Site-specific carcinogen binding to DNA - Europe PMC

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Benzo[a]pyrenediol epoxide (BPDE) is a well- studied environmental carcinogen that binds covalently to. DNA. Here we describe a photochemical technique ...
Proc. Natl. Acad. Sci. USA

Vol. 81, pp. 5623-5627, September 1984

Biochemistry

Site-specific carcinogen binding to DNA (benzo[a]pyrene diol epoxide/chicken ,B-globin gene)

T. CHRISTIAN BOLES AND MICHAEL E. HOGAN Department of Molecular Biology, Princeton University, Princeton, NJ 08544

Communicated by Walter Kauzmann, May 4, 1984

ABSTRACT Benzo[a]pyrene diol epoxide (BPDE) is a wellstudied environmental carcinogen that binds covalently to DNA. Here we describe a photochemical technique that allows us to map BPDE-binding sites within cloned gene sequences. The technique is based upon our observation that, when irradiated with laser light at 355 nm, one single-strand DNA cut is produced at each BPDE binding site. In initial experiments we have studied the distribution of such cuts in cloned DNA from the chicken adult /8-globin gene. We rind that BPDE binding in this gene sequence is distinctly nonrandom. While several prominent BPDE-binding sites are evident, a 300-basepair sequence immediately 5' to the RNA cap site is most strongly attacked by the carcinogen. This region is believed to contain important transcriptional control sequences. We discuss the possibility that sequence-specific binding to such regulatory elements may be an important feature of the mechanism of the carcinogen.

Benzo[a]pyrene (BP) is one of the most thoroughly studied chemical carcinogens. However, its detailed mode of action is still largely unknown despite 50 years of work (for a recent brief overview of the history of this field, see ref. 1). In recent years the DNA-binding forms of the carcinogen and the metabolic pathways leading to their formation have been identified. The consensus of this work is that (+ )(7R,8S,9R, 10R)-7,8,8a-dihydroxy-9a,10a-epoxy-7,8,9,10tetrahydroxybenzo[a]pyrene [(+)-BPDE] is the metabolite that forms the majority of BP adducts. The major adduct formed in vitro (2, 3), and in vivo (3), results from nucleophilic attack of the N-2 of guanine upon the C-10 position of BPDE to yield a trans opening of the 9,10-epoxide ring. This guanine derivative constitutes 80-90% of all stable adducts (2, 3) and has been proposed to be responsible for the mutagenic (4) and carcinogenic (5, 6) effects of BP. The adduct appears to distort DNA at its binding site; however, the secondary structure of the adduct is still disputed (7-10). Here, we examine the photochemistry of BPDE when bound covalently to a DNA helix. We find that, when irradiated, the carcinogen cuts the DNA strand to which it is bound. We then show that the photochemical cutting process can be developed into a powerful tool for mapping carcinogen-binding sites in a eukaryotic gene.

MATERIALS AND METHODS BPDE Modification. DNA fragments 146 ± 2 base pairs (bp) long were prepared from chicken erythrocyte nucleosomes (11), then modified with (± )-BPDE as described elsewhere (9), with the exception that unreacted BPDE and BP tetrol were removed by three phenol/chloroform extractions. The extent of modification was measured optically, using 6346 = 2.95 x 104 cm-' M` for the BPDE-DNA adduct and E258 = 1.3 x 104 cm- I M - I (base pairs) for DNA.

Poly(dG)'poly(dC) (Boehringer) was sonicated (00C), treated with S1 nuclease (Boehringer) to remove singlestrand regions, then digested with proteinase K, extracted with phenol, and fractionated over a Sepharose 6B column as described (12). The DNA fraction used for this experiment migrated on a denaturing gel as a 330 + 50 base species. DNA was modified with [3H]BPDE and bound BPDE was determined by liquid scintillation counting. Preparation of 32P-End-Labeled .-Globin DNA. A 2110-bp EcoRI/Hindll fragment from pCA(3G1 (13) was subcloned in the EcoRI/HindIII fragment of pBR322. This plasmid (pHR-16a) was used for the mapping experiments in Fig. 4 (see Fig. 5 for a schematic representation of the insert). To 32P-end-label the DNA strand with the same sense as the mRNA, referred to as the "HindIII strand," pHR-16a was cleaved with HindIII, then 3'-end-labeled at that site with the Klenow fragment of DNA polymerase I and [a32P]dNTPs (14). After purification of the labeled DNA by extraction with phenol and precipitation with ethanol, it was cleaved with EcoRI. The digest was electrophoresed in a 1% agarose gel. The 2.1-kbp band was localized by autoradiography, and the DNA was electroeluted from the excised gel band (14). The DNA was purified by Elutip (Schleicher & Schuell) chromatography. DNA was then resuspended in 10 mM Tris'HCl/l mM EDTA, pH 7.5 (TE), and modified with BPDE to give an average of 1 BPDE per 2110-base single strand. The end-labeling strategy for the "EcoRI strand" is the same, with the difference that the plasmid was first digested with EcoRI and 3'-end-labeled at that site.

RESULTS BPDE-Mediated DNA Cutting. The strategy of our work is to selectively excite BPDE with intense laser light at 355 nm, then monitor the DNA strand scission that may result. Such cutting can be quantitated in a simple manner by denaturing gel electrophoresis if the DNA samples are homogeneous with respect to molecular weight. The DNA used for our initial experiments is 146 + 2 bp chicken DNA, extracted from nucleosome core particles (11). Fig. 1 Upper illustrates the assay we use to quantitate BPDE-mediated cutting. DNA to be assayed for light-induced cutting is divided into two identical samples. One is irradiated with 355-nm laser light, then both are subjected to electrophoresis in a denaturing acrylamide gel. As seen in Fig. 1 Upper, BPDE is responsible for light-induced cutting. No cutting is detected in the absence of BPDE (slots a and b). The amount of cutting (the band intensity decrease) increases steadily with increasing carcinogen density (slots c

through h). The extent of cutting can be quantified accurately. Let Abe the integrated area of the densitometer trace of the photographic negative of an unirradiated DNA band; A + is the band area associated with a duplicate, irradiated sample. The

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Abbreviations: BP, benzo[a]pyrene; BPDE, BP diol epoxide; bp, base pair(s).

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Wavelength (nm) FIG. 1. (Upper) Gel assay for BPDE-mediated cutting. Threemicroliter samples of BPDE-modified 146-bp chicken DNA (3 x l0-4 M bp of DNA) were irradiated for 5 min with 355-nm laser light at a continuous power output of 20 mW. Irradiated samples and nonirradiated controls were electrophoresed under denaturing conditions in 5% acrylamide/7 M urea gels. After electrophoresis, gels were equilibrated in buffer without urea, stained with ethidium bromide, and photographed under UV light. Lanes a, c, e, and g are irradiated samples. Lanes b, d, f, and h are nonirradiated samples. Lanes a and b were loaded with identical amounts of unmodified DNA. Lanes c and d were loaded with identical amounts of DNA modified with BPDE to a population average of 2.6 adducts per 146-base single strand. Similarly, lanes e and f were loaded with DNA with 1.6 adducts per strand, and lanes g and h were loaded with DNA with 1.1 adducts per strand. (Lower) Bleaching of BPDEDNA complexes. Samples of BPDE-modified 146-bp chicken DNA (1.9 BPDE per strand of DNA) were irradiated for 5 min with 355-nm laser light at various intensities. Nine-microliter aliquots of irradiated samples were diluted 1:100 and absorbance spectra were recorded on a Hewlett-Packard 8504 spectrophotometer. From top to bottom, the four curves represent spectra obtained from identical samples irradiated at 0, 5, 10, and 20 mW of continuous power, respectively.

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FIG. 2. (Upper) Relationship between BPDE dose and DNA cutting. BPDE-modified 146-bp chicken DNA was irradiated for 10 min at 30 mW (e, *) or 20 mW for 5 min (o, o). Irradiated samples were electrophoresed with an identical, unirradiated control in a 5% acrylamide gel under denaturing conditions (squares): 7 M urea/83 mM Tris borate/2 mM EDTA, pH 8.3; *, 30 mM NaOH/2 mM EDTA, pH 12.5. Other BPDE complexes were assayed under nondenaturing conditions (circles): 83 mM Tris borate/2 mM EDTA, pH 8.3. The extent of BPDE-mediated cutting was quantitated from photographic negatives of the gels. The units of the axes correspond to cuts or BPDE per 146-base single strand for the squares and cuts or BPDE per 146-bp helix for the circles. The data were fit to a linear function by the method of least squares. For the denaturing gel conditions the best-fit slope is 0.925 0.030 with an intercept of 0.013 0.004. For nondenaturing conditions the slope is 0.048 0.009; intercept 0.030 0.020. (Lower) Strand specificity of BPDE cutting. BPDE-modified 300-bp poly(dG)-poly(dC) was 32P-end-labeled on either the dG strand or the dC strand. Samples were irradiated and electrophoresed with controls on 2% agarose/NaOH denaturing gels. Gels were fixed with 10%1 trichloroacetic acid and autoradiographed. BPDE-mediated cutting of the dG strand (e) or the dC strand (o) was quantitated from the autoradiograms. c,

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The quantity N can be decomposed further in terms of the average number of BPDE per strand (n) times the probability (p) that a nick occurs at a BPDE-binding site (the cutting efficiency).

In Fig. 2 Upper we display the relationship between lightinduced cutting and BPDE-binding density. When the product is assayed under denaturing conditions, the number of

Biochemistry: Boles and Hogan cuts (the log of band area change) varies linearly with increasing BPDE per strand, with a slope p = 0.92 + 0.03. This indicates that approximately one cut is made for each BPDE adduct. In contrast, when the product is assayed under nondenaturing conditions it is evident that the probability of double-strand cutting is about 5% that for single strands (lower curve in Fig. 2 Upper). The low double-strand cutting probability confirms the single-strand character of BPDE-induced cuts. It should be emphasized that increasing the irradiation dose 2-fold does not increase the efficiency of BPDE-mediated cutting and that the same cutting efficiency is observed in two different denaturing gel systems (Fig. 2 Upper). From these data, we conclude that under our irradiation conditions the cutting reaction has proceeded to completion. To determine the strand specificity of cutting we have examined BPDE-mediated cutting of poly(dG)-poly(dC). Since BPDE binds readily to G, but has little affinity for C residues (2, 3), the poly(dG)-poly(dC) helix will be modified by BPDE only on its G strand. Poly(dG)-poly(dC) was modified with BPDE, then 3'-end-labeled with the Klenow fragment of DNA polymerase I and either [a-32P]dGTP or [a32P]dCTP (14) so that cutting of each strand could be examined independently. As seen in Fig. 2 Lower, the data clearly show that only the dG strand is cut; cutting of the dC strand is not detectable even when six BPDE are bound per helix. Therefore, when irradiated, BPDE cuts only the DNA strand to which it is covalently bound. Several DNA-binding dyes are known to mediate the production of singlet oxygen (102), which in turn is capable of damaging the DNA helix (15-17). To test for the involvement of such a mechanism, we have performed BPDE cutting assays in the presence of the '02 quenchers triethylamine (18) and sodium azide (19). The data of Fig. 3 Lower show that the quenchers have no detectable effect on the cutting reaction. Thus, it seems unlikely that the BPDE cutting mechanism involves 102 production. We have also examined the fate of the pyrene moiety during the DNA cutting reaction. In Fig. 1 Lower we present absorbance spectra obtained from irradiated and control DNA samples containing 1.9 ± 0.2 BPDE molecules per strand. A marked decrease in absorbance at 346 nm is seen after irradiation. The data of Fig. 1 Lower have been plotted in Fig. 3 Upper (broken line) to show the dependence of that bleaching reaction on irradiation intensity. For comparison, we have performed cutting assays on the DNA samples used for the absorbance measurements. The intensity dependence of the cutting reaction (Fig. 3 Upper, solid line) is also first order. Both reactions appear to be single-photon processes with a quantum yield near 4 x 10at 355 nm. Such close correspondence suggests that the two reactions may be coupled. To summarize, we propose a working hypothesis to explain DNA cutting by BPDE. (i) The pyrene moiety of BPDE absorbs a single photon of light (the reaction is first order with respect to intensity, as seen in Fig. 3 Upper). (i) The excited pyrene engages in direct photochemistry with the G strand to which it is fixed (Fig. 2 Lower). As a result of the reaction, pyrene is bleached and the backbone of the helix is cut (Fig. 3 Upper). Because cutting and bleaching are stoichiometric (Fig. 2 Upper), and because diffusible singlet oxygen is not required for the cutting process (Fig. 3 Lower), it is very likely that the single DNA cut that occurs is localized to the carcinogen-binding site. Sequence Specificity ofBPDE Binding. We have used the cutting assay described above to examine BPDE-binding sites in a cloned 2.1-kbp DNA fragment containing the 5' half of the chicken adult fl-globin gene (Figs. 1-3). As described in Materials and Methods, we have labeled the ends of that

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Quencher (mM) FIG. 3. (Upper) Light intensity dependence of BPDE-mediated cutting and BPDE adduct bleaching. BPDE-modified 146-bp chicken DNA (1.9 BPDE molecules per strand) was irradiated for 5 min with 355-nm laser light of various intensities. o, Intensity dependence of BPDE adduct bleaching at 346 nm. These data were taken from the absorbance spectra of Fig. 1 Lower. *, Intensity dependence of BPDE-mediated single-strand DNA cutting. Cutting was assayed under denaturing conditions as in Fig. 1 Upper. The curves represent computer fits to the data by a generalized nonlinear least-squares method. Data were fit to the function - ln(A/A0) - Cl', in which A/AO is the fraction of material remaining uncut or unbleached after being irradiated for 5 min at the indicated intensity. C is a constant, I is the intensity of the laser in mW, and i is the order of the photochemical reaction with respect to intensity. For the bleaching reaction (---), the fit values are C = 0.056 + 0.020, and i = 1.287 + 0.127. For the cutting reaction ( ), the fit values are C = 0.112 + 0.018 and i = 1.050 0.058. (Lower) Effect of singlet oxygen quenchers on BPDE-mediated DNA cutting. BPDE-modified 146-bp DNA (1.9 BPDE per strand) was mixed with various concentrations of triethylamine (e) or sodium azide (o) then assayed for light-induced cutting as described. In all cases the supporting buffer was TE, pH 7.5. Irradiation conditions were 20 mW, 5 min at 355 nm. The broken line corresponds to 1.9 cuts per strand (100% cutting efficiency).

3-globin sequence in a strand-specific manner. Those labeled fragments were modified with BPDE, irradiated, and electrophoresed on denaturing agarose gels. Autoradiograms of such gels are shown in Fig. 4. The lanes containing modified, irradiated DNA (lanes c and e in Fig. 4 Upper; lanes c, f, and i in Fig. 4 Lower) show a series of discrete bands super-

imposed over low-level background radioactivity. The lanes containing unmodified DNA (irradiated or nonirradiated) and the lanes containing nonirradiated, modified samples

show negligible amounts of radioactive species smaller than 2.1 kilobases. Such discrete DNA cutting shows that BPDE

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-750 -500 -250 0 +250 ±500 +75 Distance From Cap Site (bp) FIG. 5. Summary of BPDE-binding sites in the pHR-16a insert. (Upper) BPDE-cutting (BPDE-binding) sites on the strand labeled at the HindIII end. (Lower) BPDE cutting sites on the other strand, labeled at its EcoRI end. Vei-tical bars in the upper section of each panel correspond to the positions of cutting sites. For consistency, position has been converted to separation, in bp, from the RNA cap site (20). The height of the bars is proportional to the optical density of bands in Fig. 4 (measured by microdengitometry). In an endlabeling experiment such heights are directly proportional to the probability that BPDE cuts (binds) at that position. The center figure in each panel is a diagram of 8-globin gene structure (20): open boxes, transcribed noncodhng sequences; shaded boxes, coding sequences. The position of the DNase I-hypersensitive site (21) is underlined. At the bottom of each panel is a computer calculation of G residue density on each strand, at 30-base resolution. The data for these calculations are the /B-globih se4uence of ref. 20. The se388 and extend through the HindIII end of quence data start at the fragment. In these calculations the nearest neighbors G-A, GG, G-T, and G-C are assigned a value 1 and all other pairing is assigned a value of 0. -

FIG. 4. Mapping BPDE-binding sites in the chicken adult f-globin (Upper) HindI11 strand. The 2110-bp EcoRI/HindIII fragment front pHR-16a was 3'-end-labeled with 32P at the findIII site and modified with BPDE to give an average of 1 BODE per 2110base single strand. Samples were irradiated at 20 mW for 10 min. DNA samples were electrophoresed on a 1.7% agarose/NaOH gel; gels were fixed with 109% trichloroacetic acid and autoradiographed. Lanes b-e are BPDE-modified DNA; lanes c and e are irradiated samples; lanes b and d are identical nonirradiated samples. Lanes g-j are unmodified DNA; lanes h and j, irradiated samples; lanes g and i, nonirradiated samples. Lanes a and f are endlabeled Taq I digests of pBR322; the fragments are 1444, 1307, 616, 368, 315, and 312 bases long (13). (Lower) EcoRI strand. The 2110bp /3-globin gene fragment from pHR-16a was 3'-end-tabeled with 32P at its EcoRI terminus and subsequently modified with BPDE to give an average of 1 BPDE per 2110-base single strand (see Materials and Methods). All Other steps were performed as for Fig. 4 Upper. Lane a is an end-labeled HindIll digest of phage X DNA. Lanes b and are end-labeled Taq I digests of pBR322. Lanes c, f, and i are BPDE-modified irradiated DNA; lanes d, g, and j are BPDEmodified nonirradiated DNA; lanes e, h, and k are nonmodified nonirradiated DNA.

gene.

binds in a distinctly nohrandom fashion to chicken j3-globin DNA. In Fig. 5, the position and relative intensity of the binding sites mapped in the two end-labeling experiments are shown. It is especially important to recognize that a great deal of cutting (BPDE binding) occurs within a 300-bp region on the 5' side of the gene. This region (0 to 300) is believed to contain important transcriptional control signals for f-globin (20, 21). Evidently, the carcinogen recognizes and selectively attacks DNA sequences within this gene control region. In an attempt to explain the nonrandom distribution of BPDE-binding sites in this gene, we have analyzed the sequence of the fragment (20) at 30-base resolution (the resolution of our electrophoresis system). These plots are also shown in Fig. 5. The plots show that the G density along the two strands is markedly nonrandom. On the EcoRI strand, the region from + 50 to -200 contains prominent G-rich segments that align with BPDE-binding sites (Fig. 5 Lower). Two other G-rich features are seen on the HindIII strand at + 650 and + 700 bp (Fig. 5 Upper) which also align with BPDE-binding sites. -

Biochemistry: Boles and Hogan However, closer examination shows that a complete correlation between G density and BPDE binding cannot be made. Intense cutting sites centered at - 100 and - 250 on the HindIII strand occur in regions of moderate to low G density. On the JcoRI strand, the prominent cutting site at - 260 falls in a region of moderate G density. On the basis of these data, we conclude that site-specific BPDE binding is not related to the density of G residues in a simple fashion. It therefore seems likely that other effects, such as sequence-specific variation of DNA secondary structure can also influence BPDE binding. In this context, we have searched the 6-globin sequence for regions capable of undergoing the B-Z transition, using a modification of the program described in the legend of Fig. 5. At 10- or 30-base resolution, there appears to be no correlation between BPDE binding and the incidence of alternating purine-pyrimidine sequences (data not shown). We would like to emphasize that our studies were carried out with linearized DNA fragments. Therefore, the observed specificity of BPDE binding cannot be attributed to the presence of alternate DNA structures stabilized by superhelical stress.

DISCUSSION The work presented in this paper constitutes direct demonstration that a carcinogen can attack a gene in a site-specific manner. In an earlier experiment, [3H]BPDE adducts were found to be distributed randomly among restriction fragments of simian virus 40 DNA (22). We believe that, even if sequence features exist on this DNA to direct BPDE binding, those experiments lacked the sensitivity and spatial resolution necessary for mapping. In a second class of experiment, BPDE binding has been mapped within a cloned fragment of the Escherichia coli lad gene (24), taking advantage of depurination and strand breakage that can be induced when BPDE binds to N-7 of guanine. Those experiments showed'that BPDE can induce depurination that is sensitive to local sequence effects. Unfortunately the N-7 adduct is rare [less than 20% of the total formed in vitro (25)], and strand breakage by depurination is inefficient. We suggest that the distribution of BPDE-binding sites may not be related to the data in a simple way in such measurements. Less direct, though highly suggestive, data on binding specificity have come from an analysis of the distribution of base-substitution mutations induced by B3PDE in the lad gene (23). That study clearly demonstrated that a few positions in the lad sequence are especially mqtable. Again, however, the relationship between binding and the pattern of mutation may be complex in those experiments. Here, we have shown in a direct way that BPDE recognizes and selectively attacks the 5' flanking regions of the chicken 8-globin gene. We believe that such' site specific binding may not be a unique property of the globin gene: many genes, including chicken 8-globin, display DNA sequence features near the origin of transcription that make the gene sensitive to nuclease digestion in chromatin (26) and in purified recombinant plasmids (27). If BPDE can recognize

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such features in the -globin gene, it may recognize and bind specifically to those same features in other parts of the genome. That hypothesis can be tested by examining BPDE binding to other genes. The plasmid pCABG1 was graciously provided by Gary Felsenfeld. We acknowledge Johnny Chang-Ning Wang for the gift of pHR16a plasmid DNA and many helpful discussions. This work was supported by American Cancer Society Grant NP352.

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8. 9. 10. 11.

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15. 16. 17. 18.

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19. Hasty, N., Merkel, P. B., Radlick, P. & Kearns, D. R. (1972) Tetrahedron Lett. 1, 49-52. 20. Dolan, M., Dodgson, J. B. & Engel, J. D. (1983) J. Biol. Chem. 258, 3983-3990. 21. McGhee, J. D., Wood, W. I., Dolan, M., Engel, J. D. & Felsenfeld, G. (1981) Cell 27, 45-55. 22. Pulkrabek, P., Leffler, S., Grunberger, D. & Weinstein, I. B. (1979) Biochemistry 18, 5128-5134. 23. Eisenstadt, E., Warren, A. J., Porter, J., Atkins, D. & Miller, J. H. (1982) Proc. Natl. Acad. Sci. USA 79, 1945-1949. 24. Haseltine, W. A., Lo, K. M. & D'Andrea, A. D. (1980) Science 209, 929-931. 25. King, H. M. S., Osborne, M. R. & Brookes, P. (1979) Chem.Biol. Interact. 24, 345-353. 26. Wu, C. (1980) Nature (London) 286, 854-860. 27. Mace, H. A. F., Pelham, H. R. B. & Travers, A. A. (1983) Nature (London) 304, 555-557.