The major, N2-dG adduct of (+)-anti-B[a]PDE induces G
A mutations in a 5'-AGA-3' sequence context
Rajiv Shukla,
Nicholas E. Geacintov1 and
Edward L. Loechler2
Department of Biology, Boston University, 2 Cummington Street, Boston, MA 02215 and
1 Department of Chemistry, New York University, New York, NY 10003, USA
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Abstract
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Previously, in a random mutagenesis study, the (+)-anti diol epoxide of benzo[a]pyrene [(+)-anti-B[a]PDE] was shown to induce a complex mutational spectrum in the supF gene of an Escherichia coli plasmid, which included insertions, deletions and base substitution mutations, notably a significant fraction of GC
TA, GC
AT and GC
CG mutations. At some sites, a single type of mutation dominated and to understand individual mutagenic pathways these sites were chosen for study by site-specific means to determine whether the major adduct, [+ta]-B[a]PN2-dG, was responsible. [+ta]-B[a]PN2-dG was shown to induce ~95% G
T mutations in a 5'-TGC-3' sequence context and ~80% G
A mutations in a 5'-CGT-3' sequence context. (+)-anti-B[a]PDE induced principally GC
CG mutations in the G133 sequence context (5'-AGA-3') in studies using both SOS-uninduced or SOS-induced E.coli. Herein, [+ta]-B[a]PN2-dG is shown to induce principally G
A mutations (>90%) either without or with SOS induction in a closely related 5'-AGA-3' sequence context (identical over 7 bp). This is the first time that there has been a discrepancy between the mutagenic specificity of (+)-anti-B[a]PDE versus [+ta]-B[a]PN2-dG. Eight explanations for this discordance are considered. Four are ruled out; e.g. the second most prevalent adduct [+ca]-B[a]PN2-dG also induces a preponderance of G
A mutations (>90%), so it also is not responsible for (+)-anti-B[a]PDE-induced G133
C mutations. The four explanations not ruled out are discussed and include that another minor adduct might be responsible and that the 5'-AGA-3' sequence context differed slightly in the studies with [+ta]-B[a]PN2-dG versus (+)-anti-B[a]PDE. In spite of the discordance, [+ta]-B[a]PN2-dG induces G
A mutations in the context studied herein and this result has proven useful in generating a hypothesis for what conformations of [+ta]-B[a]PN2-dG are responsible for G
T versus G
A mutations.
Abbreviations: (+)-anti-B[a]PDE, 7R,8S-dihydroxy-9S,10R-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene; B[a]P, benzo[a]pyrene; GHD, gapped heteroduplex; MF, mutation frequency; O-G, 5'-TTTAG133AG131ACC-3'; PAH, polycyclic aromatic hydrocarbon.
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Introduction
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One of the yet unanswered questions in mutagenesis and carcinogenesis is what is the detailed mechanism by which carcinogen adducts induce mutations? In some cases, the mutational spectrum of a carcinogen can be quite complex, e.g. in random mutagenesis studies, we showed that 7R,8S-dihydroxy-9S,10R-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene [(+)-anti-B[a]PDE], which is a metabolite of benzo[a]pyrene (B[a]P), induces all classes of mutations in Escherichia coli, including base substitutions (45%), frameshifts (24%), insertions (23%) and deletions (8%) (1,2). [B[a]P is an example of a polycyclic aromatic hydrocarbon (PAH) and a ubiquitous environmental contaminant; reviewed in ref. 3.] This level of complexity has been observed by others in many mutational systems for a variety of activated PAH derivatives (summarized in refs 1,2), as well as for the activated forms of other bulky carcinogens (4,5), perhaps the most extensive and comprehensive study being with a series of benzo[c]phenanthrene diol epoxides (6).
To gain more insight into the mechanism of mutagenesis of these carcinogens, adduct site-specific mutagenesis studies in cells have been conducted on a variety of adducts from PAHs (discussed in ref. 5). Several important conclusions have been reached, notably that the pattern of mutations can be affected by the sequence context of a single stereochemically distinct adduct (713) and by the exact stereochemistry of the adduct (1014), as well as by the cell type in which the adduct is processed (12). However, it is unclear mechanistically how these differences influence the pathway of mutagenesis.
We have focused on base substitution mutations from (+)-anti-B[a]PDE in E.coli, which occur principally at G:C base pairs and GC
TA, AT and CG mutations are all significant (57, 23 and 20%, respectively, in SOS-induced cells; 1,2). To understand this mechanistically we felt it would be essential to find examples of sites where a single type of mutation was principally induced. In our random mutagenesis study with (+)-anti-B[a]PDE (1,2), we noted that G
T mutations were observed in 5'-TG-3' sequences in general and G
A mutations were obtained at G144 (5'-CGT-3'). Adduct site-specific mutational studies with the major adduct [+ta]-B[a]PN2-dG showed that G
T mutations dominated (>95%) in a 5'-TGC-3' sequence context (7) and G
A mutations (~82%) dominated in a 5'-CGT-3' (8,9) sequence context. These correlations suggested that G
T and G
A mutations from (+)-anti-B[a]PDE are due to its major adduct [+ta]-B[a]PN2-dG. It also shows that [+ta]-B[a]PN2-dG is capable of inducing a dramatically different spectrum of mutations depending upon sequence context.
While we have evidence that [+ta]-B[a]PN2-dG induces G
C mutations, this was in a 5'-CGG-3' sequence context where G
T and G
A mutations were also prevalent (10). Thus, we wished to identify a site where [+ta]-B[a]PN2-dG induced principally G
C mutations. In our random mutagenesis studies with (+)-anti-B[a]PDE, GC
CG mutations were exclusively observed (22/22) at G133 (5'-AGA-3') in cells that were SOS-uninduced or SOS-induced (1,2). G
C mutations were also induced at G133 by the analogous diol epoxide of dibenz[a,j]anthracene (15).
Based on these findings, herein we describe the construction of a plasmid (pRB1) with [+ta]-B[a]PN2-dG in a closely related 5'-AGA-3' sequence context and, in contrast to expectations, G
A mutations were principally obtained. We show that this discrepancy is not due to: the minor adduct [+ca]-B[a]PN2-dG, which also induces G
A mutations; anything related to SOS induction, since [+ta]-B[a]PN2-dG also induced principally G
A mutations without SOS; a minor difference in pH between our studies with [+ta]-B[a]PN2-dG (pH 7.5) versus [+]-anti-B[a]PDE (pH 6.8); or a leading versus lagging strand effect.
Four other possibilities for this discordance were not investigated. (i) A minor adduct other than [+ca]-B[a]PN2-dG might be responsible. (ii) Semi-targeted mutagenesis by a [+]-anti-B[a]PDE adduct at a site near G133 might be important. (iii) The plasmid origin was closer to the adduct in pUB3 (227 bp), which was used in the studies with [+]-anti-B[a]PDE, compared with pRB1 (465 bp), which was used in the studies with [+ta]-B[a]PN2-dG, potentially affecting which DNA polymerase replicates the adduct. (iv) The sequence context in the two studies was not exactly the same (for practical reasons; see Discussion) being 5'-GTCTTTAG133AG131ACG-3' with [+ta]-B[a]PN2-dG versus 5'-AGATTTAG133AG131TCT-3' with [+]-anti-B[a]PDE, which might be important.
Regarding the latter point, we abbreviate sequence contexts with a three letter code (e.g. 5'-AGA-3' in this case), which is for convenience of discussion and does not imply that we believe that adduct mutagenesis is dictated simply by the base on the immediate 5'- and 3'-sides of the adduct. In addition, we refer to the 5'-AGA-3' sequence studied herein as `G133' for convenience and simplicity and it is important to note that distal elements of the true G133 sequence context in the supF gene in our random mutagenesis study with (+)-anti-B[a]PDE are different (see previous paragraph).
Independent of the discrepancy, the fact that [+ta]-B[a]PN2-dG induced a preponderance of G
A mutations in the 5'-GTCTTTAG133AGACC-3' sequence context has proved useful in our ongoing efforts to determine the mechanism by which [+ta]-B[a]PN2-dG induces different kinds of base substitution mutations (16).
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Materials and methods
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(+)-anti-B[a]PDE (lot no. 92-356-91-19) was purchased from the National Cancer Institute Chemical Carcinogen Reference Standard Repository (Chemsyn Science Laboratories, Lenexa, KS). All B[a]P-containing material was handled as described previously (17), including working with it under yellow light. All other materials were as described previously (810), except for BsaI (New England Biolabs, Beverly, MA) and as noted below. Strains and plasmids were as described previously (810,18); notably, the genotype for ES87 cells can be found in Rodriguez et al. (18).
Synthesis and purification of oligonucleotides
The oligonucleotide 5'-TTTAG133AG131ACC-3' (O-G) was purchased from Midland Certified Reagent (Midland, TX) and purified by reverse phase HPLC prior to adduction (17). A total of four oligonucleotides were synthesized and are designated [+ta]-B[a]PG133, [+ca]-B[a]PG133, [+ta]-B[a]PG131 and [+ca]-B[a]PG131. Each of these was synthesized from O-G by a procedure (19,20) previously outlined (810). Reverse phase HPLC purification and subsequent analysis to establish both the stereochemistry and the position of the adduct were effectively identical to those used previously (810) following published procedures (1922). After completion of the reaction, the modified oligonucleotides were separated from unmodified material by reverse phase HPLC using a 550% acetonitrile gradient over 60 min (50 mM tetraethylammonium acetate buffer, pH 7.0). The ensemble of modified oligonucleotides were then separated from each other twice using a 2030% acetonitrile gradient over 60 min (50 tetraethylammonium acetate buffer, pH 7.0). HPLC involved a Hypersil-ODS 5µ 250x4.6 mM column (Keystone Scientific, Bellefonte, PA) and a Waters HPLC system (Millipore, Milford, MA), including a Waters Model 991 photodiode array detector (254 nm). Finally, the individual adducts were desalted on the same column by extensive washing with 2 mM sodium phosphate (pH 7.0), followed by a 10 min water wash and finally the sample was eluted in 50% methanol.
CD spectra were obtained in 20 mM phosphate buffer (pH 7.0), 100 mM NaCl at room temperature using a home built linear dichroism apparatus converted to CD operation and calibrated by means of d-camphorsulfonic acid, as described previously (19,20,22).
Following HPLC purification, each oligonucleotide was 32P-5'-end-labeled and purified successively by both denaturing and native polyacrylamide (20%) gel electrophoresis as described previously (810). Visualization was done using a Bio-Rad Molecular Imager (Hercules, CA) with the software Molecular Analyst/Macintosh v.2.1.
Plasmid constructions, transformations and mutagenesis studies
The strategy (Figure 1
) to study mutagenesis by B[a]PN2-dG adducts in the G133 sequence context was effectively identical to our previous study of mutagenesis by [+ta]-B[a]PN2-dG alone in the G115 (10) and G144 sequence contexts (8,9). In brief, oligonucleotides were ligated into a gapped heteroduplex (GHD) (Figure 1
, step 9) and the plasmids designated [+ta]-B[a]PG133-pRB1 and [+ca]-B[a]PG133-pRB1 have a B[a]PN2-dG adduct of the indicated stereochemistry in the G133 sequence context. The plasmids designated [+ta]-B[a]PG131-pRB1 and [+ca]-B[a]PG131-pRB1 have a B[a]PN2-dG adduct of the indicated stereochemistry in the G131 sequence context. In order to minimize the yield of plasmids from the non-adduct-containing strand, UV damage was incorporated into it (Figure 1
, step 4) as we have done previously (710).

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Fig. 1. Structures of (+)-anti-B[a]PDE and its major adduct [+ta]-B[a]PN2-dG, as well as the strategy to situate this adduct in a 5'-AGA-3 sequence context corresponding closely to G133 in the supF gene. ([+ca]-B[a]PN2-dG, the second most prevalent adduct, is identical to [+ta]-B[a]PN2-dG, except that the adduct bond is down rather than up.) (ad) [+ta]-B[a]PG133 (or [+ca]-B[a]PG133) was formed by reaction of O-G with (+)-anti-B[a]PDE and purified by HPLC, as well as native and denaturing gel electrophoresis. Steps 1 and 2: construction of pRB1 by the introduction of a duplex oligonucleotide (5'-TTTAGAGACC-3'/5'-GGTCTCTAAA-3') into the unique HindII site in pRE0. Steps 3 and 4: single-stranded pRB1 was isolated and UV irradiated (indicated by the dashed line). Steps 58: double-stranded pRE0 was digested with HindII and mixed with UV irradiated single-stranded pRB1 and denatured/renatured to give GHD DNA, which was isolated and purified. Step 9: [+ta]-B[a]PG133 was covalently incorporated into the GHD via ligation to give [+ta]-B[a]PG133-pRB1. In addition, [+ca]-B[a]PG133 was covalently incorporated into the GHD via ligation to give [+ca]-B[a]PG133-pRB1.
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C-pRB1, the unadducted control, was constructed in parallel. O-G, used to construct C-pRB1, was isolated from the adduction reaction between 5'-TTTAGAGACC-3' and (+)-anti-B[a]PDE and, subsequently, subjected to the identical purification conditions as its corresponding adducted counterparts. This ensures that the control vector truly reflected any potential background mutagenesis at G133.
Following ligation, the vectors were purified by gel exclusion chromatography (710), which results in the plasmid being in 10 mM TrisHCl, 1 mM EDTA (pH 7.5). For one experiment, [+ta]-B[a]PG133-pRB1 was ethanol precipitated and resuspended in 100 mM phosphate buffer (pH 6.8).
Each of these plasmids was transformed into SOS-induced, ES87 cells and progeny plasmids isolated as described previously (810). Our strategy was developed such that the sequence context of G133 is embedded in a unique BsaI restriction site (5'-GAGACC-3', G133 underlined), which provided a means to isolate progeny plasmids with mutations at G133 (or G131), because they are BsaI insensitive, as described previously (810). The sequence of mutants was determined by standard dideoxy sequencing methods, as described previously (810). Mutation frequency (MF) at G133 in the BsaI site was calculated essentially identically to the way it was done for the same adduct in a PstI site (7), in a Tth111I site (8,9) and in an EaeI site (10).
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Results
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Synthesis and purification of oligonucleotides containing [+ta]-B[a]PN2-dG and [+ca]-B[a]PN2-dG in the G133 sequence context
Oligonucleotides with [+ta]- and [+ca]-B[a]PN2-dG in the G133 sequence context (designated [+ta]- and [+ca]-B[a]PG133, respectively), as well as [+ta]- and [+ca]-B[a]PN2-dG in the G131 sequence context (designated [+ta]- and [+ca]-B[a]PG131, respectively), were synthesized by reacting (+)-anti-B[a]PDE with the corresponding unadducted oligonucleotide O-G (5'-TTTAG133AG131ACC-3') essentially as described previously (810). Products were separated by reverse phase HPLC and several peaks were isolated. HPLC purification was conducted twice and Figure 2
shows a mixture of the four adduct-containing products to indicate how they separated by our HPLC methods. Peaks containing oligonucleotides with either [+ca]-B[a]PN2-dG (Figure 2
, retention times 40.2 and 42.0 min) or [+ta]-B[a]PN2-dG (Figure 2
, retention times 45.0 and 49.0 min) were identified based on their characteristic CD spectra (Figure 3
), which appear as virtual mirror images and, notably, always give a negative and positive pyrenyl signal in the 300380 nm range for [+ta]-and [+ca]-B[a]PN2-dG, respectively (1922). These assignments of adduct stereochemistry were confirmed by digestion to the mononucleoside level followed by co-chromatography with a known adduct standard (data not shown), as done previously (19,21,22). The location of the adduct in each case was determined by modified MaxamGilbert DNA sequencing with DMS (data not shown), as done previously (20). In the original HPLC purification (Figure 2
) the peaks at 45.0 and 49.0 min contained [+ta]-B[a]PN2-dG at positions G131 and G133, respectively, while the peaks at 40.2 and 42.0 contained [+ca]-B[a]PN2-dG at positions G131 and G133, respectively.

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Fig. 2. Following two rounds of reverse phase HPLC purification, the oligonucleotides O-G (32 min), [+ca]-B[a]PG131 (40.2 min), [+ca]-B[a]PG133 (42 min), [+ta]-B[a]PG131 (45 min) and [+ta]-B[a]PG133 (49 min) were mixed and separated using the same conditions as used for purification (see Materials and methods).
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Fig. 3. CD spectra of the oligonucleotide 5'-TTTAG133AG131ACC-3' containing [+ta]- or [+ca]-B[a]PdG at G133 or G131. (ad) Spectra for the peaks at 49.0, 42.0, 45.0 and 40.2, respectively, in Figure 2 . The spectra in (a) and (c) are characteristic of a [+ta]-B[a]PN2-dG adduct with a negative signal in the 300380 nm range, while the spectra in (b) and (d) are characteristic of a [+ca]-B[a]PN2-dG adduct with a positive signal in the 300380 nm range (1215). CD spectra were obtained in 20 mM phosphate buffer (pH 7.0), 100 mM NaCl at room temperature using a home built linear dichroism apparatus converted to CD operation and calibrated by means of d-camphorsulfonic acid.
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Following HPLC purification, the oligonucleotides [+ta]-B[a]PG133, [+ca]-B[a]PG133, [+ta]-B[a]PG131, [+ca]-B[a]PG131 and the unadducted control (O-G) were purified (data not shown) by both native and denaturing polyacrylamide gel electrophoresis according to our standard methods (10). We have found that each of these three steps contributes to the purification (710), e.g. in this case HPLC resolved the stereo and positional isomers (Figure 2
), while native and denaturing polyacrylamide gel electrophoresis removed other, unknown contaminants. The products were analyzed on both denaturing and native polyacrylamide gels; the latter is shown (Figure 4
). Lanes 13 show [+ta]-B[a]PG133 in serial 10-fold decreasing concentrations. Lane 1 shows one major band, as well as a minor band, which migrated just below the major band and was difficult to quantitate, but was clearly less intense than the band in lane 3, showing that it was present at a level of <1%. Only a single band was apparent in the samples for [+ta]-B[a]PG131 (lanes 46), [+ca]-B[a]PG133 (lanes 79), [+ca]-B[a]PG131 (lanes 1012) and O-G (lanes 1315). [Only limited studies were performed with [+ta]-B[a]PG131 and [+ca]-B[a]PG131 (see Discussion), so they are not extensively considered.]

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Fig. 4. Analysis of oligonucleotides O-G (unadducted control) and [+ta]-B[a]PG133 and [+ca]-B[a]PG133, as well as [+ta]-B[a]PG131 and [+ca]-B[a]PG131. Following the reaction of (+)-anti-B[a]PDE with O-G (5'-TTTAG133AG131ACC-3'), the products were purified twice by HPLC (see Materials and methods). Peaks containing a single [+ta]- or [+ca]-B[a]PN2-dG adduct at G133 or G131 were isolated (see Materials and methods) and further purified (along with O-G) by both denaturing and native polyacrylamide gel electrophoresis. Subsequent analysis by native polyacrylamide gel electrophoresis and phosphorimaging gave the results for [+ta]-B[a]PG133 (lanes 13), [+ta]-B[a]PG131 (lanes 46), [+ca]-B[a]PG133 (lanes 79), [+ca]-B[a]PG131 (lanes 1012) and O-G (lanes 1315). In each set of three, the relative amount loaded was 1.0, 0.1 and 0.01.
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Construction of C-pRB1, [+ta]-B[a]PG133-pRB1 and [+ca]-B[a]PG133-pRB1
The basic strategy for our studies (Figure 1
) follows that described in Shukla et al. (8,9) and Jelinsky et al. (10). One key feature is that an adduct at position G133 is embedded in a BsaI restriction site (5'-GAGACC-3', G133 position underlined), which must ultimately be a unique site in our construct. [+ta]-B[a]PG133, [+ca]-B[a]PG133 and O-G were each ligated individually into a vector containing an appropriate 10 base, single-stranded gap (Figure 1
, step 9) to give products designated [+ta]-B[a]PG133-pRB1, [+ca]-B[a]PG133-pRB1 and C-pRB1, respectively. The efficiency of ligation was estimated to be ~71, 59 and 73%, respectively (data not shown).
The products [+ta]-B[a]PG133-pRB1, [+ca]-B[a]PG133-pRB1 and C-pRB1 were characterized. One means to establish that a particular restriction site contains an adduct has been to show that the adduct blocks cleavage by the corresponding restriction endonuclease (BsaI in this case) (710). The presence of an adduct in [+ta]- and [+ca]-B[a]PG133-pRB1 inhibited cleavage by BsaI in the adduct-containing strand, but, interestingly, the non-adduct-containing strand was cleaved by BsaI, albeit slowly (data not shown).
A second method was used to show that [+ta]- and [+ca]-B[a]PG133-pRB1 contained a lesion. [+ta]-B[a]PG133-pRB1 was digested with PstI and BamHI, which should liberate, following denaturation, an adduct-containing, 32 nt fragment (sequence 5'-TGCAGGTCTTTAG133AG131ACCGACTCTAGAGGATC-3', where the underlining shows the sequence of O-G). When analyzed by denaturing polyacrylamide gel electrophoresis, [+ta]- and [+ca]-B[a]PG133-pRB1 each gave one major band (Figure 5
, lanes 4 and 6, respectively), which migrated more slowly than the corresponding major band from C-pRB1 (lane 2), because the presence of the adducts retards migration. Both [+ta]- and [+ca]-B[a]PG133-pRB1 gave minor bands, which are only apparent following a darker exposure than that shown in Figure 5
. In each case these minor bands appeared at positions ~26 and ~20 and are likely to be attributable to ligation of a small fraction (~2%) of the adducted oligonucleotide into the gapped duplex on the 3'- but not the 5'-side and on the 5'- but not the 3'-side, respectively. These kinds of incomplete ligation products have been observed in the past using similar construction strategies (810,17,23). [We have purposely decided not to purify B[a]P-containing closed circular vectors for reasons discussed in footnote 4 in ref. 8.] A single major band appears in the control sample (C-pRB1) as well (Figure 5
, lane 2); however, there is also a minor contaminant above the major band (~3% level) of unknown origin.

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Fig. 5. Characterization of C-pRB1, [+ca]-B[a]PG133-pRB1 and [+ta]-B[a]PG133-pRB1, as well as [+ca]-B[a]PG131-pRB1 and [+ta]-B[a]PG131-pRB1, following the liberation of an adduct-containing, 32 nt fragment. C-pRB1 (lane 2), [+ca]-B[a]PG131-pRB1 (lane 3), [+ca]-B[a]PG133-pRB1 (lane 4), [+ta]-B[a]PG131-pRB1 (lane 5) and [+ta]-B[a]PG133-pRB1 (lane 6) were each digested with PstI and BamHI, which should liberate a 32 nt fragment of sequence 5'-TGCAGGTCTTTAG133- AG131ACCGACTCTAGAGGATC-3', where the underlining shows the sequence of the original oligonucleotide prior to ligation into the GHD (Figure 1 , step 9). The samples were separated by denaturing polyacrylamide gel electrophoresis and analyzed by phosphorimaging. For size comparisons, lane 1 shows a ladder with bands at every 2 nt between 16 and 32 in length.
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Mutants derived from [+ta]-B[a]PG133-pRB1, [+ca]-B[a]PG133-pRB1 and C-pRB1
[+ta]-B[a]PG133-pRB1, [+ca]-B[a]PG133-pRB1 and C-pRB1 were each transformed into ES87 cells, which are wild-type for all known DNA repair functions (18). [ES87 cells were used so we could compare our results with our previous random adduction experiments with (+)-anti-B[a]PDE (1,2).] The progeny yield ratio [+ta]-B[a]PG133-pRB1:[+ca]-B[a]PG133-pRB1:C-pRB1 was 0.67:0.59:1.0 in one transformation. [These values are difficult to interpret for reasons discussed in Jelinsky et al. (10).] Mutations at position G133 eliminate the unique BsaI site in pRB1, rendering progeny plasmids resistant to cleavage by BsaI, which was used as the basis of the enrichment for mutations in the G133 region (810). Individual mutants were isolated and sequenced. Mutagenesis data (Table I
) are the composite of two independent constructions/transformations in all cases except the data at pH 6.8 for [+ta]-B[a]PG133-pRB1.
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Table I. Base substitution mutations in the BsaI site in progeny plasmids derived from [+ta]-B[a]PG133-pRB1 and [+ca]-B[a]PG133-pRB1 at position G133 and the corresponding non-adduct-containing control plasmid (C-pRB1) following transformation into ES87 cells
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[+ta]-B[a]PG133-pRB1 principally induced G
A base substitutions at position G133, both +SOS (~96%) and SOS (~93%) (Table I
). In one experiment, the vector [+ta]-B[a]PG133-pRB1 was placed in 100 mM phosphate buffer (pH 6.8), rather than 10 mM TrisHCl (pH 7.5), prior to transformation, but this did not affect the preference for G
A mutagenesis (Table I
). [+ca]-B[a]PG133-pRB1 also principally induced G
A base substitutions at position G133, both +SOS (~98%) and SOS (~93%) (Table I
).
MF at G133 was reasonably low with the control vector C-pRB1 (MF <~0.025% +SOS; MF ~0.05% SOS). For reasons discussed previously (10), values for MF are internally consistent and, thus, MF values at G133 for the adduct-containing vectors [+ta]-B[a]PG133-pRB1 (~1.0% +SOS; ~0.21% SOS) and [+ca]-B[a]PG133-pRB1 (~1.8% +SOS; ~0.15% SOS) are larger than the control C-pRB1, most significantly for the +SOS samples (>~40-fold for [+ta]-B[a]PG133-pRB1; >~70-fold for [+ca]-B[a]PG133-pRB1). We note that these values of MF cannot be interpreted as the inherent MF for [+ta]- or [+ca]-B[a]PN2-dG in the G133 sequence context, as discussed in Jelinsky et al. (10). Stray mutations at positions other than G133 (Table I
) are unlikely to be attributable to the adduct and are ignored.
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Discussion
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The adduct-containing oligonucleotides were purified by HPLC and native and denaturing gel electrophoresis using methods that we used previously (810). Because the four adduct-containing oligonucleotides were well resolved (Figure 2
), they were purified twice by HPLC. There was less resolution between the adducted oligonucleotides using native (Figure 4
) and denaturing (data not shown) polyacrylamide gel electrophoresis, although some separation was achieved and, importantly, these methods remove other types of impurities (data not shown; discussed in ref. 10). Adduct positioning (data not shown) and adduct assignment (e.g. the CD spectra in Figure 3
) were definitive.
Because neither [+ta]-B[a]PN2-dG nor [+ca]-B[a]PN2-dG are appreciably more mutagenic than the other when located at G133 (Table I
), it is unlikely that either is sufficiently contaminated with the other (see for example Figure 3
) to make it plausible that the mutational results attributed to each adduct in Table I
are due to cross-contamination. Aside from cross-contamination, the possibility that the results in Table I
are due to some cryptic contaminant in the oligonucleotides is remote, because there is no evidence for such a contaminant at the level of <1% (Figure 4
), which is in the range of or lower than the MF obtained for the adducted vectors (Table I
). There is also no precedent for the generation of such a putative contaminant using the relatively simple and gentle procedures of synthesis and purification that we have used over a 10 year period (see for example refs 710,17,23,24). The MF for [+ta]-B[a]PN2-dG at G133 (~1.0%; Table I
) is in the range that we have observed in the past for this lesion (0.21.0%; 710) and MF for both [+ta]-B[a]PN2-dG and [+ca]-B[a]PN2-dG is enhanced by SOS induction, each suggesting that the results in Table I
are attributable to the lesions of interest. Finally, it is unlikely that the results attributed to [+ta]-B[a]PN2-dG and [+ca]-B[a]PN2-dG in Table I
are due to vector contamination, since a higher MF was obtained in the adduct-containing versus control plasmids, especially +SOS (Table I
).
In a limited study (i.e., one construction/transformation), pRB1 with [+ta]-B[a]PN2-dG at position G131, which is also a 5'-AGA-3' sequence, gave 21 G131
A (~80%), five G131
T and no G131
C mutations (MF ~1.2%). While this result is certainly less definitive, it suggests that [+ta]-B[a]PN2-dG does indeed induce a preponderance of G
A mutations in (at least certain) 5'-AGA-3' sequence contexts. pRB1 with [+ca]-B[a]PN2-dG at position G131 gave eight G131
A, seven G131
T and three G131
C mutations (MF ~1.6%) in a parallel, limited study. This result suggests that a strong preference for G
A mutations is not absolute, which argues that the results in the study reported herein are not some grand artifact.
The discrepancy in a 5'-AGA-3' sequence context between G
A mutations obtained with [+ta]-B[a]PN2-dG versus G
C mutations obtained with (+)-anti-B[a]PDE
In our random mutagenesis studies, (+)-anti-B[a]PDE induced exclusively GC
CG mutations in the 5'-AGA-3' sequence context at position G133 in the supF gene of plasmid pUB3 (1,2), while herein [+ta]-B[a]PN2-dG induced virtually exclusively G
A mutations in a similar, but not identical, 5'-AGA-3' sequence context (Table I
).
This raises the question why did we not use a perfectly identical DNA sequence context? First, based on our three other adduct site-specific studies with [+ta]-B[a]PN2-dG (710), there was no precedent for distal changes in sequence context significantly affecting mutagenic outcome. Second, we have found that it is helpful to place an adduct in a unique restriction site, because this aids in vector characterization, in mutant isolation and in vector purification, although we have not taken advantage of the latter for a number of years (4). Embedding the adduct in a BsaI sequence context comes closest to allowing a match with the G133 sequence in supF. Finally, our adduct site-specific studies are done in double-stranded DNA vectors, because we believe that adduct conformation plays an important role in mutagenesis and our original work with (+)-anti-B[a]PDE was done in double-stranded pUB3 (1,2). This concern precludes our being able to use simpler single-stranded DNA systems.
We can think of eight possible explanations for the difference between our studies at G133 with [+ta]-B[a]PN2-dG (herein) versus with (+)-anti-B[a]PDE (1,2).
Four possibilities that we can exclude.
The cis adduct, [+ca]-B[a]PN2-dG, which is the most important minor adduct (21,25), might be preferentially mutagenic at G133. This is ruled out since [+ca]-B[a]PN2-dG also induced a preponderance of G
A mutations (Table I
).
Although we attempted to use the same level of SOS induction in our studies with [+ta]-B[a]PN2-dG (710 and herein) as we used with (+)-anti-B[a]PDE (1,2), we had no independent measure of the level of SOS induction in any case. Because G
C mutations were exclusively induced by (+)-anti-B[a]PDE at G133 in SOS cells, while G
A mutations predominated for [+ta]-B[a]PN2-dG in SOS cells, a difference in SOS induction cannot explain the difference, since these data were generated without SOS induction.
G133
C mutations dominated in random mutagenesis studies when (+)-anti-B[a]PDE-adducted pUB3 (supF target gene) was incubated at pH 8.0 or 6.8 prior to transformation (2), while pH 7.5 was used herein with [+ta]-B[a]PN2-dG. [The data in Rodriguez and Loechler (2) reported as `heat' used (+)-anti-B[a]PDE-adducted pUB at pH 8.0, while `+heat' used (+)-anti-B[a]PDE-adducted pUB at pH 6.8.] Although unlikely, we showed that pH is not a factor, given that [+ta]-B[a]PN2-dG also induced G
A mutations at pH 6.8 (Table I
).
In each of our previous three adduct site-specific studies (7,8,10), [+ta]-B[a]PN2-dG was situated in the lagging strand of a ColE1 plasmid, because each was performed to probe the (+)-anti-B[a]PDE mutagenic mechanism for a particular dG in supF that was also in the lagging strand. [Leading versus lagging strand replication is discussed in footnote 2 of Jelinsky et al. (10). The adduct site-specific study of [+ta]-B[a]PN2-dG in the 5'-TGC-3' sequence context (4) did not match any particular 5'-TG-3' sequence context in the supF gene in our studies with (+)-anti-B[a]PDE (1,2). However, in four 5'-TG-3' sequences (2), GC
TA mutations predominated: 13/15 in the lagging strand (at G99 and G102) and 12/12 in the leading strand (at G118 and G127).] Position G133 is in the leading strand of supF, so we conducted our adduct site-specific study reported herein with [+ta]-B[a]PN2-dG in the leading strand, eliminating this as a possible reason for the difference.
Four other possibilities that we cannot exclude.
(A more thorough discussion of these points is available upon request.) The ColE1 plasmid origin was farther from the adduct at G133 in our studies with [+ta]-B[a]PN2-dG using pRB1 (465 bp) than in our studies with (+)-anti-B[a]PDE using pUB3 (227 bp). Given the unique nature of ColE1 plasmid replication (2628), the closer proximity of the G133 position to the ColE1 origin in pUB3 may have resulted in a different DNA polymerase being responsible for bypass compared with our study with pRB1. An adduct mutagenic difference in the lagging versus leading strand of a ColE1 plasmid has been noted (29) and recent work suggests that these differences may indeed be due to a different DNA polymerase being responsible (R.P.P.Fuchs, personal communication). If this explanation were at the root of the discrepancy, then it is more likely that DNA polymerase III replication occurred with [+ta]-B[a]PN2-dG in the study reported herein, because the adduct was farther from the origin in pRB1.
The sequence context at position G133 in our (+)-anti-B[a]PDE mutational studies in supF (5'-AGATTTAG133AG131TCT-3') differs at the fifth base on the 5'-side and the third base on the 3'-side of the adduct compared with the study herein for [+ta]-B[a]PN2-dG (5'-GTCTTTAG133AG131ACC-3'). It is possible that, while distal changes have not led to discordance in our previous studies (710), distal changes significantly affected mutagenic outcome at G133. In fact, this is plausible, given the results at other 5'-AGA-3' sequence contexts in the supF gene (1,2). The notion that [+ta]-B[a]PN2-dG can indeed induce G
A mutations in at least some 5'-AGA-3' sequence contexts is reinforced by our limited study of [+ta]-B[a]PN2-dG in the G131 sequence context, which gave ~80% G
A mutations (see above).
Although [+ca]-B[a]PN2-dG is the best characterized minor adduct from (+)-anti-B[a]PDE, there are reports of other adducts being formed at dG and dC (3036), although these must all be very minor (<3%; 33). A minor adduct at the G:C base pair at position C133 could account for the apparent discrepancy.
The G133
C mutations observed in our random mutagenesis studies with (+)-anti-B[a]PDE could have been due to semi-targeted mutagenesis from an adduct located at a base pair other than G133. This seems unlikely given that semi-targeted mutagenesis is usually less prevalent than targeted mutagenesis (discussed in ref. 7) and virtually no mutations were observed at base pairs 130140 in supF in our random mutagenesis studies with (+)-anti-B[a]PDE, except at G133 (1,2).
Implications of [+ta]-B[a]PN2-dG-induced G
A mutations in a 5'-AGA-3' sequence context
Approximately 95% of base substitution mutations from (+)-anti-B[a]PDE were induced at G:C base pairs and GC
TA and GC
AT were most prevalent (57 and 23%, respectively, with the remainder being GC
CG) (1,2). We have now identified two sequence contexts where [+ta]-B[a]PN2-dG induces a preponderance of G
A mutations (i.e. 5'-CGT-3 and 5'-AGA-3') and one sequence context where it induces a preponderance of G
T mutations (i.e. 5'-TGC-3'). These results show conclusively that there is nothing inherent about the kinds of mutations that [+ta]-B[a]PN2-dG induces. In fact, we have shown that [+ta]-B[a]PN2-dG induced 57/58 G
T mutations in a 5'-TGC-3' sequence context (4) and 46/48 G
A mutations in a 5'-AGA-3' sequence context (Table I
). This is a dramatic difference and a change from a ratio of 57:1 to 2:46 in energetic terms amounts to a change of ~4.3 kcal/mol in something related to the mutagenic mechanism.
While it is still unknown how a single adduct, such as [+ta]-B[a]PN2-dG, is able to induce such a dramatically different pattern of mutations in different DNA sequence contexts, it is most likely due to its ability to adopt different conformations in DNA (16,37). In this regard, B[a]PN2-dG adducts of various stereochemistries have been shown to adopt a large number of adduct conformations (reviewed in ref. 38), including examples where the dG moiety of an adduct retains base pairing potential, as well as base displaced and intercalated conformations, where the adduct does not retain base pairing potential. Based on these NMR results, our mutagenesis results and our molecular modeling results (16), we have proposed a hypothesis that a base displaced conformation of [+ta]-B[a]PN2-dG with the dG moiety in the major versus minor groove is responsible for G
T versus G
A mutations, respectively.
 |
Acknowledgments
|
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We gratefully acknowledge the Cancer Research Program of the National Institutes of Health, Division of Cancer Cause and Prevention (Bethesda, MD) for providing (+)-anti-B[a]PDE. This work was supported by grants from the NIH to E.L.L. (ES03775) and to N.E.G. (CA20851). The phosphorimager used in this work was purchased with National Institutes of Health Shared Instrumentation grant no. RR11397.
 |
Notes
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2 To whom correspondence should be addressed Email: loechler{at}bio.bu.edu 
 |
References
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Received August 17, 1998;
revised September 17, 1998;
accepted September 25, 1998.