken lines) and negative for those containing 10S adducts (solid lines). For context II(A) containing a cis DE-1/10S, adduct these longwavelength bands were not detectable, and the assignment of configuration was based on enzymatic hydrolysis to the nucleoside derivative (see the text). elution order of the oligonucleotides remained the same after removal of the DMT group. Chromatography (Table 1) of the detritylated oligonucleotides provided the adducted 16-mers as early (cis DE-2/10R)- and late-eluting (cis DE-2/10S) diastereomers (1.4 and 1.8 A260 units, respectively). Circular dichroism (CD) spectra of the diastereomeric oligonucleotides (Figure 2) exhibited distinct bands in the 300-360 nm region that were opposite in sign for the early- and lateeluting diastereomers, except in the case of the adducted context II(A) oligonucleotide. For the oligonucleotides with relatively intense, long-wavelength bands, an absolute configuration was assigned (13) as 10R for the adducts with positive and 10S for the adducts with negative bands in this region. The sign of these bands is the same in the oligonucleotides as in the corresponding, monomeric nucleoside adducts (18, 19). For the context II(A) cis DE-1 oligonucleotides, the CD spectra did not provide a clear basis for assignment of configuration, and consequently, both isomers were enzymatically hydrolyzed (13, 18) to the monomeric nucleosides. The CD spectra of the adducted nucleosides obtained upon digestion were identical in shape to those of the corresponding DE-1-adenosine adducts (18) and DE-2-dAdo adducts (19) with known absolute configuration. The early-eluting diastereomer gave a single nucleoside adduct, which had 10R absolute configuration, whereas enzymatic hydrolysis of the late-eluting oligonucleotide prior to electrophoresis gave the 10S adduct as the major (∼80%) nucleoside adduct, accompanied by ∼20% of a 10R adduct. Thus, this late-eluting cis DE-1/10S adducted oligonucleotide, although homogeneous on HPLC, contained a cochromatographic impurity or impurities that gave rise to the 10R nucleoside adduct on hydrolysis. Upon further purification of the cis DE-1/10S oligomer by gel electrophoresis, the major component was well separated from the impurity (∼20%). The CD spectrum of the electrophoretically purified oligomer was featureless in the longwavelength region (Figure 2). Weak, positive long-wavelength CD bands, presumably due to the impurity, which were seen in the CD spectrum measured before electrophoresis (not shown), were absent in the spectrum of the purified material. After purification by HPLC, 32P-end-labeled aliquots were examined by polyacrylamide gel electrophoresis, and those containing detectable impurities were purified by gel electrophoresis and then phosphorylated all as described previously (11). Briefly, aliquots (∼0.5 OD unit) were purified by electro-
Page et al. phoresis on a 20% polyacrylamide gel (0.3 cm). Oligonucleotides were detected under UV light, and the corresponding area of each gel was cut out and eluted overnight at 50 °C. The purified oligonucleotide was then desalted on a reversed-phase Sep-Pak cartridge (Waters, Marlborough, MA) and recovered by elution with 50% MeOH in water (1 mL). After being dried, the eluent was washed with 90% ethanol, dried again, and resuspended in water (100 µL). The purity was confirmed by further electrophoresis after end labeling with [32P]ATP. Construction of M13mp7L2 Vectors. M13mp7L2 DNA (50 µg) was cut with EcoRI (4 units/µg of DNA) for 4 h at 30 °C, heated to 65 °C for 15 min to inactivate the enzyme, extracted with phenol/chloroform, and precipitated with ethanol. The scaffolds were synthesized such that 20 nucleotides on either end were complementary to the ends of linearized M13mp7L2, and the remaining 16 nucleotides were complementary to the oligonucleotide 16-mers [contexts I(A) and II(A)]. The cut M13 vector (2 µg) was mixed with oligonucleotide 16-mer/scaffold (25:1 molar ratio of oligonucleotide to scaffold) that had been annealed by heating to 50 °C followed by slow cooling to room temperature. The mixtures were left at room temperature for 5 h prior to the addition of T4 DNA ligase (30 units) and incubation at 16 °C overnight. Ligation reaction mixtures were then treated with uracil-DNA glycosylase (1 unit) at 37 °C for 40 min to create abasic sites in the scaffold that would lead to its degradation by endonucleases upon transfection into E. coli. Ligation efficiencies were estimated from the fraction of circular DNA in these mixtures, determined after gel electrophoresis in 1.4% agarose and Southern blotting, all as described previously (11). Transfection of E. coli SMH 77 with Constructs and Analysis of Progeny Bacteriophage. E. coli SMH 77 were SOS-induced by exposure to UV radiation (40 J/m2) immediately before they were made competent (CaCl2 method) for transfection. Aliquots of the ligation mixture (20 ng of DNA) were transfected into 100 µL of competent cell suspension, mixed with 2xYT top agarose (3 mL), poured onto 2xYT agar plates, and grown overnight at 37 °C. Plaques were screened for mutation by the use of oligonucleotide probes all as described previously (11).
Results The four isomeric B[a]P DE-dAdo adducts (Figure 1) were separately incorporated at the position labeled X into each of the two oligonucleotide 16-mers, context I(A) and context II(A), shown in Figure 1. These adducted oligonucleotide sequences, along with corresponding control sequences containing dAdo in place of adducted dAdo residues, were ligated into the bacteriophage M13mp7L2 vector. The efficiency of ligation of the control context I(A) oligonucleotide was ∼50%. For the adducted oligonucleotides, efficiencies were somewhat lower (28-34%) and were on average ∼65% of the control efficiencies. For unadducted context II(A), the ligation efficiency was 36%, and the adducted oligonucleotides derived from this context were ligated with ∼80% of this efficiency. The recoveries of plaques from the various transfections were always much lower for constructs containing adducted oligonucleotides than for those containing unadducted oligonucleotides, and these recoveries (plaque yields) were somewhat lower for the adducted context I(A) constructs (1.8-5.2% of the unadducted control) than for the adducted context II(A) constructs (4.5-8.9% of the unadducted control). However, despite the low recoveries of progeny with the adducted constructs, analysis by differential hybridization allowed substantial numbers of mutations to be analyzed for each construct (Table 2). The number of mutations analyzed ranged from 106 for the cis DE-2/10S adduct in context II(A) to 676 for the
Context Effects on Mutation by BPDE-dAdo Adducts
Chem. Res. Toxicol., Vol. 12, No. 3, 1999 261
Table 2. Number of Base Substitution Mutations Recovered from SOS-Induced Cells and the Percentage of Total Progeny Found To Be Base Substitution Mutations (MF)a I(A), 5′-TTTXGAGTCTGCTCCC
II(A), 5′-CAGXTTTAGAGTCTGC
number of mutations
number of mutations
adduct
AfT
AfG
AfC
MF (%)b
AfT
AfG
AfC
MF (%)b
cis DE-1/10R cis DE-2/10R cis DE-1/10S cis DE-2/10S
616 158 118 158
17 6 12 13
43 8 18 50
68 ( 2 49 ( 3 28 ( 2 26 ( 2
172 80 45 57
28 23 94 43
21 11 6 6
24 ( 1 9(1 13 ( 1 17 ( 2
a With unadducted constructs, no base substitution mutations were found among 19 020 progeny from context I(A) or among 13 853 progeny from context II(A). Thus, the spontaneous mutation frequency in these experiments was 20% of the total mutations
were A f C transversions in the case of the cis DE-2/ 10S adduct. These same 10S adducts gave much lower A f C mutation frequencies in context II(A). The effects of adduct structure on mutation frequency are clearly complex and, as noted above, are dependent on sequence context. In context I(A), the R adducts were more mutagenic than the corresponding S adducts and the DE-1 adducts were more mutagenic than the corresponding DE-2 adducts. Similar differences were not apparent in context II(A). It can be seen in Figure 1 that the difference between a DE-1 adduct and a DE-2 adduct with the same configuration at the 10-position lies in the different configurations of the carbons, C-7 and C-8. For adducts with a 10R configuration, the configuration of the 7,8dihydroxy function found in DE-1 gives substantially higher mutation frequencies in either sequence context. However, DE-1 and DE-2 adducts with an S configuration demonstrate similar mutagenic properties in context I(A). Mutagenicity is also similar for adducts with a 10S configuration in context II(A) even though the most frequent mutation observed is A f T in the DE-1/10S case and A f G in the DE-2/10S case. These findings show that the configuration at hydroxyl-bearing C-7 and C-8 is important in determining the mutagenicity of the 10R adducts but has little effect on that of the 10S adducts. Other structural variants can be evaluated from these data. For example, comparisons of data for DE-2/10S and DE-1/10R (or DE-2/10R and DE-1/10S) relate structures in which the 7,8-dihydroxy function is identical, but in which the configurations at the 9- and 10-positions are the inverse of one another. For these comparisons, substantial differences in mutation frequencies are found in context I(A), and while the overall frequencies are not so different in context II(A), substantial differences in the preferred mutational change are apparent.
Discussion Previously, we reported mutation studies in this same system for dAdo adducts derived from trans opening of B[a]P diol epoxides (11). To aid in the comparison of that to these studies, the data from the trans study are reproduced and shown in the inset of Figure 3. The most striking observation is that with either cis or trans adducts, substantially higher mutation frequencies were found with context I(A) than with context II(A). Also, in both studies, context I(A) favored substantially higher frequencies of A f T mutations than context II(A). A f
262 Chem. Res. Toxicol., Vol. 12, No. 3, 1999
C mutations also occurred at substantially higher frequencies in context I(A) than in context II(A), except with the trans DE-1/10R adduct where the difference in frequencies was small, 3% in context I(A) and 2% in context II(A). In contrast to these observations, context II(A) favored higher frequencies of A f G mutations than context I(A) in all cases, although the actual differences in frequencies were very small for the trans 10S adducts and the cis 10R adducts. One difference between the trans and cis B[a]P-dAdo adduct data is that in context I(A) the cis 10R adducts are 2-3-fold more mutagenic than their 10S counterparts, whereas the R and S trans adducts exhibited similar mutagenicities (11). In Figure 3, the DE-1/10R trans adducts can be readily compared with the DE-1/10R cis adducts, etc. When this comparison is made, the effects of inverting the configuration at the 7-, 8-, and 9-positions are evaluated. In general terms, the inversion of these three chiral centers does not have a dramatic effect on mutation, and interestingly, the 10S adducts in context II(A) in either the trans or cis case are the adducts for which A f G mutations constitute the highest fraction of total mutations. If one wished to compare a cis or trans adduct from a given diol epoxide metabolite, a cis adduct with an S configuration should be compared with the corresponding trans adducts with an R configuration. For the eight constructs that could be compared in this fashion, the overall mutation frequency is higher in the case of the cis adduct in five cases, higher in the case of the trans adduct in two cases, and about the same in one case. Chary et al. (6) have previously compared cis- and transopened DE-2 adducts in an N-ras sequence. In their hands, the cis-opened adducts were about 3-fold more mutagenic than the corresponding trans-opened adducts. In the studies from our laboratories, the mutation frequencies for the trans- and cis-opened adducts from the (7R,8S)-diol (9S,10R)-epoxide [(+)-DE-2] were about the same in context I(A), but in context II(A), the cisopened adduct was about twice as active as the transopened adduct. However, for adducts derived from the (7S,8R)-diol (9R,10S)-epoxide [(-)-DE-2], mutation frequencies were comparable in context II(A), but the transopened adduct was about twice as active as the cis-opened adduct in context I(A). Our ongoing studies of site-specific mutation are targeted to an evaluation of the effects of structure and sequence context on the mutagenic activity of polycyclic aromatic hydrocarbon-DNA adducts. The substantial differences in mutagenic properties found for the same adduct in the two different sequence contexts used in this study indicate that findings on mutagenic activities are only meaningful within the sequence contexts in which measurements have been made. For example, comparison of relative activities of the cis DE-2/10S and cis DE-2/ 10R adducts in Table 2 would show that the 10R adduct was about twice as active as the 10S adduct if the comparison was made in context I(A). However, comparison of these same adducts in context II(A) would lead to exactly the opposite conclusion; i.e., the 10S adduct is about twice as active as the 10R adduct. These observations serve to strongly emphasize that relative activities for different adducts in one context may not apply to other sequence contexts. Several reports have indicated that sequence context is important in determining the mutagenic consequences
Page et al.
of trans lesion synthesis (9, 10, 20-25). Our findings, first for adducts arising from trans opening (11) and, currently, for adducts arising from cis opening of the four isomeric B[a]P diol epoxides by dAdo, extend these observations on sequence dependence. Furthermore, our findings indicate that though some effects of sequence context may be fairly general, e.g., mutation frequencies for any B[a]P diol epoxide-dAdo adduct studied so far being greater in context I(A) than in context II(A), sequence context effects also can vary with the nature of the adduct being studied. To illustrate this point, Table 2 shows that context I(A) increases the mutation frequency of cis DE-2/10R by ∼5-fold over that found in context II(A), whereas for the cis DE-2/10S adduct, the corresponding increase is only ∼1.6-fold. The range of the extent of the context I(A)-mediated increase in mutation frequency in the data summarized here, together with the fact that with some benzo[c]phenanthrene diol epoxide-dAdo adduct mutation frequencies are actually higher in context II(A) than in context I(A) (16), indicates that both adduct and sequence are jointly responsible for the effects observed. Of course, interactions between the template strand and the polymerase presumably also have an important role in determining mutation frequency. Clearly, a comprehensive investigation of the intrinsic mutagenic potencies of a range of carcinogenDNA adducts in varied sequence contexts is required to define the rules that govern sequence context effects on various adducts. This study and other studies ongoing in our laboratories are aimed at the eventual elucidation of the roles of both structure and sequence context in the determination of mutagenic effects.
Acknowledgment. The research was supported in part by the National Cancer Institute, DHHS, through a contract with ABL.
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Context Effects on Mutation by BPDE-dAdo Adducts
(10)
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