Chem. Res. Toxicol. 1992,5, 773-778

773

Direct Synthesis and Identification of Benzo[ alpyrene Diol Epoxide-Deoxyguanosine Binding Sites in Modified Oligodeoxynucleotides Bing Mae,? Leonid A. Margulis,? Bin Li,+Victor Ibanez,t Hongmee Lee,* Ronald G. Harvey,* and Nicholas E. Geacintov*ft Chemistry Department and The Radiation and Solid State Laboratory, New York University, New York,New York 10003, and The Ben May Institute, The University of Chicago, Chicago, Illinois 60637 Received May 5, 1992

Adducts derived from the reaction of the benzo[alppene metabolite model compound (+)-

anti-7~,8a-dihydroxy-9a,10a-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene I(+)-BPDE] with the single-stranded oligodeoxynucleotide 5’-d(TATGCGTAT) were obtained according to direct synthesis techniques described earlier [Cosman, M., Ibanez, V., Geacintov, N. E., and Harvey, R. G. (1990) Carcinogenesis 11,1667-16721. Four major adducts, involving trans and cis addition (trans/cis adduct ratio = 4.5) of (+)-BPDE to the exocyclic amino groups of guanines Gd and Gs (the numbers denote the positions of the guanines counted from the 5’-side) were obtained. These adducts can be separated from one another by reverse-phase high-performance liquid chromatography methods. The site of BPDE binding on either Gd or Ge can be determined from the electrophoresis band patterns on 20% polyacrylamide gels of the BPDE-modified oligonucleotides subjected to the G+A and G Maxam-Gilbert strand cleavage reactions [Maxam, A. M., and Gilbert, W. (1980) Methods. Enzymol. 65,499-5601. The electrophoresis gel band patterns are different for unmodified DNA and the two different BPDE-modified oligonucleotides because (1) the strand cleavage fragments bearing BPDE residues migrate slower than the corresponding fragments derived from the unmodified oligonucleotide and (2) strand cleavage tends to be inhibited on the 5’-sides of BPDE-modified guanines in the G+A, but not the G reaction. examples of the total synthesis (9-13) and the direct synthesis (7,14-19) approaches have been published. The ultimate tumorigenic form of the ubiquitous Using the direct synthesis approach, we have recently environmentalpollutant benzo[al pyrene is the dihydrodiol synthesized adducts in which a single guanine residue in epoxide (+)-anti-7~,8a-dihydroxy-9a,l0a-epoxy-7,8,9,10-oligonucleotides9 or 11 bases long was modified with (+)tetrahydrobenzo[alpyene [(+)-BPDEll(I). This comand (-)-BPDE at the exocyclic aminogroup (20,21).These pound readily forms covalent adducts with DNA, and the procedures were used to synthesize 5 mg of a trans-BPDEc dominant type of adduct involves trans addition of BPDE oligonucleotideadduct (11bases long) whose structure, in (10-position)to the exocyclic amino group of guanine (Ithe duplex form, was recently characterized by one- and 3). The formation of such DNA lesions can lead to two-dimensional NMR techniques (22). mutagenesisand is widely believed to constitute the critical There are base sequenceswhich contain severalguaninea initial step in the multistage tumorigenesis phenomenon close to one another, which are of biological significance. (4-6). For example, codons 12 and 13 in ras oncogenes contain In order to study the relationships between adduct more than one guanine (23)and are particularly prone to structure, base-sequence dependence, and biological acmutations induced by alkylatingagents (24). In this work, tivity [e.g., site-directed mutagenesis experiments (7)1, it it is shown that the direct synthesis approach previously is essential to synthesize BPDE-oligodeoxynucleotide developed (20) for modifying oligonucleotides containing adducts with defined stereochemistry and site of modia single guanine with (+)-BPDE can also be used with fication. There are basically two types of approaches for oligonucleotides containing two guanines. Either one of synthesizingBPDE- and other carcinogen-oligonucleotide the two guanines becomes covalently linked with (+)adducts (8). In the total synthesis approach, a modified BPDE, and the site of modification can be established by carcinogen-nucleotide adduct is synthesized and is subMaxam-Gilbert sequencingmethods using high-resolution sequently incorporated into an oligonucleotide sequence. electrophoretic gels. These methods are illustrated with In the direct synthesis method, an oligonucleotide with the oligonucleotide d(TATGCGTAT1 in which the (+)a defined sequence is first synthesized and is covalently BPDE becomes covalently linked via trans addition to modified with the desired carcinogen. A number of either of the two guanines.

Introduction

* To whom correspondence should be addressed.

+ New

York University. The University of Chicago. 1 Abbreviations: BPDE, (+)-anti-7&3a-dihydroxy-9a,lOa-epoxy7,8,9,10-tetrahydrobenzo[alpy~ene; CD, circular dichroism; TEA, triethylamine. f

Experimental Section Racemic anti-BPDE waa synthesized at The University of Chicago using methods previously summarized (6) and was dissolved in tetrahydrofuran (=5 m M solution) containing 5%

0893-228~/92/2705-Q773$03.QQ~Q 0 1992 American Chemical Society

Ma0 et al.

774 Chem. Res. Toxicol., Vol. 5, No.6, 1992 triethylamine as a stabilizer. The (+)-BPDEand (-)-BPDE were separated from one another using chiral stationary-phase highperformance liquid chromatography as described by Weems and Yang (25);a 4.6- X 250-mm (R)-N-(3,5-dinitrobenzoyl)phenylglycine Pirkle type-1A column (Regis Chemical Co., Morton Grove, IL) was used, and the elution conditions were as follows: mobile phase, 85% hexane, 10% ethanol, and 5% acetonitrile; flow rate, 2 mL/min. The enantiomers thus separated were further purified with a normal-phase 4.6- X 250-mm Zorbax SIL column (Dupont Co., Wilmington, DE); the elution conditions were as follows: mobile phase, 40% tetrahydrofuran in hexane; flow rate, 1mL/min. Caution: BPDE is hazardous and should be handled with great care, auoiding contact with the skin; any spillage should be immediately treated with a dilute solution of acid in order to hydrolyze the diol epoxide. The oligodeoxynucleotided(TATGCGTAT) was synthesized by means of a Cyclone DNA synthesizer (Biosearch, Inc., San Rafael, CA), purified, and modified covalently with (+)-BPDE in 20 mM sodium phosphate buffer solution (pH = 7.0) as described (ZO),with one modification: the addition of 1.7 % TEA1 which elevates the pH of the solution to about 11and tends to give more reproducible reaction yields as mentioned earlier (21). Furthermore, it is recommended that high levels of purities of all reagents be maintained, especially the buffer solution, which should be freshly prepared. The (+)-BPDE-oligonucleotide reaction mixture was subjected to HPLC separation employing a 10- X 250-mm Hypersil-ODScolumn (Keystone Scientific, Inc., Bellefonte, PA) and a 0-90% linear gradient of a methanol/2O mM sodium phosphate buffer solution in 60 min with a flow rate of 3 mL/min. The presence of modified and unmodified oligonucleotidesin the eluates was detected via the W absorbance of the DNA samples at 254 nm; in addition, the modified adducts were detected by fluorescence methods (20) as well (excitation wavelength 350 nm; emission viewing wavelength: 400 nm). The chemical and stereochemical nature of the (+)-BPDEW-dG lesionswere established by enzyme digestion of the BPDEoligonucleotideadducb to the nucleoside and BPDE-nucleoside levels, followed by comparisons of the HPLC elution times and CD1 spectra of these digests with those of BPDE-dG standards of established stereochemistry (3,20,21,261. The (+)-BPDE-modified oligonucleotideswere labeled at the 5'-end with [y-s2P]ATP purchased from New England Nuclear Corp. (Boston, MA) and employing a T4polynucleotide kinase 5'-terminus labeling system (Bethesda Research Laboratories, Gaithersburg, MD). The modified and unmodified oligonucleotides were subjected to the Maxam-Gilbert G+A as well as G cleavage reactions in which strand scission occurs at the 5'-side of guanine and adenine (27). The protocols published by Maniatis et al. (28)were followed closely. Briefly, in the case of the G+A strand cleavage reaction, the DNA solution was heated in 80% formic acid at 37 O C for 15 min and subsequently purified by precipitation in cold ethanol twice; the DNA was then dissolved in a 1M piperidine solution and heated at 90 "C for 30 min. The G strand cleavage reaction is similar, but the formic acid step is replaced by a reaction with dimethyl sulfate (27,223). The various samples were subjected to gel electrophoresis using 20% polyacrylamide denaturing gels (7 M urea) using a Poker-Face I1 Model SE 1600gel electrophoresis apparatus 40 cm long (Hoefer Scientific Instruments, San Francisco); the applied voltage was 2500 V, and the temperature was typically 45 "C. The bands were detected by standard autoradiography techniques using Kodak film, No. XAR 5 (Eastman Kodak, Co., Rochester, NY). Absorption spectra were determined with an Aviv Model 14 DS UV-vis spectrophotometer (Aviv Associates, Lakewood, NJ). Circular dichroism spectra were determined with a home-built CD system which was calibrated with a d-camphorsulfonic acid solution (29);the magnitude of the CD signal is reported in terms of molar ellipticities (20,261,and in terms of mdeg/A (3),where A is the absorbance at the 350-nm absorption maximum.

1 .oo

0.e.O

o.e.0

I

1

1

d' 1 j(

0 o .' 4e0. o /

0.20

-

,

4

0.00

10

10

90

22

24

28

Elutlon Tlmr (mln)

Figure 1. HPLC elution profile of (+)-BPDE-U-d(TATGCG-

TAT) reaction mixtures (see the text for elution conditions and other details). Peak 1: Unmodified oligonucleotide. Peaks 2 and 3 trans-BPDE-N2-dG oligonucleotide adducts with the BPDE residues at G4 (peak 3) and Go (peak 2) as determined by gel electrophoresis in this work. Eluates 4 and 6 contain cisaddition products. Bottom trace: Detection by absorbance at 254 nm. Upper trace: Detection by fluorescence (excitation 350 nm, emission viewed at 400 nm).

Results HPLC Elution Profiles of (+)-BPDE/Oligonucleotide Reaction Mixtures. An elution profile of a (+)BPDE/oligonucleotidereaction mixture is shown in Figure 1. Five different elution peaks, characterized by their absorbances at 254 nm, are evident (lower trace, Figure 1). Peak 1 at 18 min is due to the unmodified oligonucleotided(TATGCGTAT). The next four eluates between 21 and 26 min contain (+)-BPDE-oligonucleotideadducts and emit fluorescence upon excitation at 350 nm (upper trace in Figure 1). Determination of BPDE-N-dG Adduct Stereochemistry. Each of these four eluates was subjected to enzyme digestionusing standard procedures (snakevenom phosphodiesteraseand bacterial alkaline phosphatase) to obtain (+)-BPDE-nucleoside adducts (20,211. For each eluate, a single (+)-BPDE-dG adduct was observed by reverse-phase HPLC (data not shown) which coeluted either with a cis-(+)-BPDE-W-dG adduct standard (enzyme digests of eluates 4and 5)or witha truns-(+)-BPDEW-dG adduct standard (enzyme digests of eluates 2 and 3). Furthermore, the CD spectra of the enzyme digests of eluates 4 and 5 were the same as those of authentic cis(+)-BPDE-W-dG adducts, while the CD spectra of peaks 2 and 3 coincided with those of authentic trans-(+)-BPDEW-dG adducts (3). In summary, eluates 4 and 5 contain cis-(+)-BPDE-W-dG oligonucleotide adducts, while eluates 2 and 3 contain trans-(+)-BPDE-W-dG oligonucleotide adducts. Adducts with cis-additionstereochemistry elute before those with trans-addition stereochemistry as was found earlier in the case of other (+)-BPDEoligonucleotide adducts (20, 26). UV SpectroscopicCharacteristics. The absorption, fluorescence emission, and CD spectra of each of these four adduct solutions were entirely similar to those published earlier for similar oligonucleotide adducts containingsingle BPDE residues (20,211. A representative absorption spectrum of HPLC eluate 3 (Figure 1)is shown in Figure 2,while the CD spectra of eluates 2 and 3 are depicted in Figure 3. Both CD spectra are similar to that of another trans-BPDE-"2-dG adduct in the sequence

Chem. Res. Toxicot., Vol. 5, No. 6,1992 778

BenzotaJpyrene Dihydrodiot Epoxide Binding Sites 0.9

HPLC ELVATES No.

249

I

0.6

0.3

0.0 220

250

200

310

340

370

3

AGl

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AG3

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1

2

G1

GZ

3 G q

a-

- a

-b

C-

- dd

e-

- e

f-

- f

400

Wavelength (nm)

Figure 2. Absorption spectrum of HPLC eluate 3 [containing 5’-d(TATGBPDECGTAT)].The absorption spectrum of eluate 2 is similar (data not shown). The modified oligonucleotidestrand concentration was =6 pM (estimated from the molar extinction coefficient of 29 OOO M-l cm-l at the 350-nm absorption maximum as described in ref. 20). I

2

b-

d, &-

.

1

8-

h-

- g

- h

’

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s a

30

m 0 Q

16

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-

1

0

I -16

-20 t- , 240

280

320

360

400

Wavelength, nm

240

280

320

360

400

Wavelength, nm

Figure3. Circular dichroism spectra of HPLC eluates 2 (A) and 3 (B) [5’-d(TATGCGBPD’TAT)and 5’-d(TATGBPDWGTAT), respectively].

d(CACATGTACAC) (21), thus confirming the transaddition stereochemistryof the adducts in HPLC eluates 2 and 3 as determined from the enzyme digestion experiments. Below 300 nm, these CD spectra are attributed to a superposition of excitonic pyrenyl-dG residue interactions, and to the intrinsic single-stranded DNA CD spectra. Above 300 nm, the CD spectra resemble inverted absorption spectra and are attributed to the asymmetry of the environment of the pyrenyl residues (induced CD effects). The CD spectra of both samples are quite similar, except that the magnitude of the CD signal due to the induced CD mechanism is somewhat greater in the case of HPLC eluate 3 than in the case of eluate 2. Identification of Site of Binding by Gel Electrophoresis. The site of modification at sites G4 or Gs can be established by Maxam-Gilbert gel sequencing methods.

Figure 4. Gel electrophoresis patterns of unmodified oligonucleotides (HPLC eluate 1)subjected to the Maxam-GilbertA+G and G strand cleavage reactions (lanesAG1 and G1, respectively). The analogous band patterns for HPLC eluates 2 (AG2, G2) and 3 (AG3, G3) are shown in the neighboring lanes. The letters a-i designate the different bands which are identified in Table I.

We illustrate the procedures using the two major truns(+)-BPDE-oligonucleotide adducts (eluates 2 and 3). The gel electrophoresis patterns of the unmodified oligonucleotide (HPLC eluate 1,Figure 1)and the (+)trans-BPDE-oligonucleotide adducts (eluates 2 and 3) subjected to the Maxam-Gilbert G+A (lanes AG1, AG3, and AG3) and G (lanes G1, G2, and G3) strand cleavage reaction are shown in Figure 4. The letters a-i designate the different distances of migration and are used as reference in the following discussion. On the basis of the following considerations, each of these bands has been identified as summarized in Table I. (A) HPLC Elution Peak 1 (Unmodified Oligonucleotide). As expected, the G+A strand cleavagereaction yields five bands which are identified as the 9-mer, %mer, &mer, 3-mer, and dT (bands d, f, g, h, and i, respectively, in lane AG1). The G reaction yields only three bands, which are identified as the 9-mer, 5-mer, and 3-mer (d, g, and h, respectively, in lane Gl). The 9-, 5-, and 3-mer bands occur at the same levels in the G+A and G reactions, as expected. (B) Electrophoretic Migration Patterns of Intact, BPDE-Modified 9-mers. The electrophoretic band patterns of HPLC eluates 2 and 3 subjected to the strand cleavage reactions are shown in lanes AG2, G2, and AG3, G3, respectively. The slowest mobility bands in each of these lanes are attributed to the BPDE-modified 9-mers, which migrate slower than the unmodified 9-mer (lanes AG1 and Gl). There is a slight but definite difference in electrophoretic mobilities of the BPDE-modified 9-mers in lanes 2 and 3, which is particularly evident in lanes G2 and G3. We conclude that (1) the presence of the

776 Chem. Res. Toxicol., Vol. 5, No. 6,1992

Ma0 et al.

Table I. Identification of Bands in t h e Electrophoretic Gel (Figure 4)

3.5 I

,

N, no. of Neff,effective ael level BPDE a b c d d’ e f g h i

yes ves -~ ves no yes ves no no no no I

DNA bases

fragment length”

9 9 7 9 7 5 7 5 3 1

11.3 11.0 9.3

8.8 7.7

DNA fragmentb 5’-d(pTATGCGBPDETAT) ~’-~(DTATG~’~~CGTAT) 5’-d(pTATGCGBPDETp) 5’-d(pTATGCGTAT) 5’-d (pTATGBPDECGTp) 5’-d(pTATGBPDECp) 5’-d(pTATGCGTp) 5’-d(pTATGCp) 5’-d(pTATp)

~’-~(PTP)

Nett = M(BPDE)/Mav, where Mavis the molecular weight of the unmodified fragment divided by the number of nucleic acid residues in that fragment; M(BPDE) is the effective molecular weight of the modified fragment obtained from eq 1and the data in Figure 5. The values of the constants a = 3.55 and b = 0.00767 mm-l in eq 1were obtained from a least squares fit (see text). b Terminal phosphate groups are shown only.

covalently bound BPDE residues significantly slows the electrophoretic mobilities of the modified oligonucleotide strands and (2) the electrophoretic mobility depends on the placement of the BPDE residue in the oligonucleotide sequence. This difference in the elelctrophoretic mobilities of the unmodified and (+)-BPDE-modified oligonucleotides and their cleavage fragments is the basis of the method for distinguishing between different sites of modification. (C) Electrophoretic Mobility Patterns of G-Reaction Oligonucleotide Fragments. We first consider the electrophoretic band patterns arising from the G strand scission reaction in lanes G2 and G3, obtained with HPLC eluates 2 and 3, respectively. In lane G2, three bands are observed. The bands at levels g and h comigrate with those observed in the case of the unmodified strand (lane Gl);therefore, these two bands do not contain any BPDE residues. This suggests that the BPDE residue in the modified oligonucleotide in eluate 2 is not located at the first G counted from the 5’-side (position G4); therefore, it must be located at the second G (Gd. In lane G3, only the 3-mer (level h) comigrates with the analogous band obtained from the unmodified oligonucleotide;in this lane, bands at levels b and e are attributed to the 9-mer and 5-mer fragments bearing the BPDE residue at position G4 (first G counted from the 5’4de). Therefore, HPLC eluate 3 contains modified oligonucleotides bearing the BPDE residue at G4. (D) Electrophoretic Mobility Patterns of (G+A)Reaction Oligonucleotide Fragments. The conclusion that HPLC eluate 2 contains the modified oligonucleotide 5’-d(TATGCGBPDETAT),while HPLC eluate 3 corresponds to the oligonucleotide 5’-d(TATGBPDECGTAT), can be further confirmed by consideringthe electrophoretic band patterns resulting from the G+Areaction (lanes AG2 and AG3, Figure 4). In lane AG2, only the dT and 3-mer fragments comigrate with those of the unmodified oligonucleotide fragments. Two slower bands at levels a and c are observed (the weaker bands at levels d and f are attributed to unmodified 9-mer and 7-mer degradation products resulting from the loss of BPDE residues during the Maxam-Gilbert strand cleavage reactions). In lane AG3, the band at level h is missing, but three other bands at levels b, d(d’),and e are discerned. The results shown in lanes AG2 and AG3 may be understood if it is assumed

\

2.5 2‘7 0

28

56

Distance

84

112

140

(mm)

Figure 5. Semilog plot of molecular weights of the different

unmodified oligonucleotide fragments obtained from the G+A strand cleavage reaction as a function of the relative migration distance D, according to eq 1. T h e value of D = 0 waa used as t h e distance of the slowest band in Figure 4 (band a).

that the A+G strand cleavage reaction is inhibited at the 5’-side of BPDE-modified guanines. The presence of a band at level h in lane AG2 then signifies that there is no BPDE residue at G4 and that it is therefore located at Gg; this assignment is completely consistent with the one deduced from the band pattern in the corresponding G reaction (lane G2). The modified 5-mer band is missing in this lane, while the band at level c is attributed to the retarded 7-mer fragment containing the BPDE residue at position Gg. In lane AG3 (HPLC elution peak 3), the unmodified 3-mer band is missing, suggesting that the BPDE residue is located at position Gq; again this assignment is consistent with the conclusionsderived from the G-reaction data (lane G3). The bands at levels e and d(d’) are attributed to BPDE-modified 5-mer and 7-mer fragments, respectively. (E) Relative Mobilities of BPDE-Modified DNA Fragments. In general, under identical conditions of electrophoresis, the distance D of migration of singlestranded oligonucleotide fragments follows the empirical relationship (30-32):

D = a - b log (M)

(1) where a and b are constants and Mis the molecular weight of the unmodified fragments. A semilogarithimic plot based on data for the unmodified oligonucleotide G+A reactions (lane AG1, Figure 4) is shown in Figure 5. The data points fall on a straight line (correlation coefficient: 0.992). The effective molecular weights M(BPDE) of the modified fragments (a-c, d’, and e) can be estimated from this plot. The effective lengths, Neff,of each of these modified fragments, obtained from Neff= M(BPDE)/Mav, where Ma, is the mean molecular weight of the nucleic acid bases in that particular fragment (Mav = 312, 324, and 327 g mol-’ for fragments d, f, and g), are shown in Table I. It is evident that the presence of the BPDE = Neff residue affects the mobility as if an additional - N = 1.8-2.7 bases were present, depending on the fragment (N is the number of nucleic acid residues in each fragment). The observed lower mobilities of the BPDEmodified oligonucleotidesand fragments can be attributed to the following factors: (1)the mass of the BPDE residue (ita molar mass is 319 Da, which is close to the average molar mass of the nucleotides), (2) the lack of a negative

Benzo[a]pyrene Dihydrodiol Epoxide Binding Sites

Chem. Res. Toxicol., Vol. 5, No. 6, 1992 777

charge on the BPDE residue, in contrast to the charge of -1 per nucleotide (-2 at the terminiof the oligonucleotide), and (3) the position of the BPDE residue in the sequence. The retardation is highest for fragments a, c, and e (AN = 2.3, 2.3, and 2.7, respectively) in which the center of mass is shifted closer to the 3'-end due to the presence of the BPDE residue nearer to that end. Fragments b and d', in which the BPDE residue is located more a t the center of the sequence, are characterized by the two smallest values of AN (2.0 and 1.8, respectively). These differences, however, could also result from base sequence effects. These questions will need to be investigated in greater detail.

Acknowledgment. This work was supported by the Office of Health and Environmental Research, The Department of Energy (under Grant DE-FGO288ER60674). The synthesis of the modified oligonucleotides was supported by Grant CA 20851 from the US. Public Health Service, Department of Health and Human Resources, awarded by the National Cancer Institute. The Radiation and Solid State Laboratory is supported by DOE Grant DE-FG02-86ER60405. The portion of the work performed at The University of Chicago was supported by NIH Grant ES-04732 and by American Cancer Society Grant CN-22.

Discussion

(1) Weinstein, I. B., Jeffrey, A. M., Jennette, K. W., Blobstein, S. H.,

The diol epoxide (+)-BPDE exhibits astrong preference for binding to guanines in short oligonucleotides by either cis or trans addition of the dG moieties to the C10 position of BPDE. Similar selective binding patterns are observed with native DNA (2, 3) and synthetic polynucleotides containing only GC base pairs (20,211. The overall trans/ cis adduct ratio in the case of the single-stranded oligonucleotide d(TATGCGTAT) is -4.51, which is similar to the value observed in double-stranded GC-containing polynucleotides (21),but lower than in the case of native DNA (2, 3). Thus, the proportion of trans adducts is significantly higher than that of cis adducts in native double-stranded DNA, in synthetic polynucleotides, and in single-stranded oligonucleotides. The secondary structure of polynucleotides does not seem to play a significant role in determining the trans/cis adduct ratio (20). The simple HPLC procedures employed here prove that four adducts, differing from one another only in their stereochemical characteristics and adduct placement, can be separated from one another using 0-90% methanol/ buffer gradients. This suggests that the direct synthesis method, because of its simplicity and relative speed, is useful for synthesizing (+)-BPDE-modified oligonucleotides with one or two guanines. The site of adduct placement can be determined by using the Maxam-Gilbert sequencing approach described here. The lower probabilities of strand breakage at modified G's in the G+A strand cleavage reaction help to quickly identify the site of the BPDE-modified guanine. However, the identification of the site of BPDE binding does not solely depend on differences in the probabilities of strand breakage at modified and unmodified guanine sites. The lower electrophoretic mobilities of fragments bearing BPDE residues can be used to distinguish between the two possible sites of binding of (+)-BPDE to guanines by comparing the electrophoretic migration patterns of fragments obtained from the G+A and G strand cleavage reactions of modified and unmodified oligonucleotides. One possible limitation of the technique is that the mobilities of modified and unmodified fragments approach one another as the molecular weights, or lengths of these fragments, are increased. It should be noted, however, that identification is facilitated the closer the site of binding is to the 32P-labeled end, regardless of the length of the entire fragment. Our own experience suggests that the mobilities of BPDE-modified and unmodified oligonucleotides can be easily distinguished from one another for BPDE-modified DNA sequences with as many as 23 bases.2 B. Mao, B. Li, and N. E. Geacintov, unpublished observations.

References Harvey, R. G., Harris, C., Autrup, H., Kasai, H., and Nakanishi, K. (1976) BenzoIaIpyrene diol epoxides as intermediates in nucleic acid binding in vitro and in vivo. Science 193, 592-594. (2) Meehan, T., and Straub, K. (1979) Double stranded DNA stereoselectively binds benzo[a]pyrene diol epoxides. Nature 277,410412. (3) Cheng, S. C., Hilton, B. D., Roman, J. M., and Dipple, A. (1989)

DNA adducts from carcinogenic and noncarcinogenic enantiomers of benzo[alpyrene dihydrodiol epoxide. Chem. Res. Toxicol.2,334340. (4) Conney, A. H. (1982) Induction of microsomal enzymes by foreign

chemicals and carcinogenesisby polycyclic aromatic hydrocarbons. Cancer Res. 42, 4875-4917. (5) Singer,B.,andGrunberger,D. (1983)MolecularBiologyofMutagens and Carcinogens, Plenum Press, New York. (6) Harvey, R. G. (1991) PolycyclicAromaticHydrocarbons: Chemistry and Carcinogenicity, Cambridge University Press, Cambridge, England. (7) Benasutti, M., Ezzedine, Z. D., and Loechler, E. L. (1988) Construction of an Escherichia coli vector containing the major DNA adduct of activated benzo[alpyrene at a defied site. Chem. Res. Tozicol. 1, 160-168. (8) Basu, A. K., and Essigman, J. M. (1988) Site-specifically modified oligonucleotides as probes for the structural and biological effecta of DNA-damaging agents. Chem. Res. Toxicol. 1, 1-18. (9) Harris, C. M., Zhou, L., Strand, E. A., and Harris, T. M. (1991) New Strategy for the synthesis of oligodeoxynucleotidesbearing adducts at exocyclic amino sites of purine nucleosides. J. Am Chem. SOC. 113,4328-4329. (10) Casale, R., and McLaughlin, L. W. (1990) Synthesis and properties

of an oligonucleotidecontaining a polycyclic aromatic hydrocarbon site specificallybound to the N*amino group of a 2'-deoxyguanoeine residue. J. Am. Chem. SOC. 112, 5264-5271. (11) Smith,C. A. (1991) Chemicalsyntheaisof oligonucleotidescontaining a naphthalene diolepoxide deoxycytidine adduct in solution and using a mixed chemistry semi-automated solid phase approach. Carcinogenesis 12, 631-636. (12) Stezowski,J. J., Loos-Guba,G., Schtinwklder,K. H., Straub, A., and Glusker, J. P. (1987) Preparation and characterization in solution of oligonucleotides alkylated by activated carcinogenic polycyclic aromatic hydrocarbons. J. Biomol. S t r u t . Dyn. 5, 615-637. (13) Lee, H., Hinz,M.,Stezowski, J. J.,andHarvey,R. G. (1990) Synthesis of polycyclic aromatic hydrocarbon-nucleoside and oligonucleotide adducts specifically alkylated on the amino functions of deoxyguanosine and deoxyadenosine. Tetrahedron Lett. 31,6773-6776. (14) Sanford, D. G., and Krugh, T. R. (1985) N-Acetoxy-2-acetylaminofluorene modification of a deoxyoligonucleotideduplex. Nucleic Acids Res. 13,5907-5913. (15) Johnson, D. L., Reid, T. M., Lee, M.-S., King, C. M., and Romano, L. J. (1986) Preparation of a viral DNA molecule containing a sitespecific 2-aminofluorene adduct: a new probe for mutagenesis by carcinogens. Biochemistry 26,449-456. (16) Marques, M. M., and Beland, F. A. (1990) Synthesis, characterization and conformational analysis of ras sequences modified by arylamine carcinogens at the first base of codon 61. Chem. Res. Toxicol. 3, 559-565. (17) Lasko, D. D., Basu, A. K., Kadlubar, F. F., Evans, F. E., Lay, J. O., Jr., and Essigman, J. M. (1987) A probe for the mutagenic activity

of the carcinogen 4-aminobiphenyl: synthesis and characterization of an Ml3mplO genome containing the major carcinogen-DNA adduct at a unique site. Biochemistry 26, 3072-3081.

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Registry No. (+)-BPDE, 63323-31-9; 5’-d(TATGCGTAT), 143104-43-2;cis-(+)-BPDE-Nz-dG,66141-82-0;tram-(+)-BPDEW-dG, 65437-20-9.