Unwinding and hydrodynamic flow linear dichroism characteristics of

Chem. Res. Toxicol. 1993,6, 616-624

616

Unwinding and Hydrodynamic Flow Linear Dichroism Characteristics of Supercoiled DNA Covalently Modified with Two Isomeric Methylchrysene Diol Epoxides of Different Biological Activities Luisa Balasta,+Rong Xu,t Nicholas E. Geacintov,*lt Charles E. Swenberg,t S h a n t u Amin,$ and S t e p h e n S. Hechts Chemistry Department, New York University, New York, New York 10003, Armed Forces Radiobiology Research Institute, Bethesda, Maryland, and Naylor Dana Institute for Disease Prevention, American Health Foundation, Valhalla, New York 10595 Received March 31, 199P

Adducts derived from the covalent binding of two positional monomethyl-substituted isomers of a bay region chrysene diol epoxide to supercoiled pIBI30 DNA (2926 base pairs/genome) were prepared, and their characteristics were investigated by a combination of gel electrophoresis and flow linear dichroism techniques. The 5- and 6-methyl derivatives of trans-1,2-dihydroxyanti-3,4-epoxy-1,2,3,4-tetrahydrochrysene [(+)-ti- and (+)-6-MeCDE, respectively], both with lR,2S,3S,4R stereochemistry, are characterized by significant differences in their biological activities [Melikian et al. (1988) Cancer Res. 48,1781-17871. When covalently bound to plasmid DNA, these two molecules give rise to striking differences in the gel electrophoretic and flow hydrodynamic characteristics of the modified supercoiled DNA. The hydrodynamic flow linear dichroism of linearized DNA molecules (obtained by EcoRI enzyme digestion of covalently closed supercoiled pIBI3O DNA), modified covalently with the highly tumorigenic and mutagenic (+)-5-MeCDE derivative, indicates that flexible joints, bends, or kinks are formed at the site of binding of (+)-5-MeCDE. Slab gel data, as well as ethidium bromide-titration tube agarose gel electrophoresis data, indicate that the formation of (+)-5-MeCDE-DNA lesions causes the removal of superhelical turns with an unwinding angle of 13 f 3" per covalently bound polycyclic aromatic residue. In contrast, the biological inactive (+)-6-MeCDE does not significantly alter the characteristics of supercoiled DNA, the unwinding angle is only 2.7 f lo,and the changes in persistence lengths detected by the flow linear dichroism technique are significantly smaller than in the case of (+)-5-MeCDE-DNA adducts. The observed differences in unwinding effects and alterations in persistence lengths induced by the covalent binding of (+)-5-MeCDE and the (+)-6-MeCDE isomer parallel those observed with DNA adducts derived from the covalent binding of the highly active (+)-trans-7,8-dihydroxy-ant~-9,lO-epoxy-7,8,9,lO-tetrahydrobenzo[alpyrene [(+)-BPDEI isomer and its less active (-)-BPDE enantiomer to 4x174 supercoiled DNA [Xu et al. (1992) Nucleic Acids Res. 20, 616743176].

Introduction

6-position does not enhance the biological activity of chrysene (4-12). Furthermore, the addition of a second Chrysene, like other polycyclic aromatic hydrocarbons methyl group to the 9-position of 5-MeC does not (PAH), is metabolically activated to highly reactive, significantly diminish its tumor-initiating activity on mutagenic and tumorigenic diol epoxide derivatives (1mouse skin, although the 5,8-, 5,6-, 5,7-, and 5,lO-di3). The covalent binding of diol epoxide molecules to DNA methylchrysene derivatives are less tumorigenic than is believed to be the critical event in mutagenesis and in 5-MeC (13-15). the initiation of the complex series of events in tumorIn living cells, 5-MeC and 6-methylchrysene(6-MeC), igenesis. While chrysene is considered to be a weak as well as 5,6-dimethylchrysene, are metabolically acticarcinogen, the presence of amethyl group at the 5-position vated to diols and subsequently to the highly reactive diol [as in 5-methylchrysene (5-MeC)] strongly increases its epoxide derivatives (4, 7, 9, 11, 12, 14). The ultimate tumor-initiating activity; interestingly, substitution at the mutagenic and tumorigenic derivative of 5-MeC is the diol * Address correspondence to this author at the Chemistry Department, epoxide stereoisomer (+)-trans-1,2-dihydroxy-anti-3,429 Washington Place, Room 453,New York University, New York, NY epoxy-l,2,3,4tetrahydro-5methylchrysene[(+)-5-MeCDEl 10003. which is characterized by lR,2S,3S,4R stereochemistry. t New York University. t Armed Forces Radiobiology Research Institute. Interestingly, the analogous 1R,2S,3S,4R isomeric me1 American Health Foundation. tabolite (+)-6-MeCDE,which differs from (+)-5-MeCDE e Abstract published in Advance ACS Abstracts, August 15, 1993. Abbreviations: EB, ethidium bromide; 5- and 6-MeC, 5- and only in the position of the methyl substitution (Figure 11, 6-methylchrysene, respectively; 5-MeCDE, trans-1,2-dihydroxy-anti-3,4is only weakly mutagenic or nonmutagenic and is non6-MeCDE, trans-1,2-dihyepoxy-1,2,3,4-tetrahydro-5-methylchrysene; tumorigenic ( 6 , lI). However, in the newborn mouse lung droxy-anti-3,4-epoxy-1,2,3,4-tetrahydro-6-methylchrysene [both 5MeCDE and 6-MeCDE are (+)-enantiomers with R,S,S,R stereochemand liver, the lower tumorigenicity of (+)-6-MeCDEmay istry]; BPDE, trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahybe, at least in part, due to the lower apparent extent of drobenzo[alpyrene; PAH, polycyclic aromatic hydrocarbon; LD, linear dichroism. DNA adduct formation in vivo (9). The presence of a 0893-228x/93/2706-0616$04.00/00 1993 American Chemical Society

Unwinding and Linear Dichroism of Supercoiled DNA

Chem. Res. Toxicol., Vol. 6, No. 5,1993 617

or bends at the binding sites are strikingly different for these stereochemically identical but positionally different, methyl-substituted isomers.

Materials and Methods I

CH3

(+)-5-MeCDE

(+)-6-MeCDE

Figure 1. Structures of (+)-5-MeCDE and (+)-6-MeCDE.

methyl group at the 5-position in the bay region leads to considerable structural distortion of the chrysene aromatic ring system (16) and may be an important factor in determining the relative biological activities of 5- and 6-MeCDE (10). The bay region in 5-MeCDE is sterically crowded; in order to relieve the strain, the bay region is somewhatwider, and the aromatic ring system is probably nonplanar by loo or so (16). These structural distortions do not arise in 6-MeCDE because the methyl group is more distant from the bay region. From the point of view of understanding structurebiological activity relationships on a molecular level, the structural characteristics of adducts derived from the binding of polycyclic aromatic diol epoxide isomers of different biologicalactivities of DNA are of great interest. For example, a variety of optical spectroscopictechniques have been employed to show that different trans-7,8dihydroxy-anti-9,1O-epoxy-benzo[alpyrene(BPDE) isomers with R,S,S,R or S,R,R,S steric configurations (1720),and the 5- and 6-methyl-substituted R,S,S,R chrysene diol epoxide positional isomers (21), display strikingly different adduct conformations. High-resolution NMR techniques have been recently employed to determine the detailed structural difference between the adducts formed from the binding of different BPDE stereoisomers to DNA (22-24). The binding of PAH diol epoxide enantiomers to DNA can cause decreases in the apparent persistence lengths due to increased flexibility, hinge joints (25-28), bends, or kinks (29) at the sites of the lesions. The unwinding of supercoiled DNA (30-35) may be another effect which produces changes in the tertiary structure of DNA which may be of biological significance. Recently, we have shown that, upon chemical binding of the (+)BPDE enantiomer to supercoiled 4 x 1 7 4 DNA, the unwinding angle per bound BPDE residue is 11f 1.8O, while in the case of the (-)-BPDE enantiomer the unwinding angle is 6.8 f 1.7" (33). In this work, we describe the effects of a pair of positional (rather than enantiomeric) isomers of methyl-substituted chrysene diol epoxides on the hydrodynamic characteristics and electrophoretic mobilities of supercoiled plasmid pIBI30 DNA and EcoRI enzyme-digested linearized pIBI30 DNA molecules. Gel electrophoresis techniques were used to assess the unwinding of supercoiled DNA induced by the covalently bound (+)-5-MeCDE and (+)6-MeCDE residues. The flow linear dichroism (LD) technique is useful for assessing the conformations of the covalently bound PAH diol epoxide residues (21), the unwinding of supercoiled DNA induced by drug molecules (34) and carcinogens (35),and the formation of flexible joints, bends, and/or kinks induced by these compounds in linear DNA (25,26,28). We show here that the DNA conformations of the covalent adducts formed, degrees of unwinding of supercoils, and induction of flexible joints

Diol Epoxides. The (+)-5-MeCDE and (+)-6-MeCDE enantiomers were synthesized and purified by methods which have been previously described (12, 36,37), and were dissolved in tetrahydrofuran (THF). The concentrations of these compounds in the THF stock solutions were estimated using a molar extinction coefficient of 40 900 M-lcm-lat 260nm (38). Caution:

Diol epoxide derivatives can be highly carcinogenic and mutagenic and must be handled with care; if spillage occurs, the MeCDE derivatives can be hydrolyzed using weakly acidic solutions @H 4-5). Supercoiled a n d Linearized DNA Fragments. The plasmid pIBI30 supercoiled DNA (2926 base pairs) was isolated by alkaline lysis methods from Escherichia coli (39). The pIBI3O DNA was linearized by digestion with the restriction enzyme EcoRI; one unit of this enzyme was added per microgram of pIBI30 DNA dissolved in an assay buffer solution containing 100 mM Tris-HC1 (pH 7.5))5 mM MgC12, 50 mM NaC1, and 0.1 mg of bovine serum albumin/mL. The reaction mixture was incubated at 37 OC for 4 h, then extracted with buffer-saturated phenol and ether, and subsequently stored at 4 OC. All DNA samples were dissolved in TE buffer (5mM Tris buffer solution containing 1 mM EDTA at pH 7.9). Preparation of Covalent Adducts. Small quantities of the 5- or 6-MeCDE-THF solutions were added to solutions of DNA in T E buffer such that the THF concentration did not, as a rule, exceed 2% by volume. The initial molar ratios (ri)of MeCDE/ (DNA nucleotide) concentrations were typically varied from ri = 0.05 to 0.20 in the case of (+)-5-MeCDE, and from ri = 0.10 to0.50 in the case of (+)-6-MeCDE,althoughin a few experiments higher concentrations were also employed. The solutions were incubated at room temperature for at least 1 h. The tetraol hydrolysisproducta were removed by three successiveextractions with ethyl acetate followed by exhaustive dialysis against TE buffer. The yields of covalently bound MeCDE residues (based on the concentration of added MeCDE molecules at a DNA concentration of 1.5 X l(r M) were 21 2% in the case of (+)5-MeCDE and 7 f 1%in the case of (+)-6-MeCDE. Determination of Level of Covalent Modification (h). The molar ratio, rb = (covalently bound MeCDE molecules)/ (DNA nucleotide), was routinely determined by measuring the absorbance of the bound aromatic hydrocarbon residues at 304 nm. The molar extinction coefficients e of the DNA-bound residues at 304 nm were determined by enzymatically degrading the modified DNA to the nucleotide level, followed by HPLC separation of the modified from the unmodified nucleosides (6), and by summing the absorbance of all the covalent MeCDEnucleotide elution peaks; the major adducts involved the binding of the 5-MeCDE residues to the exocyclic amino group of guanosine as shown by Melikian et al. (6);presumably the mode of binding of 6-MeCDE involves similar mechanisms, although this has not yet been demonstrated explicitly. For enzyme digestion,the modified DNA samples were suspended in solutions of 20 mM sodiumsuccinate and 10mM CaCl2 (pH6.0). Digestion was carried out for 7 h at 37 "C in the presence of 15 units of phosphodiesterase I1 (bovine spleen) and 4500 units of micrococcal nuclease. A 10 X 25 cm Vydac 201TP1010 10-pm column (Separations Group, Hesperia, CA) and a Waters Associates HPLC system (Millipore, Waters Division, Milford, MA) were used to separate the adducts from one another using a 0-100% 5 mM phosphate buffer-methanol gradient (elution time 90 min, rate of elution 3 mL/min). The concentrations of the adducts in the HPLC elution peaks were determined by their UV absorbances at 254 nm using a molar extinction coefficient for MeCDE-nucleotide adducts of 4 7 OOO M-lcm-l(40). The molar extinction Coefficients of the DNA-bound residues at 304 nm were found to be equal to 3500 300 for the (+)-5-MeCDE-

*

618 Chem. Res. Toxicol., Vol. 6, No. 5, 1993

DNA adducts and 2500 300 M-l cm-l for the (+)-6-MeCDEDNA adducts. Wedge Gel Electrophoresis. Wedge-shaped, agarose gel electrophoresis experiments were performed with an LKB 2012 Maxiphor Submarine electrophoresis unit (Pharmacia/LKB, Piscataway, NJ). The advantages of using wedge-shaped gels have been described earlier (41,42). The dimensions of the gels were 15 X 20 cm, with one end 10-13 mm, and the other 3-4 mm thick. The 1% agarose gels were cast in 89 mM Tris base, 89 mM sodium borate, and 2 mM EDTA, pH 8.0 buffer solution (TBE buffer). About 10 pL of samples containing =lo0 ng of DNA were injected into the wells. The TBE loading buffer also contained 2.5% Ficolland 0.005% bromophenolblue. The power supply was a Buchler Model 3-1500 (Haakebuchler, Saddle River, NJ) and was operated at 40 V (about 20 mA) for 18 h. After electrophoresis, the gels were washed with deionized water for 5 min. Subsequently, a 0.8 pg/mL ethidium bromide (EB) solution was added and subjected to shaking for 20-30 min. The gels were then destained using water for 30 min. The gel was then illuminated with UV light and photographed. The negatives were scanned with a Fisher Model EC910 densitometer (E-C Apparatus Corp., St. Petersburg, Corp., FL), and the signals were processed utilizing a computer and appropriate software. Tube Gel Electrophoresis. In order to verify that the observed gel electrophoretic mobilities were due to unwinding of supercoiled DNA, the EB titration tube gel electrophoresis method used by Espejo and Lebowitz (43) and DeLeyes and Jackson (44) was employed. The tube gels were prepared by heating 1% agarose solutions (TBE buffer, 0.018 M NaCl containing different amounts of EB in the range of concentrations of 0.001-0.10 pg/L EB) into 20-cm-long glass rods with one of their ends wrapped twice with parafilm. After 45 min, the ends of the tubes were unwrapped, the tubular gels were extruded, and each was cut to a length of 18 cm. The ends were wrapped with small pieces of nylon mesh stockings held in place by rubber bands. The glass rods were then inserted into the tube gel adapter used with the vertical gel apparatus, and the upper and lower chambers were filled with the 0.018 M NaC1-TBE solution.About 200 ng of modified or unmodified DNA in 10 pL of solution containing 4.5% Ficoll and 2.4 pM EB (EB/DNAnucleotide ratio =0.2) were added to the tops of the tubes. Electrophoresis was performed at 95-102 V (2.5 mA/tube, 16 tubes at a time) for 3 h a t 20 "C.After electrophoresis, the gels were extruded into 16 separate large test tubes, the bands were developed by adding a 0.8 pg/mL EB solution for 30 min with shaking, and the gels were then photographed under UV illumination. Determination of Superhelical Density. In order to determine the superhelical density of the supercoiled DNA, the pIBI30 DNA was partially relaxed by reactions with topoisomerase I (Bethesda Research Laboratories, Bethesda, MD) utilizing a protocol similar to that of Keller and Wendell (43). Reactions were carried out in solutions (pH 7.9) containingabout 1pg of DNA, 30 units of topoisomerase I, 10 mM Tris buffer, 0.2 M NaCl, and 2 mM EDTA. The reactions were carried out at 0 "C for various time intervals (5-30 min) and were stopped by adding a solution of 10% N-lauroylsarcosine and 20 mM EDTA (46). Wedge gel electrophoresis was employed to separate DNA molecules with different topological winding numbers, and the number of bands was counted visually. Flow Linear Dichroism. The DNA was placed within the annular space of a home-built quartz Couette cell (outer cylinder: 23-mm inner diameter; inner cylinder: 22 mm outer diameter), and partial orientation of the supercoiled DNA was achieved by rotating the inner cylinder at 400 rpm (flow gradient: 900 s-'), while the outer cylinder remained stationary (33,34). The linear dichroism is defined as LD = All - Al, where All and Al are the absorbances with the linearly polarized light oriented in a parallel or perpendicular direction with respect to the flow lines. Other details, involving the electronic measurement and processing of the LD signals, are described elsewhere (47). Absolute calibration of the LD apparatus was achieved by means of a polarizing crystal substituted for the sample.

Balasta et al.

7

BAND NUMBER Figure 2. Slab gel electrophoresis of supercoiled pIB130 DNA treated with topoisomerase1. Observed band positionsexpressed as a percent of the distance between the unmodified nicked relaxed and supercoiled DNA bands.

Results Determination of Superhelical Density. T h e topoisomerase-treated pIBI3O DNA was subjected to wedge gel electrophoresis. In general, besides the nicked, relaxed DNA band, 11-13 other bands (depending on the sample) could be resolved visually upon staining with ethidium bromide (data not shown). As an example, the positions of these bands as a function of t h e percent distance migrated relative t o the distances migrated by the unmodified nicked (slowest) and t h e native, fully supercoiled bands (highest mobility) are shown in Figure 2. Since there are 2926 base pairs in pIBI3O DNAmolecule, a n d t h e number of superturns per DNA molecule is 12 f 1,the superhelical density or the number of superturns per 10 base pairs, u, is calculated to be equal to -0.041 f 0.003. This number is similar in value t o those characterizing other types of supercoiled DNA (48). Gel Electrophoretic Mobilities. Typical wedge gel electrophoretic patterns of supercoiled pIBI30 DNA covalently modified with (+)-5-MeCDE and (+)-6-MeCDE are shown in Figure 3. The upper bands are due to t h e more slowly migrating nicked closed circular DNA, while the lower, faster migrating bands are due to t h e covalently closed supercoiled DNA. Faint bands below the nicked closed circular DNA bands are discernible in some of the lanes a n d are attributed to linear double-stranded DNA. Relaxed, Nickel Closed Circular DNA Bands. In the case of (+)-5-MeCDE, the mobility of the more slowly migrating nicked DNA band increases with increasing levels of modification up to rt, values near 0.06 and then decreases as t h e level of modification is increased still further (Figure 3A). Increases in the mobilities of BPDEmodified nicked closed circular DNA have been previously reported (30, 31) and attributed t o flexible hinge-like behavior at t h e alkylation sites (31). T h e diminishing mobilities at rt, > 0.06 might be due to t h e effects of increasing masses of t h e modified DNA molecules at high levels of binding. These effects were not investigated further.

In the case of t h e (+)-6-MeCDE-modified DNA, there are no observable changes in t h e mobilities of the nicked bands (Figure 3B). However, there are significant changes in the relative abundance5 of t h e nicked and supercoiled bands as rb is increased. Supercoiled DNA Band. T h e mobility of the supercoiled DNA modified covalently with (+)-5-MeCDEgradually decreases as q,is increased and coincides with that


Chem. Res. Toxicol., Vol. 6, No. 5, 1993 619

(A) (+)-5-MeCDE-pIBI30DNA 1

2

3

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t 100

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‘b Figure 4. Relative electrophoreticmobilitiesof (+)-6-MeCDEpIBI30 DNA adducts ( 0 ) and (+)-5-MeCDE-pIBI30 DNA adducts (D) as a function of binding levels (b).The lowest data points (+) represent the relative mobilities of the nicked band [(+)-5-MeCDE-DNA adducts].

Figure 3. Electrophoretic mobilities (slab gels) of MeCDEtreated covalent pIBI30 supercoiled DNA with different levels of covalently bound residues (rb = adduct/nucleotide). (A) (+)5-MeCDE-pIBI30 adducts,lanes 1-11, rbvalues: (1) 0, (2) 0.011, (3) 0.021, (4) 0.032, (5) 0.042, (6) 0.053, (7) 0.063, (8) 0.074, (9) 0.084, (10) 0.095, (11) 0.11. (B) (+)-6-MeCDE-pIBI30 adducts, lanes 1-7, rb values: (1) 0, (2) 0.014, (3) 0.028, (4) 0.042, (5) 0.056, (6) 0.07, (7) 0.084.

of the nicked band near q, = 0.06. Beyond this value of rb, the relative mobility increases and the distribution of mobilities broadens with increasing level of modification. This is due to the rewinding of relaxed closed circular DNA in the right-handed sense,an effect which is observed when intercalating drugs bind to superhelical DNA (4851),and which has also been noted for the covalent binding of BDPE to supercoiled DNA (30,31,33). Furthermore, it is evident from Figure 3A that the widths of the 5-MeCDE-modified supercoiled DNA bands are broadened significantly. In contrast, (+)-6-MeCDE produces a much smaller change in the mobilities of the supercoiled bands (Figure 3B), and there is little broadening of these bands. However, the amplitudes of the nicked bands increase significantly as rb is increased; the amount of supercoiled DNA remaining at the highest levels of modification is quite small (Figure 3B). Densitomer tracings of the data shown in Figure 3 were obtained (data not shown), and the values corresponding to the maxima in the mobility distributions are plotted as a function of the level of covalent binding in Figure 4. In the case of the (+)-5-MeCDE adducts, the minimum in the mobilities is reached at rb = 0.063 f 0.007. Since it is assumed that all superhelical turns have been removed at this value of q,,the overall apparent unwinding angle is calculated to be 8 = 12 f 2O. However, besides unwinding, other changes in the tertiary structures of the modified DNA brought about by the covalently bound (+)-5-MeCDE residues might affect the electrophoretic mobilities of supercoiled DNA. Therefore, the number of superhelical turns removed per covalently bound diol epoxide residue was also studied by the EB titration tube gel electrophoresis method. A crude estimate of the unwinding angle in the case of the (+)-6-MeCDE-pIBI30 adducts can be made by comparing the relative electrophoretic mobility at the highest

level of modification (86 % , Figure 4), with that of the topoisomers (Figure 2). It is estimated that about 3.5 superhelical turns are removed for an q,value of 0.084 f 0.01, leading to an unwinding angle of 2.6 f 0.4’. Tube Gel Electrophoresis. In this technique, identical unmodified or modified DNA samples are subjected to agarose gel electrophoresis in individual tubes, each containing EB molecules at different concentrations C. The number of superhelical turns can be estimated from the EB concentration C = C’ in the tube in which the DNA is completely unwound (coincidence of the mobilities of the nicked and covalentlyclosed circular bands) employing the formula (43,44):

+

= YmKC’/(l KC’)

(1) where Y, is the critical molar [drug]/[DNA nucleotide] ratio at the EB concentration C’, vm = 0.18 is the maximum ratio of bound EB/DNA nucleotide under saturating conditions of EB concentration, and K (M-l) is the mean association constant of EB estimated from Scatchard plots. These latter plots were found to be linear (datanot shown), and the apparent value of the binding constant K in pIBI30 DNA in the buffer used in our experiments was found to be equal to (3.0 f 0.1) X lo6 M-l with Ym = 0.18 f 0.01; the same values of K and vm were also found in the case of 4x174 DNA (33). The superhelical density ts is then calculated from the relationship (48): Y,

ts = - 1 . 4 4 ~ ~ (2) which is valid for an unwindingangle of 26O per intercalated EB molecule. Typical tube gel electrophoresis data for unmodified and (+)-5-MeCDE- and (+)-6-MeCDE-modified supercoiled pIBI30 DNA are depicted in Figures 5 and 6, respectively. The tube gel experiments were run in two stages (an example is shown in Figure 5): (A) a coarse scale of incremental increases of the EB concentration C in each gel was employed in order to determine the approximate value of C’; (B) a second set of experiments was performed with the same DNA sample using a narrower range of ethidium concentrations in the vicinity of the value of C’ estimated from the first experiment. Unmodified pIBI30 DNA. In the case of unmodified DNA, the supercoiled DNA is completely relaxed when


(A) 1

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Figure 5. Tube gel electrophoresis of unmodified supercoiled pIBI30 DNA. (A) Coarse intervals of ethidium bromide concentrations, in pg/mL: (1)0.002, (2) 0.003, (3) 0.004, (4) 0.005, (5) 0.006, (6) 0.007, (7) 0.008, (8) 0.009, (9) 0.010, (10) 0.020, (11) 0.030, (12) 0.040, (13) 0.050, (14) 0.070, (15) EB in loading buffer only, (16) no EB in loading buffer or gel. (B) Fine intervals of ethidium bromide concentrations, in pg/mL: (1)0.012, (2) 0.014, (3) 0.016, (4) 0.018, (5) 0.020, (6) 0.022, (7) 0.024, (8) 0.026, (9) 0.028, (10)0.030, (11)0.032, (12) 0.034, (13)0.036, (14) 0.038, (15) EB in loading buffer only, (16) no EB in loading buffer or gel. (A) (+)-5-MeCDE-pIBI30 DNA 1

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Figure 6. Tube gel electrophoresis of covalently modified supercoiledpIBI30 DNA adducts. (A) Covalent (+)-5-MeCDEpIBI30 DNA adducts, rb = 0.021. Ethidium bromide concentrations in pg/mL: (1)0.009, (2) 0.010, (3) 0.011, (4) 0.012, (5) 0.013, (6) 0.014, (7) 0.015, (8) 0.016, (9) 0.017, (10) 0.018, (11) 0.019, (12)0.020, (13)0.021, (14)0.022, (15)no EB in gel or buffer, (16)unmodifiedpIBI30 DNA, no EB in gel or buffer. (B) Covalent (+)-6-MeCDE-pIBI30 DNA adducts, Q, = 0.042. Ethidium bromide concentrations in pg/mL: (1)0.008, (2) 0.010, (3) 0.012, (4) 0.014, ( 5 ) 0.016, (6) 0.018, (7) 0.020, (8) 0.022, (9) 0.024, (10) 0.026, (11)0.028, (12) 0.030, (13) 0.032, (14) 0.034, (15) no EB, (16) unmodified pIBI30 DNA, no EB.

the EB concentration in the gel is C' = 0.025 f 0.002 pg/ mL (lanes 7-9, Figure 5B), or (6.3 f 0.5) X lo4 M. Some

Balasta et al.

of the individual bands, characterized by differences in the topological winding number a (51), can be resolved in some of the lanes. The correspondingvalue of v,, obtained from eq 1, is 0.029 f 0.002, and the superhelical density calculated from eq 2 is e = -0.041 f 0.003. This result is in excellent agreement with the value of e obtained by the band counting method (Figure 2). Covalent (+)-5-MeCDE-ModifiedpIBI30 DNA Adducts. In the case of the covalentlybound (+)-5-MeCDE residues, the mobilities of the relaxed (nicked) and closed circular DNA bands for a modified sample with q,= 0.021 f 0.002 coincide at an EB concentration of C' = 0.0140.016 pg/mL [(3.6-4.0) X 10-8 MI (Figure 6A, lanes 6-8). The corresponding values of vc are 0.017-0.019, and the decrease in the superhelical density is e(q,= 0.021) - e(q, = 0) = -0.026 - (-0.041) = 0.015 f 0.002. This corresponds to a removal of an average of 4.4 f 0.5 superhelical turns per DNA molecule for (0.021 residues/nucleotide) X (2926 base pairs/molecule) X (2 nucleotides/base pair) = 123 f 12 covalently bound (+)-5-MeCDE residues per DNA molecule. The effective unwinding angle is 8 = 13 f 2' per covalently bound (+)-5-MeCDE residue. This value of 8 is close to the to the value of 12 f 2' estimated from the slab gel electrophoresis experiments (Figure 4). Covalent (+)-6-MeCDE-ModifiedpIBI30 DNA Adducts. In the case of a (+)-6-MeCDE-DNA adduct sample with Q, = 0.042 f 0.004, the mobilities of the supercoiled and nicked circular DNA bands coincide at an EB concentration of C' = 0.020-0.022 pg/mL (Figure 6B, lanes 7 and 8). Carrying out the calculations as outlined in the previous paragraph yields an unwinding angle of 2.5 f 0.6' per covalently bound (+)-6-MeCDE residue, in good agreement with the estimate from the slab gel data (2.6 f 0.4'). In the above analysisof the tube gel data, it was assumed that the EB binding constant K is not significantlyaffected by the presence of the covalently bound MeCDE residues, as has been shown experimentallyin the relevant low range of values (52). Flow Linear Dichroism Characteristics. (A) Linear Dichroism of Unmodified and Modified pIBI3O DNA Titrated with EB. As previously described (34, 35), the magnitude of the linear dichroism signal of unmodified DNA measured at 258 nm is a function of the superhelicaldensity. The sign of the LD signal is negative because of the average inclinations of the planes of the base pairs with respect to the flow direction (28). As discussed elsewhere (34,35),the changes in LD parallel the variations in the sedimentation (&) coefficientswhen supercoiled DNA is titrated with EB (49),resulting in unwinding and rewinding of the DNA as the EB concentration is increased. The minimum in the LD signal occurs when r(EB) = 0.029 f 0.003 (mol of EB added/mol of DNA nucleotide),correspondingto the completerelaxation of the covalently closed DNA molecules, and the hydrodynamic characteristics of the fully relaxed DNA are the same as those of the nicked DNA molecules. Since, under the conditions of these experiments, 95% of the added EB moleculesare bound to the DNA (53),the LD minimum occurs at the critical EB binding ratio vc = 0.029 f 0.003, which is in excellent agreement with the value of vc = 0.027 f 0.002 obtained from the tube gel electrophoresis experiments (Figure 5). A flow linear dichroism EB titration curve with a (+)6-MeCDE-pIBI30 DNA adduct [q,(G-MeCDE) = 0.0251 closely resembles the EB titration curve obtained with


1

01

v

t

-2 -2.5' 0

Chem. Res. Toxicol., Vol. 6, No. 5, 1993 621

"

0.02

"

0.04

'

I

0.06 r(W

"

0.08

"

0.1

"

-2 -

0.12

Figure 7. Flow linear dichroism (LD) measured at 258 nm of supercoiledpIBI3O DNA (1.9 X 1WM)as a function of ethidium bromide concentration r (mol of EB addedhucleotide). ( 0 ) unmodified DNA; (0)(+)-6-MeCDE-DNA adduct, rt, = 0.025; (m) (+)+MeCDE-DNA adduct, rt, = 0.020.

unmodified pIBI30 DNA (Figure 7, compare open and closed squares). This result suggeststhat covalentlybound (+)-6-MeCDE residues have little apparent effect either on the subsequent binding of EB, nor on the superhelical density, or on the unwinding of the DNA induced by intercalated EB molecules. In the case of the covalent (+)-5-MeCDE-pIBI30 DNA adducts [rb(5-MeCDE) = 0.0201, however, the EB titration curve is more shallow near the minimum [r(EB) = 0.023 - 0.0291. (B) Flow Linear Dichroism of MeCDE-Modified Supercoiled and Linearized pIBI30 DNA as a Function of a.It is well established that the magnitude of the LD signal measured within the DNA absorption band (e.g., at the 258-nm maximum) is a function of its persistence length. It is known that changes in flexibilities, or the formation of bends or kinks induced by covalently bound carcinogens, can manifest themselves as decreases in the abilities of the modified DNA molecules to align themselves in the hydrodynamic flow gradient of the Couette cell (2528). Hogan et al. showed that the binding of racemicBPDE to DNA results in decreased electrophoretic mobilities of the modified DNA (29). Since bends, kinks, or increased flexibilities of the modified DNA are expected to decrease the magnitudes of the measured LD signals (25-28), while unwinding tends to produce the opposite effect (Figure 7), it was of interest to compare the effects of covalent 5and 6-MeCDE adduct formation on the LD signals of modified supercoiled and linear DNA (Figure 8). Of course, in the case of EcoRI-linearized pIBI3O DNA fragments, increases in the LD signals due to unwinding of supercoils cannot occur. The dependence of the LD signals, measured within the DNA absorption band at 258 nm, for unmodified DNA titrated with EB, and (+)-5-MeCDE-modified,and (+)6-MeCDE-modified supercoiled DNA as a function of the binding levels rb, are compared in Figure 8A. The magnitude of the LD signal of the unmodified DNA increases with increasing r(EB), while (+)-6-MeCDE residues, at least up to rb = 0.03, do not cause any measurable unwinding or changes in the persistence lengths. The lack of change of the LD signals as a funcntion of increasing rt,[(+)-6-MeCDEl is consistent with the small changes in the electrophoretic mobilities (Figure 4). In the case of covalent adduct formation with (+)-5-MeCDE, a decrease in the magnitude of the LD signal is observed with increasing rt, (Figure 8A). Since the gel electrophoresis experiments (Figures 3,4,and 6A) clearly indicate that unwinding is occurring in this range of rb values, it

-1.11

0

0.01

0.02

0.03

0.04

r "I

I

(B)

-20

0

I

I

I

0.01

0.02

0.03

I 0.04

'b Figure 8. Flow linear dichroism measured at 258 nm as a function of r (EB,or covalentlybound MeCDE residues/nucleotide).DNA concentrations: 1.5 X 10-4 M in terms of the concentration of

nucleotides. (A) SupercoiledpIBI30 DNA ( 0 )unmodified DNA titrated with EB; (m) (+)-5-MeCDE-DNAadducts with different rb values; (0)(+)-6-MeCDE-DNA adducts with different rt, values. (B) Flow linear dichroism of EcoRI-linearized pIBI3O DNA subsequently modified covalently with (+)-5-MeCDE(w) or (+)-6-MeCDE(0)as a function of level of covalent binding level n,. is evident that the effects of bending, kinking, or flexibility increases exhibit a greater effect on the LD signal than the unwinding of the supercoiled DNA induced by the (+)-5-MeCDE residues. This latter conclusion is supported by the analogous experiments performed with linear DNA (Figure 8B). Covalently closed circular pIBI30 DNA molecules were cleaved with the restriction enzyme EcoRI to produce linear DNA fragments of homogeneous and defined chain length (verified by gel electrophoresis, data not shown). As observed previously with the highly tumorigenic (+)BPDE stereoisomer, and to a much lesser extent with the inactive (-)-BPDE enantiomer (25,261, the magnitudes of the LD signals at 258 nm decrease as a function of fi (Figure 8B). However, it is quite striking that this effect is much more pronounced in the case of the (+)-5-MeCDEmodified EcoRI DNA fragments than in the case of the (+)-6-MeCDE-EcoRI linear DNA adducts. These results suggest that the formation of covalently bound adducts derived from the biologically highly active (+)-5-MeCDE stereoisomer is accompanied by the appearance of kinks, flexible hinge joints, or bends, at the site of the lesion. In contrast, the biologically less active (+)-6-MeCDE positional isomer is much less effective in generating such changes in the tertiary structure of DNA [we mention in passing that, in principle, static bends can be differentiated from flexiblehinge join& by ligation and gel electrophoresis analysis of site-specificallymodified oligonucleotides(54)l.


Balasta et al.

Table I. Summary of Unwinding Angles Due to (+)-5-MeCDEand (+)-6-MeCDEResidues Bound Covalently to pIBI30 Supercoiled DNA method

angle 6 (deg)

(A) (+)-5-MeCDE-DNA Adducts = 0.063 & 0.007 slab gela = 0.020 f 0.002 tube gel R, = 0.040 & 0.004 tube gel av value (B)(+)-6-MeCDE-DNA Adducts R, = 0.084 & 0.008 slab gelb a = 0.042 & 0.004 tube gel R, = 0.070 & 0.007 tube gel av value R, R,

b

12f2 14 f 2 13 f 3 13 f 3

2.6 f 0.4 2.5 f 0.6 3.0 f 0.6 2.7 f 1.0

From the minimum in the electrophoretic mobility (Figure 4). Estimated from Figure 4.

Discussion The superhelical densities of unmodified supercoiled pIBI30 DNA, and DNA covalently modified with (+)-5MeCDE and (+)-6-MeCDEdetermined by slab gel or tube gel techniques, as well as the associated unwinding angles, are summarized in Table I for a few values of rb. The values of the superhelical densities and the unwinding angles obtained by the slab gel method and the ethidium titration method (tube gels) are in good agreement with one another. The average unwinding angles for (+)-5MeCDE- and (+)-6-MeCDE-modified pIBI30 DNA are 13 f 3' and 2.7 f l.Oo, respectively. Differences between (+)-5-MeCDE- and (+)-6MeCDE-pIBI30 DNA Adducts. Besides the striking differences in the unwinding angles associated with the covalent DNA adducts derived from these two positionally different, but enantiomerically identical, MeCDE isomers, there are other important dissimilarities in their characteristics, as summarized in the following paragraphs. (A) Conformations of Adducts. The conformations of (+)-5-MeCDE- and (+)-6-MeCDE-modified linear DNA (21) and supercoiled pIBI30 DNA (52) have been investigated by flow linear dichroism techniques. In both types of DNA, the long in-plane axis of the phenanthrenyl ring system of the (+)-5-MeCDE residue is tilted at an angle of 45-48' relative to the DNA helix axis. In contrast, the (+)-6-MeCDE residues appear to be unoriented [or oriented at the magic angle of 4 5 ' (2111. As in the case of covalently bound (+)-trans-BPDE residues (20,22,23), the orientation angle of 45-48O observed in the case of the covalentlybound (+)-5-MeCDE residues is consistent with an external adduct conformation; however, this hypothesis remains to be verified by NMR methods. (B) Flexible Hinge Joints or Bends at the Lesion Sites. The ethidium titration tube gel experiments yield unwinding angles similar to those evaluated from the slab gel experiments (Figures 5 and 6, Table I); these results suggest that the decreased electrophoretic mobilities observed in the case of the MeCDE-modified supercoiled pIBI30 DNA adducts on slab gels are dominated by unwinding effects, rather than by changes in flexibilities or bending of the DNA a t the sites of the lesions. While the magnitude of the LD signals is also sensitive to unwinding effects when the intercalator EB is added to the solutions (Figure 7), the LD signals of supercoiled DNA modified covalently with (+)-5-MeCDE residues (in the absence of EB) reflect primarily changes in the DNA persistence length. In contrast, the covalently bound (+)6-MeCDE residues give rise to much smaller changes in

persistence length (Figure 8) and unwinding effects (Figures 3B, 4, and 6B). The difference between the two types of adducts is also evident in the tube gel data of Figure 6. The (+)-6MeCDE-pIBI30 DNA adducts exhibit resolved individual bands (Figure 6B) due to different topoisomers which are separated in the presence of EB (51), while the (+)-5MeCDE-modified DNA exhibits broadened and smeared bands (Figure 6A). (C) Formation of Nicks. While the (+)-6-MeCDEmodified pIBI30 DNA is characterized by only a small unwinding angle (Table I) and the persistence length of the modified DNA is not significantly affected, there is a prominent nicking effect. At a modification level of Q E 0.08 (+)-6-MeCDE residue per nucleotide, most of the DNA is nicked (Figure 3). While nicking is also observed in the case of (+)-5-MeCDE-modifiedDNA, this effect is much less pronounced; a more quantitative investigation of this phenomenon was beyond the scope of this work. (D) Extent of Covalent Binding to DNA. The (+)5-MeCDE isomer binds predominantly via the 4-position to the exocyclic amino group of deoxyguanosine in native DNA, and (+)-5-MeCDE is about 4 times as reactive as (+)-6-MeCDE with respect to native DNA (6); a similar mode of chemical binding of the (+)-6-MeCDE isomer to deoxyguanosine is likely, but remains to be demonstrated. In the newborn mouse lung, formation of 5-MeCDE-DNA adducts is about 20 times greater than that of 6-MeCDEDNA adducts (9). Differences in binding levels should therefore be carefully considered in assessing the differences in the mutagenic potentials and tumorigenic activities of these positional isomers in future experiments. Nevertheless, it is possible that the higher biological activity of (+)-5-MeCDE is related, at least in part, to the significant structural alterations this isomer induces upon binding to native DNA described here, in contrast to the negligiblysmall effects induced by (+)-6-MeCDE. These striking differences may be related not only to the difference in the positions of the methyl groups in these two compounds as such, but also to the pronounced distortions in the planarity of the aromatic ring system and the benzylic diol epoxide ring brought about by the presence of a methyl group in the bay region of 5-MeCDE (16). (E) Unwinding Angles. The unwinding angles of 13 f 3 O for (+)-5-MeCDE-modified supercoiled DNA is significantly smaller than the value of 26' observed for the classical noncovalent intercalator EB (48), and the values of 22 f 3" (30) and >30' (31) reported for the covalent binding of racemic anti-BPDE to SV40 supercoiled DNA. However, using the enantiomer (+)-antiBPDE covalently bound to 4x174 supercoiled DNA, we have recently reported that the unwinding angle per residue is only 11.5 f 2.4' (33);this smaller unwinding angle is consistent with the minor groove-conformation of the major (+)-anti-BPDE-N2-dG adducts (22, 55), as discussed by Xu et al. (33). The mechanism of unwinding involving the (+)-BPDE residues located in the minor groove can be accounted for (33) in terms of the widening of the minor groove which may cause changes in the repulsive interactions between nearby phosphate groups, thus allowing for a local untwisting of the double helix due to enhanced base-stacking interactions (56). The LD characteristics of the DNA adducts derived from (+)-antiBPDE and (+)-5-MeCDE are similar ( 2 1 , 5 2 ) . However,

Unwinding and Linear Dichroism of Supercoiled DNA high-resolution NMR data on the latter adducts are not yet available, and it is therefore premature to speculate whether the covalently bound (+)-5-MeCDE residues are also situated in the minor groove. However, if this turns out to be the case, then the unwinding mechanisms due to the covalent binding of (+)-anti-BPDE and (+)-5MeCDE could be similar.

Conclusions It is interesting to compare the biological effects and the DNA-binding characteristics of the stereochemically identical positional isomers (+)-5-MeCDE and (+)-6MeCDE on the one hand, and the (+)-anti-BPDE (R,S,S,R) and (-)-anti-BPDE (S,.R,.R,S) optical isomers on the other hand. Both (+)-5-MeCDE (6,37) and (+)anti-BPDE (57-62) are highly tumorigenic and mutagenic in mammalian cell and Salmonella typhimurium T A 100 systems. Both MeCDE isomers (6) and anti-BPDE enantiomers (60,63) bind predominantly to guanosine in native DNA, and the differences in adduct conformations have been discussed either here or in other publications (17-23). It is noteworthy that both (+)-5-MeCDE and (+)-anti-BPDE (33)give rise to similar unwinding angles (11-15'), which are larger than the unwinding angles observed for the covalent adducts derived from (+)-6MeCDE and (-)-anti-BPDE. In addition, the apparent formation of hinge joints or bends is much more pronounced in the case of the biologically more active (+)5-MeCDE and (+)-anti-BPDE (25,261 isomers than in the case of the less active positional [(+)-6-MeCDEl or optical [(-)-anti-BPDEI isomers. It remains to be seen whether these observed differences in DNA damage play a role in determining the differences in the biological activities of these pairs of positional or stereochemical isomers. Acknowledgment. This work was supported by the Department of Energy, Office of Health and Environmental Research (Grant DE-FG02-86ER60405), and, in part, by the U.S. Public Health Service [Grants CA20851 (NYU) and CA44377 (AHF)], awarded by the National Cancer Institute, Department of Health and Human Services.

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Unwinding and hydrodynamic flow linear dichroism characteristics of

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