©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
In vivo and in Vitro Replication Consequences of Stereoisomeric Benzoapyrene-7,8-dihydrodiol 9,10-Epoxide Adducts on Adenine N at the Second Position of N-ras Codon 61 (*)

(Received for publication, May 19, 1994; and in revised form, November 16, 1994)

Parvathi Chary (1) Gary J. Latham (1) (2) (3) Donald L. Robberson (1) (5) Seong J. Kim (4) Shin Han (4) Constance M. Harris (2) (4) Thomas M. Harris (2) (4) R. Stephen Lloyd (1)(§)

From the  (1)Sealy Center for Molecular Science, The University of Texas Medical Branch, Galveston, Texas 77555, the (2)Center in Molecular Toxicology and the (3)Departments of Biochemistry and (4)Chemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37235, and the (5)Department of Molecular Genetics, University of Texas, M. D. Anderson Cancer Center, Houston, Texas 77030

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Benzo[a]pyrene-7,8-dihydrodiol 9,10-epoxide (BPDE), a metabolite of the widespread environmental pollutant benzo[a]pyrene, is mutagenic in both bacterial and mammalian systems. Toward understanding the mutagenic effects of different stereoisomers of BPDE at specific sites in DNA, six stereochemically defined BPDE adducts were constructed on adenine N^6 at position 2 of the human N-ras 61 codon within an 11-base oligonucleotide fragment. Both the nonadducted and BPDE-adducted N-ras 61 11-mers were inserted into a unique EcoRI site in single-stranded M13mp7L2 DNA and utilized for in vivo studies. The ligation efficiencies of BPDE-adducted 11-mers into the single-stranded vector were determined by Southern hybridization and confirmed by electron microscopy. Repair-deficient AB2480 E. coli cells were transformed with adducted and nonadducted DNA samples. The resultant plaque-forming abilities were used to evaluate the replication competence of the various BPDE adducts with respect to the nonadducted 11-mer. Point mutations due to aberrant replication at the adducted site were identified by the technique of differential DNA hybridization. All of the six BPDE adducts examined were mutagenic in vivo, generating exclusively AG mutations at frequencies ranging from 0.26 to 1.20%. In vitro replication studies using these BPDE-adducted 11-mers involved primer extension assays with Klenow fragment. All of the BPDE-modified templates demonstrated distinct blockage at the adducted site and/or 1 base 3` to the adducted site, allowing essentially no translesion synthesis to form fully extended polymerization products in vitro.


INTRODUCTION

Polycyclic aromatic hydrocarbons (PAH) (^1)are pervasive in the environment, arising during combustion processes. Some of these PAH are carcinogens. Benzo[a]pyrene (BP) is one such PAH that has received intense study in an attempt to define mechanisms of genotoxicity. This compound is metabolically activated to bay region 7,8-dihydrodiol 9,10-epoxides that initiate mutagenesis and carcinogenesis by covalently binding to DNA(1, 2, 3) . The mutagenic potential of these diol epoxides is dependent on a variety of interactions including ones between the carcinogen and the template, the nature of the polymerase involved in replication past the adduct, and the efficiency of DNA repair within the cell. DNA lesions caused by such carcinogens if improperly repaired, may be converted to permanent changes in the genome during DNA replication. These changes involve base substitutions, deletions, or frameshift mutations that eventually can lead to neoplastic transformation(2) . Incubation of BPDE with DNA containing ras genes can result in oncogenesis(4, 5) . The mechanism by which these ras genes are activated in tumor cells often involves a single point mutation, usually resulting in the alteration of amino acid residue 12 or 61 of the protein encoded by these genes(4) . In vitro mutagenesis experiments showed that activating mutations could also occur at codons 13, 59, and 63(6, 7) .

There is considerable evidence to demonstrate that primary nucleotide sequence can modulate the stereo-selectivity and distribution of BPDE lesions in modified DNA(8, 9, 10) . Thus, carcinogenic species may be biased toward or against certain bases because of the stereoelectronic effects of adjacent bases or the stereochemistry of the ultimate carcinogen(11) . Furthermore, preferential site-specific mutation at a particular position could be due to the relative stability of the adduct or the lack of significant structural distortions in the nucleotide caused by the carcinogen(12) . The interaction of these PAH diol epoxides with numerous sites in DNA involves a nucleophilic attack in every case at the benzylic carbon of the epoxide resulting in a S

The heterogeneity of base adduction by BPDE is significantly reduced by synthesizing optically pure (+)- or (-) -anti or -syn enantiomers. Variations in biological activity between enantiomers within a given test system are likely to be due to different conformations assumed by these adducts. Studies that differentiate between effects due to adducts produced by cis and trans addition of anti- and/or syn-BPDE to DNA are also gaining importance(16, 17) . Studies examining the metabolism of BP in 3-methylcholanthrene-treated rats have shown four possible BPDE isomers. The ratios of formation for (+)-anti-BPDE/(-)-syn-BPDE/(-)-anti-BPDE/(+)-syn-BPDE were 214:36:24:1, respectively.(18, 19, 20, 21) . However the adduct-forming potentials of these BPDE isomers on adenine have not been firmly established. In spite of the availability of considerable information on both the binding spectra and mutational specificity of BPDE, little is known about the relationship between these two factors within specific sequences. Template-directed mutagenesis employing oligodeoxynucleotides bearing stereo-specific and site-specific lesions offer the possibility of correlating a stereochemically-defined adduct with a particular mutation spectrum. The mutagenicities of these stereoisomers, however, are different in bacterial and mammalian cells (22, 23, 24) . Single-stranded vectors carrying a defined, uniquely located lesion are powerful tools for investigating mutagenic mechanisms in vivo both in prokaryotic and eukaryotic systems(25, 26) .

The objective of this study was to correlate in vivo and in vitro replication competence with BPDE adduct chirality. Toward this goal, six stereochemically-defined BPDE adducts were constructed on adenine N^6 at position 2 of N-ras codon 61 within an 11-base oligodeoxynucleotide by the postoligomerization strategy(27, 28) .


EXPERIMENTAL PROCEDURES

Bacterial Strains and Culture Conditions

The strain of Escherichia coli used for extraction of single-stranded M13mp7L2 DNA was UT481 (met thyDelta(lac-pro) hsdR BamHI hsd M+ sup DTn10/F` tra D 36 pro AB lacI^qZDeltaM15). Cells were grown in minimal medium at 37 °C, and overnight shaking cultures (200 rpm) were diluted 1:100 in Luria-Bertani broth and grown to an A of 0.2. M13mp7L2 phage were used to transfect these cells. Bacterial cells were harvested at the end of 16 h, and single-stranded phage DNA was isolated from the supernatant. Repair-deficient AB2480 (uvrA16 recA13, courtesy of Dr. A. Ganesan, Stanford University) E. coli cells were utilized to study the replication competence and mutagenesis of N-ras 61-adenine-M13mp7L2 templates by transfecting these cells with DNA bearing different stereoisomeric adducts of BPDE. Shaking cultures were routinely grown at 37 °C to an A of 0.35 for transfection, while the feeder cells were grown to stationary phase.

Isolation of Single-stranded DNA and Insertion of N-ras 61-containing Oligonucleotide

Single-stranded M13mp7L2 DNA (a gift from Dr. C. Lawrence, University of Rochester) was isolated as described by Sambrook et al.(29) . A unique EcoRI site within an engineered hairpin loop was utilized to linearize the phage DNA. This restriction digested DNA was passed through a Nensorb column (Bio-Rad) for subsequent purification. A fraction of the endonuclease-digested DNA was analyzed by electrophoresis through a 1.4% agarose gel and visualized by ethidium bromide staining. The distinctive greater mobility of linearized single-stranded DNA relative to the corresponding circular DNA confirmed that the reaction proceeded to completion. The linearized M13mpL2 DNA was reconstituted in 10 mM Tris-HCl (pH 8.0), 1 mM EDTA at a concentration of 0.5 µg/µl and stored at -20 °C.

BPDE-adducted N-ras 61-containing 11-mers were synthesized by the method of Kim et al. (28) and purified as described by Latham et al.(30) . Both the nonadducted and the BPDE-adducted N-ras 61-containing oligonucleotides were phosphorylated with T4 polynucleotide kinase (New England Biolabs Inc., Beverly, MA). A 100-fold molar excess of the different N-ras 61 11-mers relative to the amount of linearized vector were individually ligated together in the presence of a 2-fold excess of a 51-mer scaffold(25, 30) . Each reaction was incubated overnight at 16 °C with a total of 400 units of T4 DNA ligase (New England Biolabs Inc.).

Determination of Ligation Efficiencies

The efficiency of incorporation of the nonadducted and BPDE-adducted N-ras 61 11-mer into the unique EcoRI site of M13mp7L2 DNA was monitored by electrophoresis on a 1.4% agarose gel and subsequent Southern blot analysis. A P-end labeled probe was used that was complementary to the N-ras 61 11-mer with the flanking M13mp7L2 sequences. Both singly and doubly ligated DNA molecules were identified by autoradiography, but only doubly ligated molecules were utilized to calculate ligation efficiencies. Prehybridization and hybridization was performed at approximately 25 °C for 16 h. Densitometric scanning of autoradiographs (Hyperfilm MP from Amersham Corp.) was performed with the VISAGE gel electrophoresis analysis system (BioImage, Ann Arbor, MI).

Ligation efficiencies were further confirmed by electron microscopy subsequent to the removal of the 51-mer scaffold. Samples were prepared using the formamide modification of the basic protein (Kleinschmidt) technique as described by Davis et al.(31) . Grids were rotary shadowed with platinum/palladium in a ratio of 80:20 and examined in either a Philips 300 or 410 electron microscope. Projected images were traced from photographic negatives, and lengths were determined with a map measure.

Bacterial Replication and Mutagenesis of N-ras 61-Ade-M13mp7L2

Repair-deficient AB2480 (uvrA, recA) E. coli cells were transfected with 0.5 µg of the N-ras 61-Ade-M13mp7L2 DNA by the calcium chloride/rubidium chloride procedure(29) . The 51-mer scaffold was displaced from the ligated template prior to transfection by the addition of a 5-fold excess of a sequence complementary to the 51-mer (c51-mer) followed by heat denaturation for 2 min at 95 °C and rapid cooling on ice. After correcting for ligation efficiencies, the plaque-forming abilities of different N-ras 61 adducts in M13mp7L2 represented the frequency of replication bypass through the BPDE lesions. The error rate of bypass and the spectrum of point mutations resulting from in vivo replication was determined by differential hybridization. Plaques were transferred onto nitrocellulose filters by successively lifting each plate 4 times. Each set of filters was hybridized with about 100 pmol of one of four P-labeled 17-mer oligonucleotides constituting an 11-base complementary region to the N-ras 61 sequence. Each probe differed by only 1 base at the site opposite the adducted position and was represented by either A, T, C, or G. Hybridization was performed at 39 °C for 16 h, a stringency at which only perfect matches in sequence could be scored as positives. Prehybridization and hybridization solutions constituted 5 times saline/sodium phosphate/EDTA (SSPE), 5 times Denhart's solution, 50 µg/ml fish milt DNA, 0.1% SDS, and 20% formamide. Excess radiolabel was removed by washing the filters 3 times with 2 times SSPE at 39 °C for 10 min on each wash. Representative samples of wild-type and mutant plaque DNA that had been identified by the technique of differential hybridization were confirmed by dideoxy sequencing(32) . Control assays were performed in vivo utilizing M13mp7L2 DNA containing 11-mer inserts with N-ras codon 61, wherein the adenine at position 2 (designated 61^2) was modified to C, G, and T successively and hybridized with their complementary 17-mer probes. Parallel in vitro control assays involved dot blot assays with 51-mers bearing the 11-mers, wherein the N-ras 61^2 adenine was replaced by C, G, and T and correspondingly hybridized with the complementary probes.

Enzymatic Construction of BPDE-adducted 33-mers for Primer Extension Assays

To facilitate in vitro primer extension of the 11-mer oligonucleotide bearing various stereochemically-defined BPDE adducts, 33-mers were constructed for the stable alignment of the primer. As detailed by Latham et al.(30) , a 5-fold molar excess of a 22-mer bridge was utilized relative to the various N-ras 61 11-mers. The 5` phosphorylated 22-mer bridge included a 17-base terminal region complementary to the M13 sequencing(-40) primer and a portion of the EcoRI site from the M13mp7L2 sequence. The N-ras 61 11-mers with the remaining sequence of the restriction site were 5`-end labeled with 1:100 labeled/unlabeled ATP. A 27-mer scaffold utilized in 3-fold excess to the two oligonucleotides was designed to align the 5`-PO(4) of the 22-mer in proximity to the 3`-OH of the N-ras 61 11-mer. Ligation reactions were performed in the presence of 2000 units of T4 ligase at 16 °C for 48 h. Ligated 33-mer products were separated on 10% polyacrylamide gels and detected by subsequent autoradiography. The corresponding bands were excised and DNA eluted in water. Excess urea was eliminated, and the oligonucleotides precipitated in absolute alcohol.

In Vitro DNA Synthesis

M13 sequencing(-40) primer was end-labeled with [-P]ATP (3000 Ci/mmol) (DuPont NEN) and added to BPDE-adducted or nonadducted 33-mer templates in an equimolar ratio of 0.5 pmol each. The primer was annealed to various templates at 65 °C for 2 min and slow-cooled to <30 °C. One unit (0.5 pmol) of Klenow fragment was utilized per reaction in a volume of 20 µl. Reactions were performed at 37 °C under conditions specified by Latham et al.(30) . Aliquots of reactions were terminated at various time points ranging from 2 to 30 min. Primer-extended products were resolved on 15% denaturing polyacrylamide sequencing gels with 8.3 M urea (30 cm times 35 cm times .04 cm) and visualized by autoradiography. Various exposures of the gel to Hyperfilm MP (Amersham Corp.) enabled quantification of the percentage of extended primers by densitometry using the VISAGE gel electrophoresis analysis system (BioImage, Ann Arbor, MI). Band intensities were further confirmed by the use of the PhosphorImager (Molecular Dynamics, Sunnyvale, CA).


RESULTS

Purity of Stereochemically-defined BPDE Adducts

Oligodeoxynucleotides containing stereo-specifically and site-specifically placed lesions are of great value in studying single adduct mutagenesis in bacterial and mammalian systems(17, 28) . DNA templates with defined adducts can also be utilized in in vitro studies to evaluate the replication capability of various DNA polymerases in the vicinity of the lesion(16, 23) . To investigate the effect of individual stereoisomers in vivo and in vitro, six stereochemically-specific BPDE adducts on the N^6 of adenine at position 2 of N-ras codon 61 on an 11-mer sequence were synthesized. The six modified N-ras codon 61 oligodeoxynucleotides contained (+)- or (-)-anti-trans-, (+)- or(-)-syn-trans-, or (+)- or(-)-anti-cis-BPDE adducts (Fig. 1). Samples were analyzed and purified by HPLC, polyacrylamide gel electrophoresis, and capillary gel electrophoresis. In addition, the oligodeoxynucleotides were digested with nuclease P1 followed by snake venom phosphodiesterase and alkaline phosphatase, and the resulting nucleoside mixture was analyzed by HPLC. Only the expected nucleosides were observed (data not shown).


Figure 1: Structures of adenine N stereoisomers placed at the second position of N-ras codon 61 within an 11-mer oligodeoxynucleotide.



The purities of 11-mers containing the isomeric dA-BPDE adducts were also analyzed on 15% polyacrylamide sequencing gels subsequent to labeling the 5` terminus using T4 polynucleotide kinase and [-P]ATP. Electrophoretic migration of BPDE-modified oligomers was distinctly slower than that of unmodified 11-mers (Fig. 2). Slight differences in mobility pattern were also observed among the various adducted 11-mers. Gels exposed for longer periods revealed no traces of unmodified 11-mer contaminants in the six dA-modified oligodeoxynucleotides.


Figure 2: Polyacrylamide gel electrophoresis of 11-mers containing a single isomeric dA N^6-BPDE adduct. Oligodeoxynucleotides were labeled with P at the 5` terminus and subjected to electrophoresis through 15% polyacrylamide gels (33 times 44 times 0.04 cm). The BPDE-adducted oligomers are shown in lanes2-7. Lane1, unmodified 11-mer; lane2, (-)-syn-trans-BPDE 11-mer; lane3, (+)-syn-trans-BPDE 11-mer; lane4, (+)-anti-cis-BPDE 11-mer; lane5, (-)-anti-cis-BPDE 11-mer; lane6, (+)- anti-trans-BPDE 11-mer; lane7, (-)-anti-trans-BPDE 11-mer.



Control Assays for Detection of Specific Point Mutations at Position 2 of N-ras Codon 61

Prior to presenting the data concerning the biological fate of various BPDE-adducted 11-mers that were introduced into the unique EcoRI site in M13mp7L2 DNA, it was important to determine if all point mutations could be detected with the same frequency at position 2 of N-ras codon 61. Given that the ras gene is known to exhibit high secondary structure, 11-mers bearing the N-ras codon 61, differing only by AC or AG or AT at position 2, were inserted into the phage DNA and detected by differential hybridization and then plaque purified (Fig. 3). High titer phage stocks were readily produced, indicating that there was no difficulty associated with replication of each of these genomes. Using a reconstruction experiment to ensure that we detect any point mutation in a background of wild type plaques, the following experiments were performed. Mutant phage particles were mixed with wild-type phage approximately at a ratio of 1:25 (mutant/wild-type, respectively) and transfected into repair-deficient E. coli cells. As shown in Fig. 4A, differential hybridization was performed on plaques transferred to nitrocellulose filters by four successive lifts of each plate followed by incubation of each filter with one of the four P-labeled 17-mers differing by only 1 base at position 2 of N-ras 61. Hybridization was highly specific as determined by a positive signal only with the complementary probes. Fig. 4B indicates the total number of phage that hybridized with their corresponding complementary sequences, and these values approximately equaled the expected ratio of 25:1, wild-type/mutant phage, respectively.


Figure 3: Schematic representation for the insertion of N-ras 61 oligodeoxynucleotides in the M13mp7L2 genome and the detection of point mutations.




Figure 4: Determination of specific point mutations at position 2 of N-ras codon 61. 11-mers containing the N-ras 61 codon, differing only by AC or AG or AT at position 2, were inserted into the unique EcoRI site of M13mp7L2 phage DNA. A shows a mixture of mutant/wild-type plaques (1:25, approximately) that hybridized specifically only to their complementary probes. B is a tabular form of the actual number of wild-type and mutant phage that give a positive signal, and these values approximately equal the original number of plaques plated.



To further determine the limits of sensitivity of this differential hybridization, four 51-mer sequences bearing the N-ras 61 codon within them but differing only by a single nucleotide at position 2 were constructed. Dot-blot assays were performed with these DNAs ranging from 1 pg to 5 µg. DNA amounts as low as 1 ng hybridized with their complementary 17-mers with high specificity and were distinctly detected after an overnight exposure. DNAs ranging from 0.5 to 5 µg further exhibited an intense signal with their corresponding probes under exposures as short as 15 min (data not shown).

These control experiments firmly establish the viability of phage containing any type of point mutation and our ability to identify any of these mutations resulting as a consequence of replication past a BPDE-adducted site.

Ligation Efficiencies and Replication Competence of N-ras 61 BPDE-adducted DNA in Vivo

Plaque-forming abilities by the various BPDE-adducted and nonadducted N-ras 61-M13mp7L2 DNA were taken as a measure of their replication competence in repair-deficient AB2480 E. coli cells (Fig. 3). For each of the stereoisomers in this study, at least three separate ligation reactions were performed followed by a minimum of two transfections/ligation. To facilitate screening of plaques, experiments were performed in three batches, each involving a pair of enantiomers. The number of plaques screened for each of the adducted samples was taken as a percentage of the total number of plaques obtained by transfection with the nonadducted sample, the latter value being specific to each individual set of stereoisomers. These results are compiled in Table 1Table 2Table 3. Linearized M13mp7L2 DNA was used as a control to detect the background levels of plaque formation by these DNA molecules. The ligation efficiency with each of the six stereochemically-defined BPDE-adducted DNAs was markedly below that of the unmodified N-ras 61-11-mer template. As tabulated, the percentage of ligation efficiency relative to the nonadducted DNA ranged from 8.5% for the (+)-anti-trans-BPDE adduct to 31.2% for the (-)-syn-trans-BPDE adduct ( Table 1and Table 2). Besides doubly-ligated molecules, Southern hybridization indicated the presence of singly-ligated molecules that had a greater mobility than the covalently closed circular molecules on a 1.4% agarose gel (data not shown). However, the contribution of these linear molecules to the percentage of plaques formed could be no more than 0.1-0.3% of the nonadducted sample based on the data derived from the plaque-forming ability of the linearized nonadducted M13mp7L2 DNA (Table 1Table 2Table 3).







In concert with the results of Southern blot analyses for the determination of ligation efficiencies, independent confirmation was obtained through direct visualization of doubly-ligated molecules by electron microscopy. Using denaturing electron microscopic methodologies, the appearance of circular single-stranded DNA molecules was direct evidence for the presence of doubly ligated 11-mers into the EcoRI restriction site of M13mp7L2 DNA. Linear single-stranded DNA molecules represented either the vector DNA with no insert or those that were singly ligated to an 11-mer. At least 200 molecules were scored for each of the six stereoisomerically-defined BPDE-adducted DNAs and the nonadducted N-ras 61-11-mer template samples. The average full-length circular forms of ligated vector DNA with the insert was 1.76 ± 0.18 µm (Fig. 5). As discussed above, although full-length linear DNA molecules were detected by electron microscopy, it was not possible to distinguish singly-ligated molecules from the linearized vector alone due to the small insert size. This electron microscopic study provides an alternative methodology for verification of the double ligation event.


Figure 5: Ligated circles of BPDE-adducted 11-mer within the single-stranded M13mp7L2 vector as determined by electron microscopy. The BPDE-modified 11-mers were inserted into an unique EcoRI site within M13mp7L2 single-stranded DNA, resulting in covalently closed circular molecules.



Similar to the ligation efficiencies, the plaque-forming abilities of all the six stereochemically defined BPDE-adducted DNAs were distinctly lower than the corresponding values of the unmodified template. Furthermore, a wide spectrum of plaque-forming abilities was observed, ranging from 5.8% for the(-)-anti-trans-BPDE adduct to 22.8% for the (+)-anti-cis-BPDE adduct ( Table 1and Table 3).

In Vivo Mutagenesis of Stereochemically Defined BPDE Adducts

The plaques formed by the replication of various BPDE-adducted and nonadducted N-ras 61-M13mp7L2 DNAs in AB2840 E. coli cells were screened by differential hybridization. To determine the spectrum and frequency of point mutations as a result of replication past the adducted site, four probes differing by only a base opposite the modified site were utilized. As shown in Table 1-3, AG transitions were the sole mutations observed with all six stereo-specific BPDE adducts analyzed. The mutational frequency ranged from 0.26 to 1.20%, indicating subtle changes in the incidence of point mutations. Similar mutation rates were observed even on lowering the concentrations of the BPDE-adducted 11-mers in the ligation reactions such that their molar ratios relative to the phage DNA was 10:1 and 2:1 respectively. This observation strengthened the evidence for the fact that the frequency of single base substitution was a true reflection of the impact of these bulky adducts on the cell's replication machinery.

Qualitative and Quantitative Analyses of in Vitro DNA Replication of BPDE-adducted and Nonadducted Oligodeoxynucleotide Templates

In vitro replication of a given template is influenced by the polymerase utilized in the reaction. In the present investigation, Klenow fragment was chosen due to its precedence of widely being utilized for kinetic studies, although polymerase III is normally the key enzyme involved in in vivo replication. To understand the role of this polymerase in in vitro primer extension assays for the bypass of BPDE lesions, 33-mers were constructed from each of the adducted oligodeoxynucleotides. Using this methodology, individual adducts were studied in isolation from all of the others. Similar to the in vivo studies, six adducted N-ras 61^2 templates were synthesized for in vitro analysis; i.e. 33-mers containing (+)- or(-) -anti-trans-, (+)- or(-)-syn-trans-, and (+)- or(-)-anti-cis-BPDE adducts. Since de novo synthesis of a molecule of this length with stereochemically-defined BPDE adducts in the N-ras 61 codon is technically difficult, the templates were constructed enzymatically as schematically represented in Fig. 6and as detailed under ``Experimental Procedures.'' More than 50% of the ligated material was recovered with each of the six BPDE adducted 33-mers.


Figure 6: Synthesis of site-specific BPDE-adducted 33-mers employed for in vitro studies.



Equimolar ratios of templates (nonadducted and adducted) and P-end-labeled primer were utilized for the polymerization reactions. A time course study was performed that included primer extension reactions for 2, 5, 10, and 30 min. Sequence analyses of extended primers replicated on DNA templates were carried out by subjecting the reaction mixtures to 15% polyacrylamide gel electrophoresis. Following electrophoresis, positions of the oligomers were established by autoradiography as shown in Fig. 7. Sequences for both the template and primer are depicted above the results of primer extension (Fig. 7). No qualitative differences were observed within individual extension reactions for any of the adducted templates, ranging from 2 to 30 min. The reactions appeared to be complete within the first 2 min, indicating no further translesion synthesis over longer incubation times. The P-end labeled 17-mer primer employed in each of the reactions was completely utilized as indicated by the absence of a band at the 17-mer position (Fig. 8). The nonadducted 33-mer template exhibited a full-length product at the end of 2 min of polymerization. In contrast, none of the six adducted oligodeoxynucleotides accumulated full length products even after 30 min of synthesis. All six BPDE-modified 33-mers served as poor templates resulting in blockage of in vitro replication due to synthesis being stopped opposite and/or 1 base 3` to the adducted site. Replication of (+)-anti-trans-, (+)-syn-trans-, and (-)-anti-cis-BPDE-adducted templates was completely blocked at 1 base 3` to the adducted site. With(-)-anti-trans-, (-)-syn-trans-, and (+)-anti-cis-BPDE-modified 33-mers, a nucleotide was placed opposite the adducted site in each case but no replication occurred beyond that point (Fig. 7).


Figure 7: A kinetic analysis of primer extension reactions with templates containing various BPDE stereoisomers. The sequence of the template-primer complex is represented at the top. The adducted site is designated by an asterisk. Chain elongation studies were performed at 2, 5, 10, and 30 min. Lane1, N-ras 61-33-mer; lane2, (+)-anti-trans-BPDE-33-mer; lane3, (-)-anti-trans-BPDE-33-mer; lane4, (-)-syn-trans-BPDE-33-mer; lane5, (+)-syn-trans-BPDE-33-mer; lane6, (+)-anti-cis-BPDE-33-mer; lane7, (-)-anti-cis-BPDE-33-mer.




Figure 8: Differential blockage of replication at the adducted site and 1 base 3` to the site of lesion. Lanes2-7 contain templates with different stereoisomeric BPDE adducts. Lane1, N-ras 61-33-mer; lane2, (+)-anti-trans-BPDE-33-mer; lane3, (-)-anti-trans-BPDE-33-mer; lane4, (-)-syn-trans-BPDE-33-mer; lane5, (+)-syn-trans-BPDE-33-mer; lane6, (+)-anti-cis-BPDE-33-mer; lane7, (-)-anti-cis-BPDE-33-mer. These data were taken from a 30-min primer extension reaction with the Klenow fragment. The 27-mer and 28-mer represent the partially extended products up to 1 base 3` to the adducted site and opposite the site of lesion, respectively. Percentages of fully extended primer and blockage at various positions were analyzed by VISAGE gel electrophoresis analysis system and were tabulated as shown below.



Extended products were quantitated by densitometric analysis of the autoradiographs. A 30-min time point was chosen to measure the amount of fully-extended or truncated products formed with nonadducted and adducted templates (Fig. 8). Based on the autoradiographic signal, 99.3% of the extended product of the nonadducted template was of full length (Fig. 8, lane1). With the adducted templates that inhibited replication beyond 1 base 3` to the site of lesion, approximately 99.7% of the partially extended products were represented by this premature termination (Fig. 8, lanes2, 5, and 7). Two of the lesions ((-)-anti-trans- and (-)-syn-trans-) responsible for significant pause sites 1 base downstream of the adduct revealed polymerized products amounting to two-thirds of all the extended products (Fig. 8, lanes3 and 4). The remaining one-third was represented by those product molecules that terminated opposite the corresponding adduct. The (+)-anti-cis-BPDE enantiomer exhibited roughly a 14:1 ratio of partially extended products 3` to the adducted position to those opposite the lesion (Fig. 8, lane6). In essence, all of the BPDE-adducted templates proved to be poor substrates for in vitro replication with Klenow fragment in contrast to the relatively efficient replication competence of four of these six adducted sequences in repair-deficient E. coli cells after adjusting for ligation efficiencies.


DISCUSSION

Specific chiral interactions of different diastereomers of BPDE with nucleophilic sites in DNA cause genotoxic lesions that lead to consequences such as mutations, which can initiate a cancerous response in cells, subsequently leading to alterations in gene expression(23, 34, 35) . The focus of this study was to observe the role of six stereochemically-defined BPDE adducts both in vivo and in vitro when anchored at a specific position on DNA. The mutagenic potential of dA-BPDE adducts that were determined by in vivo studies included the relative lethality of these lesions as well as the type and incidence of point mutations arising from replication of the damaged DNA. An additional parameter investigated by in vitro observations involved the role of Klenow fragment in translesion synthesis on encountering the various stereospecific BPDE adducts attached to the DNA template.

To assess the relative abilities of these stereoisomers to adversely influence replication, it is critical that the modified oligonucleotides be of utmost purity(33) . Toward this goal, synthesis of oligodeoxynucleotides bearing adducts at exocyclic amino sites of adenine was carried out by the postoligomerization method, and the integrity of the samples was determined by a variety of analytical techniques(27, 28) . The resultant high purity of the adducted oligonucleotides establishes that within our ability to determine, the mutational frequencies observed were due to the adducts alone and not as a result of contaminants (Fig. 2).

In vivo studies involved repair-deficient cells that had a recA, uvrA genotype. This eliminated the effect of inducible responses attributed to DNA repair. Furthermore, the choice of a single-stranded vector was advantageous in two ways. First, single-stranded DNA are poorer substrates than double-stranded genomes for repair, consequently aiding in a better understanding of template-directed mutagenesis(36, 37) . Second, the ease of introducing nonadducted or adducted oligodeoxynucleotides into single-stranded M13mp7L2 is far greater than inserting oligomers into a gapped duplex(38, 39, 40, 41) . However it is not uncommon to obtain poor ligation efficiencies with modified DNA as observed in this study(40) . This could be attributed to the interaction of the bulky BPDE adduct with the template, thus causing structural distortions. The broad range of ligation efficiencies from 8.5 to 31.2% exhibited by the different stereoisomers is likely to be a result of the chirality of the molecule. The presence of a distinct population of singly- ligated molecules in the ligation reaction could be either because of the formation of a secondary structure by the vector DNA or because the physical presence of the adduct makes it difficult to form a closed molecule. The recovery of relatively good yields of the 33-mer constructs for in vitro analyses suggests that the ligation at least at the 3`-OH of the 11-mer is reasonably efficient. Therefore, the perturbations inhibiting ligation are more likely to occur at the 5` end of the 11-mer.

A wide spectrum of lethality was observed within the various adducted templates examined even after adjusting for ligation efficiencies. When considering ligation efficiencies and plaque-forming abilities as contributors to survival, the data suggest that percentage lethality follows the rank order of(-)-anti-trans- > (-)-syn-trans- > (+)-anti-trans- > (+)-syn-trans- > (+)-anti-cis- (-)-anti-cis-BPDE-adducted templates (Table 1-III). It is possible that there is either direct blockage of polymerase III activity or apparent loss of processivity by this holoenzyme after bypassing the(-)-anti- and(-)-syn-trans-BPDE adducts that lead to decreased survival. Similar diminished levels of enzyme processivity were observed in replicative bypass of an abasic DNA lesion(42) . However, the low to nonlethality of the remaining four BPDE isomers examined could be due to very little or no blockage of DNA replication in vivo(43) . Thus, differences in spatial configuration influence the template properties of lesions toward DNA replication and survival of the cell(23) .

In vivo mutagenesis of all of the BPDE adducts studied revealed only AG transitions. These findings are in contrast to earlier reports both in prokaryotic and mammalian cells wherein AT transversions were prevalent when adenine was the site of lesion for BPDE or other bulky adducts such as 9,10-dimethy-1,2-benzanthracene and cis-diamminedichloroplatinum II(4, 5, 11, 44, 45, 46, 47) . Previous studies using a styrene oxide DNA adduct in the same sequence context as this work, however, resulted in AG base substitutions(30) . These mutations do not follow the ``A-rule'' put forth to explain the mutational behavior of abasic and bulky, ``noninstructional'' lesions. DNA polymerases that preferentially insert adenine opposite these sites of lesions are believed to be subject to an A rule. Therefore, in contrast, dA may be directly miscoding or misinstructional rather than requiring a ``default'' mutation mode. This misinstructional lesion effect could possibly be influenced by local sequence context. In addition, it could be a consequence of the structural distortion of the adducted base that preferentially allows AG transitions alone to occur. Results presented in this study exhibited a frequency of error spanning from 0.26 to 1.20%, indicating a 5-fold difference. Furthermore, decreasing the molar concentrations of the adducted 11-mers by 10-50 fold in the ligation reaction caused no change in the percentage mutations, thus attributing the mutagenecity to the BPDE adducts rather than to any contaminants. The small yet significant changes in the mutation frequency among the BPDE lesions studied, could be a consequence of adduct conformational polymorphism resulting in varying interactions with cellular enzyme systems. The adducts could directly be in contact with the polymerase involved in replication, leading to stabilization of a mispaired configuration, as proposed for one of the mitomycin C/DNA lesions(48) . Alternatively, they could cause subtle structural changes in the polymerase or the template such that optimal base pairing with the incoming dNTP does not occur. The fact that more than approximately 98% of the time these bulky adducts were not mutagenic in this study, implies that DNA polymerases can be flexible without completely compromising fidelity(17) . These enzymes may have an additional ``sensor'' to bypass any structural distortions the adducts make in the major groove, similar to the observations made with the N^2-dG BPDE adducts(49) . Furthermore, they may also identify bases in spite of improper hydrogen bonding between base pairs, as observed with the DNA lesions induced by vinyl chloride(50) . However, the impact of DNA repair enzymes substantially altering our in vivo results in the present study was curtailed by allowing replication to occur in a repair-deficient environment.

In vivo analyses of translesion synthesis of the BPDEadducted templates were complemented by in vitro studies. Previous in vitro primer extension assays with BPDE-adducted templates involved almost exclusively guanine residues (16, 51, 52) . These bulky DNA adducts are known to block DNA replication with certain enzyme systems either at or 1 base prior to the site of the adduct in the template(53) . Recent in vitro studies with oligonucleotides containing stereospecific trans-adducts of anti- and syn-BPDE on adenine indicated that the polymerase (Sequenase) was completely arrested at 1 base 3` to the adduct(54) .

The present study is a further effort to investigate how BPDE-adenine adducts behave upon encountering polymerases in an in vitro system. Although replication in E. coli cells is predominantly performed by DNA polymerase III, the difficulty of assembling all the core proteins involved in the proper functioning of this holoenzyme is a major limiting factor. Therefore, the alternate choice of Klenow fragment was made due to its wide usage with various adducted templates. Kinetic analyses in this investigation revealed that the Klenow fragment had no capacity to perform translesion synthesis even after an incubation period of 30 min. Temporal (from 2-30 min) comparison of the patterns of the partially extended products from each adducted template indicated that the primer extensions were completed as early as 2 min. All of the template and primer were utilized completely as indicated by the fact that no primer was left at the 17-mer position when equimolar substrates were employed in the reactions (Fig. 8).

Quantitative analyses of the truncated products exhibited almost total termination 3` to the adducted site with (+)-anti-trans-, (+)-syn-trans-, and (-)-anti-cis-adducted templates, whereas with (-)-anti-trans-,(-)-syn-trans- and (+)-anti-cis-BPDE adducts a stop site was observed 3` to the adducted site, followed by complete blockage opposite the lesion (Fig. 8). Besides the bulky nature of the adducts that could be responsible for this resistance to in vitro replication, the orientation and tilt of the pyrenyl ring relative to the modified strand could be a causal factor for stalling and abrupt cessation of primer extension with the Klenow fragment. It is not unlikely that those adducts impeding the enzyme progress beyond 1 base 3` to the lesion site (Fig. 8, lanes2, 5, and 7) could be stereo-selectively distinct in angle positioning from the isomers that allow incorporation of a nucleotide opposite the adduct (Fig. 8, lanes4 and 6). This dichotomy is further exacerbated by the fact that isomers categorized under the first group exhibit a S configuration at the C-10 of BPDE, whereas the isomers encompassing the second group revealed a R conformation at the same position (Fig. 1). There is evidence to show that PAH with 10S configuration point in the opposite direction to those with the 10R configuration (3) . The precedence of exonuclease digestion controlled by adduct orientation relative to the 5` 3` strand polarity indicates its influence on enzyme action(55) . Likewise it is probable that polymerization by a specific enzyme is dictated by the bulky adduct orientation relative to the site-specifically modified single-stranded DNA. NMR studies on the duplex structure of adducted oligonucleotides at N^2 of dG determined the directionality of various stereoisomers of BPDE(49, 56, 57) . Similar observations on spatial positioning were made with styrene oxide adducts that showed a pattern of in vitro blockage parallel to those obtained in this study(30, 58) . In spite of BPDE lesions forming effective blocks to DNA synthesis by Klenow fragment in vitro, it is clear that in vivo replication, which is necessary for the survival of a cell, occurred beyond the adducted site, probably through polymerase III.


FOOTNOTES

*
This work was supported by United States Public Health Service Grants ES05355 and ACS FRA 381. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel: 409-772-2179; Fax: 409-772-1790.

(^1)
The abbreviations used are: PAH, polycyclic aromatic hydrocarbons; BP, benzo[a]pyrene; BPDE, benzo[a]pyrene-7,8-dihydrodiol 9,10-epoxide; Ade, adenine; HPLC, high performance liquid chromatography.


ACKNOWLEDGEMENTS

We thank Dr. M. L. Dodson for helpful comments throughout the course of this study and M. L. Augustine for technical assistance.


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