the jol~nai. of chemistry vol. no. issue september 25, pp ... · were checked on sds polyacrylamide...

0 1993 by The American Society for Biochemistry and Molecular Biology, Inc. THE JOL~NAI. OF B I O L ~ ~ ~ C A L CHEMISTRY Vol. 268, NO. 27, Issue of September 25, PP. 20620-20629, 1993

Printed in U.S.A.

Covalent Binding of the Carcinogen BenzoCulpyrene Diol Epoxide to Xenopus Zueuis 5 S DNA Reconstituted into Nucleosomes*

(Received for publication, May 5, 1993, and in revised form, June 9, 1993)

Bettye L. Smith$ and Michael C. MacLeod8 From the Department of Carcinogenesis, University of Texas M. D. Anderson Cancer Center, Smithville, nxas 78957

The sequence-specific interactions of bulky carcinogens with purified DNA might reasonably be expected to be altered when the DNA is organized into chromatin. We have approached this subject by studying the covalent binding of a potent carcinogen, 7r,8t-dihydroxy- 9t,1Ot-oxy-7,8,€J,lO-tetrahydrobenzo[u]pyrene (BPDE-I) to nucleosomal and free DNA, using a defined fragment of DNA derived from the 5’-end of the 5 S rRNA gene of Xenopus laeuis, reconstituted into nucleosomes by salt exchange with unlabeled chicken mononucleosomes. Micrococcal nuclease and hydroxyl radical “footprinting“ experiments demonstrated the formation of a uniquely positioned nucleosome covering the transcriptional start point of the gene. The reconstituted nucleosomes or control DNA samples were modified with BPDE-I. DNA was repurified from the nucleosomal and control modification reactions and then irradiated with laser light at 355 nm, causing strand breaks at the positions of adducts. Nucleosomal DNA exhibited a marked decrease in the level of carcinogen binding, especially in the central 80-90 base pairs of the nucleosomal region. Interestingly, although overall binding was inhibited about 2-fold, the sequence-specific pattern of binding to deoxyguanosine residues seen with purified DNA was maintained in the nucleosomal DNA.

Benzo[a]pyrene is a potent environmental pollutant that, once ingested or absorbed, is metabolically activated to reactive electrophilic intermediates (Miller and Miller, 1976; Harvey, 1991), which bind covalently with nucleophiles such as DNA, RNA, or proteins. “he results of mouse skin carcinogenesis studies suggest that the (+)-enantiomer of BPDE-I1 is the ul- timate carcinogenic form leading to malignant transformation (Slaga et al., 1979). Consistent with this suggestion, the major DNA adducts formed from either benzo[alpyrene or racemic

* This work was supported by Grant CA 35581 from the National Cancer Institute and by National Service Award T32 HD07296 GT from the National Institute of Child Health and Human Development. The laser-induced strand scission work was performed at the Center for Fast Kinetics Research, University of Texas at Austin. The Center is supported jointly by the Biomedical Research Technology Program of the Division of Research Resources of the National Institutes of Health through Grant RR00886 and by the University of Texas at Austin. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduer- tisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ Present address: Dept. of Pharmacology and lbxicology, Virginia Commonwealth UniversitylMedical College of Virginia, Box 230 MCV Station, Richmond, VA 23298.

6 To whom correspondence and requests for reprints should be di- rected.

The abbreviations used are: BPDE-I, 7r,8t-dihydroxy-9t,lOt-oxy-

RSB, reticulocyte swelling buffer; THF, tetrahydrofuran; bp, base 7,8,9,1O-tetrahydrobenzo[ulpyrene; PCR, polymerase chain reaction;

pairb).

BPDE-I result from addition of the (+)-enantiomer of BPDE-I to the exocyclic amino group of dGuo, with the (-)-enantiomer of BPDE-I accounting for less than 5% of the total adducts (Mee- han and Straub, 1979; Harvey, 1991; Slaga et al., 1979). Based in part on this stereoselectivity, it has been suggested that perhaps noncovalent intercalation complexes are precursors to the final covalent adduct (MacLeod and Zachary, 1985; Geacin- tov, 1988; MacLeod, 1990).

In an effort to understand the mechanisms of carcinogen interaction, extensive studies have probed the binding of BPDE-I to DNA in vitro. Kinetic studies on the mechanism of carcinogen interaction with DNA suggest that rapid intercalation (noncovalent binding) occurs between the base pairs of the DNA, followed by hydrolysis to nonreactive products (tetrols) or alternatively by covalent adduct formation (Geacintov, 1988; Harvey, 1991). Intercalation and rate of hydrolysis have been correlated, and it has been suggested that intercalation is an important factor in hydrolysis. Furthermore, the hydrolysis reaction predominates over covalent binding by 15:l (MacLeod and Zachary, 19851, suggesting that the catalysis of hydrolysis by DNA plays a major role in limiting the amount of covalent damage the DNA suffers. Although covalent binding of BPDE-I to DNA only accounts for a small fraction of the total reaction, it is enough to induce malignant transformation and is considered the major pathway leading to mutagenesis and carcinogenesis (Miller and Miller, 1976; Harvey, 1991).

In eukaryotic nuclei, genomic DNA is complexed with a variety of proteins. The most abundant of these proteins are the histones which function to compact DNA into chromatin, the basic subunit being the nucleosome. The structural organization of DNA into nucleosomes is considered an important ele- ment in the control of gene expression. In fact, several studies have shown that addition of nucleosomes to DNA severely in- hibits transcription in vitro (Rhodes, 1986; van Holde, 1989; Lee and Garrard, 1991; Hayes and Wolffe, 1992). More recently, it has been shown that nucleosomal placement is distinctly non-random both in vivo and in vitro (Simpson, 1978; Simpson and Stafford, 1983; Drew and Travers, 1985; Ramsay, 1986; Rhodes, 1986; Gottesfeld, 1987; Drew and Calladine, 1987; Shrader and Crothers, 1989; Moyer et aZ., 1989; Powers and Bina, 1991; Hayes et al., 1991). Nucleosomes are positioned in a sequence-specific manner that in some cases correlates with important regulatory regions. For example, a nucleosome is positioned around the start site of the 5 S rRNA gene extending into the internal control region in a number of species including Lytechinus variegatus (Simpson and Stafford, 1983; Moyer et al., 1989) and Xenopus borealis (Rhodes, 1986; Drew and Cal- ladine, 1987; Hayes et al., 1991). A nucleosome also appears to be uniquely positioned over the glucocorticoid response ele- ments of the mouse mammary tumor virus (MMTV) promoter, and its position is disrupted by receptor binding (Richard-Foy and Hager, 1987; Bresnick et al., 1990; Pina et al., 1990).

Because of the size and hydrophobicity of the BPDE-I mol- ecule, one might expect that the formation of a nucleosome on

20620

Benzo[a]pyrene Diol Epoxide Binding to Nucleosomes 20621

DNA would drastically alter the interaction of that DNA with BPDE-I. Initial low resolution studies based on nuclease digestion and using mixed sequence nucleosomes suggested that BPDE-I covalently binds to DNA between nucleosomes, the linker region, with a 3-fold greater affinity than to nucleosomal DNA (Kootstra et al., 1979; Jack and Brookes, 1982). In addition, the rates of hydrolysis and covalent binding of BPDE-I to DNA in mixed sequence nucleosomal cores (MacLeod et al., 1989) were significantly decreased compared to free DNA, and this was correlated with a large decrease in intercalation. To further clarify this issue, we have analyzed the covalent

binding of BPDE-I to sequence-positioned nucleosomes. These nucleosomes were prepared by the exchange of histones from chicken erythrocyte core particles to a cloned DNA fragment containing the 5 S rRNA gene from Xenopus laeuis, which has been shown to position nucleosomes precisely over the start site of the gene. Using a homogeneous population of nucleosomes allowed us to identify sequence- and structure-specific modulation of covalent binding of BPDE-I to DNA in nucleosomes at the nucleotide level.

MATERIALS AND METHODS Chemicals-7r,8t-Dihydroxy-9t,10t-oxy-7,8,9,l0-tetrahydrobenzoIal-

pyrene diol epoxide (BPDE-I) was obtained from the Chemical Carcino- gen Reference Compound Repository, Division of Cancer Cause and Pre- vention, National Cancer Institute and was found to be >95% pure by high pressure liquid chromatography analysis. Concentrated stocks of BPDE-I were stored in anhydrous tetrahydrofuran (THF) at -20 "C. Stocks were quantitated spectrophotometrically, using molar extinction coefficients supplied by the Repository. The integrity ofthe BPDE-I stock solutions was checked routinely by spectroscopic methods (MacLeod and Lew, 1988). DNA concentrations are expressed in moles of base pairs/ liter, determined spectroscopically using c260 = 13,200. BPDE-I is a potent carcinogen and must be handled carefully. Wherever possible, dis- posable labware was used, and all vessels contacting the carcinogen were decontaminated by treatment with dilute H,SO,.

Plasmid Construction-A 370-bp NaeYHindIII restriction fragment derived from the plasmid pHs11 (a gift from Joel M. Gottesfeld) containing X. laeuis somatic type 5 S rRNA (Peterson et al., 1980) was subcloned into the BamHI site of pGEM-3ZK-) using synthetic linkers. The clone used in this study, pGXlsl4, had the orientation indicated in Fig. 2B as verified by dideoxy sequencing. The plasmid is numbered with respect to the natural transcription start point of the 5 S rRNA gene.

Preparation of DNA Fragments for Reconstitution-The region containing the insert of pGXlsl4 was amplified using the polymerase chain reaction (PCR) with Taq DNA polymerase and primers specific for pUC/ m13 sequencing vectors. Amplified DNAwas purified by extraction with phenol, phenolkhloroform, chloroform, and ether and then precipitated with ethanol. Plasmid DNA or PCR-generated fragments were digested with the appropriate restriction endonucleases. The restricted DNAwas purified by agarose gel electrophoresis in 1 x TAE buffer (40 mM Tris, 5 mM sodium acetate, 1 mM EDTA, pH 7.8) containing 0.5 pg/ml ethidium bromide. The desired DNA fragment was excised from the gel, electro-

fill-in with the appropriate [32PldNTP. eluted, and repurified. DNA fragments were end-labeled by Klenow

Preparation of Nucleosomal Core Particles-Nuclei were isolated from chicken erythrocytes as described previously (Olins et al., 1976; MacLeod et al., 1989) with the following modifications. Upon arrival, fresh chicken blood was stored in 50-ml aliquots in 10% glycerol at -20 "C. For a typical preparation, 100 ml ofblood was thawed on ice and centrifuged at 10,000 rpm for 15 min. The supernatant was decanted, the pellet was resuspended in approximately 30 ml of RSB (10 mM Tris, pH 7.4, 10 mM NaCl, 3 IIIM MgCl,, 0.1 mM phenylmethylsulfonyl fluoride), and the resulting suspension was centrifuged at 6000 rpm for 10 min. The supernatant was decanted, and the pellet was resuspended in approximately 30 ml of RSBN (RSB plus 0.5% Nonidet P-401, then centrifuged as before. The nuclei were washed with RSBN until the

RSBN. pellet was white (usually 4-5 times) and finally resuspended in 5 ml of

Nuclei (-100A2,drnl) were digested to mono- and oligonucleosomes following the procedures described by Lutter (1978). The preparation was further purified by sedimentation through a continuous 5-25% sucrose gradient (Beckman SW27 rotor) for 17 h at 141,000 x g, 4 "C,

and the 11 S mononucleosomal peak was collected. Contaminating HI- containing chromatosomes (Olins et al., 1976, Simpson, 1978) were removed by addition of KC1 to a final concentration of 0.1 M. The precipitated material was pelleted by centrifugation, and the supernatant was dialyzed extensively against 10 mM Tris, pH 8.0,O.Z mM EDTA, 0.1 mM phenylmethylsulfonyl fluoride. The resultant mononuchsomal core particles were concentrated to -1 mg/ml using an Amicon ultra- filtration device. Agarose gel electrophoresis showed the DNA fragments be - 146 bp in length. The integrity and purity of core histones were checked on SDS polyacrylamide slab gels by the method of Laemmli (1970). They were routinely found to be free of contaminating H1 and non-histone proteins.

To prepare core length DNA, core particles were adjusted to 10 mM NaC1, 10 mM EDTA, 10 mM "is, pH 7.4, 0.5% SDS and digested with 100 pg/ml proteinase K at 37 "C for 1 h. The DNA was purified by organic extraction and precipitation as described above.

32P-end-labeled 5 S DNA by the exchange of histones from chicken Salt Exchange Reconstitution-Nucleosomes were reconstituted on

erythrocyte mononucleosomes (Moyer et al., 1989). Core particles and linear 5 S DNA (at an approximate molar ratio of 1001) were combined in 300 pl of TE buffer (10 mM Tris, pH 8.0, 0.2 mM EDTA, 0.1 mM phenylmethylsulfonyl fluoride). 5 M NaCl was added by dropwise addition to a final concentration of 0.8 M NaC1. After 20 min on ice, reaction tubes were tightly covered with Spectropor No. 1 dialysis tubing, then inverted into TE buffer containing 0.6 M NaCl and dialyzed overnight at 4 "C. This was followed by a 3-h dialysis against TE containing 0.05 M NaCl. Mock-reconstituted samples were prepared concurrently as described above in the absence of core histones by substituting purified core length chicken DNA for the core particles. The efficiency of reconstitution was monitored by an electrophoretic mobility shift assay. Ali- quots of reconstituted and mock-reconstituted samples were loaded on nondenaturing gels (0.7% agarose, 0.5 x TBE (0.09 M Tris, 0.09 M boric acid, 0.002 M EDTA, pH 8.3)) and electrophoresed at 100 V. The gels were analyzed by autoradiography.

Nucleosomal Positioning-The translational positioning of nucleosomes on 5 S DNA was determined using light micrococcal nuclease digestions (Olins et al., 1976; Lutter, 1978; Simpson, 1978; Drew and Calladine, 1987). Reconstituted or mock-reconstituted preparations were digested using 2 units of micrococcal nucleaselpg of DNA at 4 "C for increasing times of digestion (usually 0, 1, 5, 10, and 30 mid. After digestion with proteinase K, the DNA samples were purified and analyzed on denaturing gels (8% polyacrylamide, 8 M urea; Maniatis et al., 1982). The rotational positioning was determined by hydroxyl radical footprinting as described (Tullius et al., 1987).

Modification of DNA with BPDE-Z-Reconstitution mixtures were treated with 1/20 volume of 200 or 400 p~ BPDE-I in THF or with 1/20 volume of THF only at 4 "C overnight. These preparations were digested with proteinase K and extracted sequentially with ethyl acetate, phenol, ch1oroform:isoamyl alcohol (24:1), and ether. The DNAwas precipitated with ethanol, rinsed with ether, and redissolved in 10 mM Tris, pH 7.4. This treatment effectively removes both the protein components of the reconstitution mixes and the BPDE-I hydrolysis products formed during the modification reaction. The level of modification was determined spectrophotometrically using the molar extinction coefficient of BPDE-I-DNA adducts (€346 = 29,500; Hogan et al., 1981).

BPDE-Z Photolysis-BPDE-I-modified DNA was irradiated for 0, 10, 20, 60, 90, or 120 s in siliconized capillary tubes in the beam of a neodymium:YAG (yttrium, aluminum, garnet) laser (Quantel YG 581), operated at 10 Hz with frequency tripling optics. The output of the laser at 355 nm was -20 mJ/pulse, and the effectiveness of the laser irradiation was routinely monitored by bleaching of the pyrene chromophore in a sample of BPDE-I-modified salmon sperm DNA (previously treated with 1/20 volume of 1.2 mM BPDE-I in THF). After irradiation, samples of 32P-labeled 5 S DNA were analyzed on standard denaturing gels as described above, except that the positions and intensities of the radio- active bands were determined with a Molecular Dynamics 400A Phos- phorImager. Control DNA samples containing no BPDE-I adducts were always irradiated and analyzed in parallel with the modified samples. No specific strand scissions were seen in the control preparations. Marker lanes were prepared according to the method of Maxam and Gilbert (1980).

RESULTS

Reconstitution-End-labeled 5 S DNA fragments were reconstituted into nucleosomes by the method of salt exchange (Moyer et al., 1989). As a control, DNA was purified from chicken erythrocyte core particles and substituted for the core

20622 Benzo[a]pyrene Diol Epoxide Binding to Nucleosomes

particles in the reconstitution protocol. The eficiency of reconstitution was routinely monitored by an electrophoretic mobility shift assay. As shown in Fig. 1, the reconstitution mixture (R) or the mock-reconstituted DNA (MI, incubated in the absence of core histones, was analyzed by agarose electrophoresis in nondenaturing conditions. In these gels, DNA associated with histones exhibits a lower mobility than free DNA. Greater than 95% of the 32P-end-labeled 5 S DNA was reconstituted into nucleosomes as detected by this gel shift assay. Identical results were obtained using 5% polyacrylamide gels (data not shown).

Based on previous results (Simpson and Stafford, 1983; Rhodes, 1986; Drew and Calladine, 1987; Hayes et al., 1991). we expected the formation of a uniquely positioned nucleosome surrounding the transcriptional start point of this gene. The translational positioning of nucleosomes on the end-labeled DNA was determined using light micrococcal nuclease digestions (Olins et al., 1976; Lutter, 1978; Simpson, 1978; Drew and Calladine, 1987). At low levels of digestion, this enzyme cleaves the linker DNA between nucleosomes, but DNA complexed with core histones is more resistant to digestion resulting in a footprint in denaturing gels. Fig. 2 shows experiments designed to determine the nucleosomal positions. In Fig. 2A, a PCR-generated DNA fragment was restricted with EcoRI and PstI, and the noncoding strand was end-labeled at the EcoRI site. The resulting 430-bp fragment was then reconstituted and digested with micrococcal nuclease. The pattern of micrococcal nuclease digestion of free DNA is shown in lane 2, while that of reconstituted DNA is in lane 1. Comparison of these lanes revealed a region of DNA that was protected from digestion in the reconstituted sample relative to the mock-reconstituted DNA. Visual inspection of the autoradiogram suggested that the protected region encompassed about 100 nucleotides of 5”flanking sequence and extended about 40 nucleotides into the coding region. This footprinted region was designated nucleosome 1 (vl). To more precisely determine the position of this region, the autoradiogram was quantitated by densitometry. The results for v 1 are shown in the graph in Fig. 2 A . The bands in lane 2, representing micrococcal nuclease-generated fragments of free DNA, were assigned numbers, and the same numbers were assigned to corresponding regions in the reconstituted lane. To correct for sequence-dependent variations in micrococcal nuclease cutting efficiency, the integrated density of each band in the R lane was normalized to the integrated density of the analogous band in the M lane, and the results were plotted as In

M R FIG. 1. Electrophoretic mobility shift assay. The association of

DNA with core histones was monitored by electrophoresis under nondenaturing conditions. Complex formation resulted in a decreased electrophoretic mobility compared to the free DNA fragment. M = mock- reconstituted; R = reconstituted.

(FVM) uersus band position (Rhodes, 1986; Drew and Calladine, 1987). Locations of reduced cutting by micrococcal nuclease appear as distinct minima, and the region located below the dashed line is identified as the preferred nucleosome position. The region above the line represents the end of the nucleosomal DNA as it leaves the core particle. From this analysis, we determined the nucleosome to reside over the 5’ end of the gene from position 40 to -95.

According to the length of end-labeled DNA fragment used in this study, a second nucleosome was expected to lie in the area 3‘ of the gene (Drew and Calladine, 1987). Using the methods described above, we determined the second nucleosome to be positioned in the flanking region of the gene, extending to the end of the fragment (data not shown). This region was not studied further.

Based on micrococcal nuclease digestions, two nucleosomes reside on the 5 S DNA fragment, as shown schematically in Fig. 2 B . This finding is in agreement with Drew and Calladine’s placement (1987) of a nucleosome over the start site of the X. borealis 5 S rRNA gene, and with that found by Simpson and Stafford (1983) on the 5 S rRNA gene of the sea urchin, L. variegatus. The positioning of v l shown in Fig. 2A would place the dyad axis of the nucleosome close to position -23, one of several possible locations of the dyad axis deduced by Drew and Calladine (1987) for the X. borealis gene on the basis of DNase I data. A canonical nucleosome of 146 bp centered at -23 would extend from -96 to +50, consistent with the micrococcal nuclease data (Fig. 2 A ) and with hydroxyl radical positioning data (see below).

The rotational positioning of v 1 was determined by hydroxyl radical footprinting. Using a variant of the Fenton reaction (Tullius et al., 1987), hydroxyl radicals are generated in solu- tion and subsequently react with the sugar backbone of DNA, producing single strand breaks at each nucleotide with little sequence selectivity. However, for nucleosomal DNA there is a modulation of hydroxyl radical cutting based on whether the backbone is facing outward from the nucleosome surface and therefore exposed to cutting, or facing inward and therefore protected. This results in a 10.0- to 10.6-bp periodicity in the cutting frequency (Tullius et al., 1987; Hayes et al., 1990,1991; Pina et al., 1990). When applied to the reconstituted 5 S rRNA gene, a clear periodicity in hydroxyl radical cutting was observed (Fig. 3A), extending from about position +58 to -77, with somewhat less distinct periodicity extending for another 20 base pairs in both directions. As shown in Fig. 3B, when the data were corrected by normalization to the hydroxyl radical- induced band distribution in the control, a periodic pattern covering the entire region judged to be nucleosomal on the basis of micrococcal nuclease digestion was seen. This pattern is similar to that reported for X. borealis by Hayes et al. (1991). Assuming the dyad is at -23, the periodicity of the central 100 bp of the nucleosome, calculated from hydroxyl radical cutting maxima, is 10.5 bp, similar to the periodicity reported for X. borealis on the basis of DNase I digestion (10.6 bp; Drew and Calladine, 1987). The existence of a 10.5 bp periodicity in hydroxyl radical cutting strongly supports our suggestion that a nucleosome is formed in this region, and the extent of the periodic pattern is closely similar to the extent of protection from micrococcal nuclease digestion.

BPDE-I Photolysis-To determine the sequence-specific distribution of adducts on nucleosomal versus free DNA, it was necessary to employ a highly sensitive technique. Boles and Hogan (1986) observed that when irradiated with laser light at 355 nm, BPDE-I-modified DNA is cleaved at (primarily) dGuo residues. I t was suggested (Boles and Hogan, 1984, 1986) that the absorption of a photon by the pyrene moiety of a BPDE-I- deoxyguanosine adduct initiated a photochemical reaction that


0 5 10 15 20 25 30 35 40 45 band position

M E WA 5s RNA gene PH

-200 -100 1 100 200 300

0 5s RNA gene nucleosome positions

IIIIUO pUCM13 promoters T7 promoter EZI Sp6 promoter

mock-reconstituted (M) fragments were analyzed on 8% polyacrylamide, 8 M urea gels. The resulting autoradiographs were scanned by densi- FIG. 2. Determination of translational positioning. A, analysis of nucleosome 1. Micrococcal nuclease digestions of reconstituted (R) and

tometry using a Visage 60 Bio Image. The integrated densities were calculated using soRware supplied with the Visage 60. The bands obtained with the mock-reconstituted fragment were assigned band numbers, and corresponding numbers were assigned to the same regions in the reconstituted lane. To correct for sequence-dependent variations in micrococcal nuclease cutting efficiency, the integrated density of each band in the R lune was normalized to the integrated density of the analogous band in the M lune, and the results were plotted as In (R/M) uersus band position (4, 19). Quantitation of this gel placed vl in the region of band 5 to band 35, which corresponds to nucleotide 40 to -95 in the sequence. B, schematic diagram of nucleosomal positions. The positions of the vector-derived promoters are noted by hatched boxes. The 5 S rRNA gene is noted by the white box; the flanking regions extend from -1 to -113 5' of the gene, and from 122 to 281 3' of the gene. Nucleosomal positions are shown as checkered boxes. M = MueII; E = EcoRI; KIA = KpnI and AuuI (which occupy the same position in the sequence); P = PstI; and H = HindIII. The position of ul extends from -96 to 50, and the u2 extends from position 160 to the end of the fragment (310).

cleaved the phosphodiester backbone of the DNA. The absorbance of the pyrene moiety was bleached in the process, and the adjacent phosphodiester bond was broken either by direct oxi- dation or production of a short-lived free radical. I t was shown that diffusible singlet oxygen was not required for the cutting reaction, and that bleaching and cutting were stoichiometric. Therefore, it was postulated that the single strand scission occurs at the site of the adduct (Boles and Hogan, 1984,1986).

Since bleaching of the pyrene chromophore and strand scission are stoichiometric, the efficiency of the laser treatment can be monitored by measuring the decrease in absorbance of the pyrene chromophore with time of irradiation. As shown in Fig. 4, the absorbance of BPDE-I-modified DNA at 347 nm decreased with increasing times of irradiation. The top line represents the absorbance of the pyrene moiety at t = 0 s, the subsequent lines below it are t = 5, 20, and 60 s, respectively.

The fraction of adducts remaining unbleached can be determined by calculating: [A347 ( i ~ a d i a t e d ) I l [ A ~ ~ ~ (unirradiated)]. When the fraction of molecules remaining unbleached is plotted versus time of irradiation (Fig. 4, inset ), it can be easily seen that at least 80% of the molecules are bleached by 60 s. The residual absorbance at 347 nm appears to be due to a shoulder rather than a local maximum in the spectrum and may be due to photoproducts of the reaction rather than unbleached pyrene chromophore. If this interpretation is correct, at least 90% of the chromophores are estimated to be bleached by 20 s of irradiation.

To determine the binding pattern in nucleosomal versus free 5 S DNA, reconstituted and mock-reconstituted samples were modified with equivalent amounts of BPDE-I (10 PM). The overall levels of modification were determined to be 2.0 adducts/ strand for mock-reconstituted DNA and 1.2 adductshtrand for


-66 -18

1300 1660 1800 2060 2300 2660 2800 3060 3300 3660 n "

I

1300 1650 1800 2060 2300 2660 2800 3060 3300 3550

Position FIG. 3. Determination of rotational positioning. Hydroxyl radical digests of reconstituted and mock-reconstituted, end-labeled 5 S DNA

fragments were analyzed on polyacrylamide gels, and the distribution of radioactivity in the gel was determined by exposure to a phosphorimaging plate (Molecular Dynamics) and digitization. A, individual lanes in the digitized image were analyzed with the area integration function of the ImageQuant software package. This results in the digital equivalent of a densitometer scan of each lane. The lane containing a 2-min digest of the reconstituted DNA is shown. A distinct periodicity in cutting frequency is seen; numbers above the scan indicate the approximate positions of maximum cutting. B, to correct for alterations in cutting frequency in the free DNA, the corresponding mock-reconstituted lane was also analyzed. The natural logarithm of the point-by-point ratio of the recon8tituted:mock-reconstituted density is plotted.

reconstituted DNA. In Fig. 5, TCIAG are Maxam-Gilbert marker lanes, control (mock-reconstituted) lanes are shown (left to right) in increasing times of irradiation (in seconds); reconstituted are shown in decreasing times of irradiation. The banding pattern in the mock-reconstituted lanes corresponded to dGuo residues in the sequence. The strong band present in all lanes at position -24 is due to a minor contaminant (-0.5%) in the original end-labeled DNA preparation. As previously described for other sequences (Boles and Hogan, 1984, 1986; Dittrich and Krugh, 1991a, 1991b), the distribution of adduct- derived bands among dGuo residues in the irradiated samples was not uniform. In this gel, the integrated density of individual residues in the region of interest (calculated for the mock-reconstituted, 60-s irradiated sample) varied by a factor of 2-3 from strongly labeled bands (e.g. -42 and -65) to weakly labeled bands (e.g. -13 and -50). The pattern obtained was reproducible from experiment to experiment (cf: Fig. 8). Al- though analysis of the binding pattern itself is beyond the scope of the present studies, it was noted that clusters of dGuo residues 3 to 4 nucleotides in length were generally more highly labeled than were isolated dGuo residues (data not shown), in agreement with previous studies (Boles and Hogan, 1984,1986; Dittrich and Krugh, 1991a, 1991b). Interestingly, the pattern above nucleotide 40 in the reconstituted lanes (the region of linker DNA) was quite similar to the pattern seen in the control. However, in the nucleosomal region (+40 to -84 in this gel), bands present in the control lanes were absent or much

less intense in the reconstituted lanes. This footprint was approximately coincident with the footprints obtained after micrococcal nuclease digestion. This distinctive difference indicates that the nucleosomal DNA was protected from BPDE-I binding. Laser irradiation of DNA, either reconstituted or mock-reconstituted, that had not been treated with BPDE-I, did not produce specific strand scissions.

The precise interactions of BPDE-I within the nucleosome (Fig. 5) were quantitated on a Molecular Dynamics Phospho- rImager at single base resolution. The integrated density of each observable band was calculated and divided by the total integrated density of the appropriate lane. This number represents the fraction of the total molecules that were cut at that nucleotide. To control for sequence-specific variation in the binding of BPDE-I to individual dGuo residues, we followed the methods used by Drew and Calladine (1987). Thus, the fractional density at each nucleotide position in the reconstituted preparation was normalized to the fractional density at that position in the mock-reconstituted preparation. This ratio, which represents the decrease in BPDE-I binding relative to free DNA, is plotted versus nucleotide position in Fig. 6 for data obtained after 60 s of irradiation. Comparison of the fractional densities at each nucleotide as a function of irradiation time indicated that the strand scission reaction was virtually com- plete after 20 s of irradiation. The 60-s data were chosen for this figure as a representative data set. The graph clearly shows a significant decrease of cutting in the central 80-100 bp


0.090

0.0260

FIG. 4. Bleaching of the pyrene chromophore. The effectiveness of laser irradiation was monitored by measuring bleaching of the pyrene chromophore. 0.020 Salmon sperm DNA was treated with 60 PM BPDE-I, and repurified DNA samples were irradiated for 0, 5, 20, and 60 s; absorbance spectra from 31Ck390 nm are ' 0.0160 shown. Bleaching was monitored by the decrease in absorbance at A3,,, character- istic of the pyrene chromophore. Approxi- 4 mately 50% bleaching was observed after 5 s of irradiation, and 80% occurred after 0.010 20 or 60 s of irradiation. Inset, the fraction remaining unbleached is determined by:

radiated), where is the background (A,,, - A,,, irradiated)/(A347 - A,,, unir-

absorbance outside the spectrum of the chromophore. The fraction is plotted as a function of time of irradiation.

*

0.0060

0.0

d \

8 %

of the nucleosomal region (from about -67 to 19) compared to the linker region (52 and beyond). A transitional region appears between the nucleosomal and linker region (-81 to -95 and 28 to 44), indicating intermediate levels of BPDE modification toward the ends of the nucleosome. The ratios of integrated densities in these regions were averaged for three time points of irradiation (20, 60, and 90 s ) , and the means for the three regions are shown in Table I. The binding density of the central nucleosomal region is about 2.5-fold lower than the non-nucleo- soma1 region, consistent with the generalized decrease in BPDE-I binding to the reconstituted preparation.

Individual dGuo residues in purified DNA exhibit sequence- dependent differences in BPDE-I binding (Boles and Hogan, 1984, 1986; Kootstra et al., 1989; Reardon et al., 1989, 1990; Dittrich and Krugh, 1991a, 1991b; Tang et al., 1992). Since binding of BPDE-I to the 5 S rRNA gene sequences in reconstituted nucleosomes was not completely inhibited, it was of interest to determine whether there was a modulation of sequence dependence upon nucleosome formation. To facilitate this analysis, reconstituted samples were modified with twice as much BPDE-I (20 p ~ ) as were mock-reconstituted samples (10 PM). This led to approximately equivalent levels of modification. Fig. 7 shows the cutting pattern of BPDE-I-modified DNA at high resolution produced by laser-induced strand scission. TC/AG are Maxam-Gilbert marker lanes, the control and reconstituted lanes are indicated with corresponding times of irradiation (in seconds). As expected, the positions of bands in this gel corresponded to the positions of dGuo residues in the sequence. Visual inspection of the gel indicated a very similar pattern of laser-induced strand scission in the control and reconstituted samples. This similarity covered the region from -68 to +20, shown above to be nucleosomal.

To further elucidate this pattern, the density in the lanes of Fig. 7 corresponding to the 60-s time point for the control and reconstituted lanes was determined and plotted versus the relative position in the gel (Fig. 8). The density is proportional to the level of radioactivity in a particular band and corresponds to the level of cutting at that site. It is clear from Fig. 8 that the

g i 3 3 Q Q i ? E 8 8

Wavelength (nm)

local pattern of covalent binding of BPDE-I to DNA is the same in nucleosomal (heavy line) and free (light line) DNAmolecules, suggesting that sequence specificity of BPDE-I interaction dominates over structural specificity, and that it is therefore a critical variable in the mechanism of binding.

DISCUSSION

Covalent binding of BPDE-I to DNA is considered the initial event in the pathway leading to malignant transformation. Consequently, BPDE-I-DNA interactions have been studied extensively in vitro, leading to the suggestion that BPDE-I binds predominantly to regions rich in deoxyguanosine (Boles and Hogan, 1984, 1986; Kootstra et al., 1989; Dittrich and Krugh, 1991a, 1991b). Since DNAis complexed with proteins in vivo, it will ultimately be necessary to elucidate the mechanism of BPDE-I binding to DNA in chromatin in vitro and in vivo. As a first step in this direction, we have chosen to study the least complex level of chromatin organization, the nucleosome. Nucleosomal structure has been extensively studied in an effort to understand its' role in the in vivo mechanisms of gene regulation, transcription, replication, and DNA repair. In particular, the structural organization of the nucleosomal core particle is known, and the arrangement of the core particle with respect to DNA sequence has been studied. For some sequences, specific positions are favored by nucleosomes. Al- though specific nucleosome positioning sequences have not been clearly identified, it has been shown that short runs of (A, T) are preferentially positioned with minor grooves facing toward the octamer, while short runs of (G, C) tend to be positioned with minor grooves facing away from the octamer (Drew and "ravers, 1985). Furthermore, deletion of sequences within nucleosomes (particularly near the dyad axis) leads to loss of positioning (FitzGerald and Simpson, 1985; Ramsay, 1986).

One of the most studied regions, the 5 S rRNA gene, from a variety of species has been shown to precisely position nucleosomes (Simpson and Stafford, 1983; Rhodes, 1986; Drew and Calladine, 1987; Gottesfeld, 1987; Hayes et al., 1990, 1991; Lee et al., 1993). Several tools have been used to map the precise


locations of nucleosomes on these sequences. DNase I and hydroxyl radicals have been used to determine the rotational setting of nucleosomes (i.e. the orientation of DNA on the surface of the octamer; Thoma, 1992). These agents cleave the DNA within a nucleosome at accessible points in the minor groove, thereby displaying a 10-bp periodicity within the nucleosome compared to random cleavage in naked DNA. Dif- ficulties are associated with determining the exact limits of a nucleosome by DNase I digestion, since the ends are less firmly

A reconstituted reconstituted mock-

C G 0 1020609090602010 0

-40

-60 -50 iz -7(

-81

FIG. 5. High resolution mapping of BPDE-I-modified nucleosomal DNA. A 430-bp PCR-generated fragment was labeled a t the AuaI site (Fig. 2B), and a portion was reconstituted into nucleosomes. Mock- reconstituted and reconstituted samples were modified with 10 PM BPDE-I as described under “Materials and Methods.” BPDE-I-modified mock-reconstituted or reconstituted DNA was irradiated with laser light for 0, 10, 20, 60, or 90 s, then analyzed on denaturing polyacrylamide gels. Lanes TC and AG represent Maxam-Gilbert marker lanes. Spectrophotometrically determined binding levels revealed a 2-fold difference in the overall formation of adducts in the nucleosomal compared to control DNA.

BPDE-I binding patterns. The inte- FIG. 6. Quantitative comparison of

grated density of each dGuo-associated band in the 60-s time point of Fig. 5 was divided by the total integrated density of the appropriate lane, to give the fraction of the total represented by each band (fractional density). At each position, the ratio of the fractional density for the reconstituted sample ( R ) to the fractional density for the mock-reconstituted sample (M) is plotted. Horizontal lines indicate the average values for the regions defined in Table I. The rectangle in the upper left of the figure indicates the position of the nucleosome, with the central 100 bp indicated by shading; the dyad axis is indicated by a “+.”

attached to the octamer and thus exhibit only partial resistance to digestion. Difficulties are also encountered in hydroxyl radical footprinting, since the region apparently contacting histones (-180 bp; Hayes et al., 1990, 1991) is longer than the canonical nucleosome length of 146 bp. Micrococcal nuclease, on the other hand, has been used to determine the translational setting (the position of the octamer on the DNA) of the nucleosome, because this enzyme preferentially cleaves between nucleosomes.

In the current studies, we have used micrococcal nuclease digestion and hydroxyl radical footprinting to establish the region between positions -96 and +50 as the most likely translational position of our reconstituted nucleosome. The data in Fig. 6 indicate that this is the same region that is protected from attack by the bulky electrophile BPDE-I. This placement agrees fairly well with the placement of a nucleosome over the transcriptional start point in X. borealis 5 S DNA reported by Rhodes (1986) and Drew and Calladine (1987). This agreement is plausible since the X. laevis and X. borealis genes differ by only 20 out of 150 nucleotides in this region (Peterson et al., 1980). Formation of a nucleosome with the dyad axis close to the transcriptional start point of the 5 S rRNA gene has also been observed in the sea urchin, L. variegatus, both in vitro (Simpson and Stafford, 1983) and in vivo (Thoma and Simpson, 1985). This unique positioning of a nucleosome around the beginning of the gene between these diverse species suggests that this placement may be important in the regulation of the gene. Experimental alterations in the position and extent of histone interactions with the 5 S rRNA gene in vitro have indeed been found to alter the ability of the transcription factor TFIIIA to bind to the internal control region sequences of this gene (Rhodes, 1986; Gottesfeld, 1987; Lee et al., 1993).

Previous studies of nucleosomal positioning using shorter fragments of the X. laevis 5 S rRNA gene have identified several alternative placements of a nucleosome on this gene (Got- tesfeld, 1987; Lee et al. 1993). The differences in apparent translational positioning probably result from differences in the positions of the ends of the fragments and in the techniques used for reconstitution and analysis, rather than from differences in the source of the histones (Gottesfeld, 1987; Lee et al.

TABLE I Inhibition of BPDE-I binding to different DNA regions

Regions W M ” Nucleosome, center (-67 to +19) 0.27 f 0.04 Nucleosome, ends (-95 to -81; 28 to 44) 0.48 f 0.07 Linker (52 to 112) 0.73 f 0.13

integrated density of the corresponding bands in the reconstituted and a For each dGuo residue in the indicated regions, the ratio of the

mock-reconstituted lanes of Fig. 5 (WM) was calculated for the 20-, 60-, and 90-s time points and averaged.

0 -100 -15 -50 -25 0 25 50 15 100

Position

FIG. 7. High resolution mapping of

modified DNA. BPDE-I-modified mock- sequence-specific binding of BPDE-I-

reconstituted and reconstituted DNA were irradiated with laser light for 0, 10, 20, or 60 s. To obtain comparable levels of modification, control samples (mock-reconstituted) were modified with 10 PM BPDE-I, and reconstituted samples were modified with 20 PM BPDE-I. TClAG represent Maxam-Gilbert marker lanes. The banding pattern corresponds to dGuo residues in the marker lanes.


mock-

T A reconstituted reconstituted

I

20

1

-1 0

-20 1

-30

-40

-50

C G

d

.

0 r”.r

-60 4

10 20 60 60 20 10 ” -

I

0 --

I

1993). However, it is interesting that the nucleosome position found by Lee et al. (1993) using histone tetramers covers the entire TFIIIA binding site (+45 to +95) while the position we report does not. I t is possible that interactions with other chromatin components in nuclei may influence nucleosomal positioning. The existence of two alternative positions that differ in the accessibility of the TFIIIA control region might then function as a “molecular switch” (Meersseman et al., 1992).

After determining the translational setting of v l , we analyzed the levels of binding of BPDE-I to nucleosomal and free

DNA. When reconstituted and mock-reconstituted samples were modified with equivalent amounts of BPDE-I, regional inhibition of BPDE-I binding by nucleosomes was observed. Binding in the central 100 bp of the nucleosome was decreased about 2.5-fold. Furthermore, a transition to intermediate levels of binding was apparent toward the ends of the nucleosomes, suggesting that the central region of the nucleosome is maxi- mally protected from attack by BPDE-I. These results are consistent with the results of Moyer et al. (19891, who found that another bulky carcinogen, aflatoxin B1, bound to the central

20628 Benzo(a1pyrene Diol Epoxide Binding to Nucleosomes

-23 FIG. 8. BPDE-I binding pattern in

DNA. The mock-reconstituted (M; light free versus nucleosomal regions of

line) and reconstituted lanes (R; dark

of Fig. 7 were quantitated using software .I supplied by Molecular Dynamics (Image- Quant) which constructs the equivalent of CI

a densitometer tracing. To aid in visual comparison of the two data sets, the R tracing has been arbitrarily shifted 200 units in the vertical direction. Electropho- resis is from left to right, and the positions of selected bands are noted by urrows.

line) corresponding to 60 s of irradiation P

+

100 bp of sequence-positioned nucleosomes about 2.4-fold less than to non-nucleosomal regions. An intriguing pattern emerged upon closer inspection of ad-

duct formation within the region that is footprinted by nucleosome formation. The data in Figs. 7 and 8 demonstrate visually that the local sequence-dependent pattern of BPDE-I binding to free DNA is mirrored in nucleosomes. This can also be in- ferred from the apparent lack of periodicity in cutting seen in the central portion of the nucleosome in Fig. 7. From this observation we suggest that the rotational setting has little effect on binding of this carcinogen to DNA, although the overall binding to nucleosomes is 2-3-fold lower compared to non- nucleosomal regions. This observation was surprising, since we expected that the bulky nature of the carcinogen would limit its accessibility to dGuo residues that had their N2-amino group oriented toward the histone core. To verify a lack of periodicity in the adduct distribution, the cutting data for dGuo residues between -67 and +19 in Fig. 7 were analyzed further in terms of the orientation of the minor groove and therefore of the target exocyclic amino group. Residues within 2 nucleotides of each local maximum in the hydroxyl radical cutting distribution (Fig. 31, and therefore expected to be relatively accessible from the minor groove, were compared to residues halfway between the maxima and therefore less accessible. The mean ratio of band densities (RIC, Fig. 6) for “accessible” dGuo residues (0.26 * 0.04, n = 5) was not significantly different from the mean for “inaccessible” dGuo residues (0.25 * 0.04, n = 11).

The observation that the target specificity is maintained in nucleosomes suggests that simple accessibility of the reactive site of deoxyguanosine is not the rate-limiting step in covalent binding of BPDE-I to DNA. Rather, the rate-limiting step must involve the noncovalent interaction of the diol epoxide with the DNA, either by intercalation of BPDE-I or direct interaction with the minor groove prior to covalent adduct formation (Gea- cintov, 1988; MacLeod, 1990). These results, as well as those of Moyer et al. (1989) with aflatoxin B1, can be rationalized in terms of known features of nucleosomal structure. The struc- tures of mixed sequence nucleosomes obtained at 7-A resolution by x-ray diffraction analysis (Richmond et al., 1984; Uber- bacher and Bunick, 1989) indicate that the central turn of the DNA superhelix (-80 bp) interacts with the histone H3-H4 tetramer, while the remainder of the DNA toward the ends of the particles interacts with H2A-H2B dimers. Biochemical studies, including exonuclease digestion and thermal melting profiles, have suggested that the 20-35 bp of DNA at each end of the nucleosome is less firmly bound than the central region (Weischet et al., 1978; Simpson, 1979; Prunell, 1983). Indeed, one H2A-H2B dimer is lost from core particles at low levels of intercalative binding of ethidium bromide (McMurray and van Holde, 1986). Interestingly, the intercalation of ethidium bro-

Relative Position

mide in nucleosomal DNA preferentially involves only about 25 bp at the end of the particle (McMurray and van Holde, 1991). If intercalation of BPDE-I is necessary for covalent binding, on the basis of the ethidium bromide results, one might expect enhanced intercalation and therefore enhanced covalent binding to the terminal 20-30 bp of nucleosomal DNArelative to the central portion, in accord with our experimental data (Fig. 6). Within the central region of the nucleosomal core, the probabil- ity of formation of an intercalation site between adjacent base pairs would not be expected to depend on the rotational orientation of those base pairs. Therefore, if intercalative binding is a necessary prerequisite for covalent binding, this would ex- plain the lack of correlation of BPDE-I binding with minor groove accessibility noted above. Kinetic studies of BPDE-I binding to purified DNA have not provided a definitive answer to whether or not intercalation is necessary for covalent binding (Geacintov, 1988; MacLeod, 1990). Although the current studies are consistent with an important role for intercalation, it remains possible that noncovalent interaction of the carcinogen in the minor groove is rate-determining, both in free DNA and in nucleosomal DNA. Further studies will be necessary to clarify the mechanism of covalent binding.

Although genetic mechanisms are clearly important in carcinogenesis, the relative importance of nucleosomal positioning in gene expression suggests that epigenetic mechanisms may play a role in carcinogenesis, in that conformational changes during critical times of replication may be passed from one generation to the next. Two possible nonmutational mechanisms by which DNA binding could affect the process of carcinogenesis are: 1) a carcinogen-DNA adduct could alter the positioning of nucleosomes by increasing or decreasing the affinity of the octamer for DNA, thereby altering gene expression, andor 2) a carcinogen-DNA adduct could alter the transcription factorhinding site interaction.

Transcriptionally active RNA polymerase I1 genes have been shown to contain a more open chromatin structure, suggesting repression through inaccessibility of pertinent regulatory sequences (Lee and Garrard, 1991). These open regions are considered nucleosome-free and, therefore, more susceptible to attack by a carcinogen. However, previous results from other laboratories (Kootstra et al., 1979; Jack and Brookes, 19821, and directly confirmed in the present study, suggest that this difference in binding only amounts to a factor of 2-3. Differ- ences in binding due to sequence selectivity can be of greater magnitude. Furthermore, as shown in Fig. 8, the sequence selectivity of binding is maintained within the nucleosome. Since important regulatory regions of eukaryotic genes are of- ten GC-rich, it is conceivable that a sequence of high BPDE-I binding potential could be more highly modified in a nucleosome than a sequence of low binding potential in the linker

Benzo[alpyrene Diol Epoxide Binding to Nucleosomes 20629

region. Nevertheless, differential binding of these regions could deleteriously alter the expression of these genes. Since nucleosomal positioning is thought to control gene expression, then structural localizations would also determine binding of BPDE-I to these regions. The effect of BPDE-I binding to nucleosomes on transcription, replication, and assembly has yet to be elucidated.

Acknowledgments-We thank D. O'Connor for providing technical expertise in the use of the YAG laser, A. Beceiro for expert technical assistance, A. Wolffe for communication of data prior to publication, J. Gottesfeld for providing the 5 S DNA-containing plasmid, J. Riley and J. Ing for graphics support, and M. Gardiner for manuscript preparation.

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