Chem.- Biol. Interactions, 23 (1978) 201--213
201
© Elsevier]North-Holland Scientific Publishers Ltd.
BENZO(a)PYRENE 7,8-DIHYDRODIOL-9,10-OXIDE MODIFICATION OF DNA: RELATION TO CHROMATIN STRUCTURE AND RECONSTITUTION
HIROSHI YAMASAKI,THOMAS W. ROUSH and I. BERNARD WEINSTEIN Department of Medicine and Institute of Cancer Research, Columbia University College of Physicans and Surgeons, 701 West 168th Street, New York, N.Y. 10032 (U.S.A.)
(Received September 30th, 1977) (Revision received May 22rid, 1978) (Accepted May 24th, 1978)
SUMMARY
Purified duck reticulocyte DNA was incubated in vitro y~ith a 7,8dihydrodiol-9,10-oxide derivative of benzo(a)pyrene (BPDE). The carcinogen-modified DNA was somewhat more susceptible to partial digestion by the single strand specific endonuclease $1 than unmodified DNA, suggesting slight denaturation of the helix at sites of modification. Chromatin was reconstituted in vitro utilizing this carcinogen-modified DNA and unmodified-chromatin associated proteins. This reconstituted chromatin showed the same kinetics and extent of digestion by S t a p h y l o c o c c a l nuclease, and similar nucleosome profiles on sucrose density gradient centrifugation, as those obtained with native chromatin or chromatin reconstituted with unmodified DNA. Moreover, polyacrylamide gel electrophoresis of DNA fragments obtained from nuclease digests gel electrophoresis of DNA fragments obtained from nuclease digests of the reconstituted chromatins suggested t h a t the chromatin containing carcinogen-modified DNA had the same subnucleosome structure as that reconstituted with unmodified DNA. In a separate set of studies intact duck reticulocyte chromatin was reacted directly with BPDE. Nuclease digestion studies indicated that 65% of the carcinogen was bound to the 'open' regions of chromatin, and 35% to 'closed' regions. These results indicate that although covalent binding of a benzo(a)pyrene (BP) derivative to DNA produces local distortions in conformation of the helix, this modification does n o t appear to interfere with the ability of the DNA to associate with histones to form nucleosome structures. In addition, although DNA in the open regions of chromatin is more susceptible to reaction with the BP derivative, there is appreciable reaction with the DNA associated with histones. Abbreviation: AAF, N-2-acetylaminofluorene; BP, benzo(a)pyrene; BPDE, benzo(a)pyrene 7,8-dihydrodiol-9,10-oxide.
202 INTRODUCTION It is n o w well established that several carcinogens, or their metabolites, bind covalently to cellular DNA following in vivo administration [1--4]. It is likely that the quaternary structure of DNA as it exists in vivo, i.e. its association with proteins, is an important determinant in this process. Benzo(a)pyrene (BP} is a potent and ubiquitous environmental carcinogen that requires metabolic activation to exert its cytotoxicity, mutagenicity and carcinogenicity [5--7]. Recent studies [8--14] indicate that the most reactive metabolite of BP is benzo(a)pyrene 7,8-dihydrodiol-9,10-oxide (BPDE). Several studies indicate that this derivative is the major metabolite responsible for the in vivo binding of BP to RNA and DNA in rodent, bovine and human cells [9--12,14]. In addition, the complete structures of the major BP-nucleoside adducts found in cellular R N A and DNA have been determined [15,16] and these reveal that the 10 position of BPDE is linked to the 2 amino group of guanine. Certain stereochemical aspects of this reaction have also been elucidated [17]. Related studies from other laboratories are consistent with this structure [12,18,19]. In eukaryotes, cellular DNA occurs as a nucleoprotein complex, or chromatin. It has been found that chromatin consists of a series of nucleoprotein subunits (nucleosomes) each containing a b o u t 200 base pairs of DNA and 2 molecules of each of the 4 major classes of histones [20--24]. Digestion of nuclei with Staphylococcal nuclease first releases multimers and monomers of nucleosomes. As digestion proceeds, the DNA of the nucleosome is reduced in size to a 'core' containing 140 base pairs. Finally, the DNA of the nucleosome core is itself attacked to produce a limit digest consisting of a series of DNA fragments to discrete sizes ranging from a b o u t 140--20 base pairs [20,22,25--27]. These fragments presumably reflect the organization of DNA and histones within the subnucleosome structure. It was of interest, therefore, to determine to what extent the modification of DNA with BPDE would interfere with its ability to participate in the formation of nucleosome structures, as well as to investigate the possibility that regions of DNA associated with histones in intact nucleosomes might be protected from modification,by BPDE. In this paper, we have modified DNA by in vitro reaction with BPDE and then examined the, effects of this modification on in vitro reconstitution of nucleosome structures. We have also reacted intact chroma~in with BPDE and then examined the distribution of the covalently bound BP residues in 'open' and 'closed' regions of the DNA, i.e. regions highly susceptible to digestion by Staphylococcal nuclease versus regions relatively resistant to digestion by this nuclease. MATERIALS AND METHODS
Chemicals [14C]BPDE (Sp. act. 53.6 ~Ci/~mol) was obtained from the National
203 Cancer Institute, Bethesda, Maryland. It was a racemic mixture of what we have previously designated 'Isomer I' [14]. Staphylococcal nuclease (EC 3.1.4.7) and Proteinase K were obtained from Worthington Biochemical Corporation, Freehold, New Jersey, and E. Merck, Rahway, New Jersey, respectively. Endonuclease S~ (EC 3.1.4. X) from Aspergillus oryzae was from Miles-Pentex Laboratories, Kankakee, Illinois.
Preparation of duck reticulocyte chromatin and DNA. Duck reticulocyte chromatin and DNA were prepared as previously described [28] from reticulocytes of phenylhydrazine hydrochloride-treated ducks. Modification of DNA and chromatin of [14C]BPDE. The modification of DNA was carried out under conditions similar to those previously described for the modification of DNA with N-acetoxy AAF [29]-. The reaction mixture contained 10 mM sodium cacodylate buffer, pH 7.2; 30% (v/v) ethanol; 500--600 pg of native DNA/ml; and 0.3 pmol/ml of [~4C]BPDE (53.6 Ci/mol). The mixture was incubated at 37°C f~r 30 min. Unbound radioactivity was extracted with an equal volume of ether. Ether extractions were repeated 8 times and after the final extraction the remaining ether was evaporated under nitrogen gas. After the addition of 0.1 vol. of 2 M sodium acetate (pH 5.0) and 2 vols. of cold ethanol, the modified DNA was spooled out of the solution and then dissolved in 0.1 mM EDTA; 1 mM Tris--HC1 (pH 7.9). Additional extractions did not remove radioactivity indicating that we were dealing with radioactive material covalently bound to the DNA. The modification of intact chromatin was carried out in a reaction mixture containing in 1 ml: lmM Tris--HC1, pH 7.9; 5% (v/v) ethanol; chromatin equivalent to 0.8--1.0 mg of DNA; and 0.3 pmol of [~4C]BPDE (53.6 Ci/mol). Incubation and extraction of non-covalently bound carcinogen was carried out as described for DNA modification. There was no evidence that these procedures altered the nucleosome structure of chromatin when analyzed by susceptibility to nuclease digestion, sucrose density gradient centrifugation of gel electrophoresis of DNA fragments after partial digestion of chromatin samples by Staphylococcal nuclease. The extent of DNA modification was determined by the ratio of radioactivity to A260 and expressed as the percent of total base residues modified by the BP derivative, assuming that 1 mg of DNA/ml equals 20 A260 units. The extent of chromatin modification was also expressed as the extent of DNA modification, after purification of the DNA as described [28]. In the present studies BPDE-modified samples refers to DNA or chromatin preparations in which 0.4--0.7% of the DNA bases contained covalently bound BP adducts. Reconstruction of chromatin from DNA and chromosomal proteins. Chromosomal proteins were prepared from duck reticulocyte chromatin, as described by Stein et al. [30], by suspension in 3 M NaC1, 5 M urea,
204 10 mM Tris--HC1 (pH 7.9). The DNA was removed by centrifugation at 150 000 g for 15 h. Protein was determined by the m e t h o d of Lowry et al. [31]. Native or carcinogen-modified DNA was reconstituted with native chromosomal proteins as described previously [ 29].
Nuclease digestion o f DNA and chromatin The digestion of DNA by S, endonuclease from A. oryzae, the digestion of chromatin b y Staphylococcal nuclease and the quantitation of extents of digestion were carried o u t as described previously [29]. Partially digested chromatin was prepared by stopping the digestion with 5 mM EDTA, added at the indicated time. Gel electrophoresis o f DNA Electrophoresis of DNA purified from undigested regions of chromatin was carried out as previously described [20] using an E-C Slab gel apparatus. DNA samples were purified from partially digested chromatin by incubating the chromatin with 100 pg/ml of proteinase K in the presence of 0.4 M NaC1, 10 mM Tris--HC1 (pH 7.9), 0.2% SDS and 5 mM EDTA for 1 h at 37°C. Protein was extracted with equal vols. of phenol/chloroform (1 : 1) solution. After shaking vigorously for 15 min, the aqueous phase was extracted 4 times with equal vols. of ether and the DNA precipitated with 2 vols. of cold ethanol. After overnight precipitation at - 3 0 ° C , DNA was collected by centrifugation at 15 000 g for 30 min at -20°C. The precipitated and dried DNA was dissolved in 50--100 pl of 0.5X Peacock's buffer [32] and electrophoresed in 6% acrylamide slab gels containing 0.32% bisacrylamide. After electrophoresis the DNA in the gel was stained with 0.017% (v/v) ethidium bromide solution. RESULTS
Susceptibility of [~4C]BPDE-modified regions of DNA to $1 nuclease digestion Previous studies have revealed that binding of the potent hepatocarcinogen N-2-acetylaminofluorene (AAF) to nucleic acids causes a marked distortion in the DNA helix which we have termed 'base displacement' [3,33,34]. The distortion produces localized regions of denaturation at the sites of AAF modification [33--36] whose size can be estimated with the aid of $1 nuclease, an endonuclease which under appropriate conditions preferentially digests single stranded regions of DNA [29]. Prior to using BPDE-modified DNA in chromatin studies it was of interest to determine the extent of denaturation of the modified DNA utilizing endonuclease .$1. Fig. 1 shows the time course of digestion by S~ of [~4C]BPDE-modified DNA compared to unmodified native and heat denatured DNA. The heatdenatured DNA was completely hydrolyzed within 20 mi~, at which time the control native DNA and the BPDE modified DNA were hydrolyzed less than 8%. The digestion of the BPDE modified DNA proceeded slightly more
205
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Fig. ]. Digestion of D N A samples with S 1 nt~e]ease. Native, heat denatured or [ '4C ]BPDRmodified D N A samples (50 /~g/m]) were incubated wif~ S, hue]ease ( ] 0 000 units/m]) for the indicated period of time at 37°C. The extent of digestion o f DNA samples and release of ['4C]BPDE-modified residues were determined as described in M e t h o d s . . , native DNA; ~, heat-denatured DNA; x, BPDE-modified DNA; o, release o f ["C]BPDEmodified residues from BPDE-modified DNA. Similar results were obtained when the entire study was repeated using 5000 units/ml of S, nuclease.
rapidly than the c o n t r o l native DNA. With the modified DNA the release of ~C was considerably faster than the release of total nucleotides (A~60). Almost 80% of the 14C residues were released after 1 h incubation with S, nuclease, whereas only 30% of the nucleotides were rendered acid soluble. These results suggest that the sites to which the [ I~C]BP residues were bound represented localized regions of denaturation. Separate more extensive studies indicate that the size of these localized denatured regions is about 1--2 base plates, which is considerably smaller than the regions of denaturation associated with AAF modification of DNA [37].
Reconstitution o f nucleosomes with ['4C]BPDE-modified DNA and unmodified chromatin associated protein Control native DNA, [~4C]BPDE-modified DNA or heat-denatured DNA were mixed with native chromosomal proteins to reconstitute chromatin. The extent of reconstitution was then determined by digestion with Staphylococcal nuclease. As shown in Fig. 2, chromatin reconstituted from control and BPDE-modified DNA were digested by Staphylococcal nuclease in a similar manner. As was reported previously for native chromatin [ 3 8 ] , about 50% of the DNA, in both the reconstituted chromatin containing unmodified DNA and in the material containing the [14C]BPDE-modified DNA, was protected from extensive digestion. Moreover, the release of A:60 and of radioactivity from the reconstituted chromatin containing [~C]BPDEmodifed DNA showed similar kinetics. The material 'reconstituted' from heat-denatured DNA was, however, rapidly and almost completely digested by Staphylococcal nuclease (Fig. 2). These results indicate that although
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Fig. 2. Kinetics of Staphylococcal nuclease digestion of reconstituted chromatin. Chromatin reconstituted with control native DNA (o--o), chromatin reconstituted with DNA containing a 0.65% modification of the bases with ['4C]BPDE ( , - ~ ) , and chromatin reconstituted with heat-denatured DNA (~.--a), were digested by Staphylococcal nuclease for the indicated periods of time and the release of total nucleotides was determined after terminating the reaction with 2 vols. of 2 M PCA/2 M NaCl mixture. The release of ['4C]BPDE-modified residues during the digestion of ['4C]BPDE-modified chromatin (x--x) was determined as in Fig. 1. The data presented are typical of those obtained in 5 similar independent experiments. co mp lete l y denatured DNA cannot associate with histones t o f o r m chromatin-like material, [14C]BPDE-modified DNA can interact with histones t o reconstitute chromatin, in spite of the presence of localized regions of denaturation and the bulky substituent. In order t o determine w he t he r the repeating subunits of chromatin, or nucleosomes, were also f o r m e d in chrom at i n reconstituted w i t h ' B P D E modified DNA, the above chromatin preparations were partially digested with Staphylococcal nuclease and then subjected t o sucrose density gradient centrifugation. As is shown in Fig. 3, similar profiles of nucleosome monomers (tube 18), dimers (tube 25) and trimers (tube 30) were f o u n d in the chromatin reconstituted with unmodified native DNA and BPDE-modified DNA. These profiles are similar t o those f o u n d in previous studies of partially digested native chromatin, in which evidence has been presented t hat these peaks correspond to nucleosome m o n o m e r s , dimers and trimers [ 2 0 , 2 9 ] . With the BPDE preparation the profile o f radioactivity, representing the distribution of the ['4C]BP adduct, paralleled t h a t o f the A:~0. This provides f ur t her evidence t h a t the [ 1~C]BPDE modified regions of DNA had participated in the f o r m a t i o n o f nucleosome structures during the reconstitution procedure. As expected, the "reconstituted' material obtained with heat d en atu r ed DNA failed to display nucleosome-like structures when similarly analyzed (not shown here).
Integration of [~4C]BPDE-modified regions o f DNA into the subnucleosome structure of chromatin during reconstitution When DNA is isolated from a limit Staphylococcal nuclease digest o f either native or reconstituted chromatin, a series of double-stranded DNA
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Fig. 3. Sucrose density gradient centrifugation of chromatin subunits. After approximately 20% digestion of chromatin by Staphylococcal nuclease, the reaction was stopped by the addition of 5 mM Na-EDTA. The suspension was then layered on a 5--10% linear sucrose density gradient in 5 mM Na-EDTA (pH 7.9) and centrifuged 16 h at 25 000 rev/min in an SW41 rotor at 4°C. The direction of sedimentation is from left--right. (a) The profile of a sample of control reconstituted chromatin. (b) The profile of a sample of chromatin reconstituted with ['4C]BPDE modified DNA. , A~0 profile; _[- , c.p.m, in each fraction. Similar profiles were obtained in three additional independent studies. f r a g m e n t s is o b s e r v e d w h i c h are spaced at a l m o s t regular intervals and range in size f r o m a b o u t 1 6 0 - - 2 0 base pairs [ 2 0 , 2 2 , 2 5 - - 2 7 ] . It was o f interest, t h e r e f o r e , t o see w h e t h e r similar D N A fractions are o b t a i n e d f r o m c h r o m a t i n reconstituted with [ 14C]BPDE-modified DNA. Fig. 4 s h o w s that t h e slab gel e l e c t r o p h o r e s i s pattern o f t h e D N A fragm e n t s o b t a i n e d f r o m the limit digest o f c h r o m a t i n r e c o n s t i t u t e d w i t h t h e [ ~ 4 C ] B P D E - m o d i f i e d D N A was essentially the s a m e as that o b t a i n e d f r o m a c o n t r o l preparation o f c h r o m a t i n . In order t o see w h e t h e r [ ~ C ] B P D E - m o d i f i e d regions o f D N A w e r e distrib u t e d e v e n l y a m o n g s u b n u c l e o s o m e fractions t h e slab gel was s u b j e c t e d t o f l u o r o g r a p h y [ 3 9 ] . F l u o r o g r a p h y o f t h e gels o b t a i n e d w i t h [ I ~ C ] B P D E D N A d e m o n s t r a t e d that radioactivity was distributed in m a n y o f t h e D N A bands. T h e r a d i o a c t i v i t y was n o t sufficient, h o w e v e r , t o d e t e r m i n e t h e specific activities o f individual bands.
Covalent binding of [14C]BPDE to 'open' and 'closed' regions of intact chroma tin In order t o see w h e t h e r [14C]BPDE can bind c o v a l e n t l y t o D N A w h i c h is
208
Fig. 4. Polyacrylamide gel electrophoresis of DNA extracted from Staphylococcal nuclease digests of chromatin. Gels represent DNA extracted from control reconstituted chromatin (right), and from chromatin reconstituted with [~4C]BPDE-modified DNA (left). Extent of digestion was 40--50%. Migration is from top to bottom.
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already a part of the nucleosome structure, a separate set of experiments was undertaken. Intact chromatin was incubated with ['4C]BPDE under conditions which did n o t produce detectable changes in the overall structure of the chromatin, and the noncovalently bound radioactivity was extracted. The [~4C]BPDE-modified chromatin was then incubated with Staphylococcal nuclease, as in Fig. 2, and at various time points DNA was extracted and purified from the undigested fraction of the chromatin and its specific radioactivity determined. Fig. 5 shows the kinetics of Staphylococcal nuclease digestion of the modified chromatin and the specific radioactivity of the DNA in the resistant regions during the course of digestion. The digestion of the total DNA followed a pattern typical for chromatin, reaching a plateau at a b o u t 50% digestion. This indicates that the [~4C]BPDE modification procedure and the subsequent ether extractions had n o t grossly disturbed the chromatin structure. It is of interest that during the digestion there was an appreciable decrease in the specific radioactivity of the undigested fraction of the DNA, particularly at the early time points. This indicates that the 'open' regions of the DNA, i.e. those which are most susceptible to Staphylococcal nuclease digestion have a higher specific activity than the 'closed' regions of DNA which are more resistant to digestion. At 60 min, which represents a limit digest of the DNA (about 50% total digestion), a b o u t 65% of the total [ ~ C ] B P adducts had been removed and the remaining 35% of the carcinogen residues were left on the nuclease resistant DNA regions of chromatin. Thus, when incubated with intact chromatin [ 1~C]BPDE preferentially modified the 'open' regions of DNA. It should be noted, however, that there was also substantial reaction with
210 'closed' regions of DNA, i.e., those protected by histones from Staphylo-
coccal nuclease digestion. DISCUSSION The present results indicate that modification of native DNA by reaction with BPDE leads to small localized regions of denaturation which are excised by $1 nuclease, a single strand specific endonuclease. The kinetics of release of BP-modified nucleotides and their ratio to total nucleotides released during S, nuclease digestion suggest that these regions of denaturation are considerably smaller than those associated with the covalent binding of AAF to DNA. A more detailed analysis of the conformation of the BP-modified regions is presented elsewhere [37]. We have found that despite these localized changes in DNA structure and the covalent attachment of the bulk BP residue to the 2 amino group of guanine [ 1 4 , 1 5 ] , the modified DNA can still reassociate with histones to form a chromatin-like structure which by the criteria employed in the present study resembles that of normal nucleosomes. Our results do not, of course, exclude the possibility that the chromatin reconstituted with the modified DNA may have subtle distortions which are n o t revealed by the present methods. In separate studies we have found that the covalent attachment of AAF residues to DNA also does n o t grossly interfere with its ability to function during chromatin reconstitution [ 2 9 ] . On the other hand, completely denatured DNA cannot function in the reconstitution o f chromatin. These results suggest that although the DNA must have a structure which is predominantly double stranded, localized distortions in base pairing and the presence of fairly bulky substituents do n o t interfere with the DNAhistone associations involved in nucleosome formation. This is consistent with other evidence indicating that in the formation of nucleosomes histone molecules do not appear to recognize specific base sequences in the DNA [201. Utilizing intact chromatin as a substrate, we obtained elsewhere that BPDE reacts preferentially with the DNA present in the 'open' regions of the chromatin, i.e. regions~ readily~ s u s c e p t i b l e ~ o digestion by Staphylococcal nuclease. Substantial amounts of the carcinogen residue were, however, also b o u n d to DNA in 'closed' regions, i.e., those associated with histones in nucleosome structures and more resistant to digestion by this nuclease. The number of BP residues bound to 'closed' regions was a b o u t half that bound to 'open' regions. Thus, although the closed regions are protected from digestion with Staphylococcal nuclease digestion, they are susceptible to chemical modification. Rather similar results have been obtained by Metzger et al. [40] in studies on the in vitro modification of intact chromatin with N-acetoxy-AAF. 'Closed' regions of chromatin DNA also appear to be susceptible to modification by chemical carcinogens in vivo since we have found that when either hamster or human cell cultures were incubated with [3H]BP there was covalent binding of radioactivity to both 'open' and
211
'closed' regions of nuclear DNA, as defined by susceptibility and Staphylococcal nuclease digestion {unpublished studies). Similar results have recently been reported with rat liver following in vivo administration of [14C]AAF [41]. BPDE reacts predominantly with the 2 amino group of guanine [14,15], which lies in the minor groove of the helix, and N-acetoxy-AAF reacts predominantly with the C-8 position of guanine [42,43], which lies in the major groove. It appears that in nucleosomes the DNA is wrapped around the outside of a histone core [21,44,45] and our evidence indicates that neither the major nor the minor grooves of the double helix are completely shielded in terms of susceptibility to chemical modification. Whereas activated derivatives of BP and AAF bind preferentially to the Staphylococcal nuclease susceptible fraction of chromatin DNA, it is curious that Ramanathan et al. [46] and Metzger et al. [41] have reported that in vivo AAF binds preferentially to the DNAase I resistant fraction of chromatin. These apparent discrepancies may relate to differences in specificities and sites of action between Staphylococcal and DNAase I nuclease. It is also possible that interpretation of the in vivo results obtained with DNAase I is complicated by the process of DNA repair. Further studies are required to determine the possible biologic significance of findings in these model systems. The results predict that all regions of chromatin associated DNA are susceptible to attack by activated derivatives of chemical carcinogens, although the attack may not be totally random. In addition, DNA modification by carcinogens is unlikely to disturb the gross structure of cellular nucleosomes. It is, of course, still possible that carcinogens might act by disturbing more subtle aspects of chromatin structure and gene expression. ACKNOWLEDGEMENTS
The authors wish to thank Richard Axel for valuable discussions and suggestions. We are indebted to the Carcinogen Repository of the National Cancer Institute for providing the [14C]benzo(a)pyrene 7,8-dihydrodiol 9,10-oxide used in these studies. This research was supported by the National Cancer Institute CA-02332 and National Cancer Institute Contract CP2-3234, D.H.E.W. REFERENCES 1 J.A. Miller, Carcinogenesis by chemicals: An overview -- G.H.A. Clowes Memorial Lecture, Cancer Res., 30 (1970) 559. 2 C.C. Irving, Interaction of chemical carcinogens with DNA. In H. Busch (Ed.), Methods in Cancer Research, Vol. 12, Academic Press, New York, 1973, pp 189-244. 3 I.B. Weinstein and D. Grunberger, Structural and functional changes in nucleic acids modified by chemical carcinogens. In P. Ts'o and J. DiPaolo (Eds.), Chemical Carcinogenesis, Part A, Marcel Dekker, New York, 1974, pp 217--235.
212 4 D.S.R. Sarma, S. Rajalakshmi and E. Farber, Chemical Carcinogenesis: Interactions of carcinogens with nucleic acids. In F.F. Becker (Ed.), Cancer, Vol. 1, Plenum, N e w York, 1975, pp 235--287. 5 H.V. Gelboin, E. Huberman and L. Sachs, Enzymatic hydroxylation of benzo(a)pyrene and its relationship to cytotoxicity~ Proc. Natl. Acad. Sci. (U.S.A.), 64 (1969) 1188. 6 N. Kinoshita and H.V. Gelboin, Aryl hydrocarbon hydroxylase and polycyclic hydrocarbon tumorigenesis. Effect of the enzyme inhibitor 7,8-benzoflavone on tumorigenesis and macromolecular binding, Proc. Natl. Acad. Sci. (U.S.A.), 69 (1972} 824. 7 E. Huberman and L. Sachs, Cell-mediated mutagenesis of mammalian cells with chemical carcinogens, Int. J. Cancer, 13 (1974} 326. 8 A. Borgen, H. Darvey, N. Castagnoli, T.T. Crocker, R.E. Rasmussen and I.Y. Wang, Metabolic conversion of benzo(a)pyrene by Syrian hamster liver microsomes and binding of metabolites to deoxyribonucleic acid, J. Med. Chem., 16 (1973) 502. 9 P. Sims, P.L. Grover, A. Swaisland, K. Pal and A. Hewer, Metabolic activation of benzo(a)pyrene proceeds by a diol-epoxide, Nature, 252 (1974) 326. 10 P. Daudel, M. Duquesne, P. Vigny, P.L. Grover and P. Sims, Fluorescence spectral evidence that benzo(a)pyrene-DNA products in mouse skin arise from diol-epoxides, FEBS Lett., 57 (1975) 250. 11 V. Ivanovic, N.E. Geacintov and I.B. Weinstein, Cellular binding of benzo(a)pyrene to DNA characterized by low temperature fluorescence, Biochem. Biophys. Res. Commun., 70 (1976) 1172. 12 H.W.S. King, S.R. Osborne, F.A. Beland, R.G. Harvey and P. Brookes, (-+)-7~,8~Dihydroxy-95,105-epoxy-7,8,9,10-tetrahydobenzo(a)pyrene in an intermediate in the metabolism and binding to DNA of benzo(a)pyrene. Proc. Natl. Acad. Sci. (U.S.A.), 73 (1976) 2679. 13 E. Huberman, L. Sachs, S.K. Yang and H.V. Gelboin, Identification of mutagenic metabolites of benzo(a)pyrene in mammalian cells. Proc. Nat. Acad. Sci. U.S.A., 73 (1976) 607. 14 I.B. Weinstein, A.M. Jeffrey, K.W. Jennette, S.H. Blobstein, R.G. Harvey, C. Harris, H. Autrup, H. Kasai and K. Nakanishi, Benzo(a)pyrene diol-epoxides as intermediates in nucleic acid binding in vitro and in vivo, Science, 193 (1976) 592. 15 A.M. Jeffrey, K.W. Jennette, S.H. Blobstein, I.B. Weinstein, F.A. Beland, R.G. Harvey, H. Kasai, I. Miura and K. Nakanishi, Benzo(a)pyrene-nucleic acid derivative found in vivo: structure of a benzo(a)pyrenetetrahydrodiol epoxide-guanosine adduct, J. Am. Chem. Soc., 98 (1976} 5714. 16 A.M. Jeffrey, K.W. Jennette, I.B. Weinstein, K. Grzeskowiak, K. Nakanishi, C. Harris, F.A. Beland and R.G. Harvey, Structure of benzo(a)pyrene-nucleic acid adducts formed in human and bovine bronchial explants, Nature (1977) (in press). 17 K. Nakanishi, H. Kasai, H. Cho, R.G. Harvey, A.M. Jeffrey, K.Wo Jennette and I.B. Weinstein, Absolute configuration of a ribonucleic acid adduct formed in vivo by metabolism of benzo(a)pyrene, J. Amer. Chem. Soc., 99 (1977) 258. 18 M.R. Osborne, F.A. Beland, R.G. Harvey and P. Brookes, Reaction of (+-)-7~,8~3dihydroxy-9~3,10~-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene with DNA, Int. J. Cancer, 18 (1976) 362. 19 H. Yagi, H0 Akagi, D.R. Thakker, H.D. Mah, M. Koreeda and DoM. Jerina, Absolute stereochemistry of the highly mutagenic 7,8-diol 9,10-epoxides derived from the potent carcinogen trans-7,8-dihydroxy 7,8-dihydrobenzo(a)pyrene, J. Am. Chem. Soc., 99 (1977) 2358. 20 R. Axel, W. Melchior, B. Sollner-Webb and G. Felsenfeld, Specific sites of interaction between histones and DNA in chromatin, Proc. Natl. Acad. Sci. ~U.S.A.), 71 (1974) 4101. 21 R.D. Kornberg, Chromatin structure: a repeating unit of histones and DNA, Science, 184 (1977) 868. 22 M. Noll, Subunit Structure of Chromatin, Nature, 251 (1974) 249.
213 23 24 25 26 27 28 29 30 31 32 33
34 35 36 37 38 39 40 41 42 43 44 45 46
AoL. Olins and D.E. Olins, Spheroid chromatin units, (~ bodies), Science, 183 (1974) 330. H. Weintranb, A. Worcel and B. Alberts, A model for chromatin based upon two symmetrically paired half-nucleosomes, Cell, 9 (1976) 409. H. Weintraub and F. VanLente, Dissection of chromosome structure with trypsin and nucleases. Proc. Natl. Acad. Sci. (U.S.A.), 71 (1974) 4249. B. Sollner-Webb and G. Feisenfeld, A comparison of the digestion of nuclei and chromatin by staphylococcal nuclease, Biochemistry, 14 (1975) 2915. B.R. Shaw, T.M. Herman, R.T. Kovacic, G.S. Beandreau and K.E. Van Holde, Analysis of subunit organization in chicken erythrocyte chromatin, Proc. Natl. Acad. Sci. (U.S.A.), 73 (1976) 505. R. Axel, H. Cedar and G. Felsenfeld, Synthesis of globin ribonucleic acid from duckreticulocyte chromatin in vitro, Proc. Natl. Acado Sci. (U.S.A.), 70 (1973) 2029. H. Yamasaki, S. Leffler and I.B. Weinstein, Effect of N-2-acetylaminofluorene modification on the structure and template activity of DNA and reconstituted chromatin, Cancer Res., 37 (1977) 684. G.S. Stein, R.J. Mans, E.J. Gabby, J.L. Stein, J. Davis and P.D. Adwadker, Evidence for fidelity of chromatin reconstitution, Biochemistry, 14 (1975) 1859. O.H. Lowry, N.J. Rosebrough, A.L. Farr and R.J. Randall, Protein measurement with folin phenol reagent, J. Biol. Chem., 193 (1951) 265. A. Peacock and C. Dingman, Resolution of multiple ribonucleic acid species by polyacrylamide gel electrophoresis, Biochemistry, 6 (1967) 1818. J.H. Nelson, D. Grunberger, C.R. Cantor and I.B. Weinstein, Modification of ribonucleic acid by chemical carcinogens. IV. Circular dichroism and proton magnetic resonance studies of oligonucleotides modified with N-2-acetylaminofluorene, J. Mol. Biol., 62 (1971) 331. A.F. Levine, L.M. Fink, I.B. Weinstein and D. Grunberger, Effect of N-2-acetylaminofluorene modification on the conformation of nucleic acids, Cancer Res., 34 (1974) 319. R.P.P. Fuchs, In Vitro Recognition of carcinogen-induced local denaturation sites in native DNA by S 1 endonuclease from Aspergillus oryzae, Nature, 257 (1975) 151. H. Yamasaki, P. Pulkrabek , D. Grunberger and I.B. Weinstein, Differential excision from DNA of the C-8 and N 2 guanosine adducts of N-acetyl-2-aminofluorene by single-strand specific endonucleases, Cancer Res., 37 (1977) 3756. P. Pulkrabek, S. Leffler, I.B. Weinstein and D. Grunberger, Conformation of DNA modified with a dihydrodiol epoxide derivative of benzo(a)pyrene, Biochemistry, 16 (1977) 3127. R.J. Clark and G. Felsenfeld, Structure of chromatin, Nature New Biol., 229 (1971) 101. W.M. Bonnet and R.A. Laskey, A film detection method for tritium-labeled proteins and nucleic acids in polyacrylamide gels, Eur. J. Biochem., 46 (1974) 83. G0 Metzger, F.X. Wilhelm and M.L. Wilhelm, Distribution along DNA of the bound carcinogen N-acetoxy-N-2-acetylaminofluorene in chromatin modified in vitro. Chem.-Bioh Interact., 15 (1976) 257. G. Metzger, F.X. Wilhelm and M.L. Wilhelm, Non-random binding of a chemical carcinogen to the DNA in chromatin, Biochem. Biophys. Res. Commun,, 75 (1977) 703. E. Kriek, Carcinogenesis by aromatic amines, Biochim. Biophys. Acta, 355 (1974) 177. E. Kriek, J.A. Miller, U. Juhl and E.C. Miller, 8(N-2-Fluorenylacetamide)-guanosine, an arylamidation reaction product of guanosine and the carcinogen N-Acetoxy-N-2fluorenylacetamide in neutral solution, Biochemistry, 6 (1967) 177. K.E. Van Holde, C.G. Sahasrabuddhe and B. Shaw, A model for particular structure in chromatin, Nucl. Acid Res., 1 (1974) 1579. J.P. Baldwin, P.G. Boseley, M. Bradbury and K. Ibel, The subunit structure of the eukaryotic chromosome, Nature, 253 (1975) 245. R. Ramanathan, D.S.R. Sarma and E. Farber, Non-random nature of in vivo interaction of 3H-N-hydroxy-2-acetylaminofluorene and its subsequent removal from rat liver chromatin-DNA, Chem.-Biol. Interact., 14 (1976) 375.
201
© Elsevier]North-Holland Scientific Publishers Ltd.
BENZO(a)PYRENE 7,8-DIHYDRODIOL-9,10-OXIDE MODIFICATION OF DNA: RELATION TO CHROMATIN STRUCTURE AND RECONSTITUTION
HIROSHI YAMASAKI,THOMAS W. ROUSH and I. BERNARD WEINSTEIN Department of Medicine and Institute of Cancer Research, Columbia University College of Physicans and Surgeons, 701 West 168th Street, New York, N.Y. 10032 (U.S.A.)
(Received September 30th, 1977) (Revision received May 22rid, 1978) (Accepted May 24th, 1978)
SUMMARY
Purified duck reticulocyte DNA was incubated in vitro y~ith a 7,8dihydrodiol-9,10-oxide derivative of benzo(a)pyrene (BPDE). The carcinogen-modified DNA was somewhat more susceptible to partial digestion by the single strand specific endonuclease $1 than unmodified DNA, suggesting slight denaturation of the helix at sites of modification. Chromatin was reconstituted in vitro utilizing this carcinogen-modified DNA and unmodified-chromatin associated proteins. This reconstituted chromatin showed the same kinetics and extent of digestion by S t a p h y l o c o c c a l nuclease, and similar nucleosome profiles on sucrose density gradient centrifugation, as those obtained with native chromatin or chromatin reconstituted with unmodified DNA. Moreover, polyacrylamide gel electrophoresis of DNA fragments obtained from nuclease digests gel electrophoresis of DNA fragments obtained from nuclease digests of the reconstituted chromatins suggested t h a t the chromatin containing carcinogen-modified DNA had the same subnucleosome structure as that reconstituted with unmodified DNA. In a separate set of studies intact duck reticulocyte chromatin was reacted directly with BPDE. Nuclease digestion studies indicated that 65% of the carcinogen was bound to the 'open' regions of chromatin, and 35% to 'closed' regions. These results indicate that although covalent binding of a benzo(a)pyrene (BP) derivative to DNA produces local distortions in conformation of the helix, this modification does n o t appear to interfere with the ability of the DNA to associate with histones to form nucleosome structures. In addition, although DNA in the open regions of chromatin is more susceptible to reaction with the BP derivative, there is appreciable reaction with the DNA associated with histones. Abbreviation: AAF, N-2-acetylaminofluorene; BP, benzo(a)pyrene; BPDE, benzo(a)pyrene 7,8-dihydrodiol-9,10-oxide.
202 INTRODUCTION It is n o w well established that several carcinogens, or their metabolites, bind covalently to cellular DNA following in vivo administration [1--4]. It is likely that the quaternary structure of DNA as it exists in vivo, i.e. its association with proteins, is an important determinant in this process. Benzo(a)pyrene (BP} is a potent and ubiquitous environmental carcinogen that requires metabolic activation to exert its cytotoxicity, mutagenicity and carcinogenicity [5--7]. Recent studies [8--14] indicate that the most reactive metabolite of BP is benzo(a)pyrene 7,8-dihydrodiol-9,10-oxide (BPDE). Several studies indicate that this derivative is the major metabolite responsible for the in vivo binding of BP to RNA and DNA in rodent, bovine and human cells [9--12,14]. In addition, the complete structures of the major BP-nucleoside adducts found in cellular R N A and DNA have been determined [15,16] and these reveal that the 10 position of BPDE is linked to the 2 amino group of guanine. Certain stereochemical aspects of this reaction have also been elucidated [17]. Related studies from other laboratories are consistent with this structure [12,18,19]. In eukaryotes, cellular DNA occurs as a nucleoprotein complex, or chromatin. It has been found that chromatin consists of a series of nucleoprotein subunits (nucleosomes) each containing a b o u t 200 base pairs of DNA and 2 molecules of each of the 4 major classes of histones [20--24]. Digestion of nuclei with Staphylococcal nuclease first releases multimers and monomers of nucleosomes. As digestion proceeds, the DNA of the nucleosome is reduced in size to a 'core' containing 140 base pairs. Finally, the DNA of the nucleosome core is itself attacked to produce a limit digest consisting of a series of DNA fragments to discrete sizes ranging from a b o u t 140--20 base pairs [20,22,25--27]. These fragments presumably reflect the organization of DNA and histones within the subnucleosome structure. It was of interest, therefore, to determine to what extent the modification of DNA with BPDE would interfere with its ability to participate in the formation of nucleosome structures, as well as to investigate the possibility that regions of DNA associated with histones in intact nucleosomes might be protected from modification,by BPDE. In this paper, we have modified DNA by in vitro reaction with BPDE and then examined the, effects of this modification on in vitro reconstitution of nucleosome structures. We have also reacted intact chroma~in with BPDE and then examined the distribution of the covalently bound BP residues in 'open' and 'closed' regions of the DNA, i.e. regions highly susceptible to digestion by Staphylococcal nuclease versus regions relatively resistant to digestion by this nuclease. MATERIALS AND METHODS
Chemicals [14C]BPDE (Sp. act. 53.6 ~Ci/~mol) was obtained from the National
203 Cancer Institute, Bethesda, Maryland. It was a racemic mixture of what we have previously designated 'Isomer I' [14]. Staphylococcal nuclease (EC 3.1.4.7) and Proteinase K were obtained from Worthington Biochemical Corporation, Freehold, New Jersey, and E. Merck, Rahway, New Jersey, respectively. Endonuclease S~ (EC 3.1.4. X) from Aspergillus oryzae was from Miles-Pentex Laboratories, Kankakee, Illinois.
Preparation of duck reticulocyte chromatin and DNA. Duck reticulocyte chromatin and DNA were prepared as previously described [28] from reticulocytes of phenylhydrazine hydrochloride-treated ducks. Modification of DNA and chromatin of [14C]BPDE. The modification of DNA was carried out under conditions similar to those previously described for the modification of DNA with N-acetoxy AAF [29]-. The reaction mixture contained 10 mM sodium cacodylate buffer, pH 7.2; 30% (v/v) ethanol; 500--600 pg of native DNA/ml; and 0.3 pmol/ml of [~4C]BPDE (53.6 Ci/mol). The mixture was incubated at 37°C f~r 30 min. Unbound radioactivity was extracted with an equal volume of ether. Ether extractions were repeated 8 times and after the final extraction the remaining ether was evaporated under nitrogen gas. After the addition of 0.1 vol. of 2 M sodium acetate (pH 5.0) and 2 vols. of cold ethanol, the modified DNA was spooled out of the solution and then dissolved in 0.1 mM EDTA; 1 mM Tris--HC1 (pH 7.9). Additional extractions did not remove radioactivity indicating that we were dealing with radioactive material covalently bound to the DNA. The modification of intact chromatin was carried out in a reaction mixture containing in 1 ml: lmM Tris--HC1, pH 7.9; 5% (v/v) ethanol; chromatin equivalent to 0.8--1.0 mg of DNA; and 0.3 pmol of [~4C]BPDE (53.6 Ci/mol). Incubation and extraction of non-covalently bound carcinogen was carried out as described for DNA modification. There was no evidence that these procedures altered the nucleosome structure of chromatin when analyzed by susceptibility to nuclease digestion, sucrose density gradient centrifugation of gel electrophoresis of DNA fragments after partial digestion of chromatin samples by Staphylococcal nuclease. The extent of DNA modification was determined by the ratio of radioactivity to A260 and expressed as the percent of total base residues modified by the BP derivative, assuming that 1 mg of DNA/ml equals 20 A260 units. The extent of chromatin modification was also expressed as the extent of DNA modification, after purification of the DNA as described [28]. In the present studies BPDE-modified samples refers to DNA or chromatin preparations in which 0.4--0.7% of the DNA bases contained covalently bound BP adducts. Reconstruction of chromatin from DNA and chromosomal proteins. Chromosomal proteins were prepared from duck reticulocyte chromatin, as described by Stein et al. [30], by suspension in 3 M NaC1, 5 M urea,
204 10 mM Tris--HC1 (pH 7.9). The DNA was removed by centrifugation at 150 000 g for 15 h. Protein was determined by the m e t h o d of Lowry et al. [31]. Native or carcinogen-modified DNA was reconstituted with native chromosomal proteins as described previously [ 29].
Nuclease digestion o f DNA and chromatin The digestion of DNA by S, endonuclease from A. oryzae, the digestion of chromatin b y Staphylococcal nuclease and the quantitation of extents of digestion were carried o u t as described previously [29]. Partially digested chromatin was prepared by stopping the digestion with 5 mM EDTA, added at the indicated time. Gel electrophoresis o f DNA Electrophoresis of DNA purified from undigested regions of chromatin was carried out as previously described [20] using an E-C Slab gel apparatus. DNA samples were purified from partially digested chromatin by incubating the chromatin with 100 pg/ml of proteinase K in the presence of 0.4 M NaC1, 10 mM Tris--HC1 (pH 7.9), 0.2% SDS and 5 mM EDTA for 1 h at 37°C. Protein was extracted with equal vols. of phenol/chloroform (1 : 1) solution. After shaking vigorously for 15 min, the aqueous phase was extracted 4 times with equal vols. of ether and the DNA precipitated with 2 vols. of cold ethanol. After overnight precipitation at - 3 0 ° C , DNA was collected by centrifugation at 15 000 g for 30 min at -20°C. The precipitated and dried DNA was dissolved in 50--100 pl of 0.5X Peacock's buffer [32] and electrophoresed in 6% acrylamide slab gels containing 0.32% bisacrylamide. After electrophoresis the DNA in the gel was stained with 0.017% (v/v) ethidium bromide solution. RESULTS
Susceptibility of [~4C]BPDE-modified regions of DNA to $1 nuclease digestion Previous studies have revealed that binding of the potent hepatocarcinogen N-2-acetylaminofluorene (AAF) to nucleic acids causes a marked distortion in the DNA helix which we have termed 'base displacement' [3,33,34]. The distortion produces localized regions of denaturation at the sites of AAF modification [33--36] whose size can be estimated with the aid of $1 nuclease, an endonuclease which under appropriate conditions preferentially digests single stranded regions of DNA [29]. Prior to using BPDE-modified DNA in chromatin studies it was of interest to determine the extent of denaturation of the modified DNA utilizing endonuclease .$1. Fig. 1 shows the time course of digestion by S~ of [~4C]BPDE-modified DNA compared to unmodified native and heat denatured DNA. The heatdenatured DNA was completely hydrolyzed within 20 mi~, at which time the control native DNA and the BPDE modified DNA were hydrolyzed less than 8%. The digestion of the BPDE modified DNA proceeded slightly more
205
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Fig. ]. Digestion of D N A samples with S 1 nt~e]ease. Native, heat denatured or [ '4C ]BPDRmodified D N A samples (50 /~g/m]) were incubated wif~ S, hue]ease ( ] 0 000 units/m]) for the indicated period of time at 37°C. The extent of digestion o f DNA samples and release of ['4C]BPDE-modified residues were determined as described in M e t h o d s . . , native DNA; ~, heat-denatured DNA; x, BPDE-modified DNA; o, release o f ["C]BPDEmodified residues from BPDE-modified DNA. Similar results were obtained when the entire study was repeated using 5000 units/ml of S, nuclease.
rapidly than the c o n t r o l native DNA. With the modified DNA the release of ~C was considerably faster than the release of total nucleotides (A~60). Almost 80% of the 14C residues were released after 1 h incubation with S, nuclease, whereas only 30% of the nucleotides were rendered acid soluble. These results suggest that the sites to which the [ I~C]BP residues were bound represented localized regions of denaturation. Separate more extensive studies indicate that the size of these localized denatured regions is about 1--2 base plates, which is considerably smaller than the regions of denaturation associated with AAF modification of DNA [37].
Reconstitution o f nucleosomes with ['4C]BPDE-modified DNA and unmodified chromatin associated protein Control native DNA, [~4C]BPDE-modified DNA or heat-denatured DNA were mixed with native chromosomal proteins to reconstitute chromatin. The extent of reconstitution was then determined by digestion with Staphylococcal nuclease. As shown in Fig. 2, chromatin reconstituted from control and BPDE-modified DNA were digested by Staphylococcal nuclease in a similar manner. As was reported previously for native chromatin [ 3 8 ] , about 50% of the DNA, in both the reconstituted chromatin containing unmodified DNA and in the material containing the [14C]BPDE-modified DNA, was protected from extensive digestion. Moreover, the release of A:60 and of radioactivity from the reconstituted chromatin containing [~C]BPDEmodifed DNA showed similar kinetics. The material 'reconstituted' from heat-denatured DNA was, however, rapidly and almost completely digested by Staphylococcal nuclease (Fig. 2). These results indicate that although
206
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Fig. 2. Kinetics of Staphylococcal nuclease digestion of reconstituted chromatin. Chromatin reconstituted with control native DNA (o--o), chromatin reconstituted with DNA containing a 0.65% modification of the bases with ['4C]BPDE ( , - ~ ) , and chromatin reconstituted with heat-denatured DNA (~.--a), were digested by Staphylococcal nuclease for the indicated periods of time and the release of total nucleotides was determined after terminating the reaction with 2 vols. of 2 M PCA/2 M NaCl mixture. The release of ['4C]BPDE-modified residues during the digestion of ['4C]BPDE-modified chromatin (x--x) was determined as in Fig. 1. The data presented are typical of those obtained in 5 similar independent experiments. co mp lete l y denatured DNA cannot associate with histones t o f o r m chromatin-like material, [14C]BPDE-modified DNA can interact with histones t o reconstitute chromatin, in spite of the presence of localized regions of denaturation and the bulky substituent. In order t o determine w he t he r the repeating subunits of chromatin, or nucleosomes, were also f o r m e d in chrom at i n reconstituted w i t h ' B P D E modified DNA, the above chromatin preparations were partially digested with Staphylococcal nuclease and then subjected t o sucrose density gradient centrifugation. As is shown in Fig. 3, similar profiles of nucleosome monomers (tube 18), dimers (tube 25) and trimers (tube 30) were f o u n d in the chromatin reconstituted with unmodified native DNA and BPDE-modified DNA. These profiles are similar t o those f o u n d in previous studies of partially digested native chromatin, in which evidence has been presented t hat these peaks correspond to nucleosome m o n o m e r s , dimers and trimers [ 2 0 , 2 9 ] . With the BPDE preparation the profile o f radioactivity, representing the distribution of the ['4C]BP adduct, paralleled t h a t o f the A:~0. This provides f ur t her evidence t h a t the [ 1~C]BPDE modified regions of DNA had participated in the f o r m a t i o n o f nucleosome structures during the reconstitution procedure. As expected, the "reconstituted' material obtained with heat d en atu r ed DNA failed to display nucleosome-like structures when similarly analyzed (not shown here).
Integration of [~4C]BPDE-modified regions o f DNA into the subnucleosome structure of chromatin during reconstitution When DNA is isolated from a limit Staphylococcal nuclease digest o f either native or reconstituted chromatin, a series of double-stranded DNA
207
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Fig. 3. Sucrose density gradient centrifugation of chromatin subunits. After approximately 20% digestion of chromatin by Staphylococcal nuclease, the reaction was stopped by the addition of 5 mM Na-EDTA. The suspension was then layered on a 5--10% linear sucrose density gradient in 5 mM Na-EDTA (pH 7.9) and centrifuged 16 h at 25 000 rev/min in an SW41 rotor at 4°C. The direction of sedimentation is from left--right. (a) The profile of a sample of control reconstituted chromatin. (b) The profile of a sample of chromatin reconstituted with ['4C]BPDE modified DNA. , A~0 profile; _[- , c.p.m, in each fraction. Similar profiles were obtained in three additional independent studies. f r a g m e n t s is o b s e r v e d w h i c h are spaced at a l m o s t regular intervals and range in size f r o m a b o u t 1 6 0 - - 2 0 base pairs [ 2 0 , 2 2 , 2 5 - - 2 7 ] . It was o f interest, t h e r e f o r e , t o see w h e t h e r similar D N A fractions are o b t a i n e d f r o m c h r o m a t i n reconstituted with [ 14C]BPDE-modified DNA. Fig. 4 s h o w s that t h e slab gel e l e c t r o p h o r e s i s pattern o f t h e D N A fragm e n t s o b t a i n e d f r o m the limit digest o f c h r o m a t i n r e c o n s t i t u t e d w i t h t h e [ ~ 4 C ] B P D E - m o d i f i e d D N A was essentially the s a m e as that o b t a i n e d f r o m a c o n t r o l preparation o f c h r o m a t i n . In order t o see w h e t h e r [ ~ C ] B P D E - m o d i f i e d regions o f D N A w e r e distrib u t e d e v e n l y a m o n g s u b n u c l e o s o m e fractions t h e slab gel was s u b j e c t e d t o f l u o r o g r a p h y [ 3 9 ] . F l u o r o g r a p h y o f t h e gels o b t a i n e d w i t h [ I ~ C ] B P D E D N A d e m o n s t r a t e d that radioactivity was distributed in m a n y o f t h e D N A bands. T h e r a d i o a c t i v i t y was n o t sufficient, h o w e v e r , t o d e t e r m i n e t h e specific activities o f individual bands.
Covalent binding of [14C]BPDE to 'open' and 'closed' regions of intact chroma tin In order t o see w h e t h e r [14C]BPDE can bind c o v a l e n t l y t o D N A w h i c h is
208
Fig. 4. Polyacrylamide gel electrophoresis of DNA extracted from Staphylococcal nuclease digests of chromatin. Gels represent DNA extracted from control reconstituted chromatin (right), and from chromatin reconstituted with [~4C]BPDE-modified DNA (left). Extent of digestion was 40--50%. Migration is from top to bottom.
209 I
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already a part of the nucleosome structure, a separate set of experiments was undertaken. Intact chromatin was incubated with ['4C]BPDE under conditions which did n o t produce detectable changes in the overall structure of the chromatin, and the noncovalently bound radioactivity was extracted. The [~4C]BPDE-modified chromatin was then incubated with Staphylococcal nuclease, as in Fig. 2, and at various time points DNA was extracted and purified from the undigested fraction of the chromatin and its specific radioactivity determined. Fig. 5 shows the kinetics of Staphylococcal nuclease digestion of the modified chromatin and the specific radioactivity of the DNA in the resistant regions during the course of digestion. The digestion of the total DNA followed a pattern typical for chromatin, reaching a plateau at a b o u t 50% digestion. This indicates that the [~4C]BPDE modification procedure and the subsequent ether extractions had n o t grossly disturbed the chromatin structure. It is of interest that during the digestion there was an appreciable decrease in the specific radioactivity of the undigested fraction of the DNA, particularly at the early time points. This indicates that the 'open' regions of the DNA, i.e. those which are most susceptible to Staphylococcal nuclease digestion have a higher specific activity than the 'closed' regions of DNA which are more resistant to digestion. At 60 min, which represents a limit digest of the DNA (about 50% total digestion), a b o u t 65% of the total [ ~ C ] B P adducts had been removed and the remaining 35% of the carcinogen residues were left on the nuclease resistant DNA regions of chromatin. Thus, when incubated with intact chromatin [ 1~C]BPDE preferentially modified the 'open' regions of DNA. It should be noted, however, that there was also substantial reaction with
210 'closed' regions of DNA, i.e., those protected by histones from Staphylo-
coccal nuclease digestion. DISCUSSION The present results indicate that modification of native DNA by reaction with BPDE leads to small localized regions of denaturation which are excised by $1 nuclease, a single strand specific endonuclease. The kinetics of release of BP-modified nucleotides and their ratio to total nucleotides released during S, nuclease digestion suggest that these regions of denaturation are considerably smaller than those associated with the covalent binding of AAF to DNA. A more detailed analysis of the conformation of the BP-modified regions is presented elsewhere [37]. We have found that despite these localized changes in DNA structure and the covalent attachment of the bulk BP residue to the 2 amino group of guanine [ 1 4 , 1 5 ] , the modified DNA can still reassociate with histones to form a chromatin-like structure which by the criteria employed in the present study resembles that of normal nucleosomes. Our results do not, of course, exclude the possibility that the chromatin reconstituted with the modified DNA may have subtle distortions which are n o t revealed by the present methods. In separate studies we have found that the covalent attachment of AAF residues to DNA also does n o t grossly interfere with its ability to function during chromatin reconstitution [ 2 9 ] . On the other hand, completely denatured DNA cannot function in the reconstitution o f chromatin. These results suggest that although the DNA must have a structure which is predominantly double stranded, localized distortions in base pairing and the presence of fairly bulky substituents do n o t interfere with the DNAhistone associations involved in nucleosome formation. This is consistent with other evidence indicating that in the formation of nucleosomes histone molecules do not appear to recognize specific base sequences in the DNA [201. Utilizing intact chromatin as a substrate, we obtained elsewhere that BPDE reacts preferentially with the DNA present in the 'open' regions of the chromatin, i.e. regions~ readily~ s u s c e p t i b l e ~ o digestion by Staphylococcal nuclease. Substantial amounts of the carcinogen residue were, however, also b o u n d to DNA in 'closed' regions, i.e., those associated with histones in nucleosome structures and more resistant to digestion by this nuclease. The number of BP residues bound to 'closed' regions was a b o u t half that bound to 'open' regions. Thus, although the closed regions are protected from digestion with Staphylococcal nuclease digestion, they are susceptible to chemical modification. Rather similar results have been obtained by Metzger et al. [40] in studies on the in vitro modification of intact chromatin with N-acetoxy-AAF. 'Closed' regions of chromatin DNA also appear to be susceptible to modification by chemical carcinogens in vivo since we have found that when either hamster or human cell cultures were incubated with [3H]BP there was covalent binding of radioactivity to both 'open' and
211
'closed' regions of nuclear DNA, as defined by susceptibility and Staphylococcal nuclease digestion {unpublished studies). Similar results have recently been reported with rat liver following in vivo administration of [14C]AAF [41]. BPDE reacts predominantly with the 2 amino group of guanine [14,15], which lies in the minor groove of the helix, and N-acetoxy-AAF reacts predominantly with the C-8 position of guanine [42,43], which lies in the major groove. It appears that in nucleosomes the DNA is wrapped around the outside of a histone core [21,44,45] and our evidence indicates that neither the major nor the minor grooves of the double helix are completely shielded in terms of susceptibility to chemical modification. Whereas activated derivatives of BP and AAF bind preferentially to the Staphylococcal nuclease susceptible fraction of chromatin DNA, it is curious that Ramanathan et al. [46] and Metzger et al. [41] have reported that in vivo AAF binds preferentially to the DNAase I resistant fraction of chromatin. These apparent discrepancies may relate to differences in specificities and sites of action between Staphylococcal and DNAase I nuclease. It is also possible that interpretation of the in vivo results obtained with DNAase I is complicated by the process of DNA repair. Further studies are required to determine the possible biologic significance of findings in these model systems. The results predict that all regions of chromatin associated DNA are susceptible to attack by activated derivatives of chemical carcinogens, although the attack may not be totally random. In addition, DNA modification by carcinogens is unlikely to disturb the gross structure of cellular nucleosomes. It is, of course, still possible that carcinogens might act by disturbing more subtle aspects of chromatin structure and gene expression. ACKNOWLEDGEMENTS
The authors wish to thank Richard Axel for valuable discussions and suggestions. We are indebted to the Carcinogen Repository of the National Cancer Institute for providing the [14C]benzo(a)pyrene 7,8-dihydrodiol 9,10-oxide used in these studies. This research was supported by the National Cancer Institute CA-02332 and National Cancer Institute Contract CP2-3234, D.H.E.W. REFERENCES 1 J.A. Miller, Carcinogenesis by chemicals: An overview -- G.H.A. Clowes Memorial Lecture, Cancer Res., 30 (1970) 559. 2 C.C. Irving, Interaction of chemical carcinogens with DNA. In H. Busch (Ed.), Methods in Cancer Research, Vol. 12, Academic Press, New York, 1973, pp 189-244. 3 I.B. Weinstein and D. Grunberger, Structural and functional changes in nucleic acids modified by chemical carcinogens. In P. Ts'o and J. DiPaolo (Eds.), Chemical Carcinogenesis, Part A, Marcel Dekker, New York, 1974, pp 217--235.
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