Chem. Res. Toxicol. 1993,6, 59-63
59
Base-Sequence Dependence of Covalent Binding of Benzo[a]pyrene Diol Epoxide to Guanine in Oligodeoxyribonucleotides Leonid A. Margulis, Victor Ibanez, and Nicholas E. Geacintov’ Chemistry Department and Radiation and Solid State Laboratory, New York University, New York,New York 10003 Received August 6, 1992 The base-sequence dependence of the yield of the covalent binding reaction of (+)-anti7~,8a-dihydroxy-9a,10a-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene [(+)-anti-BPDE] with the exocyclic amino group of guanine surrounded by different flanking bases X and Y in the singlestranded oligonucleotide d(CTATXGYTATC) was investigated. With an initial ratio of [(+)anti-BPDE]/[oligonucleotidestrand] = 2, the percentage of modified strands varied from 20 f 2 % when the modified dG was surrounded by pyrimidines to 5-7 % when the central dG was surrounded by purines. The trans/cis ratio of (+)-anti-BPDE-N2-dG adducts was in the range of 3-5. The lower reaction yields observed when the modified guanine residues in singlestranded oligonucleotides are surrounded by purines rather than by pyrimidines is tentatively attributed (1) to steric effects arising from the presence of the bulkier purines flanking the reacting dG moieties on the 5’- and 3’4des and/or (2) to noncovalent interactions between anti-BPDE and neighboring purines which decrease the probability of optimal alignment for covalent binding between the interacting moieties in the bimolecular transition-state complex. Noncovalent intercalation of (+)-anti-BPDE prior to the covalent binding reaction is not a relevant process in the case of single-stranded oligonucleotides and is therefore not a critical requirement for obtaining high yields of covalent trans- and cis-(+)-anti-BPDE-N2-dG adducts in these oligonucleotide sequences. Osborne (17)has investigated the overallreaction yields of covalent adducts resulting from the reactions of (+IThe polycyclic aromatic hydrocarbon benzo[al pyrene, anti-BPDE with different self-complementary oligonua ubiquitous environmental pollutant, is metabolized in cleotides of different base compositions and sequences. vivo to a variety of oxygenated derivatives, including the However, the effects of sequence on the yields of formation highly reactive, mutagenic, and tumorigenic diol epoxide of site-specific and stereochemically defined BPDE-N2(+)-anti-7~,8a-dihydroxy-Qa,lOa-epoxy-7,8,9,lO-tetr~~dG adducts in deoxyoligonucleotides have not yet been drobenzo[a]pyrene [(+)-anti-BPDE)l (1-3). This meinvestigated. When (+)- and (-)-anti-BPDE is reacted tabolite binds predominantly via trans and cis addition to with single-strandedoligonucleotidescontainingthymine, the exocyclic amino group of guanine in native DNA (4cytosine, adenine, and a single guanine residue, reactions 8). We have recently employed a direct synthesisapproach of (+)- or (-)-anti-BPDE with the exocyclic amino group (9) to produce stereospecific and positionally defined (N2) of the single guanine by cis or trans addition are adducts derived from the binding of (+I- and (-)-antidominant (9,10,18). The direct synthesis of positionally BPDE to guanine residues in single-stranded oligonucleand stereochemically defined BPDE adducts with oligootides 9-11 bases long (9, 10). The chemical and physical nucleotidesequences containingtwo (19) or three m i n e s 2 characteristics of these BPDE-oligonucleotideadducts can is also feasible. In our previous work, we have shown that be characterized by a variety of chemical, spectroscopic relatively large yields of (+)- and (-)-anti-BPDE-dG (9, lo), and NMR (11,12) techniques. adducts can be obtained when single-stranded oligonuA detailed characterization of the conformations of cleotides containing a single guanine surrounded by different BPDE-DNA adducts in general is of great pyrimidines, either thymines (9,10,18) or cytosines (11, significance for an understanding of the mechanisms of 12),are reacted with (+)- and (-)-anti-BPDE in aqueous mutations induced by these bulky polycyclic aromatic solutions. In this work, we have investigatedthe influence compounds on a molecular level (13,141. The availability of neighboring bases flanking a dG residue on the 5’- and of positionally and stereochemically defined BPDE3’4des on the yields of (+)-anti-BPDE-N2-dGadducts in modified oligonucleotides in different sequence contexts single-stranded oligonucleotides 9-1 1 bases long. offers the possibilities of correlating the structural characteristicsof these bulky mutagen-DNA adducts with their Experimental Section biologicalcharacteristicsin site-directed mutagenesis (15) and in in vitro site-directed mutagenesis experiments (16). Different oligonucleotides were synthesized by the phosphor-
Introduction
Corresponding author. 1 Abbreviations: (+)-anti-BPDE, (+)-anti-78,8a-dihydroxy-9a,lOaepoxy-7,8,9,lO-tetrahydrobenzo[aIpyrene; TEA, triethylamine;BPT, cisand trans-7,8,9,10-tetrahydroxytetrahydrobenzo[alpyrene: Pu, purine; Py, pyrimidine.
amidite technique using a Cyclone DNA synthesizer (Biosearch, Inc., San Rafael, CA) and purified as described earlier (9). The
*L.A. Margulis, B. Mao, V. Ibanez, and N. E. Geacintov, to be published. 0 1993 American Chemical Society
60 Chem. Res. Toxicol., VoZ. 6, No. I, 1993
I
MarguZis et al.
1 0.
20
1
r
40
d(CTATXG*YTATC)
TIME (minutes)
Figure 1. Typical HPLC elution profile of an equilibrated (+)-
anti-BPDE/d(CTATCGTTATC) reaction mixture; absorbance
(254-nm) peaks (solid trace A) with retention times of 19.3,22.7, and 24.4 min are attributed to the unmodified oligonucleotide, adducts, respecand to cis- and trans-d(CTATCGBPDETTATC) tively (the trans- and cis-tetraols are represented by the two peaks at retention times of 43.1 and 45.1 min). The fluorescence signal (excitation at 350 nm, emission viewed at 400 nm) in the elution region of the unmodified and modified oligonucleotide region (traceF)can be used to distinguishthe fluorescent adducts from the unmodified oligonucleotide. The areas under the curves are represented by trace I (integral);the ratios of areas under the peaks due to unmodified and cis- and trans-modified oligonucleotides are approximately 17:1:5. oligonucleotides were dissolved in 20 mM sodium phosphate buffer solution (pH 11)containing 1.5% TEA (IO),and thestrand concentration was =O.l mM ( e l 0 A z M units at the absorption maximum of 260 nm). The (+)-anti-BPDE enantiomer was purchased from the National Cancer Institute Chemical Carcinogen Reference Standard Repository (Midwest Research Institute, Kansas City, MO). The (+)-anti-BPDE was dissolved in tetrahydrofuran (3-5mM) containing 5% TEA as a stabilizer. Aliquots of this (+)-anti-BPDE solution were added to the aqueous oligonucleotide solutions so that the ratio [BPDE]/ [oligonucleotidestrand] = 2, unless otherwise specified. The tetrahydrofuran concentration did not exceed 15% in any experiment and was typically 5% ;there was no noticeable effect of tetrahydrofuran on the reaction yields in the range of concentrationsspecified. After mixing, the reaction mixture was stored overnight at room temperature in the dark. This reaction mixture, containing unmodified oligonucleotides,adducted oligonucleotides, and the tetraol hydrolysis products BPT, was subjected to reverse-phase HPLC analysis and separation employing a Hypersil-ODS 250- X 10-mm column (Keystone Scientific, Inc., Bellefonte, PA) and, unless otherwise noted, a 0-9096 linear methanol gradient in 20 mM sodium phosphate buffer solution (pH = 7.0) in 60 min. The flow rate was 3.0 mL/min, and a 254-nm absorbance detector and a fluorescence signal detector were employed.
Results Adduct Separation. A representative HPLC elution profile of a (+)-anti-BPDEdigonucleotidereaction mixture, obtained with the 11-mer d(CTATCGTTATC), is shown in Figure 1;the labels A, F, and I refer to the 254nm absorbance, fluorescence, and cumulative integrals of the absorbance signals, respectively. The eluates with retention times of 19.3, 22.7, and 24.4 min contain unmodified oligonucleotide and cis- and trans-modified (+)-anti-BPDE-dGoligonucleotide adducts, respectively (9, 18), and were collected for further analysis. The fluorescencesignalsdistinguishthe BPDE-oligonucleotide
Figure 2. Percent of d(CTATXGYTATC)stands modified by formation of covalent (+)-anti-BPDE-N2-dG adducts at the starred G residue as a function of different flanking bases X and Y. Clear bars: (+)-trans; dark bars: (+)-cis addition products. The yield of cis adducts was not determined for the ...GG*G... sequence (see text).
adducts from the unmodified oligonucleotide, since the latter is not fluorescent. The reaction yields, and relative proportions of trans/cis adducts, can be determined from the relative areas (area integrals) of each peak in Figure 1. The reaction yields are defined in terms of the fraction of oligonucleotide strands which had undergone reaction with (+)-anti-BPDE to form covalent BPDE-N%lG oligonucleotide adducts, and the results obtained with different sequences are summarized in the form of a bar graph (Figure 2). The two different adduct fractions obtained for each sequence were subjected to enzyme digestion as described earlier (9,lO)in order to ascertain the nature of the adducts (data not shown). In all cases, only the unmodified deoxynucleosides and modified cis- and trans-(+)-antiBPDE-N2-dG nucleosides were detected as described elsewhere (9,10,18),thus allowing for the assignments of the (+)-anti-BPDE-oligonucleotidestereochemistry in each case. In order to determinethe effects of neighboring guanines on the reactivities of a central dG residue, the deoxyoligonucleotide d(CTATGGGTATC)was reacted with (+)anti-BPDE under conditions identical to those described above; however, because all three adjacent dG's were modified, a more elaborate HPLC separation procedure was developed. Full details will be published elsewhere.2 Briefly, the reaction mixture was subjected to an initial HPLC separation procedure as described above, using a +90% methanol/20 mM sodium phosphate buffer (pH 7.0) gradient in 60 min, but with a flow rate of 1.5 mL/min. A set of three overlapping maxima were observed with retention times between 22.5 and 25 min; this fraction, containing a mixture of (+)-anti-BPDE-N2-dGadducts situated at either of the three guanines, was collected and evaporated to dryness. The overall reaction yield was 24 % . The residue was redissolved in a 13% acetonitrile mixture in 50 mM triethylammoniumacetate solution (pH = 7.0) and subjected to a second HPLC separation under isocratic conditions. Three distinct eluates with retention times of 4.9,5.4, and 6.0 min (with reaction yields of approximately 6%, 12%, and 6%, respectively) were observed and collected separately. Using the high-resolution gel electrophoresis methods in conjunction with the G+A MaxamGilbert cutt'ng reactions as described elsewhere (19),it
f
Chem. Res. Toxicol., Vol. 6, No. 1, 1993 61
Base-Sequence Dependence of Binding 3 . d(ATACGCATA1 0.dICTATGTATC1 .d(CATAGCATC)
i
0
O
2
4
6
IO
8
40
20:
t ’
/
b
was established that these eluates corresponded to the trans-(+)-BPDE-N2-dG adducts 5’-d(CTATGGBPDEGTATC)-3’, 5’-d(CTATGGGBPDETATC)-3’, and 5’-d(CTATGBPDEGGTATC)-3’ adducts, respectively. Overlapping eluates containing cis adducts in lower yields were also observed, but no attempts were made to separate the different cis adducts and to determine the site of BPDE fixation in each one. Effects of Initial BPDE/Oligonucleotide Ratio on Reaction Yields. The total yields of BPDE-oligonucleotide adducts (both cis and trans), as a function of the initial reaction ratio r = [BPDEl/ [strand] in four different 9-mer oligonucleotides, are depicted in Figure 3a. The yields of covalent adducts increase with increasing (+)anti-BPDE concentration, but in a nonlinear manner. The overall adduct yields are highest in d(ATACGCATA) and d(CTATGTATC1, in which the modified single guanine is surrounded by two d T or two dC bases. The yields are smaller when there is one adenine flanking the dG base [d(CATAGCATC) and d(CTACGATAC)I. In all cases, the trans/& adduct ratio is about 5 (data not shown). The total reaction yields obtained with oligonucleotides containing two guanines [d(TATGCGTAT) and d(GATACATAG)] as a function of the reaction ratio rare depicted in Figure 3b. In the first oligonucleotide, both guanines are surrounded by pyrimidines and are located at the fourth positions counted from the 5‘-end and the 3‘-end, respectively. The overall reactivityper dG residue is lower than in oligonucleotidescontaining single guanines (Figure 3a). It is shown elsewhere that the yields of trans-BPDEN2-dG adducts (and of cis adducts) are the same at the two different guanines in d(TATGCGTAT) and that the trans/cis adduct ratio is about 4.5 (19). In d(GATACATAG), in which both dG residues occupy terminal positions, the overall reaction yield is significantly lower than in the oligonucleotide d(TATGCGTAT) (Figure 3b). Two eluates with similar absorbance signals and with retention times of 31.9 and 33.9 min were observed (data not shown), which suggests that the yields of adducts at the two terminal guanines are similar. Effects of Flanking Bases on Reaction Yields. The reaction yields obtained when (+)-anti-BPDE was reacted with the sequence d(CTATXGYTATC), where X and Y
are different nucleotides, is summarized in Figure 2. The highest reaction yields (=20% of the trans adduct) were obtained when both of the flanking bases X and Y were pyrimidines. The lowest yields were observed when both flanking bases were adenines or guanines. Intermediate yields were observed when only one of the bases was adenine. In the oligonucleotide d(CTATGGGTATC) the yield of d(CTATGBPDEGGTATC)and d(CTATGGBPDEGTATC) adducts was 6 % (thislatter result is also depicted in Figure 2), while the yield of d(CTATGGGBPDETATC) adducts was 12 % ;in all three cases, these yields are lower than in the case of the oligonucleotide d(CTATXGBPDEYTATC) in which both X and Y are pyrimidines. In general, in different sets of experiments, the overall absolute reaction yields varied within about five percentage points. However, the relative yields were reproducibly arranged in the order Py-G-Py > Pu-G-Py or Py-G-Pu > Pu-G-Pu, as shown in Figure 2. The yields of cis adductswere always significantly lower than those of trans adducts. The trans/cis adduct ratios were in the range of 3-5 in the modified d(CTATXGBPDEYTATC) oligonucleotides, with X and Y being either T, C, or A (Figure 2).
Discussion Effects of Base Sequence on Reaction Yields. The results presented here demonstrate that the substantial reaction yields of BPDE-N2-dG adducts previously obtained with single-stranded oligonucleotides containing a single guanine surrounded by two thymines (9,181or two cytosines (11, 12) are not unusual. The yield of transBPDE-N2-dGadducts in the oligonucleotide d(CTATXGYTATC) varies from a high of about 20 7% (based on the fraction of modified strands obtained) to a low of about 5 % ,depending on the nature of the flanking bases X and Y. The yield of adducts with cis stereochemistry is consistently lower under these reaction conditions, varying from about 2% to about 4%. The yields of cis adducts is also lower when the reactive guanine is flanked by adenines (rather than by pyrimidines on either the 5’side, 3’-side, or both the 5’- and the 3’-sides (Figure 2). The relative yields of cis adducts in the sequence containing three adjacent guanines were not determined. The reactivity of (+)-anti-BPDE with the exocyclic amino group of guanine is lower when it is flanked on either the 5’- or the 3’-side by adenine rather than by pyrimidines. When this G is flanked by adenines on both sides, the yield is lowered still further (Figure 2). When guanines, rather than pyrimidines, flank the targeted G, the reaction yields are also lower; the yields are lowest (6%)with G’s flanking the modified guanine moiety on both sides, or a G located on the 3’-side of the target guanosyl residue. With a G residue on the 5’-side of the modified guanine, the yield is higher (12%)than with G residues on both the 5’- and the 3‘-sides (6%). These results may be understood in terms of simple steric effects if it is assumed that the transition-state complex involving (+)-anti-BPDE and the exocyclic amino group on the target guanine residue is characterized by a defined orientation of the BPDE molecule relative to the 5’ 3’ strand direction. The formation of such a complex may be less favored when another guanine is present at the 3’-rather than at the 5‘4de of the reacting guanine residue. The generally lower reaction yields in the presence of
-
62 Chem. Res. Toxicol., Vol. 6, No. 1, 1993
neighboringadenines or guanines suggest that both purines might hinder the proper alignment of the C10 end of the (+)-anti-BPDE molecule with respect to the exocyclic amino group of the reacting dG residue. In addition, noncovalent interactions between the pyrenyl residues and the nucleotides, especially basestacking interactions between (+)-anti-BPDE and the target dG and adjacent purine/pyrimidines bases, might also be important in hindering the correct alignment of the reacting moieties in the transition-state complex. We speculate that such interactions between the pyrenyl residues and neighboring purines, which give rise to unfavorable reaction geometries, may be greater than with neighboring pyrimidines, thus accounting for the lowered reaction yields in the presence of neighboring purines. Indeed, it has been shown that both purine and pyrimidine nucleotides (20,21)and nucleosides (22)form complexes with pyrene (20)and with BPT (21,221in aqueous solutions and that the association constants with purines are significantly larger than those with pyrimidines (20,22). Stereoselectivityof Adduct Formation. When (+)anti-BPDE reacts with native DNA, trans adduct formation at the exocyclic amino group of guanine is the major product [>go% (6-811. It has been suggested that this stereoselectivity of adduct formation is due to the formation of noncovalent (probably intercalative) BPDEDNA complexes prior to the covalent binding reaction (6). The existence of such noncovalent and unstable BPDE-DNA and BPT-DNA complexes has been demonstrated experimentally (23-291, and it has been shown that these complexes form on time scales of several milliseconds or less after mixing (23, 30). The possible relationships between intercalation and the formation of covalent reaction products have been discussed (31-35). Even though intercalation lis the dominant mode of noncovalent binding of anti-BPDE (23-26) and other polycyclic aromatic hydrocarbon metabolites (27, 35) to native DNA, its importance in terms of the subsequent covalent binding reactions is not established (32). The relatively high efficiency of the covalent binding of (+Ianti-BPDE to dG in single-stranded oligonucleotides (Figures 2 and 3) suggests that intercalation in these reactions is not a critical requirement for efficient covalent binding. The trans/cis product ratio appears to be strongly affected by the secondary structure and base sequence; in native DNA this ratio is >90 (7), while in poly(dG-dC)-poly(dG-dC) and poly(dG).poly(dC) it is only 6 f 1 (10). In our experiments with single-stranded oligonucleotides,the trans/cis adduct ratio varies from 3 to 5. The striking differences in the trans/cis adduct ratios in doublestranded native DNA and in poly(dG-dG).poly(dG-dC) and poly(dG).poly(dC) suggest that base-sequence effects may be quite important in determining the levels of cis adduct formation in double-stranded nucleic acids.
Concluding Remarks Effects of base sequence on the yield of covalent (+)anti-BPDE-N2-dG adducts when (+)-anti-BPDE is reacted with single-stranded deoxyoligonucleotides of different sequences can account for differences in reaction yields by a factor of about 4. Base-sequence effects on reaction yields in double-stranded oligonucleotidesremain to be investigated in detail. Using self-complementary oligonucleotides, in the duplex form, Osborne ( 17) found
Margulis et al. that the formation of (+)-anti-BPDE-N2-dG adducts is enhanced at G residues with adjacent cytosines and guanines; however, the yields per G residue and as a function of position in oligonucleotides containing two or three guanines were not reported. In general, the yield patterns were different ( 1 7)from those reported here using single-stranded oligonucleotides. There is evidence that, in the case of native duplex DNA, the effects of base sequence extend beyond those flanking the modified G since enhanced binding of (A)-anti-BPDE was found at some, though not all, guanine-rich sequences (36-39), possibly because of sequence-dependent local microconformation effects (36, 40).
Acknowledgment. This work was supported by the Department of Energy, Office of Health and Environmental Research (Grant DE-FG02-88ER60674). The Radiation and Solid State Laboratory at New York University is supported by the Department of Energy (Grant DE-FG02-86ER604051, and the oligonucleotide synthesis facility is supported by Grant CA 20851 from The National Cancer Institute (NIH). References (1) Conney, A. H. (1982)Induction of microsomal enzymes by foreign chemicals and carcinogenesis by polycyclic a~ Jatic hydr&arbo&. Cancer Res. 42,4875-4917. Singer, B., and Grunberger,D. (1983)MolecularBiologyofMutagens and Carcinogens, Plenum Press, New York. Harvey, R. G. (1991)PolycyclicAromatic Hydrocarbons: Chemistry and Carcinogenicity, Cambridge University Press, Cambridge, England. Weinstein, I. B., Jeffrey, A. M., Jennette, K. W., Blobstein, S. H., Harvey, R. G., Harris, C., Autrup, H., Kasai, H., and Nakanishi, K. (1976)Benzo[alpyrene diol epoxides as intermediates in nucleic acid binding in uitro and in uiuo. Science 193, 592-594. Koreeda, M., Moore, P. D., Wislocki, P. G., Levin, W., Conney, A. H., Yagi, H., and Jerina, D. M. (1978)Binding of benzo[alpyrene 7,8-diol-9,10-epoxidesto DNA, RNA, and protein of mouse skin occurs with high stereoselectivity. Science 199,778-781. Meehan, T., and Straub, K. (1979)Double stranded DNA stereoselectively binds benzo[alpyrene diol epoxides. Nature 277,410412. Cheng, S.C., Hilton, B. D., Roman, J. M., and Dibble, A. (1989) DNA adducts from carcinogenic and noncarcinogenic enantiomers of benzo[a]pyrene dihydrodiol epoxide. Chem.Res. Toxicol. 2,334340. Jeffrey, A. M., Jennette, K. W., Blobstein, S. H., Weinstein, I. B., Beland, F. A,, Harvey, R. G., Kasai, H., Miura, I. and Nakanishi, K. (1976)Benzo[a]pyrenenucleic acid derivative found in vivo: structure of benzo[alpyrenetetrahydrodiol epoxide-guanosine adduct. J. Am. Chem. SOC. 98, 5714-5715. Cosman, M., Ibanez, V., Geacintov, N. E., and Harvey, R. G. (1990) Preparation and isolation of adducts in high yield derived from the binding of two benzo[a]pyrene-7,8-dihydroxy-9,10-oxide stereoisomers to the oligonucleotide d(ATATGTATA). Carcinogenesis 11, 1667-1672. Geacintov, N. E., Cosman, M., Mao, B., Alfano, A., Ibanez, V., and Harvey, R. G. (1991)Spectroscopic characteristics and site I/site I1 classsification of cis and trans benzo[alpyrene diol epoxide enantiomer-guanosine adducts in oligonucleotides and polynucleotides. Carcinogenesis 12,2099-2108. Cosman, M., de 10s Santos, C., Fiala, R., Hingerty, B. E., Ibanez, V., Margulis, L. A., Live, D., Geacintov, N. E., Broyde, S., and Patel, D. J. (1992)Solution conformation of the major adduct between the carcinogen (+)-anti-benzo[alpyrene diol epoxide and DNA. h o c . Natl. Acad. Sci. U.S.A. 89, 1914-1918. de 10s Santos, C., Cosman, M., Hingerty, B. E., Ibanez, V., Margulis, L. A., Geacintov, N. E., Broyde, S., and Patel, D. J. (1992)Influence of benzo[a]pyrene diol epoxide chirality on solution conformations of DNA covalent adducts: the (-)-trans-anti-[BPlGC adduct structure and comparison with the (+)-trans-anti-[BPlG.C enantiomer. Biochemistry 31, 5245-5252. Loechler, E. L. (1989)Adduct-induced base-shifts: a mechanism by which the adducts of bulky carcinogens might induce mutations. Biopolymers 28, 909-927.
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